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Page 100 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POSTERS MONDAY & TUESDAY
POS-MON-01
DIGESTIVE VACUOLE GENESIS AND ENDOCYTIC
PROCESSES IN THE EARLY INTRAERYTHROCYTIC
STAGES OF PLASMODIUM FALCIPARUM
Abu Bakar N.1, Klonis N.1, 2, Hanssen E.1, 2, Chan C.1 and Tilley L.1, 2
1
Department of Biochemistry, La trobe University. 2Centre of
Excellence for Coherent X-ray Science, La Trobe University.
The digestive vacuole of the malaria parasite Plasmodium falciparum
is the site of hemoglobin digestion and heme detoxification and is the
target of chloroquine and other antimalarials. The mechanisms for
genesis of the digestive vacuole and transfer of hemoglobin from the
host cytoplasm are still debated. In the present study, we use live-cell
imaging and photobleaching to monitor the uptake of the pH-sensitive
fluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in
ring-stage and trophozoite-stage parasites. We compare these results
with electron tomography of serial sections of parasites at different
stages of growth. We show that uptake of erythrocyte cytoplasm is
initiated in mid-ring-stage parasites. The host cytoplasm is internalised
via cytostome-derived invaginations and concentrated into several
acidified peripheral structures. Hemoglobin digestion and hemozoin
formation take place in these vesicles. The ring-stage parasites can
adopt a deeply invaginated cup shape but do not take up hemoglobin via
macropinocytosis. As the parasite matures, the hemozoin-containing
compartments coalesce to form a single acidic digestive vacuole that
is fed by hemoglobin-containing vesicles. There is also evidence for
hemoglobin degradation in compartments outside the digestive vacuole.
The work has implications for the stage specificity of quinoline and
endoperoxide antimalarials.
POS-MON-03
L1CAM ACTS AS A SOX10 MODIFIER GENE DURING
ENTERIC NERVOUS SYSTEM DEVELOPMENT
Anderson R.B.1, Wegner M.2 and Wallace A.S.1
1
University of Melbourne, Australia. 2University of Erlangen-Nurnberg,
Germany.
During development, the enteric nervous system (ENS) is derived from
neural crest cells that emigrate from the hindbrain, enter the foregut and
migrate caudally to colonise the entire gut. Failure of neural crest cells
to fully colonise the gastrointestinal tract results in an ‘aganglionic zone’
that lacks an enteric nervous system over a variable length of the distal
bowel, a condition in humans known as Hirschsprung’s disease. The
variability observed in the penetrance and severity of Hirschsprung’s
disease strongly suggests a role for modifier genes. Human clinical and
animal model studies have suggested that the X-linked gene, L1CAM,
may act as a modifier gene for the development of Hirschsprung’s
disease. To examine whether L1cam interacts with the Hirschsprung’s
associated gene, Sox10, we used a two-locus complementation
approach. To assay the effects of an interaction, we examined whether
the migration of enteric neural crest cells was altered in L1cam null
mutant mice when combined with a heterozygous mutation in Sox10.
We show that interactions between L1cam and Sox10 significantly delay
neural crest migration within the developing gut, and that neural crest
cells undergo excessive cell death prior to gut entry. Using a doxycycline
inducible system, we show that Sox10 can regulate the expression of
L1cam. Thus, L1cam can act as a modifier gene for the Hirschsprung’s
associated gene, Sox10, and is likely to play a role in the aetiology of
Hirschsprung’s disease.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 101
POS-TUE-02
ROLES OF WNT SIGNALLING IN SPERMATOGENESIS
Abud H.E.1, Kerr G.1, Horvay K.1 and Loveland K.L.1, 2, 3
1
Monash University, Department of Anatomy and Developmental
Biology, Clayton Vic 3800. 2Monash University, Department of
Biochemistry, Clayton Vic 3800. 3ARC Centre of Excellence in
Biotechnology and Development.
Male infertility is a worldwide problem with increasing incidence and
may be caused in part by disruptions in the development of male
germ cells and their supporting somatic cells. We have discovered
that testicular cell communication via the Wnt signalling pathway is
required for sperm development, because genetically altered mice
with perturbed Wnt signalling exhibit interrupted spermatogenesis and
appear to have reduced fertility. We are currently using two unique
mouse models to understand the precise functional role of Wnt signalling
during spermatogenesis. We are studying the effect of blocking Wnt
signalling by conditional mutation of beta-catenin, a key mediator
of canonical Wnt signalling. In contrast, the effects of constitutively
active Wnt signalling will be studied by conditional mutation of the
negative regulator, Adenomatous Polyposis Coli (APC). In addition, the
endogenous Wnt signalling components and downstream target genes
present in the mouse testis are being examined using a combination
of immunohistochemical and qRT-PCR techniques. Our results to date
implicate canonical Wnt signalling in post-mitotic germ cell development,
and ongoing work with human clinical specimens will reveal the potential
contribution of disturbances in this pathway to male infertility.
POS-TUE-04
SEXUALLY-DIMORPHIC EXPRESSION OF MIR202* IS
ASSOCIATED WITH TESTIS DIFFERENTIATION IN THE
MALE CHICKEN EMBRYO
Bannister S.C.1, 3, Smith C.A.2, Buermans H.4, Doran T.J.1, Sinclair A.H.2, 3
and Tizard M.L.V.1
1
CSIRO Livestock Industries, Australian Animal Health Laboratory,
Geelong, Australia, 3220. 2Early Development & Disease, Murdoch
Children’s Research Institute, Royal Children’s Hospital, Parkville,
Victoria, Australia, 3052. 3Department of Paediatrics, The University of
Melbourne, Parkville, Victoria, Australia, 3052. 4Center for Human and
Clinical Genetics, Leiden University Medical Centre, Leiden, Netherlands.
In the chicken, sex is determined genetically by the inheritance of sex
chromosomes at fertilization. In birds the sex chromosomes are Z and
W and assort as ZW in females and ZZ in males. Establishment of the
sexual phenotype of the embryonic gonads is subsequently driven by the
expression of sex-determining genes. In the chick, the gonads begin to
differentiate into testes and ovaries at embryonic day 6.5 (E6.5). After this
point, testes develop bilaterally in the male, whilst asymmetric development
proceeds in the female, with only the left gonad forming a functional ovary.
We are studying the chicken embryo as a model for vertebrate gonadal
development and aim to identify how microRNAs (miRNAs) may be involved
in defining, or maintaining the differentiation of embryonic testes and
ovaries. MiRNAs are 21-24nt non-coding RNAs which potentiate sequence-
specific, post-transcriptional gene repression during development. We
have used microarray and next-generation sequencing technologies to
compare male and female miRNA expression profiles across three stages
of gonadal sex differentiation in the chick. We have since focused our
studies on chicken MIR202*, which was found to be up-regulated in testes
cords of male gonads, from the onset of sex differentiation. Our recent
work shows MIR202* expression is suppressed during estrogen-induced
feminization of male embryonic gonads and promoted in female gonads
when estrogen synthesis is blocked by an Aromatase inhibitor. These
findings suggest MIR202* expression is associated with male embryonic
testes development. Our continuing studies are focused on elucidating the
cellular and molecular function of MIR202* during sexual differentiation.
We are currently using in ovo retroviral-mediated delivery to optimise
over-expression of MIR202* during early gonadogenesis. This will allow
us to identify and validate MIR202* gene targets and understand the sex-
specific regulation of MIR202* expression and processing.
POSTERS MONDAY & TUESDAY
POS-MON-05
TWIST1 IS REQUIRED BOTH IN THE CRANIAL NEURAL
CREST AND THE CRANIAL MESODERM FOR PROPER
HEAD DEVELOPMENT
Bildsoe H.1, 2 , Loebel D.A.F.1, 2, Jones V.1, Hor A.1, Steiner K.1, Chen Y.T.3,
Beringer R.R.3 and Tam P.P.1, 2
1
Embryology Unit, Children’s Medical Research Institute, Locked Bag
23 Wentworthville, NSW 2145, Australia. 2Sydney Medical School,
University of Sydney, NSW, Australia. 3Department of Molecular
Genetics, University of Texas, M.D.Anderson Cancer Center, 1515
Holcombe Blvd, Houston, TX 77030, USA.
The basic helix-loop helix transcription factor Twist1 is a key regulator
of craniofacial development. Twist1-null mouse embryos exhibit failure
of cephalic neural tube closure and abnormal head development. The
mutant embryos die at E11.0 preventing studies beyond midgestation.
We have used Cre-loxP conditional deletions to dissect the function of
Twist1 specifically in the cranial neural crest (CNC) and cranial mesoderm
(CM). Deletion of Twist1 in CNC cells resulted in loss or malformations
of the CNC-derived skeleton, including the frontal bone, upper jaw and
snout, due to impaired CNC cell survival and differentiation. Parietal
and interparietal bones are also affected although these structures
are primarily of mesodermal origin. Loss of Twist1 in the CM leads to
incomplete closure of the cephalic neural tube but normal development
of 1st branchial arch and frontonasal tissues. The majority of these
embryos died at mid-gestation. Embryos developing past midgestation
show loss or reduction of parts of the chondrocranium and poor
development of the posterior skull base, while the viscerocranium was
not affected. Taken together, the phenotypes of these two tissue-specific
conditional deletions recapitulate that of the null mutation. Our results
further show that tissue-specific loss of Twist1 function in one of the two
cell populations that make up the craniofacial skeleton can impact on
both tissues, suggesting that interaction between primordial tissues is
critical for proper craniofacial development.
POS-MON-07
SIDEROPHORE DISCOVERY BY APPLICATION
OF IMMOBILIZED METAL ION AFFINITY
CHROMATOGRAPHY
Braich N.E. and Codd R.
School of Medical Science (Pharmacology) and Bosch Institute, The
University of Sydney, NSW 2006, Australia.
Siderophores are low-molecular-weight Fe(III) binding secondary
metabolites produced by bacteria and plants. Siderophores are
pharmacologically important and have numerous applications. Currently
they are clinically used for the treatment of iron overload diseases.
However, they are difficult to synthesize and/or purify. Thus, studies
were conducted on the use of Ni2+-charged immobilized metal ion
affinity chromatography (IMAC) for the capture of hydroxamate-based
siderophores as a fast and effective means of biodiscovery. Results
showed the successful capture of monohydroxamate, dihydroxamate,
and trihydroxamate based siderophore standards and optimal capture
conditions were also established. IMAC was successfully used for the
purification of desferrioxamine B (DFO B), a trihydromate, from crude
samples of culture supernatant of the DFOB-producing bacterium
Streptomyces pilosus. RP-HPLC traces indicate a significant purification
of >50 species reduced to 3 species, of which 2 are media derived.
Salinispora triopica is an actinomycete bacteria which is predicted by
bioinformatics to produce multiple siderophores including DFO and
yersiniabactin like siderophores. Preliminary results show that S. tropica
produces DFOB, but also produces several unidentified siderophore-
like molecules. Currently, investigations are underway on identifying the
remaining siderophores. This work opens the door for the use of IMAC
in discovering a range of secondary metabolites with metal ion binding
affinity.
Page 102 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-06
FGF9 ANTAGONISES FEMALE AND PROMOTES MALE
GERM CELL FATE IN MICE
Bowles J., Feng C.-W., Davidson T.-L., Spiller C. and Koopman P.
Institute for Molecular Bioscience, University of Queensland, Australia.
In the fetal gonad, germ cells commit to a female or male sexual fate
on the basis of environmental cues, rather than XX or XY chromosome
constitution. Germ cells in a developing ovary enter meiosis, hence
committing to the female fate or oogenesis. In a developing testis, germ
cells do not enter meiosis during fetal life but they do stop proliferating
and arrest in G0/G1 (mitotic quiescence), hence committing to the male
fate or spermatogenesis. In recent years we showed germ cells in a
female mouse embryonic gonad are triggered to enter meiosis by the
potent signaling molecule retinoic acid (RA). RA induces germ cells to
express a key gene, Stra8, which encodes a protein essential for initiation
of meiosis. In the developing testis, germ cells avoid entering meiosis
because RA is actively degraded by a cytochrome P450 enzyme,
CYP26B1. Hence, CYP26B1 is a ‘meiosis-inhibiting’ substance by virtue
of its ability to degrade the meiosis inducer, RA. Here, we report that a
second ‘meiosis-inhibiting’ substance, FGF9, acts directly on germ cells
to prevent up-regulation of Stra8 and hence their entry into meiosis.
In addition, FGF9 maintains germ cell pluripotency and promotes
expression of male fate markers. Since RA is more abundant in the
developing ovary and FGF9 is more abundant in the developing testis,
the model we propose allows for ‘back-up’ and hence robustness in the
oogenic/spermatogenic fate decision. Antagonistic interplay between
FGFs and RA is proving to be a recurring theme in development, in each
instance being associated with key cell lineage decisions. Our findings
provide a new example of this phenomenon.
POS-TUE-08
TWIST FUNCTION IN CRANIOFACIAL MORPHOGENESIS
Brinas I.B., Wade C.W. and Farlie P.F.
Murdoch Childrens Research Institute, Royal Children’s Hospital,
Flemington Rd, Parkville, Victoria, 3052.
Twist1 has been demonstrated to play critical roles in the early
development of neural crest and mesodermally derived tissues. Twist2
has been less well characterised but its relatively late onset of expression
suggests specific roles in the development of a number of sites. We
have used RCAS-mediated overexpression to investigate the function of
Twist genes in craniofacial development. Sustained expression of Twist1
or 2 results in failure of fusion between the maxillary, lateral nasal and
frontonasal processes. The Twist1 phenotype tends to be more severe
than the Twist 2 phenotype. Analysis of the craniofacial skeleton reveals
a failure of fusion between the premaxilla and the maxillary/palatine
bones. In addition, the premaxilla, maxilla and palatine bones were
hypoplastic. These facial clefts were either uni- or bilateral in nature and
exhibited substantial variation in the severity in terms of the width of the
cleft and the extent of hypoplasia. Apart from some skeletal hypoplasia,
the mandible appears to be predominantly unaffected by Twist1 or 2
overexpression. There are no consistent changes in either Fgf or Shh
or Bmp signalling around the time of facial fusion. The molecular basis
of this phenotype remains unclear. Apoptosis and proliferation was
quantified to determine the cause of facial clefts in Twist1 overexpressed
embryos. There was no change in apoptosis of Twist1 overexpressed
and control embryos. There was, however, an unexpected 3-fold
increase in the number of proliferating cells within the lateral nasal and
frontonasal masses of Twist1 overexpressed embryos. It is likely that
Twist2 overexpression is also upregulating proliferation but we will test
this. Overall, Twist1 and 2 appear to have important roles in craniofacial
morphogenesis and particularly in the fusion of the facial processes.
POSTERS MONDAY & TUESDAY
POS-MON-09
DISCOVERY OF A NOVEL A-TYPE LAMIN
INTERACTING TRANSCRIPTION FACTOR, LITF,
REQUIRED FOR MYOGENESIS
Ahmady E.1, Deeke S.A.1, Rabaa S.1, Kouri L.1, Krotneva S.1, Kenney
L.1, Blais A.2, Stewart A.F.R.1 and Burgon P.G.1
1
University of Ottawa Heart Institute, Ottawa ON Canada. 2University
of Ottawa, Ottawa ON Canada.
Lamins are intermediate filament proteins of the inner nuclear
membrane fundamentally important for nuclear architecture, chromatin
organization and transcriptional regulation of gene expression.
However, the molecular mechanisms that couple lamins to transcription
are poorly understood. Here, we report the identification of an A-type
Lamin-interacting Transcription Factor, LITF, a unique single copy
amniota gene. Chromatin immunoprecipitation and gel mobility shift
assays demonstrated that LITF binds to DNA within close proximity of
genes encoding many transcription factors that control tissue-specific
differentiation. Transient transfections of GAL4-LITF fusion constructs
with a GAL4 responsive reporter showed that LITF contains a strong
transactivation function within its conserved C-terminal domain. LITF
protein is primarily expressed in cardiac and skeletal muscle and
is expressed as early as E8.5 in the mouse embryo. Using the well-
characterized C2C12 model of in vitro muscle differentiation, we
found that blocking LITF expression in myoblasts also prevented the
expression of myogenic transcription factors (Mef2C, MyoD, Myf6 &
MyoG) and subsequently inhibited myogenic differentiation. In addition,
LITF mediated chromatin immunoprecipitation studies revealed that
LITF interacts with many DNA regions in close proximity of transcriptions
factors (GATA4, Pitx2, Mef2c, RXRα and Sox4) that are important for
normal muscle formation and differentiation. Data will be presented
from the phenotypic analysis of a muscle specific LITF null. These
observations coupled with the molecular uniqueness of LITF suggest
that LITF lies in a prominent position within the regulatory process of
muscle development. Our discovery of LITF provides the first direct
molecular link between the Lamin A/C gene and gene expression.
POS-MON-11
POSITIONAL CLONING OF LYMPHATIC MUTANTS IN
ZEBRAFISH
Coxam B.1, Neyt C.1, Shulte-Merker S.2 and Hogan B.1
1
Institute for Molecular Bioscience, The University of Queensland, 306
Carmody Road, St Lucia, 4072, Brisbane, QLD, Australia. 2Hubrecht
Institute Uppsalalaan 83584 CT UTRECHT,The Netherlands.
Lymphatic vessels play an essential role in fluid homeostasis, immune
responses, fat absorption, and in pathological processes such as
lymphedema and cancer metastasis. Despite recent breakthroughs
in unravelling the complexity of lymphatic vessel development (called
lymphangiogenesis), a lot is left to discover about the early steps of this
process and its underlying cellular dynamics and molecular regulation.
Using a genetic screen in zebrafish, we have identified two mutants
presenting novel lymphatic phenotypes. One mutant presented a block
in lymphangiogenesis associated with larval stage cardiac dysfunction
and the other exhibited pleiotropic developmental defects encompassing
a block in lymphangiogenesis, blood vasculature hyper-branching
and a dysmorphic head. Initial molecular genetic data and a detailed
phenotypic analysis at the cellular level using vascular transgenic
fish lines will be presented. Keywords: Lymphatic system, Zebrafish,
Positional Cloning.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 103
POS-TUE-10
PROLIFERATION OF NEURONS AND NON-NEURONAL
CELLS IN THE DEVELOPING MOUSE STELLATE
GANGLION
Gonsalvez D.G., Cane K.N. and Anderson C.R.
University of Melbourne. Parkville VIC, 3010.
Mouse sympathetic ganglia appear around embryonic day (E)9.5,
with differentiated neurons present at E10.5 and the first glial cells
at E11.5. From E10.5 onwards, the number of cells in the ganglion
increases dramatically. We have used immunoreactivity to Sox10, to
mark uncommitted neural crest cells and glia, and tyrosine hydroxylase
(TH), to mark sympathetic neurons in E10.5 to E18.5 stellate ganglia
(n=4 in each case). We have also estimated cell division rates with
bromodeoxyuridine (BrdU) and S-phase length with BrdU and
ethyldeoxyuridine(EdU). At E10.5, 99% of all cells are positive for Sox10
expression, with half of this population also expressing TH; only 1% of
cells express TH alone. By E11.5, 19% of cells express Sox10 alone,
6% express both Sox10 and TH and 76% of cells express TH only.
The proliferation rate of both TH-IR and Sox10-IR cells was highest
around E12.5, when around 50% of both neuronal and non-neuronal
cells in the ganglion contained BrdU after a two hour pulse. We have
determined that S-phase lengths for neuronal and non-neuronal cells
differ during this period (TH+-6hrs, Sox10+-3hrs). Total cell cycle lengths
for the two cell populations do not differ greatly at either of the two time
points considered. Proliferation of non-neuronal cells overtook that of
neurons on E16.5, when neuronal division had dropped to a low rate.
Between E11.5 and E14.5, the growth of the ganglion is largely due to
the division of existing neurons. The increasing disparity between the
number of neurons and non-neuronal cells during this period does not
depend on differences in proliferation rate or cell cycle length, but solely
on the relative starting numbers of neurons versus non-neuronal cells,
which is established on E10.5 when most, but not all, of the neural crest
precursor cells differentiate into neurons.
POS-TUE-12
CHARACTERISATION OF THE DEVELOPMENTAL
ROLES OF THE DROSOPHILA MACPF PROTEIN
TORSO-LIKE
Crossman T.1, 2 , Johnson T.K.1, 2, Herr A.1, 2, Whisstock J.C.2 and Warr
C.G.1
1
School of Biological Sciences, Monash University, Clayton, Victoria,
Australia 3800. 2Department of Biochemistry and Molecular Biology,
Monash University, Clayton, Victoria, Australia 3800.
Torso-like (tsl) is the sole Drosophila member of the Membrane Attack
Complex and Perforin (MACPF) protein superfamily. While members
of the MACPF family are typically involved in immunity and defence,
tsl has long been established as a maternally expressed gene and key
component of the pathway responsible for patterning the anterior and
posterior poles of the developing oocyte. However, our recent research
has indicated additional roles for tsl in development. Expression studies
using in situ hybridization experiments and a reporter gene line have
shown that tsl is expressed in a number of specific tissue and cell types.
In the developing embryo we see tsl expression in the midline glia of the
central nervous system, a specialized set of glia that are analogous to
the vertebrate floor plate. During larval stages we see tsl expression in
the prothoracic gland, a part of the ring gland, which is the hormonal
control centre of the developing fly. In adults we see expression of tsl in
a subset of cells in the retina. Functional studies on tsl mutant strains
have shown serious central nervous system defects, indicating a role
for tsl in midline glia survival or function. To study the role of tsl in the
prothoracic gland we are performing tissue-specific RNA interference
experiments. The role of tsl in the adult retina is being characterised
using available mutant strains and the many markers available for
different cell types in the retina. Overall these approaches aim to enable
the complete characterisation of the roles of tsl from embryogenesis to
adulthood, and to provide insight into the roles of MACPF proteins in
development.
POSTERS MONDAY & TUESDAY
POS-MON-13
INTEGRIN LINKED KINASE (ILK) IS REQUIRED
FOR LENS EPITHELIAL CELL SURVIVAL AND
PROLIFERATION
Teo J.1, McQueen-Miscamble L.1, Turner K.1, Martinez G.1, Reneker L.2,
Dedhar S.3, Robinson M.L.4 and De Iongh R.U.1
1
Anatomy & Cell Biology, University of Melbourne, Parkville, Victoria,
Australia. 2Ophthalmology, University of Missouri. 3British Columbia
Cancer Research Center, Vancouver, Canada. 4Zoology, Miami
University, Oxford, Ohio.
The role of growth factors in lens development has been investigated
extensively; the role of ECM signaling is less well understood. The laminin-
binding integrins (α3β1, α6β1) are cooperatively required for epithelial cell
survival during embryonic development. In this study we investigated
the role of integrin linked kinase (ILK), a downstream mediator of ECM-
integrin signaling, in lens development. We generated mice that were
conditionally null for Ilk in the lens or expressed a hyperactive ILK in the
lens. Consistent with its epithelial expression, Ilk deletion only resulted in
a phenotype if deleted in lens epithelium, not when deleted in fibres only.
The phenotype was characterised by thinning of the epithelium by E17.5,
loss of central epithelial cells by P2 and extensive fibre cell degeneration
and apoptosis by P10. At E17.5 there was significant inhibition (~50%)
of epithelial cell cycle progression (reduced BrdU, cyclin D1, cyclin D2
and phospho-histone3 staining), but no significant changes in epithelial
markers (E-cadherin, Pax6). Loss of Ilk also affected deposition of ECM
(laminin and collagen IV), with retention of collagen in exocytic pathway.
Erk and Akt phosphorylation were markedly decreased in Ilk null lenses.
Postnatally, there was reduced and delayed expression of fibre cell
markers, β-crystallin, c-Maf, p57kip2 and p27kip1, but not Prox1, and abnormal
accumulation of fibronectin and α-smooth muscle actin. Ectopic expression
of hyperactive kinase ILK (S343D) did not affect lens development
with only subtle changes in epithelial cell morphology detected in vitro
(increased formation of lamellipodia, enriched ILK at focal contacts). The
hyperactive ILK transgene completely rescued lens morphology in Ilk null
mice cell, but proliferation was incompletely rescued. These data indicate
IK is required for epithelial proliferation, survival and subsequent fibre cell
differentiation. ILK is required for epithelial proliferation and survival, but is
insufficient to initiate cell proliferation.
POS-MON-15
REGULATION OF INTERNEURON DEVELOPMENT BY
SUPPRESSOR OF CYTOKINE SIGNALLING-2 (SOCS2)
Faux C.H. and Turnley A.M.
Centre for Neuroscience, The University of Melbourne.
Gamma-aminobutyric acid (GABA)ergic interneurons play a vital role in
modulating the activity of the cerebral cortex. Comprising approximately
20% of the total cortical neuron population, they are an extremely
diverse population of cells, differing in their morphology, physiology and
molecular characteristics. Recent studies have shown that a disruption
to the function of GABAergic interneurons can directly contribute to
neurological and mental health issues such as schizophrenia, epilepsy
and depression. Often such disruptions arise during development, with
alterations to interneuron number, distribution or connectivity leading to
functional neural impairment. Currently, however, little is known about
the signalling mechanisms that regulate interneuron specification,
migration or maturation. We have previously shown that the regulatory
protein suppressor of cytokine signalling-2 (SOCS2) is a key player
in interneuron development. SOCS2-overexpressing mice have large
increases in numbers of calbindin and calretinin-expressing interneurons
in the adult cortex. In contrast, SOCS2 knockout mice show a reduction
in the number of parvalbumin positive cells. We have found that during
development SOCS2 is highly expressed in the ganglionic eminence
(GE) of the ventral telencephalon, which is the primary source of cortical
interneurons. In addition, SOCS2 is expressed in the developing cortical
plate and in the subventricular zone, one of the main regions through
which interneurons tangentially migrate to enter the cortex. These data
suggest that SOCS2 is involved in the early specification and/or in the
migration of particular interneuron subtypes. Further elucidation of the
precise role that SOCS2 plays in interneuron development will greatly
enhance our understanding of the complex mechanisms underlying
various neurological disorders.
Page 104 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-14
NARINGIN PROMOTES ANTICARCINOGENIC EFFECTS
ON RATS BEARING WALKER 256 TUMOR
Camargo C.A.1, Wutzki N.C.1, Bueno L.G.M.1, Gomes-Marcondes
M.C.C.2, Da Silva M.E.F.3 and Aoyama H.1
1
Departamento de Bioquímica, Instituto de Biologia, UNICAMP –
Campinas, São Paulo, Brazil. 2Departamento de Anatomia, Biologia
Celular e Fisiologia, Instituto de Biologia, UNICAMP – Campinas, São
Paulo, Brazil. 3Instituto de Ciências da Saúde, UNIP - Campinas, São
Paulo, Brazil.
Naringin (NAR), a plant polyphenolic compound, is a naturally occurring
citrus flavonone (oranges and grapefruits). Flavonoids have been
reported to exhibit numerous biological and pharmacological effects, such
as antioxidant, anticarcinogenic, antiinflammatory and cardioprotective.
Cachexia is a poorly understood syndrome characterized by generalized
host wasting, anorexia and a variety of metabolic alterations that result
in death. One characteristic of Walker 256 tumor (W256) is to provoke
cachexia in infected animals. More than 80% of the patients with
malignant disease present features of malnutrition and cachexia as the
cause of death. This study was designed to describe an in vivo naringin
therapeutic treatment of rats bearing W256. Rats bearing W256 were
treated with different daily doses (10, 25 and 35 mg/kg) of naringin
i.p. for 50 days or until the obit. Survival, dose-response (ED50) and
tumor regression curves were obtained and 25 mg/kg was taken as the
effective dose. Our studies showed that daily administrations of naringin,
in low doses (10-25 mg/kg), had inhibited tumors growth (about 50%)
in comparison with the control animals. Moreover, two animals of this
group had presented complete tumor regression and when inoculated
again with the carcinosarcoma, no tumor development was observed.
However, at a higher concentration (35 mg/kg) this flavonoid provoked
corporal weakening and cachexia process in the animals. Our studies
suggest that naringin can be used as an effective drug to treat cancer
and to prevent against the effects of cachexia. Financial Support:
CAPES, CNPq and FAPESP.
POS-TUE-16
REPAIRING HYPEROXIA-MEDIATED RESPIRATORY
DEFICIT WITH ENDOTHELIAL PROGENITOR CELLS
Firsova A.B., Cole T.J. and Mollard R.A.
Monash University, Victoria, Australia.
High tension oxygen treatment is performed to assist breathing of
very prematurely born babies. However, such treatment can result in
disruptions to lung vascularization and alveolarization and eventually lead
to chronic lung disease. Steps are taken to reinforce cell differentiation
and repair lung tissue injured following oxygen treatment, but these
steps remain sub-optimal. Previous results suggest that administration of
endothelial progenitor cells (EPCs) from bone marrow to the diabetic foot
can stimulate neovascularisation and thus lead to beneficial functional
outcomes. We have hypothesized therefore that EPCs may engraft the
site of hyperoxia-induced lung damage to similarly offer benefit. In this
study, a model of hyperoxia-mediated deficit of the neonatal mouse lung
has been established to study such EPC engraftment and investigate
associated functional outcomes. Newborn mice were treated with 90%
oxygen for four days at birth and parameters of lung development were
monitored for an eight week period. Relative to untreated controls, we
observed (i) temporary changes in lung vascularization (blood vessel
number and Pecam1 protein levels decreased) and (ii) persistent
changes in lung septation (alveolar number and tissue area decreased
and alveoli diameter increased). Treated animals also displayed an
accelerated increase in the number of secondary septa between one day
and eight weeks after hyperoxia exposure, such that an early secondary
septal number deficit was later returned to control levels. Data from
bone marrow sorting and subsequent culture have identified Kdr and
Tek as key cell surface markers for vessel-like structure formation in
vitro. This model recapitulates many aspects of mechanical ventilation-
associated alterations to the very preterm birth human lung and permits
study of the effects of attenuations to neovascularisation upon high
oxygen damaged alveoli.
POSTERS MONDAY & TUESDAY
POS-MON-17
GENETIC INTERACTION BETWEEN TRANSCRIPTION
FACTOR (Lhx1) AND WNT INHIBITOR (Dkk1) IN HEAD
FORMATION IN MOUSE EMBRYOS
Fossat N., Khoo P.-L., Jones V., Lewis S.L. and Tam P.P.L.
Embryology Unit, Children’s Medical Research Institute, University of
Sydney, Westmead, NSW 2145, Australia
In mouse embryos, Lhx1 gene encoding the LIM homeobox 1 transcription
factor is expressed in the anterior visceral endoderm (AVE) and the
anterior axial mesendoderm (AME). The expression pattern of Lhx1
partly overlaps with that of Dkk1 (Dickkopf-1) that encodes an inhibitor of
the WNT signalling pathway. Loss of Lhx1 function in the whole embryo
leads to truncation of the head at the mid- to hindbrain level. However,
chimeric embryos containing wild type visceral endoderm (including
AVE) and Lhx1-deficient embryonic tissues also display head defects,
suggesting that Lhx1 function is required more than in the AVE, which
is likely to involve the AME. To better characterise the non-AVE related
function of Lhx1, the transcriptome of Lhx1-/flox;Meox2+/Cre (Lhx1 CKO)
embryos in which Lhx1 is inactivated in the embryo proper but not in the
visceral endoderm was analyzed. In the Lhx1-deficient tissues, the gene
expression profile was reminiscent of an increase of the WNT signalling
activity. Testing the expression of the BATGal transgene where LacZ
expression reports WNT signalling, ectopic activation of WNT activity
was found in the anterior region of the Lhx1 CKO mutant embryos. A
functional connection between the transcriptional function of Lhx1 and
WNT signalling was further demonstrated by the manifestation of the
head truncation phenotype in compound heterozygous Dkk1+/-;Lhx1+/CKO
mutant embryos which was not found in either Dkk1+/- or Lhx1+/CKO simple
heterozygous mutants. The synergistic interaction between Lhx1 and
Dkk1, principally in the AME, is therefore essential for the formation of
the embryonic head through the negative modulation of WNT signalling
activity.
POS-MON-19
DNA DAMAGE-INDEPENDENT FUNCTIONS OF ASCIZ
AS AN ESSENTIAL REGULATOR OF PULMONARY
ORGANOGENESIS
Jurado S.1, Smyth I.2, Van Denderen B.1, Tenis N.1, Hewitt K.1, Cole
T.J.2 and Heierhorst J.1, 3
1
St. Vincent’s Institute of Medical Research. 2Monash University. 3Dept
of Medicine, The University of Melbourne.
Zn2+-finger proteins comprise one of the largest protein superfamilies
with diverse biological functions. The Zn2+-finger protein ASCIZ (ATMIN/
ZNF822) was originally discovered as a Chk2-interacting ATM substrate
with functions in the DNA base damage response and also proposed to
be an essential cofactor of the ATM kinase. Here we show that absence
of ASCIZ leads to p53-independent late embryonic lethality in mice.
ASCIZ-deficient primary fibroblasts exhibit increased sensitivity to DNA
base damaging agents MMS and H2O2, but ASCIZ deletion or knock-
down does not affect ATM levels and activation in mouse, chicken or
human cells. Unexpectedly, ASCIZ-deficient embryos also exhibit
severe respiratory tract defects, where lung buds never emerge from the
respiratory endoderm and where ascending separation of the tracheal
bud from the ventral foregut stalls early. Genetically, the complete
pulmonary agenesis and severe tracheal atresia place ASCIZ between
Wnt/ß-catenin and FGF10 signaling pathways. The data indicate that, in
addition to its role in the DNA damage response, ASCIZ has separate
developmental functions as an essential regulator of respiratory
organogenesis.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 105
POS-TUE-18
DOES GENETIC VARIATION IN METABOLITE
PROFILES CORRELATE WITH SUCROSE CONTENT?
Glassop D.1, 2 , Zwart A.3 and Bonnett G.D.1, 2
1
CSIRO Plant Industry, St Lucia, Queensland, 4067. 2CRC Sugar
Industry Innovation through Biotechnology, St Lucia, Queensland,
4067. 3CSIRO Mathematics Informatics and Statistics, Acton,
Australian Captial Territory, 2601.
Sugarcane provides most of the world’s supply of sugar. Sugarcane
is able to accumulate sucrose in mature stem tissue, reaching
concentrations of ~50% of the dry weight. In recent years the yield of
sucrose from sugarcane has only increased due to increases in the yield
of biomass rather than increased sucrose concentration of the biomass.
This has lead the search for new approaches to break this upper limit
for the concentration of sucrose stored. The evolution of the ‘omics’
sciences provides an opportunity to extensively examine sugarcane
via genomics, transcriptomics, proteomics and metabolomics in
the search for alternative strategies for increasing sucrose content.
Previous research revealed metabolic profiles specific to stem tissues
of different developmental age. Those results encouraged research
to determine the variation in metabolite profiles between sugarcane
progeny segregating for sucrose content. Using gas chromatography
mass spectrometry we have detected and quantified 105 metabolites
in sugarcane stem tissue from internodes four (immature) and nine
(mature) from five high and five low sucrose genotypes. Of the 105
metabolites detected and measured, 46 could be assigned an identity on
the basis of mass spectra and retention time; 59 remained unidentified.
Some of the identified metabolites have not been previously identified in
sugarcane metabolite profiles. This poster will report on the relationship
between metabolites and sucrose content and identify pathways which
could potentially be manipulated for increased sucrose production.
POS-TUE-20
FUNCTIONAL IDENTITY OF THE GAMMA
TROPOMYOSIN (γ-TM) GENE: SPECIFIC ROLES IN
EMBRYONIC DEVELOPMENT, REPRODUCTION AND
CELL VIABILITY
Hook J.1, Schevzov G.1, Hardeman E.C.2 and Gunning P.W.1
1
Oncology Research Unit, School of Medical Sciences, University of
New South Wales. 2Neuromuscular & Regenerative Medicine Unit,
School of Medical Sciences, University of New South Wales.
Tropomyosins (Tm) are highly conserved components of actin
filaments which differentially regulate filament stability and function.
The mammalian Tm family consists of four genes; α-Tm, β-Tm, γ-Tm
and δ-Tm. Multiple Tm isoforms (>40) are generated by alternative
splicing, and expression of these isoforms is highly regulated during
development. We tested the specificity of function of products from the
γ-Tm gene in mice using a series of gene knockouts. Mice homozygous
for the knockout of all cytoskeletal products from the γ-Tm gene
(Tm5NM1-11) were not viable and could not be detected as early as
blastocysts. Elimination of just two cytoskeletal products from the γ-Tm
gene (NM1,2) resulted in a 50% reduction in embryo viability. We re-
targeted the γ-Tm gene in embryonic stem (ES) cells hemizygous for the
knockout of all or subsets of isoforms from this gene. It was not possible
to create knockout ES cells for the targets which eliminated or reduced
embryo viability in mice. In contrast, homozygous ES cells were created
for a different set of isoforms (NM3,5,6,8,9,11) which were not required
for embryogenesis. We also observed that males hemizygous for the
knockout of all cytoskeletal products from the γ-Tm gene preferentially
transmitted the minus allele with 80-100% transmission. Since all four
Tm genes are expressed in early embryos, ES cells and sperm, we
conclude that isoforms of the γ-Tm gene perform specific functions in
embryogenesis, ES cell viability and sperm function that cannot be
compensated by the other Tm genes.
POSTERS MONDAY & TUESDAY
POS-MON-21
AMOUNT OF DNA METHYLTRANSFERASE 1
TRANSCRIPTS IN VARIOUS DEVELOPMENTAL
STAGES OF MOUSE TISSUES AND THEIR
EXPRESSION SITES
Isokane T.1, Ghanem.E M.2, Nishibori M.1, Hiraiwa N.3 and Yasue H.4
1
Grad. Sch. of Bio Sci., Hiroshima Univ., Hiroshima, Japan. 2Suez
Canal Univ, Egypt. 3RIKEN, Japan. 4Nat. Inst. Agrobiol. Sci, Japan.
DNA cytosine methylation in mammals influences many cellular events,
including gene transcription, genomic imprinting, X chromosome
inactivation, carcinogenesis and genome stability. A member of DNA
methyltransferase (Dnmts) has been identified that catalyzes the
reaction of cytosine methylation in DNA. When the maintenance-type
DNA methyltransferase, designated as Dnmt1 is absent in mouse,
homozygous mutant embryos cannot survive after mid-gestation as their
genomic imprinting being canceled. Epigenetic modifications of chromatin
consist of DNA methylation and post- translational modifications to
histones like acetylation, phosphorylation, glycosylation, ribosylation.
These modifications are key regulators of gene expression during
growth and differentiation in all tissues including brain. In the present
study, to elucidate the functions of Dnmt1, the amounts Dnmt1 sense/
antisense transcripts in the mouse’s tissue of different developmental
stages were measured by real-time RT-PCR. The localization of Dnmt1
sense transcripts as well as the existence of antisense transcripts was
also investigated. In results, mouse brain cells showed a remarkable
Dnmt1 gene expression values during different stages of embryonic
development. Antisense transcript was largely unaltered. The localization
of Dnmt1 transcripts in adult medulla oblongata, sense transcript was
observed remarkably in neuron, however antisense transcript observed
faintly around glia cell. In Cerebral cortex, the expression of these
transcripts was observed. These findings indicated that Dnmt1 has a
great role in brain development of adulthood. However, its role during
the embryonic stages still needs more investigation.
POS-MON-23
THE ROLE OF THE DROSOPHILA MEMBRANE
ATTACK PROTEIN TORSO-LIKE IN EARLY EMBRYONIC
PATTERNING
Johnson T.K.1, 2, Bennett M.2, Herr A.1,2, Warr C.G.2 and Whisstock J.C.1
1
Department of Biochemistry and Molecular Biology, Monash
University, Clayton VIC 3800, Australia. 2School of Biological
Sciences, Monash University, Clayton VIC 3800, Australia.
Membrane Attack Complex/Perforin-like (MACPF) proteins have
important roles in vertebrate immunity and often function by forming
oligomeric pores in cell membranes causing cell lysis. However, several
MACPF proteins play crucial yet poorly characterised roles in embryonic
patterning and neural development. In Drosophila, correct formation of
the terminal anterior and posterior structures of the embryo is governed
by Torso-like (Tsl), the only known Drosophila MACPF. Tsl is expressed
maternally in specific ovarian follicle cells at the poles of the developing
oocyte, where it is secreted and deposited in the perivitelline space
between the oocyte and future eggshell. After fertilisation Tsl mediates
localised activation of Trunk (Trk), a noggin-like growth factor, and
putative ligand of the Torso receptor tyrosine kinase. The mechanism
of Trk activation, and the role of Tsl, remains elusive, however one
hypothesis is that Trk is proteolytically cleaved by an unidentified
protease. We have used in vivo functional studies such as RNAi and
overexpression approaches to screen Drosophila serine proteases
and serine protease inhibitors for roles in terminal patterning. In so
doing we have found that over-expression of Serpin 4 from follicle cells
causes eggshell and possibly terminal patterning defects. We are also
expressing predicted cleavage site mutants of Trk in vivo to test whether
Trk is proteolytically cleaved and whether this is Tsl-dependent. Finally,
using a known and related MACPF structure, we have designed and
are testing mutations in Tsl that target the putative membrane inserting
region to determine if membrane insertion is crucial for Tsl-mediated Trk
activation and embryonic patterning.
Page 106 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-22
REGULATION OF WNT SIGNALLING IS CRITICAL IN
EARLY EYE DEVELOPMENT
Chen Y.1, Storen R.1, 2, 3, Greenlees R.1, 3, Grigg J.2, 3, Tam P.1, 3 and
Jamieson R.1, 2, 3
1
Eye Genetics Group, Embryology, Children’s Medical Research
Institute. 2The Children’s Hospital at Westmead & Save Sight Institute.
3
Sydney Medical School, University of Sydney.
Aberrations in optic vesicle and cup formation lead to disorders affecting
vision including microphthalmia (small eyes) and abnormal iris and retinal
development. Distinct domains are specified in the distal part of the optic
vesicle for retinal progenitors and the dorsal part for retinal pigment
epithelium (RPE). In optic vesicles of E9.5 mouse embryos, WNT activity
is detected in prospective RPE via assaying the expression of TopGal
and BatGal reporters. In contrast, WNT reporter activity is absent in
prospective retina, where Dkk1, an inhibitor of canonical/beta-catenin
WNT signalling, is expressed. Hence, regionalization of WNT signalling
activity may be critical for tissue specification in early eye development.
While loss of Dkk1 function and increased canonical WNT signalling
leads to head truncation precluding investigation of WNT signalling in eye
development, reduction of Wnt3 in compound Dkk1-/-;Wnt3+/- mutant
mice rescues the head phenotype and reinstates eye development.
Optic vesicle examination of E9.5 compound mutant embryos (Dkk1-
/-;Wnt3+/-) harbouring a WNT reporter revealed ectopic response of
cells in prospective retina to WNT signalling. Concurrently, there was
ectopic expression of RPE marker (Mitf) and reduced expression of
neuroretinal marker (Chx10) in WNT-responding cells. Subsequently,
iris and retinal development was disrupted and small eyes formed.
These results indicate that active canonical WNT pathway is required
in specification of RPE fate of optic vesicle cells, and suppression of
this signalling pathway in prospective retina is essential for retinal cell
fate. Our results demonstrate the role of a key inhibitor, Dkk1, in precise
spatial and temporal regulation of canonical WNT signalling in early eye
development.
POS-TUE-24
SOX18-REGULATED GENES IMPLICATED IN
LYMPHATIC DEVELOPMENT
Kartopawiro J., Neyt C., Francois M. and Hogan B.
Institute for Molecular Bioscience, University of Queensland, Brisbane
4072, Queensland, Australia.
Recent discoveries have demonstrated the pivotal role of lymphatic
vessels in numerous pathological states, including inflammatory
diseases, tumor metastasis and lymphedema. Identification of new
molecular markers implicated in lymphatic vascular development
(lymphangiogenesis) is therefore essential in order to increase our
understanding of the mechanisms that underlie these disease conditions.
Recently, the transcription factor Sox18 has been shown to initiate
lymphatic endothelial cell specification during mouse embryogenesis.
The loss of Sox18 function leads to a complete loss of lymphangiogenesis
and embryos die in utero completely devoid of lymphatic vasculature.
However, the cascade of Sox18-dependent genes that modulates early
lymphatic vascular development is yet to be identified. In order to uncover
molecular targets of SOX18, a micro-array analysis has been performed
from purified lymphatic endothelial cells, comparing wild type and Sox18
mutant mice. This study has revealed a subset of Sox18-regulated genes
with no known function in lymphangiogenesis. Using a zebrafish model
system, our goal is to investigate on a large scale the in vivo expression
patterns and function of conserved lymphatic modulators regulated by
Sox18. In order to perform this analysis we utlize in situ hybridization
analysis, heat-shock-inducible transgenic zebrafish and transient gene
knock-down (loss-of-function). We expect to identify novel genes with
critical functions in lymphatic vascular development. These discoveries
may subsequently form the basis for novel therapeutic avenues for
lymphatic disorders.
POSTERS MONDAY & TUESDAY
POS-MON-25
CHIROPTERA TRANSTHYRETIN
Khwanmunee J. and Prapunpoj P.
Department of Biochemistry, Faculty of Science, Prince of Songkla
University,Hat Yai, Songkhla 90112, Thailand.
Thyroid hormone (TH) is known playing an important role in development,
reproduction and basal metabolism of vertebrates. In the plasma of
larger mammals, thyroxine-binding globulin, transthyretin (TTR) and
albumin take responses to transport/distribute the hormones throughout
body of the animals. Among of these plasma proteins, TTR is becoming
the most of interest because of its multifunction and association to a
genetic metabolic disorder called amyloidosis. In Chiroptera, TH is
crucial not only on basal metabolism but also the reproductive cycle.
However, the TH transporter/distributor and gene expression have not
been studied in this eutherian. In this study, the nucleotide sequence of
Chiroptera liver TTR cDNA was first reported. The deduced amino acid
sequence showed highly conservation in comparison to those of TTRs
from human and other eutherians, except two amino acid residues in
the N-terminal region was absent. Parsimony analysis revealed the
amino acid sequence of the flying mammal TTR is most similar to that of
pig TTR. Sections of genomic DNA in the regions coding for the splice
sites between exons 1 and 2 were synthesized and sequenced, and
the location and mRNA splicing in Chiroptera TTR gene was identified.
Moreover, the recombinant Chiroptera TTR was produced by using the
heterologous expression system of Pichia pastoris. The recombinant
protein had general physicochemical properties similar to those of
TTRs from human and other eutherians. Its functions including binding
to retinol binding protein (RBP) and proteolytic cleavage were also
demonstrated.
POS-MON-27
SOX3 OVEREXPRESSION LEADS TO DEFECTIVE
SUBCOMMISSURAL ORGAN DEVELOPMENT WITH
CONGENITAL HYDROCEPHALUS
Lee K.P.Y., Cheah P.S., Piltz S.G., Rogers N.A. and Thomas P.Q.
The University of Adelaide, Adelaide, SA, Australia.
Abnormalities in central nervous system (CNS) development that impede
the rostral to caudal flow of cerebrospinal fluid (CSF) cause congenital
hydrocephalus (CH) in mammals. Blockage of the sylvian aqueduct
(SA), a narrow constriction connecting the third and fourth ventricles,
is a common cause of CH. Genetic studies in mice have indicated that
the subcommissural organ (SCO), a brain gland located at the dorsal
midline immediately anterior of SA, is critical for clear passage of the
SA. However, the development of the SCO is poorly understood. To
investigate the function of the CNS transcription factor Sry-related HMG
box transcription factor 3 (Sox3), we have generated two independent
BAC transgenic mouse lines to overexpress Sox3 in developing CNS
using native control elements. These transgenic lines develop overt
CH (dome-shaped cranium) with 20% and 99% penetrance in single
and double transgenic, respectively. We found endogenous Sox3
expression in the SCO from inception (approximately 11.5 dpc) through
to adulthood and its pattern is recapitulated in Sox3 transgenic mice
spatially and temporally. Double transgenic embryos invariably exhibit
profound SCO dysmorphology at 14.5 dpc and agenesis at 18.5 dpc.
BrdU analysis indicated increased SCO primordium proliferation at 12.5
dpc in double transgenic embryos, suggesting Sox3 overexpression
may inhibit progenitor differentiation. Comparison of expression profiles
of wild type and double transgenic SCO at 12.5 dpc through microarray
analysis showed increased dosage of Sox3 alters expression of genes
implicated in SCO and/or dorsal midline development, including
members of the Wnt signalling pathway. We are extending these studies
using qRT-PCR and in situ hybridisation analyses. Ultimately, our work
will provide a better understanding of the genetic and molecular basis
for CH pathogenesis.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 107
POS-TUE-26
PLATELET DERIVED GROWTH FACTOR RECEPTOR
ALPHA (PDGFRα) AND CORONARY VASCULATURE
FORMATION
Biben C.1, Ooi F.1, Hartley L.1 and Prall O.W.J.1, 2
1
Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3052 VIC,
Australia. 2Royal Melbourne Hospital, Grattan Street, Parkville 3052
VIC, Australia.
Coronary artery disease is one of the leading causes of death in the
western world. Formation of coronary arteries and veins is still poorly
understood. Classical embryology experiments have suggested that
they derive from an extra cardiac tissue, the proepicardium (PE), which
is located posterior to the heart. PE cells cover the myocardium, forming
the epicardium, from which cells invade the subepicardial space and
then the myocardium. Whether they contribute most or only some of the
lineages of the coronary vasculature is unclear, as well as whether they
give rise to additional cardiac lineages, including muscle. We have found
that the gene encoding the receptor tyrosine kinase Platelet Derived
Growth Factor Receptor Alpha (PDGFRα) is expressed throughout the
mouse PE. Pdgfrα is required at multiple steps of embryonic development
but its role in the heart is uncharacterized. Pdgfrα remains expressed
in epicardial and subepicardial cells but becomes downregulated as
those cells migrate out of the subepicardium. To understand the role
of this gene in formation and maintenance of the coronary vasculature,
we have engineered mouse models with either low Pdgfrα expression
or specifically lacking this gene in various tissues, such as the PE,
endothelium or cardiac muscle. Our preliminary observations suggest
that Pdgfrα is specifically required in subepicardial cells and involved in
coronary smooth muscle development.
POS-TUE-28
THE ROLE OF FYN KINASE IN THE DEVELOPMENT OF
PRE-IMPLANTATION EMBRYOS
Levi M.1, Maro B.1, 2 and Shalgi R.1
1
Department of Cell and Developmental Biology, Sackler Faculty of
Medicine, Tel Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
2
CNRS, Paris, France.
Fertilization in mammals triggers the arrested oocytes to exit from
meiotic metaphase. The extrusion of the second polar body and the
formation of the pronuclei mark the completion of meiosis and the
beginning of pre-implantation embryo development. Several lines of
evidence imply the involvement of Fyn, a Src family kinase, in somatic
cell cycle control. In the current study we demonstrated, using live cell
confocal imaging and microinjection of Fyn cRNA, the co-localization
of Fyn with tubulin at the spindle poles of mouse oocytes. During the
exit from meiotic metaphase the amount of phosphorylated Fyn was
reduced, Fyn disappeared from the spindle poles and concentrated
around the spindle midzone and co-localized with filamentous actin
at the cleavage furrow and contractile ring area during meiosis and
mitosis. Inhibition of Fyn by exposure to SFKs inhibitor, SU6656, or
by microinjection of DN-Fyn cRNA inhibited the exit from metaphase,
nuclear envelope breakdown and preimplantation cell mitosis. Moreover,
although microinjection of cortical DN-Fyn did not affect the initiation of
the ingression of the cleavage furrow, it prolonged the average duration
of ingression, decreased the rates of polar body extrusion and the first
cleavage and enlarged the average volume of the polar body and the
length of the meiotic spindle. We propose that Fyn regulate several
key pathways leading to the exit from metaphase, nuclear envelope
breakdown, and in the ingression of the cleavage furrow during meiosis
and mitosis.
POSTERS MONDAY & TUESDAY
POS-MON-29
IDENTIFYING NOVEL GENES IN SEX DEVELOPMENT
Alankarage D.1, 2 , Ludbrook L.1, Svingen T.3, Bagheri-Fam S.1,
Koopman P.3 and Harley V.1
1
Prince Henry’s Institute, Clayton, VIC, Australia. 2Biochemistry
and Molecular Biology, Monash University, Clayton, VIC, Australia.
3
Institute for Molecular Bioscience, University of Queensland,
Brisbane, QLD, Australia.
Disorders of sexual development (DSDs) are surprisingly common (e.g.
XX males, XY females), and often result in infertility, genital abnormalities,
gender mis-assignment and long-term psychological trauma. The main
pathway of sex development in males centres on the regulation of SOX9
in the developing XY gonad, which is regulated by SF1, SRY and SOX9,
as well as PGD and FGF9 signalling pathways. However, most DSD
conditions remain unexplained genetically suggesting the presence of
additional genes involved in gonadal development that have not yet been
identified. A microarray expression study where SF1, SOX9, SRY and
DAX1 were over expressed separately in NT2 cells, a human pluripotent
testicular EC line, was recently performed in the lab. Microarray analysis
of FACS-sorted, transiently transfected SOX9 cells identified candidate
genes responsive to SOX9 overexpression. A bioinformatic filtering
process reduced the list from 2626 genes to 10. Promising candidate
genes such as CLCN7, COL18A1 and ETV5 are being evaluated further
in terms of their gene expression in XY Sox9 knockout embryonic
mouse gonads, and by CHIP-seq and promoter analysis. This study
can potentially lead to the discovery of novel causative DSD genes and
regulatory mechanisms during sex development.
POS-MON-31
RAPAMYCIN AND ISCHEMIA REPERFUSION INDUCE
HEME OXYGENASE-1 AND PEROXIREDOXIN-1 IN
LIVER
Kist A.1, Wakkie J.1, Nikolic A.1, Zeile S.1, Versteeg R.1, Ten Berge J.1,
Wilson C.H.1, Nieuwenhuijs V.B.2, Padbury R.T.A.3 and Barritt G.J.1
1
Department of Medical Biochemistry, School of Medicine, Flinders
University, Adelaide, South Australia. 2Department of Surgery,
University Medical Centre, Groningen, The Netherlands. 3The HPB
and Liver Transplant Unit, Flinders Medical Centre and School of
Medicine, Flinders University, Adelaide, South Australia.
Liver surgery is associated with ischemia and reperfusion (IR) injury
resulting in loss of liver function and hepatocyte death. The onset of
hepatocyte damage is chiefly due to the deleterious actions of reactive
oxygen species (ROS). Experimental induction of two anti-oxidant
enzymes, heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1),
reduces ROS-induced damage to the liver. Rapamycin, employed
clinically in liver transplantation as an immunosuppressant has also
been shown to induce the synthesis of HO-1 in non-liver cell types. The
aim of this study was to test whether rapamycin will induce HO-1 and
Prx-1 in hepatocytes. Incubation of rat hepatocytes with rapamycin or
cobalt protoporphyrin (CoPP) increased HO-1 (assessed by quantitative
PCR). Maximal effects were observed at 36 h, and half-maximal at
10-100 ng/ml. Rapamycin and CoPP increased HO-1 expression in
livers removed immediately after laparotomy and in livers subject to
laparotomy and a sham operation. Livers subject to a sham operation
exhibited higher relative expression of HO-1 mRNA than livers rapidly
removed. 2D-DIGE revealed two abundant cytosolic proteins which
showed a 3-fold increase in IR (P≤0.05). These were identified as
reduced and hyperoxidised Prx-1. A 1.5-fold increase in expression of
Prx-1 mRNA was observed in IR. It is concluded that rapamycin and IR
induce expression of HO-1 and Prx-1 in hepatocytes. Clinical application
of rapamycin pre-treatment may offer protection against IR damage.
Page 108 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-30
THE CREB1 TRANSCRIPTION FACTOR MODULATES
SCD1 EXPRESSION DURING MOUSE LUNG
DEVELOPMENT
Antony N., Bird A.D., Tan K.H. and Cole T.J.
Department of Biochemistry & Molecular Biology, Monash University,
Clayton, Victoria, 3800, Australia.
Transcriptional factors play a crucial role during lung development
in regulating gene expression programs which influence branching
morphogenesis and cellular proliferation and differentiation. Cyclic AMP
Response Element-Binding Protein1 (Creb1) is a transcription factor that
mediates cyclic adenosine 3’,5’-monophosphate (cAMP) signaling in
target tissues. Creb1-null mice die at birth due to respiratory failure and
are phenotypically smaller than their wild type litter mates. The aberrant
lung morphology in Creb1-null mice is established by E16.5 and is
clearly detected at E17.5 as unexpanded distal and proximal airways. We
investigated whole genome expression profiles on embryonic day E17.5
in Creb1-null fetal mice lungs to identify novel gene targets under the
control of Creb1 and to elucidate the molecular mechanisms underlying
the role of cAMP signaling during airway development. Microarray
analysis has identified several gene targets that are down-regulated
compared to wild type mice lungs; these included, genes involved in
lipid metabolism such as, enzyme fatty acid synthase, paraoxonase-1
(Pon-1) and a lipogenic enzyme, stearoyl-CoA desaturase 1 (Scd-1). In
Creb1-null mice lungs the gene expression was significantly reduced,
Pon-1 (1.6 fold, p=0.0005, n=4) and Scd-1 (8.6 fold, p=0.0001, n=4); this
was also confirmed by quantitative RT-PCR, Pon1 (p=0.0005, n=4) and
Scd-1 (p=0.0317, n=4). The gene expression profiles of Pon-1 and Scd-
1 are developmentally regulated and the expression were increased
dramatically from E16.5 to E18.5, following which Pon-1 levels remained
elevated, while Scd-1 levels decreased after birth. The expression of
Scd-1 protein was restricted to type II pulmonary epithelial cells and the
levels were significantly lower in Creb1-null when compare to wild type
mice lungs. Expression levels of Pon-1 and Scd-1 are also affected in
other transcription factor knockout mice models suggesting overlapping
roles in transcriptional regulatory pathways during lung development.
POS-TUE-32
EXOSOMAL MICRORNA PROFILING IN
NEURODEGENERATIVE DISEASE
Bellingham S.A.1, 2, 3, Kuehlich J.1, 2, Coleman B.M.1, 2, Sharples R.1, 2
and Hill A.F.1, 2, 3
1
Department of Biochemistry and Molecular Biology, The University
of Melbourne, Victoria 3010, Australia. 2Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Victoria 3010,
Australia. 3Mental Health Research Institute of Victoria, Melbourne,
Victoria, Australia.
Exosomes are small membrane vesicles of endosomal origin that are
released from a variety of cell types into the microenvironment. The
secretion of exosomes was initially thought to be a mechanism for
removing unnecessary proteins. However, our research implicates
exosomes in the potential pathogenesis of neurodegenerative disorders
such as prion and Alzheimer’s diseases. We have demonstrated that
neuronal cells infected with prions release normal and infectious forms
of the prion protein in associated in exosomes. We have also shown
that exosomes contain the Alzheimer’s disease precursor protein (APP),
the amyloid-β peptide (Aβ), and secretase components required for Aβ
generation from APP. These studies identified previously unknown
pathways for the conversion and propagation of prion infection and
the processing of APP that may contribute to AD pathogenesis. More
recently, exosomes have been shown to contain both mRNA and miRNA,
termed exosomal RNA (esRNA) that can be transferred between cells
in a novel mechanism of cell-cell communication of genetic signals. The
transferred esRNA is also functional, as the mRNA can be translated
into new proteins in recipient cells, while the miRNA, a class of small
RNA ~ 22nt long, play important roles in gene regulatory networks by
binding to and repressing the activity of specific target mRNAs. We
therefore hypothesised that esRNA can contribute to the pathogenesis
of prion and Alzheimer’s diseases. Utilising our cell culture models, we
aim to identify disease specific signatures from isolated exosomes by
miRNA profiling with “next-generation deep sequencing”, miRNA Array’s
and quantitative PCR. This research has significant diagnostic potential
for prion, Alzheimer’s and other human diseases since circulating
exosomes can be isolated from a variety of biological fluids including
blood, urine, saliva and CSF.
POSTERS MONDAY & TUESDAY
POS-MON-33
CYCLIN-DEPENDENT KINASE-LIKE 3 REGULATES
ACTIN REMODELLING AND CELL MIGRATION
Brown M.K.1, Forrest A.2, Kerr M.3, Wani S.1, Cloonan N.1, Gabrielli B.4
and Grimmond S.1
1
Queensland Centre for Medical Genomics, Institute for Molecular
Bioscience, University of Queensland, Australia. 2Omics Science
Centre, RIKEN, Yokohama Institute, Japan. 3Institute for Molecular
Bioscience, University of Queensland, Australia. 4Cell Cycle
Laboratory, University of Queensland Diamantina Institute, Princess
Alexandra Hospital, Australia.
Organisation of the actin cytoskeleton is central to cell migration,
and is largely regulated by members of the mammalian Rho GTPase
family. Members of this family have recently been implicated in dendrite
formation and branching, with alterations in Rho GTPase-signalling
reportedly contributing to mental retardation (MR) disorders. Cyclin-
dependent kinase-like 3 (Cdkl3) is a poorly characterised member of
the serine/threonine family, with homology to the cell cycle regulator,
Cdk1. Recently, a role for Cdkl3 in neuronal morphogenesis has been
demonstrated, whereby Cdkl3 knock-down has been shown to decrease
dendrite formation and branching. We report the characterisation of
two alternative transcripts derived from the human Cdkl3 locus. While
the canonical Cdkl3 product contains two putative ERK binding motifs
and a Nuclear Localisation Sequence (NLS), the second isoform has a
single putative ERK binding motif and no NLS, suggesting an alternative
function for this variant. We report a significant decrease in cell migration
with Cdkl3 knock-down, as well as hyperphosphorylation of ERK in
HeLa cells. Additionally, Cdkl3 knock-down leads to actin remodelling
and altered cell morphology. We propose a role for Cdkl3 in the MAPK
signalling pathway in a manner that regulates actin cytoskeleton
organisation, which affects the ability of cells to migrate correctly. We
suggest that alternative splice variants from the Cdkl3 locus encode
proteins that differ in their biological activities, and that these alternative
components should be considered when investigating Cdkl3 functions.
POS-MON-35
INVESTIGATING THE RNA BINDING PROPERTIES OF
KRÜPPEL-LIKE FACTORS
Burdach J.G.1, 2 , Mackay J.1 and Crossley M.2
1
School of Molecular Bioscience, University of Sydney, Australia.
2
School of Biotechnology and Biomolecular Sciences, University of
NSW, Australia.
The Krüppel-like factors (KLFs) are a family of mammalian transcription
factors involved in the regulation of a diverse range of biological
processes including proliferation, apoptosis, differentiation and
development (Pearson et al., 2008). These proteins bind to a common
CACCC box element within GC-rich regions of DNA to activate or repress
transcription (Suske et al., 2005). DNA interaction is mediated by three
C-terminal Cys2His2 zinc finger domains which are highly conserved
across the family. The N-termini are highly variable and can contain
either transcriptional activation or repression domains. Cys2His2 zinc
finger domains are known to have a variety of functions including DNA,
RNA and protein binding (Iuchi, 2001). As transcription factors, the DNA
binding characteristics of the KLFs are well established. Preliminary in
vitro data from our laboratory suggests that these proteins may also
be capable of binding RNA, though the specificity and affinity of these
interactions is still unclear. We are using RNA immunoprecipitation
coupled with next generation sequencing technology (RIP-seq)
to determine whether these proteins are capable of specific RNA
interactions in a cellular context. This powerful technique may shed
light on the biological role of such an interaction, leading to a better
understanding of the role of KLFs in gene regulation. Iuchi, S. (2001).
Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci 58, 625-
635. Pearson, R., Fleetwood, J., Eaton, S., Crossley, M., and Bao,
S. (2008). Kruppel-like transcription factors: a functional family. Int J
Biochem Cell Biol 40, 1996-2001. Suske, G., Bruford, E., and Philipsen,
S. (2005). Mammalian SP/KLF transcription factors: bring in the family.
Genomics 85, 551-556.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 109
POS-TUE-34
TAXOL AND BORTEZOMIB INDUCE APOPTOSIS
SYNERGISTICALLY IN CHRONIC MYELOGENOUS
LEUKEMIA CELLS
Bucur O.1, 2 , Stancu A.L.1, Petrescu S.M.2 and Khosravi-Far R.1
1
Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA, USA. 2Institute of Biochemistry of the Romanian
Academy, Bucharest, Romania.
Bcr-Abl fusion protein plays a critical role in the pathogenesis and
progression of Chronic Myeloid Leukemia (CML) and in some Acute
Lymphocytic Leukemia cases. We and others have previously
established Bortezomib (FDA approved for the treatment of Multiple
Myeloma) as a potential important treatment in Bcr-Abl positive
leukemias. Taxol (Paclitaxel), a mitotic inhibitor drug (FDA approved for
the treatment of a number of cancers) is also now in clinical trials for
the treatment of CML. However, to our knowledge, there are no studies
regarding the combined treatment of Bortezomib and Paclitaxel in Bcr-
Abl positive CML. RESULTS: Combined treatment of Bortezomib and
Paclitaxel synergistically induces cell death in human Bcr-Abl positive
K562 cell line, by activating the initiator caspase 9 and effector caspase
3, which leads to the cleavage of a number of substrates, such as Poly
ADP Ribose Polymerase (PARP), and results in apoptosis. Additionally,
the exposure of the Bcr-Abl positive leukemia cells to the Bortezomib/
Paclitaxel regimen results in an increase in the activation of the
stress-related MAP kinases (such as p38 MAPK and JNK) versus the
cytoprotective kinases (ERK1, ERK2), suggesting a possible implication
of JNK and p38 MAPK in mediating the effect of the combined treatment.
Moreover, we also show that the combined treatment is effective in
inducing apoptosis in the murine Baf3 Bcr-Abl and Imatinib-resistant
Baf3 Bcr-Abl T315I cell lines. CONCLUSION: Taken together, these
findings underline that the combined treatment with Bortezomib and
Paclitaxel represents a potentially promising strategy for the treatment
of Chronic Myelogenous Leukemia in general, and of Imatinib-resistant
CML in particular.
POS-TUE-36
IDENTIFICATION OF NFIL3 AS A DIRECT
GLUCOCORTICOID-REGULATED GENE TARGET IN
T-LYMPHOCYTES
Carey K.T.1, Tan K.1, Ng J.1, Liddicoat D.R.1, 2, Godfrey D.I.2 and Cole T.J.1
1
Department of Biochemistry & Molecular Biology, Monash University,
Clayton, Victoria, 3800. 2Department of Immunology and Microbiology,
University of Melbourne, Parkville, Victoria, 3010.
Glucocorticoids (GCs) are homeostatic steroid hormones with
essential roles in the regulation of development, integrated metabolism,
immune and neurological responses. In the immune system the strong
lymphocytolytic actions of GCs are central in treatment of lymphocytic
leukemias and lymphomas such childhood acute lymphoblastic leukemia
(ALL). GCs act via the glucocorticoid receptor (GR) which is expressed
from multiple untranslated exon 1s to yield at least 11 alternatively spliced
transcripts in humans and at least five in mice (1A-1H). GR transcripts
initiating from the GR1A promoter have previously only been localised to
T-lymphocytes and brain cortex. In T-lymphocytes the GR1A promoter
is implicated in increasing sensitivity to Glucocorticoid Induced Cell
Death (GICD). CD4+CD8+ Double Positive (DP) cells as well as NK cells
in particular have been shown to be hypersensitive to GICD. To explore
the molecular pathway driving GCID in T-cells we have performed whole
genome microarray analysis in mouse GR null T-cells. Interesting direct
GR targets included P21 and Bim, in addition to many not previously
well characterised, such as Nfil3. Regulation of these targets by GCs
has been validated using qRT-PCR in WT T-cells. Nfil3 in particular has
been studied further. Previous studies suggest that the development and
functional maturation of NK cells requires Nfil3 expression, in addition
it has been demonstrated that GC-mediated upregulation of Nfil3 is
dependent on intracellular calcium levels, and correlates with GCID
of GC-sensitive leukemic cells. In silico promoter analysis revealed a
putative Glucocorticoid Response Element in the Nfil3 promoter region
which was confirmed by ChIP. Immunohistochemical staining of Nfil3 in
whole thymus has localised Nfil3 protein primarily to the medullary region,
which contains developing NK cells. It will be of interest to investigate
the involvement of GICD in negative selection of sensitive DP cells and
correlate this to expression of Nfil3.
POSTERS MONDAY & TUESDAY
POS-MON-37
A NOVEL FUNCTION OF TRISTETRAPROLIN :
INHIBITING THE ACTIVITY OF POLY(A) POLYMERASE
(PAP) BY DIRECTLY INTERACTING WITH PABPN1 AND
PAP
Wang S.-C.1, Su Y.-L.2, Chiang P.-Y.2, Lin N.-Y.1, Shen Y.-F.2 and
Chang C.-J.1, 2
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
2
Graduate Institute of Biochemical Sciences, National Taiwan
University, Taipei, Taiwan.
Tristetraprolin (TTP) is one of AU-rich element (ARE)-binding proteins,
which facilitates mRNA destabilization. To realize the detailed
mechanism of TTP upon mRNA degradation control, it is necessary
to find out its direct interaction proteins. In this report, we performed
phage-display biopanning to search the TTP-interacting proteins. One
of them, Pabpn1 (poly (A) binding protein nuclear 1), was identified and
further characterized. Moreover, their interacting domains were verified
and determined with their deletion constructs by pull-down assays.
Pabpn1 plays a major role in the polyadenylation of mRNAs, which binds
to poly(A) RNA and increases the affinity of the poly(A) polymerase
(PAP) to promote the formation of the polyadenylate tail of the 3’ end
of mRNA. Interestingly, we found that TTP could bind to both Pabpn1
and PAP and inhibit polyadenylation of ARE-containing RNA in vitro.
The poly(A) tail is important for the mRNA export, mRNA stabilization
and translation efficiency. Consequently, except the mRNA-degrading
function, inhibition of polyadenylation activity of PAP is a novel function
of TTP to regulate ARE-containing mRNA expression.
POS-MON-39
ANALYSIS OF THE RELATIONSHIP BETWEEN
BIOENERGETIC SIGNATURES AND DIFFERENTIATION
POTENTIALS OF HUMAN MESENCHYMAL STEM
CELLS FROM DIFFERENT ORIGINS
Chen C.T.1, Lan Y.W. 1, Hsu S.H.1, Hwang S.M.2 and Wei Y.H.1, 3
1
Department of Biochemistry and Molecular Biology, National Yang Ming
University, Taipei, Taiwan. 2Bioresource Collection and Research Center,
Food Industry Research and Development Institute, Hsinchu, Taiwan.
3
Department of Medicine, Mackay Medical College, Taipei, Taiwan.
Our previous studies indicate that the metabolic shift from anaerobic
glycolysis to mitochondrial respiration plays critical roles during
osteogenic differentiation of human mesenchymal stem cells (hMSCs).
Hypoxic signals suppressed the activation of mitochondria and attenuated
differentiation of hMSCs. Recent studies also showed the necessity of
mitochondrial function for stem cells to differentiate into cardiomyocytes
and other types of somatic cells. Therefore, we have hypothesized that
the metabolic signatures, namely, the relative contribution of aerobic and
anaerobic metabolism to energy production, of stem cells may affect
their differentiation potentials. We isolated hMSCs from four different
origins including cord blood, bone marrow, amniotic membrane and
amniotic fluids. Their metabolic features were analyzed by an automatic
bioenergetic analyzer, Seahorse XF24 Extracellular Flux Analyzer. We
found that hMSCs form different origins displayed distinct metabolic
signatures as revealed by their oxygen consumption rate (OCR) and
extracellular acidification rate (ECAR) despite their expression of similar
panels of surface markers. These hMSCs also showed distinct preference
in differentiating into multiple lineages of progenies such as osteoblasts
and adipocytes. Manipulation of their aerobic and anaerobic metabolism
by inhibitors against either mitochondrial or glycolytic activities affected
their differentiation abilities. Taken together, the distinct metabolic
signatures of hMSCs from different origins may serve as an additional
biomarker for the evaluation of stem cell properties in terms of their
differentiation potentials. Characterization of metabolic signatures could
be of great value in the selection of stem cells with superior quality to
facilitate future clinical application of stem cells in tissue regeneration.
Page 110 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-38
INTESTINAL STEM CELL GENE LGR5 IS REGULATED
BY SYNERGISTIC C-MYB AND β-CATENIN
COOPERATION
Cheasley D.A.1, 2 , Malaterre J.1, Vincan E.3, Lightowler S.1, Pereira L.1
and Ramsay R.G.1
1
Differentiation and Transcription Laboratory, Peter MacCallum
Cancer Institute, East Melbourne and the Pathology Department,
The University of Melbourne. 2The Department of Genetics, La Trobe
University, Bundoora. 3The Department of Anatomy and Cell Biology,
The University of Melbourne, Parkville.
The regulation of tissue development and the establishment of
homeostasis is achieved through the orchestration of transcription
factor action and co-ordination of stem and progenitor cell populations.
The transcription factor c-Myb has emerged as a key regulator of stem/
progenitor cells within the gastrointestinal tract crypt. These structures
line the epithelium of the colon and small intestine. This conclusion
is based upon our exploitation of c-myb knock-out and hypomorphic
mutant mouse models. Recent studies have found that the Wnt target
gene, Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor
5), along with being an important intestinal and colon crypt stem cell
marker, is also expressed in colon cancer cells. Based on co-incidental
expression of lgr5 and c-myb in the GI, we investigated whether lgr5 is
a c-Myb target gene. Using both in vitro and in vivo studies, we show
that the proto-oncogene c-Myb in combination with β-catenin, is bound
to (by chromatin immunoprecipitation studies), and is a more potent
regulator of, the lgr5 promoter (by reporter experiments) in the presence
of activated β-catenin. These observations parallel our previous studies
of the regulation of the c-myc gene by both of these transcription
factors (Ciznadija et al 2009) suggesting this is a more general mode
of transcriptional co-operation. How c-Myb and β-catenin co-operate
at the genetic and physical level will be presented. Collectively our data
indicate that the Wnt pathway through β-catenin converge with c-Myb in
regulating lgr5 expression in the GI stem/progenitor cells.
POS-TUE-40
REGULATION OF SPLICEOSOMAL GENES IN
SACCHAROMYCES CEREVISIAE
Chen S.-C.E., Kornfeld G.D. and Dawes I.W.
School of Biotechnology and Biomolecular Sciences, University of
New South Wales, NSW Australia.
Sm-like (Lsm) proteins function in a variety of RNA-processing
events, including splicing, post-transcriptional modification and RNA
degradation. In yeast, the proteins Lsm2-Lsm8 form a heteroheptameric
ring complex and interact with the spliceosomal U6 snRNA to facilitate U4/
U6 snRNP formation during the mRNA splicing process; whilst another
complex comprised of Lsm1-Lsm7 is involved in mRNA degradation
via decapping in the cytoplasm. An increasing number of studies have
suggested that there are important functions of introns acting in parallel
with the protein-based regulatory systems, possibly related to the
complexity of organism. Studies from our laboratory showed that the
intron of the LSM7 gene along with the coding sequence is required for
the regulation of LSM1-LSM8 expression on different carbon sources.
The aim of our study is to identify the mechanism whereby the LSM7
intron regulates the expression of LSM genes in Saccharomyces
cerevisiae. The approach involved targeted mutagenesis of the LSM7
intron sequence, in particularly, the splicing elements in the intron, and
qRT-PCR analysis for the expression profiles of the LSM genes in these
mutants. In addition, microarray analysis has also conducted to examine
the genome-wide expression profiles in response to deletion of LSM7
intron.
POSTERS MONDAY & TUESDAY
POS-MON-41
DISTINCT ROLES OF TTP, HNRNP K AND HUR IN COX-
2 MRNA STABILITY INDUCED BY LPS
Chiang P.Y.1, Shen Y.F.1 and Chang C.J.1, 2
1
Graduate Institute of Biochemical Sciences, National Taiwan
University, Taipei, Taiwan. 2Institute of Biological Chemistry, Acdemic
Sinica, Taipei, Taiwan.
Cyclooxygenases (COXs) are key regulatory enzymes catalyzing
the rate-limiting step of prostaglandin formation in various biological
processes, such as inflammation, and overexpression of the inducible
isoform COX-2 is associated with carcinogenesis. In macrophage Raw
264.7 cells, lipopolysaccharide (LPS) induces COX-2 expression at both
transcription and posttranscriptional level. To understand the mechanism
of posttranscriptional regulation, the RNA binding proteins (RNA-BPs)
bound to the 3’ untranslated region (3’UTR) of COX-2 mRNA were
studied firstly. By using RNA pull-down and RNA immunoprecipitation
assay, tristetraprolin (TTP), heterogeneous nuclear ribonuclear protein
K (hnRNP K) and HuR were proven to bind to the distinct regions of
COX-2 mRNA 3’UTR respectively. Further reporter assay indicated that
LPS-induced TTP destabilizes COX-2 mRNA by binding to the AU-rich
element (AREs) located within the first sixty nucleotides of 3’UTR in
COX-2 mRNA. On the other hand, HuR binds to both ARE and distal
region of COX-2 mRNA 3’UTR and increases mRNA stability. Moreover
the RNA stability and translation modulated by hnRNP K are mediated
by binding to the distal region of COX-2 mRNA 3’UTR. Although these
RNA-BPs function in distinct ways, the combination effect mediated by
TTP, hnRNP K and HuR causes the tight control of COX-2 expression
during inflammation reaction.
POS-MON-43
BIOPHYSICAL AND STRUCTURAL EXAMINATION OF
THE LER TRANSCRIPTION FACTOR
Stone R.D.1, 2, Dogovski C.1, 2 , Bailey M.F.1, 2, Ji Y.3, Robins-Browne
R.M.3 and Perugini M.A.1, 2
1
Department of Biochemistry and Molecular Biology, The University of
Melbourne, Parkville VIC 3010, Australia. 2Bio21 Molecular Science
and Biotechnology Institute, The University of Melbourne, Parkville,
VIC 3010, Australia. 3Department of Microbiology and Immunology,
The University of Melbourne, Parkville, VIC 3010, Australia.
Pathogenic strains of Escherichia coli cause acute and persistent
diarrhoea in humans. Several pathotypes express specific virulence
factors that mediate adhesion to the intestinal epithelium and
production of distinctive pathological changes in the intestine mucosa
(A/E phenotype). Virulence genes in pathogenic E. coli are typically
organised in operons that are tightly controlled by a network of regulatory
proteins. The genes involved in the A/E phenotype are encoded by a 36
kb chromosomal pathogenicity island, named the locus of enterocyte
effacement (LEE). LEE carries more than 40 genes which form five
transcriptional units, termed LEE1 to LEE5. A number of global and
specific regulatory proteins are known to play an essential role in
gene expression and pathogenesis. The transcription factor Ler (LEE
encoded regulator) a homologue of H-NS, is essential for anti-silencing
of LEE operons LEE2-LEE5. Ler is considered to be a key regulatory
element and novel antibiotic target. Although much work has been done
to establish the role of Ler in E. coli pathogenicity, little is known about
the solution properties of the protein and how it prevents silencing of the
LEE pathogenicity island. The purpose of this study is to characterize the
solution properties and structure of Ler via analytical ultracentrifugation,
circular-dichroism spectroscopy and X-ray crystallography.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 111
POS-TUE-42
PHOSPHORYLATION ADJACENT TO THE BZIP
DOMAIN OF FOS RELATED ANTIGEN-1 REGULATES
DNA BINDING
Diesch J.1, Low D.1, Morrice N.2, Tulchinsky E.3, Hannan R.D.4 and
Dhillon A.S.1
1
Department of Biochemistry and Molecular Biology, University
of Melbourne, Vic, Australia. 2MRC Protein Phosphorylation Unit,
University of Dundee, UK. 3Department of Cancer Studies and
Molecular Medicine, University of Leicester, UK. 4MacCallum Cancer
Centre, Melbourne, Vic, Australia.
The Activator Protein-1 (AP-1) transcription factor is a sequence-
specific DNA binding protein complex that orchestrates gene
expression programs directing cell fate decisions (e.g. proliferation,
survival, differentiation, migration). It has a dimeric core, consisting
mainly of members of the Fos, Jun and ATF protein families, and
operates downstream of many cancer-associated signal transduction
pathways. The Fos related antigen-1 (Fra-1) is a Fos family protein
frequently over-expressed in cancers of epithelial origin, and is linked
to increased tumour cell migration and invasion. There is thus much
interest in understanding how the functions of Fra-1 are regulated.
Using mass spectrometry, we have identified a novel phosphorylation
site in Fra-1 (Ser101) that lies adjacent to the region of the protein
involved in DNA binding. We substituted Ser101 with alanine (S101A)
or aspartic acid (S101D) residues, and examined the effects of these
mutations on the subcellular localisation of Fra-1, its capacity to form
dimers with c-Jun in cells, and its ability to bind to oligonucleotides that
contain an AP-1 consensus sequence (TRE). While the localization and
dimerisation of Fra-1 with c-Jun were not affected by the substitutions,
we noted a significant increase in the binding of Fra-1S101A, and decrease
in the binding of Fra-1S101D, to the TRE. As these Fra-1 proteins were
part of dimer containing endogenous c-Jun, our results suggest that
phosphorylation of Ser101 can disrupt the recognition or binding of Fra-
1/c-Jun complexes to consensus sites in AP-1 target genes.
POS-TUE-44
PROTEOLYSIS OF AN ANTI-SIGMA FACTOR
CONTROLS SIDEROPHORE SYNTHESIS AND UPTAKE
IN PSEUDOMONAS AERUGINOSA
Draper R.C. and Lamont I.L.
University of Otago, Dunedin, New Zealand.
Pseudomonas aeruginosa is an important human pathogen that
requires iron for growth and pathogenesis. To acquire iron from the
environment or host tissues, this bacterium secretes the iron-binding
molecule pyoverdine. Pyoverdine is also a signalling molecule, inducing
the expression of genes for pyoverdine synthesis and uptake. In
the absence of pyoverdine, sigma factor proteins required for gene
expression are inhibited by an anti-sigma factor, FpvR, which spans
the inner membrane. When pyoverdine binds to a cell surface receptor,
this inhibition is relieved, allowing sigma activity and the expression of
target genes. This project investigated the role of FpvR in this signalling
pathway, and sought to determine the molecular mechanism underlying
this system. We have shown that the FpvR protein is only detectable in
strains where sigma factor activity is inhibited. When pyoverdine was
added we observed a rapid disappearance of FpvR, suggesting that
proteolysis regulates FpvR activity. Candidate proteases were screened
for involvement in the degradation of FpvR. The intra-membrane
protease RseP was implicated in this process by the accumulation of
intermediate FpvR fragments in strains lacking RseP. These strains also
exhibited reduced sigma activity as measured by qRT-PCR. Together
these data suggest a model whereby the binding of pyoverdine at the
cell surface causes the degradation of FpvR, resulting in changes in
gene expression.
POSTERS MONDAY & TUESDAY
POS-MON-45
MECHANISM OF SYNERGISTIC INDUCTION OF GLUT1
TRANSCRIPTION BY ENDOTHELIN-1 AND CYCLIC AMP
IN 3T3-L1 ADIPOCYTES
Fong J.C. and Kao Y.S.
Institute of Biochemistry and Molecular Biology, School of Life
Sciences, National Yang-Ming University, Taipei, Taiwan, ROC.
We have shown previously that chronic exposure to both endothelin-1
(ET-1) and cAMP results in a synergistic increase in Glut1 transcription
in 3T3-L1 adipocytes. In the present study, we have further examined
the molecular mechanism involved. Experimental results indicate that
ET-1 through ETAR/Gi/PKCε interacts with cAMP to enhance Glut1
transcription. Although p42/p44 MAPK is required for the stimulatory
effect of ET-1 alone, it is not involved in the synergistic effect of ET-1
and cAMP on Glut1 transcription. Further investigation demonstrated
that a ternary complex containing transcription factors Sp1, pCREB
and AP-1 was mediating the synergistic effect of ET-1 and cAMP on
Glut1 transcription. While an increase in nuclear AP-1 can be achieved
by either ET-1 or cAMP, the accumulation of nuclear pCREB is only
sustainable by stimulation with cAMP. An increase in nuclear Sp1, on
the other hand, is dependent on the activation of both PKCε and CREB.
Thus it seems that chronic exposure to both ET-1 and cAMP provides
a circumstance favoring increases in nuclear Sp1, pCREB and AP-1,
and their interaction to form a ternary complex which in turn can greatly
facilitate the transcription of glut1 gene.
POS-MON-47
ARABIDOPSIS GLYCINE-RICH RNA BINDING
PROTEIN 8 (ATGRP8) EXPRESSION IN RESPONSE TO
PHOSPHATE STARVATION
Gaza H.L.1, Jost R.1, Ludwig M.2 and Finnegan P.M.1
1
School of Plant Biology, UWA. 2School of Biomedical, Biomolecular
and Chemical Sciences,UWA.
Plant growth and development is restricted by phosphate (Pi) deficiency.
This leads to an adaptive response by altering gene expression and
metabolism as a result of cell signaling (Schmidt et al, 2010). The
expression of the Arabidopsis glycine-rich RNA-binding protein 8
(AtGRP8) gene is regulated by a number of external stimuli including
cold, circadian rhythm, ABA, drought and salinity (Carpenter et al., 1994;
Kwak et al, 2005). The altered gene expression of AtGRP8 under different
stress conditions has led to the hypothesis that it may be involved in the
response of plants to phosphate starvation. Using quantitative reverse
transcription polymerase chain reaction (qRT-PCR) the expression of
AtGRP8 gene during phosphate deficiency was determined. Roots
from A. thaliana plants grown under phosphate-deficient conditions for
seven days showed a decrease in expression compared to the plants
grown continuously in phosphate-sufficient media. The transcript level
of GRP8 in the shoots however, remained unchanged. GRP8 may
function similarly to WRKY6 and WRKY75 transcription factors as a
negative regulator of genes whose expression is up-regulated during
phosphate starvation. Experiments using knock-out lines to determine
the role of GRP8 in the regulation of the phosphate starvation response
are underway.
Page 112 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-46
FUNCTIONAL ANALYSIS OF THE
PROTEIN L-ISOASPARTYL/D-ASPARTYL
O-METHYLTRANSFERASE (PIMT) GENE PROMOTER
Furuchi T.1, Harada S.1, Shimizu Y.1, Kosugi S.2, Katane M.1, Sekine M.1
and Homma H.1
1
Sch. of Pharm. Sci., Kitasato Univ., Tokyo, Japan. 2Sch. of Med.,
Kanazawa Univ., Kanazawa, Japan.
L-Aspartyl (L-Asp) and L-asparaginyl residues in proteins spontaneously
isomerize or racemize to D, L-isoaspartyl (D, L-isoAsp) or D-aspartyl
(D-Asp) residues under physiological conditions. These atypical Asp
residues can interfere with the protein function and lead to malfunction
of cells. Protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) is a
cytosolic protein repair enzyme that initiates the conversion of L-isoAsp
and D-Asp to normal L-Asp residues. Mice lacking this enzyme suffer
from epileptic seizures and die at early age; thus, formation of the
abnormal residues and their subsequent correction by PIMT is widely
believed to constitute an important pathway of protein damage and
repair. However, regulatory mechanisms of PIMT gene expression have
remained unclear. To analyze the regulatory mechanisms controlling the
expression of human PIMT, we characterized the 5’-flanking region of
the gene. About a 1 kbp fragment of the putative promoter region was
PCR-amplified and ligated into a luciferase-expression vector, pGL3-
basic. Transfection in HEK293 cells indicate that the 5’-flanking region
contains regulatory elements for constitutive expression of PIMT. The
minimal region required for the basal activity of the PIMT promoter
were determined by generating a series of deletion and point mutation
constructs, and were found to be encoded by a sequence around –190
relative to the translation initiation codon. Electrophoretic mobility shift
assay suggested the presence of factors that bind to the minimal region
in a sequence-specific manner in the nuclear extract of HEK293 cells.
Further study is currently in progress to identify the binding factors using
liquid chromatography/tandem mass spectrometry (LC-MS/MS).
POS-TUE-48
FUNCTIONAL REGULATION OF THE FRA-1/AP-1
TRANSCRIPTION FACTOR VIA INTERACTIONS WITH
PROTEIN PHOSPHATASE 2A
Gilan O.1, Jastrezebski K.2, Diesch J.1, Verrills N.3, Hannan R.D.2 and
Dhillon A.S.1
1
Bio21 Institue Department of Biochemistry and Molecular BIology.
2
Research, Peter MacCallum Cancer Centre. 3Faculty of Health,
University of Newcastle.
The Activator Protein-1 (AP-1) transcription factor complex regulates
gene expression downstream of signal transduction pathways activated
by a variety of growth factors, cytokines, hormones and cellular stresses.
The products of AP-1-regulated genes are required for the execution
of fundamental cellular processes, including proliferation, survival and
differentiation. AP-1 complexes consist of a dimeric core, formed mainly
by members of the Fos, Jun and ATF protein families. Fos related
antigen-1 (Fra-1) is a Fos family protein that is frequently over-expressed
in cancers, and is strongly linked to cancer progression. The major post-
translational modification regulating Fra-1 is phosphorylation. Using a
proteomics-based approach, we have found that Fra-1 associates with
components of the hetero-trimeric protein phosphatase 2A (PP2A)
complex. This complex consists of a holoenzyme formed by the binding
of a 36 kDa catalytic subunit (PP2Acat) to a 65 kDa scaffold subunit,
whose activity and targeting to substrates is specified by one of at least
25 regulatory subunits. We specifically identified the PR65a scaffold and
PR55alpha regulatory subunits in Fra-1 complexes and have confirmed
that both proteins, as well as the catalytic subunit, interact with Fra-1 in
HEK293 cells and in vitro. We show that the PR55alpha regulates the
recruitment of the PP2A catalytic subunit to Fra-1, and that inhibition of
PP2A catalytic activity, or silencing of B55alpha expression perturbs
proteasome-mediated degradation of Fra-1. Our results provide new
insights into how the functions of Fra-1 are regulated in cells, and reveal
a novel mechanism regulating Fra-1 turnover.
POSTERS MONDAY & TUESDAY
POS-MON-49
HEPARANASE AS A SIGNALING MOLECULE IN
CANCER AND INFLAMMATION
Goodall K.J., Poon I.K.H. and Hulett M.D.
Department of Biochemistry, LaTrobe University, Bundoora, VIC,
Australia
Heparanase is a β-D-endoglucuronidase which degrades heparan
sulfate, a key component of the extracellular matrix and basement
membrane. The degradation of the ECM is essential for both
physiological and pathological processes, including inflammation,
wound healing, tumour angiogenesis and metastasis. Heparanase is
also known to have non-enzymatic functions by regulating cell adhesion,
cell signalling and differentiation. Although heparanase has been
proposed to facilitate leukocyte migration through degradation of the
ECM and basement membrane, its role in generating an inflammatory
response by mediating the release of chemokines, cytokines and growth
factors have not been well established. In this study, peripheral blood
monocyte cells were stimulated with Heparanase, and cytokine release
was examined. Heparanase treatment of cells resulted in the release
of a range of pro-inflammatory cytokines including IL-8, IL-10, TNF,
IL-6 and IL-1β. Similar results were obtained following the treatment
of MyD88-/- mouse spleen cells with Heparanase, suggesting that
the cytokine release was not simply due to endotoxin contamination.
These data suggests that Heparanase can promote inflammation via
the release of proinflammatory cytokines. Whether cytokine release is
due to the enzymatic or signalling role of Heparanase is currently being
investigated.
POS-MON-51
EVIDENCE THAT 4E-BP, KNOWN AS A COMPLETELY
DISORDERED PROTEIN, PARTIALLY FOLDS UPON
COMPLEX FORMATION : NEW PERSPECTIVES FOR
THE REGULATION OF TRANSLATION
Gosselin P.1, Oulhen N.1,3, Czjzek M.2, Cormier P.1and Cosson B.1
1
UPMC Univ Paris 06, CNRS, UMR 7150, Traduction Cycle Cellulaire
et Développement, Station Biologique de Roscoff, 29682, Roscoff,
France. 2UPMC Univ Paris 06, CNRS, UMR 7139, Végétaux marins
et biomolécules, Station Biologique de Roscoff, 29682, Roscoff,
France.3Current address: Department of Molecular and Cell Biology and
Biochemistry Brown University, Providence RI 02912, USA.
Control of translation is a critical step in the regulation of gene expression
involved in embryonic development, and mechanisms responsible for
human pathologies. The eukaryotic Initiation Factor 4E (eIF4E) interacts with
the 5’ cap structure (m7GTP) and controls the cap-dependant translation.
The three eIF4E family members (eIF4E1, 2, 3) have been identified in
deuterostomes and interact specifically with different partners, extending
the biological impact of this translation factor. Only one isoform of each
actor has been found in sea urchin, making this organism a strong model
to study the regulation of translation. Release of eIF4E from his repressor
4E-Binding protein (4E-BP) is a key process in translation initiation and
is triggered by the phosphorylation of 4E-BP. The understanding of the
interactions between these different actors, with functional and structural
approaches, is a very important step for cellular biology. Classical structural
approaches suggest that 4E-BP is mostly or completely unstructured in
both free and bound states, and only the central domain of 4E-BP is known
to interact with eIF4E during the complex formation. These data are not
sufficient to explain the sharp phosphorylation mechanisms that control
the association of 4EBP and eIF4E. Using an original structural technic
that consists in measurement of small angles X-ray scattering (SAXS),
we show for the first time that 4E-BP adopts a folded structure upon the
binding to eIF4E but also that this inhibitor has a transitory structure when
it’s free in solution. SAXS allows us to see the rest of 4E-BP chain that is
missing in the crystallography complex structure, and reveals a « fuzzy
complex », involving a larger surface of interaction between these two
actors. The results that we obtained with sea urchins proteins, adopting
this new dynamic view of 4E-BP structure, open new perspectives for the
understanding of the sharp mechanisms of gene regulation by translation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 113
POS-TUE-50
A NOVEL ROLE OF DIPEPTIDYL PEPTIDASE 9 IN PI3K/
AKT PATHWAY REGULATION
Yao T.W.1, 2, Xia P.1, 2, Kim W.S.3, Yu D.M.T.1, 2, Choi K.Y.3 and Gorrell M.D.1, 2
1
Centenary Institute. 2Sydney Medical School, University of Sydney.
3
Yonsei University, Seoul, Korea.
Dipeptidyl peptidase (DP) IV, DP8, DP9 and FAP (fibroblast activation
protein) are DPIV gene family members of particular interest due to
their peptidase and extra-enzymatic activities that have been implicated
in various diseases. We report here a novel role of DP9 in cell signal
transduction. We found that DP8 and DP9, but not DPIV or FAP, interact
with H-Ras, a key signalling molecule that mediates multiple signalling
pathways, especially of growth factors (GFX), in both liver tissue and
human cervical carcinoma cell line, HeLa. The ERK1/2 and Akt pathways
are two major downstream effectors of Ras. Interestingly, Akt activation
(determined by Akt phosphorylation status) was significantly inhibited
by DP9 overexpression in human hepatoma cells (HepG2), whereas
ERK1/2 activity was unaffected, revealing a pathway-specific effect.
DP9 induced a significant reduction in GFX - induced Akt activation in
HepG2 cells. This is consistent with the observation that DP9 markedly
attenuated GFX-dependent mitogenic effects, including apoptosis and
proliferation. These findings, along with our previous observation that
DP9 overexpression is pro-apoptotic, suggest an important signalling
role of DP9 in the regulation of a GFX - dependent survival and/or
proliferation pathway.
POS-TUE-52
ARABIDOPSIS POLLEN TRANSCRIPTION FACTORS
Gibalova A.1, 2, Renak D.1, 2, 3, Duplakova N.1, 2, Solcova K.1, 2 and Honys D.1, 2
1
Laboratory of Pollen Biology, Institute of Experimental Botany ASCR,
Rozvojová 263, 165 02 Praha 6, Czech Republic. 2Department of
Plant Experimental Biology, Faculty of Science, Charles University in
Prague, Viničná 5, 128 44 Praha 2, Czech Republic. 3Department of
Plant Physiology and Anatomy, Faculty of Science, University of South
Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic.
Haploid male gametophyte plays a key role in plant fertility and crop
production. Despite of signifiant progress in recent years, we still
have very limited understanding of the regulatory mechanisms that
have evolved to specify the gametophytic developmental programs.
Therefore, it is necessary to identify transcription factors that are part of
such haploid regulatory networks. In our studies, we have focused on
basic-leucine zipper and heat shock transcription factors knowing to be
involved in stress response and developmental processes. We report the
functional characterization of members of these gene families that are
expressed in both gametophytic and surrounding sporophytic tissues
during flower development. The respective T-DNA insertion mutants
showed reduced transmission through male and female gametophytes,
pollen morphological defects, lower pollen germination efficiency and
slower pollen tube growth both in vitro and in vivo conditions. Transient
expression revealed the subcellular localization analysed proteins and
in addition to other data, the nucleolar localisation of the heat shock
transcription factor suggested its possible involvement in regulation
of mRNA/rRNA transcription. Acknowledgment: Authors gratefully
acknowledge the financial support from the Czech Science Foundation
(grant 522/09/0858) and Ministry of Education, Youth and Sports of the
Czech Republic (grants LC06004 and OC10054).
POSTERS MONDAY & TUESDAY
POS-MON-53
IN SILICO SEARCH OF POTENTIAL FUNCTIONAL
SITES WITHIN PROMOTERS OF MOUSE O6-
METHYLGUANINE-DNA METHYLTRANSFERASE GENE
Iatsyshyna A.P., Pidpala O.V. and Lukash L.L.
Institute of Molecular Biology and Genetics, 150 Zabolotnogo Str.,
03680, Kyiv, Ukraine.
The modulation of expression and activity of O6-methylguanine-DNA
methyltransferase, MGMT, in tumors and normal tissues is currently
being investigated as possible strategies for improving cancer therapy.
At present little is known about organization and expression of mouse
Mgmt gene, so the aim of our work was to search potential regulatory
sequences within the promoters of this gene. Sequences of the mouse
Mgmt gene promoters were taken from the database TRED. Identification
of functional sites was performed by using program TFSEARCH.
There are 3 promoters of mouse Mgmt gene in TRED database: 77428
(known), 77429 (refseq, predicted), 77430 (refseq). In sequences of
given promoters we found gomology with functional sites for 15, 9 and
19 transcription factors (TF) respectively. Common for all promoters
were such TFs: Nkx-2.5, Gata-1, SRY, USF. In the known promoter,
except characterized by others AP-1 and E2F TFs, we detected binding
sites (BS) for such TFs: CRE-BP, CREB, C/EBP, SRF, Oct-1, Tst-1, NF-
E2, Lyf-1, c-Myc. Such exclusivity of revealed sites can be an evidence
of tissue specificity of the Mgmt expression regulation. The predicted
promoter contains BSs for such TFs: LYF-1, HSF2, c-Ets-1, C/EBPβ, Ik-
2, except common TFs for all studied promoters. In the refseq promoter
sequence we revealed unique BSs for TFs: CRE-BP, CREB, C/EBP,
AP-1, c-Ets-1, Ik-2, Oct-1, YY1, Sox-5, TATA, STATx, c-Rel, Ik-1, NF-κB,
N-Myc. Thereby, by using in silico analysis of three promoters of the
mouse Mgmt gene we revealed individual BSs that can be associated
with the tissue-specific regulation of gene expression, and common
sites which could play a key role in regulation of this gene.
POS-MON-55
COPPER CHLORIDE INDUCED EXPRESSION OF
CYTOGLOBIN AND HIF-1α IN VIVO
Jusman S.W.A.1, 2, Prijanti A.R.1, 2, Iswanti F.C.1, Ferdinal F.3, Suyatna F.D.2, 4,
Wanandi S.I.1, 2 and Sadikin M.1, 2
1
Department of Biochemistry & Molecular Biology, Faculty of Medicine,
University of Indonesia. 2Biomedical Science Program, Faculty of
Medicine, University of Indonesia. 3School of Medicine, Tarumanegara
University. 4Department of Pharmacology, Faculty of Medicine,
University of Indonesia.
Copper is known to stabilize HIF-1α under normoxic condition, resulting
in induction of HIF-1α target genes. Cytoglobin, the novel globin
from vertebrate is suggested as one of HIF-1α regulated genes. This
study observed the liver tissue response to copper chloride induction
in vivo. Male Sprague-Dawley rats were given 1.25 μmol of copper
chloride solution intraperitoneally. The observation are made at 2, 6,
24, 48 and 72 hours after treatment and compared to control group.
Liver tissue were analyzed for HIF-1α protein using ELISA technique,
cytoglobin mRNA with real time RT-PCR and cytoglobin protein with
ELISA. It is showed that cytoglobin protein was up-regulated 48 hours
after treatment compared to control group, while the HIF-1α protein
and cytoglobin mRNA showed tendencies to be stimulated 24 and 48
hours respectively after treatment. It is concluded that copper chloride
stimulated expression of cytoglobin protein, which might be mediated
by stabilization of HIF-1α protein due to inhibition of its degradation by
prolil hydroxylase.
Page 114 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-54
DEMONSTRATION OF AUTOPHOSPHORYLATION OF
THE LYN PROTEIN TYROSINE KINASE A NEW SITE
IN THE UNIQUE DOMAIN – THE ROLE OF THE NEW
AUTOPHOSPHORYLATION IN LYN KINASE FUNCTION
Jarasrassamee B., Chong Y.P., Williamson N.A., Purcell A.W. and
Heung-Chin C.
Department of Biochemistry and Molecular Biology, Bio21 Molecular
Science and Biology, University of Melbourne.
Lyn is a member of the Src family of protein kinases (SFKs). Since SFKs
are important signaling enzymes participating in a variety of cellular
processes, activation of SFKs is transient and tightly regulated. It is
well known that autophosphorylation of Lyn at the conserved tyrosine
(YA) in activation loop of the kinase domain results in activation. In
addition to autophosphorylation at this site, we discovered a novel
autophosphorylation site (Y32) in the unique domain of Lyn. In this study,
mutants of Lyn carrying mutations in one or both autophosphorylation
sites including Y32F Lyn, Y32, 397F Lyn, and Y397F Lyn were generated.
Biochemical analysis revealed that unlike autophosphorylation in
most protein kinases, autophosphorylation of Lyn at this site occurs
intramolecularly. Using a phosphospecific antibody against Tyr-32 of Lyn,
we were able to demonstrate that Lyn undergoes autophosphorylation
at Tyr-32 in rat tissues, confirming the physiological relevance of our
findings. To investigate the role of Tyr-32 autophosphorylation in the
oncogenic action of Lyn, we set out to study the regulation and function of
Lyn in Chronic myelogenous leukaemia (CML). Aberrant activation and
expression of Lyn has been reported to contribute to the development of
drug resistance in CML – some CML patients fail to sustain hematologic
remission with the drug Gleevec. We have initiated studies of the role of
the Tyr-32 autophosphorylation in the oncogenic action and regulation
of the kinase activity of Lyn in both the Gleevec-sensitive and Gleevec-
resistant CML cells. Results of our studies will shed light on how Lyn
contribute to the development of drug-resistant CML.
POS-TUE-56
INVESTIGATING THE CONTRIBUTION OF GENOMIC
INSTABILITY TO ALTERED mircoRNA EXPRESSION IN
OVARIAN CANCER CELL LINES
Kan C.W.S.1, Hahn M.A.1, Huh J.Y.1, Dykema K.2, Howell V.M.1 and
Marsh D.J.1
1
Functional Genomics Laboratory, Hormones and Cancer Group,
Kolling Institute of Medical Research. 2Van Andel Research Institute,
Grand Rapids, MI, USA.
Ovarian cancer is the most lethal gynaecological malignancy and
the sixth most common cause of cancer death in Australian women.
microRNAs (miRNAs) are small non-coding RNAs that regulate gene
expression and are often aberrantly expressed in cancer. This project
investigated whether chromosomal losses or gains may contribute to
changes in expression of miRNA in ovarian cancer cell lines. Gene and
miRNA expression microarrays were performed on four ovarian cancer
cell lines and a cell line model of normal ovarian surface epithelial cells
(OSEs). Predicted regions of chromosomal loss or amplification were
identified by comparative genomic microarray analysis (CGMA) of gene
expression data. CGMA predicted chromosomal loss at 5q in all four
cancer cell lines but not in OSEs. Loss of heterozygosity in chromosome
5q has been reported to be associated with early development of
ovarian cancer. Investigation of miRNA located on chromosome 5q
revealed miR-146a to be significantly decreased (ANOVA, P<0.01)
in expression across all four cell lines compared to OSEs. Predicted
targets of miR-146a include CCBP2, chemokine-binding protein 2.
CCBP2 is reported to be over-expressed in vascular tumours and may
drive tumour development and growth. In summary, we have shown
that ovarian cancer cell lines have gene and miRNA expression profiles
that are distinct to those of OSEs. These results suggest that genomic
instability may contribute to the altered expression of a subset of miRNA
and their target genes in ovarian cancer. Supported by an Australian
Postgraduate Award, University of Sydney Cancer Research Fund and
the Cancer Institute NSW.
POSTERS MONDAY & TUESDAY
POS-MON-57
EXTENSION OF DROSOPHILA LIFESPAN BY
OVEREXPRESSION OF HUMAN CLUSTERIN
Kang B.H.1, Lee Y.N.2, Park J.J.2 and Min B.H.1
1
Department of Pharmacology and BK21 program in Biomedical
Sciences, College of Medicine, Korea University, 136-705, Seoul, Korea.
2
Department of physiology and BK21 program in Biomedical Sciences,
College of Medicine, Korea University, 136-705, Seoul, Korea.
Clusterin (CLU) is a disulfide-linked heterodimeric glycoprotein
implicated in diverse biological processes. Expression levels of CLU
increase during cellular senescence or normal aging, but it is uncertain
whether this is protective against aging, or is a consequence of aging.
To better understand the role of clusterin in organismal aging, we
generated transgenic Drosophila alleles to induce expression of the
secretory form of human clusterin (hCluS) by using the Gal4/UAS
system. The hCluS protein sized 50-60 kDa was detected in both
adult homogenate and larval hemolymph of the flies over-expressing
hCluS ubiquitously (da-Gal4>UAS-hCluS) or in motoneurons (D42-
Gal4>UAS-hCluS). Life spans of these hCluS-over-expressing flies
were significantly extended (39 days) than control flies that presented
no hCluS induction. The mean life-span of CS10 (+/+), D42-Gal4/+,
and UAS-hCluS/+ was 31, 33, and 35 days, respectively. In addition,
the hCluS-over-expressing flies showed enhanced tolerance to heat
shock, wet starvation, and oxidative stress. Furthermore, the amount
of reactive oxygen species (ROS) in the whole body was significantly
reduced in hCluS-over-expressing flies, compared to control flies. Over-
expression of hCluS in a group of neurons was sufficient to recapitulate
its effects of whole body expression, implying that hCluS works cell
nonautonomously. However, the patterns of their feeding behavior were
not affected by hCluS expression. Taken together, these results suggest
that hCluS may function as an antioxidant to reduce ROS levels and
delay the organismal aging in fruit flies. [This work was supported by the
Korea Science and Engineering Foundation (KOSEF) grant funded by
the Korea government (MEST) (No. 2009-009-1418) and BAERI (Basic
Atomic Energy Research Institute) grant from the National Nuclear R&D
Program (20090078713) funded all by the Korean Ministry of Education,
Science and Technology (MEST)].
POS-MON-59
SUPPRESSOR OF CYTOKINE SIGNALING (SOCS)5 IS
A POTENTIAL REGULATOR OF THE IL-4-JAK/STAT
PATHWAY
Kolesnik T.B., Colombus R.E., Chakravorty A., Wilson T.A., Sprigg N.S.,
Carter W., Zhang J.-G., Babon J.J., Nicola N.A. and Nicholson S.E.
The Walter & Eliza Hall Institute of Medical Research, Parkville,
Victoria, Australia.
SOCS5 has been implicated in regulation of the Th1/Th2 balance by
inhibition of IL-4 signaling in Th1 cells through interaction with the IL-4
receptor alpha chain (IL-4Rα)1. However, in SOCS5-deficient mice, Th1/
Th2 cells differentiate normally and the mice mount normal B and T cell
responses to mitogenic stimuli2. Analogous to the Th1/Th2 paradigm,
macrophages differentiate into classically activated or alternate in
response to IFNγ or IL-4, respectively. To dissect the role of SOCS5 in
IL-4 signaling in macrophages, we initially examined regulation of the
SOCS proteins. Socs5 mRNA as well as Socs1, Socs2 and Cis, was
rapidly induced in response to IL-4, however, only SOCS1, SOCS5 and
CIS could inhibit IL-4 signaling in a STAT6-reporter assay. We further
demonstrated that, like SOCS1, SOCS5 was able to directly inhibit
JAK1 enzymatic activity. Extensive mutagenesis analysis showed that
the SOCS5 N-terminus, SH2 domain and SOCS box were required for
inhibition of JAK1 activation, whereas the N-terminus was essential for
interaction with the IL-4Rα. Western-blot analysis revealed no differences
in the level of IL-4-stimulated JAK1 and STAT6 phosphorylation in
SOCS5-deficient macrophages compared to wild-type cells. To address
possible redundancy between SOCS1 and SOCS5, we analyzed IL-4
signaling in macrophages lacking both SOCS proteins. JAK1 and STAT6
phosphorylation were up-regulated in SOCS1-deficient cells to the same
extend as in double knock-out cells indicating that, at least in primary
macrophages, SOCS1 and SOCS5 are not functionally redundant. The
biological role of SOCS5 in IL-4 signaling remains to be elucidated.
1
Seki et al., PNAS 2002, 99(20): 13003-8; 2Brender et al., 2004 MCB,
24(13): 6094-6103.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 115
POS-TUE-58
THE CELLULAR SIGNALING PATHWAY MEDIATING
INDUCTION OF HBV REPLICATION AND GENE
EXPRESSION BY ETHANOL
Kim Y.H.1, 2 , Kim B.K.1, 2 and Park Y.G.1, 2
1
Dept. of Biochemistry. 2Division of Brain Korea 21 Program for
Biomedical Science, Korea University College of Medicine, Anam-
dong, Seongbuk-Gu, Seoul, 136-170, Korea.
Even though alcohol intake has been implicated in the etiology of HBV-
related diseases, neither ethanol effect on HBV replication nor the
cellular signaling pathway mediating the effect are known. This study is
designed to elucidate the effect of alcohol on HBV replication and gene
expression. Ethanol treatment increased the HBV promoter/enhancer
activity and the levels of HBV transcripts, DNA and viral antigens. In
contrast, acetaldehyde, a product of hepatic ethanol metabolism, had no
effect on the synthesis of HBV mRNA. Moreover, the ethanol-induced
increase in HBV mRNA synthesis was not affected by pretreatment with
inhibitors of alcohol dehydrogenase or acetaldehyde dehydrogenase.
However, CYP2E1 did not make an effect on ethanol-induced synthesis
of HBV mRNA even though ethanol-induced ROS generation is well
known to be mainly dependent upon CYP2E1. Ethanol-induced
synthesis of HBV mRNA was abolished by pretreatment with DPI and
Trolox. Ethanol increased the synthesis of IL-6 mRNA and the release
of IL-6 protein via ROS generation, and these events are necessary for
ethanol-enhanced synthesis of HBV mRNA and DNA as well as increase
of HBV promoter/enhancer activity. Ethanol-induced HBV replication
and gene expression was dependent upon IL-6/JAK2 signaling but not
upon STAT1 and 3. This study indicates that the enhancement of HBV
replication by ethanol treatment requires IL-6 production and JAK2
activation through ROS generation.
POS-TUE-60
FUNCTIONAL ANALYSIS OF TRANSCRIPTION
FACTORS REGULATING FRUCTOSYLTRANSFERASES
INVOLVED IN THE FRUCTAN SYNTHETIC PATHWAY IN
WHEAT
Kooiker M., Xue G.P. and McIntyre C.L.
CSIRO, 306 Carmody road, St. Lucia, Australia.
Water soluble carbohydrates (WSCs) are accumulated in the stems
and leaf sheath of several cereals like wheat, barley and oats.
The main WSCs found in wheat stems at the grain filling stage are
fructans, which are linear or branched oligosachharides, synthesised
from sucrose in the vacuole. For the synthesis the enzymes
sucrose:fructan 6-fructosyltranferase (6-SFT) and sucrose:sucrose
1-fructosyltransferase (1-SST) are essential. Fructans are an important
temporary carbon reserve and are hydrolysed by fructan exohydrolases
when the grain needs sugars for grain filling. Under normal conditions
stem WSCs can contribute to up to 20% of the grain yield, but under
stress conditions (like drought stress) this percentage can increase to
greater than 50%. Under water limited conditions a positive correlation
between grain yield and WSC content in the stem at anthesis is often
observed. Some enzymes that play an important role in stem fructan
accumulation are sucrose:fructan 6-fructosyltranferase (6-SFT) and
sucrose:sucrose 1-fructosyltransferase (1-SST), which are positively
associated with genotypic variation in stem WSCs in recombinant
inbred lines Seri/Babax (SB). We have recently identified a number of
candidate transcription factors that are potentially involved in controlling
the expression of these fructosyltransferases in wheat and have been
evaluating the regulatory role of these candidate genes in transgenic
wheat.
POSTERS MONDAY & TUESDAY
POS-MON-61
TLR REGULATION OF SPSB1 REGULATES INOS
INDUCTION
Lewis R.S., Kuang Z., Kolesnik T.B., D’Cruz A., Low A., Norton R.S.
and Nicholson S.E.
Walter and Eliza Hall Institute.
The mammalian innate immune system has evolved to recognise foreign
molecules derived from pathogens via the Toll-like receptors (TLRs).
TLR3 and TLR4 can signal via the TIR domain-containing adapter
inducing interferon (IFN) β (TRIF), which results in the transcription
of a small array of genes, including IFNβ. iNOS is an enzyme that is
rapidly induced by a range of stimuli, including cytokines and microbes,
and catalyses the production of nitric oxide (NO). NO is a potent source
of reactive nitrogen species that play an important role in the killing
of intracellular pathogens and forms a crucial component of the host
defence. We have recently identified iNOS as a target of the mammalian
SPRY domain-containing SOCS box (SPSB) -2 protein. The SOCS box
is a peptide motif, which in conjunction with elongins B and C recruits
cullin-5 and Rbx-2 to form an active E3 ubiquitin complex. Here we
show that SPSB1 is the only SPSB family member to be regulated by
the same TLR pathways that induce iNOS expression and characterise
the interaction between SPSB1 and iNOS. Through the use of SPSB-1
transgenic macrophages and shRNA knockdown of SPSB-1 we have
shown that SPSB-1 controls the induction of iNOS and the subsequent
production of NO downstream of TLR3 and 4. Further, we demonstrate
that regulation of iNOS by SPSB-1 is dependent on the proteasome
via the SPSB-1 C-terminal SOCS box. These data suggest that SPSB-
1 acts through a negative-feedback loop that together with SPSB2
controls the extent of iNOS induction and NO production.
POS-MON-63
INTRACELLULAR TRAFFICKING OF RICIN TOXIN:
COMPUTATIONAL MODELLING AND KINEMATICS
Chong D., Walker A. and Skvortsov A.
Defence Science and Technology Organisation, 506 Lorimer St.,
Fishermans Bend, VIC 3207, Australia.
To explain the efficacy of inhibitors against toxins in various cell types,
the intracellular trafficking of the inhibitors and indeed, the agent itself
must be understood. In this study, live cell 3D/4D confocal microscopy
was used to visualise ricin toxin transport in human small airway
epithelial cells to develop a computational model of toxin trafficking. In
order to predict the rate at which the toxin will reach various subcellular
compartments, such as the juxtanuclear Golgi, the trafficking route of the
toxin is modelled as a diffusive transport process. An analytic solution
of the diffusion equations is then constructed by treating the boundaries
of the cell wall and the nucleus as conformally invariant fractals. The
driving parameters of the subcellular trafficking of ricin (i.e. mean
squared displacement) were derived from confocal microscope image
analyses and were used to calibrate the transport model. This work
ultimately aims to establish a computational model of the interactions
between inhibitor, agent and target cell.
Page 116 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-62
GENERATION OF A GRANZYME B/EGFP REPORTER
FOR LIVE-CELL IMAGING
Bird C.H.1, Prescott M.1, Harper I.2 and Bird P.I.1
1
Department of Biochemistry and Molecular Biology, Monash
University, Australia. 2Monash Micro Imaging, Monash University,
Australia.
Granzyme B (GrB) is a serine proteinase found in the cytolytic granules
of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). Upon
target cell engagement, GrB and other granule proteins are released
into the intercellular space. It is thought that perforin facilitates the entry
of GrB into the target cell cytosol where GrB activates the apoptotic
pathway. However, there is currently no direct evidence of GrB release
into the cytosol of target cells, and there is increasing evidence that it is
involved in extracellular remodelling. Here we describe the generation
and characterisation of a GrB/GFP fusion protein which is expressed
stably in NK cells and trafficks correctly to the granule. The GrB is
inactive so will not induce cell death, but rather act as a tracer. Using
this fusion protein we wish to follow GrB during target cell engagement
and lymphocyte migration.
POS-TUE-64
SIDEROPHORE CONJUGATES AS ANTIMICROBIAL
AND IRON-OVERLOAD AGENTS
Obando D., Brites L.J., Shi C., Liu J. and Codd R.
School of Medical Sciences (Pharmacology), University of Sydney,
NSW 2006, Australia.
Under iron-deprived conditions, pathogenic and non-pathogenic
bacteria produce low-molecular-weight molecules called siderophores
to solubilise Fe(III), which is sparingly soluble under the oxic, aqueous
and pH neutral conditions in the environment and in the mammalian host.
An avid recognition event between the Fe(III)-loaded siderophore and
receptors at the bacterial cell surface is followed by the complex traversing
the cell to ultimately deliver iron to the cytoplasm for incorporation into
key Fe-containing molecules, such as cytochromes and ribonucleotide
reductase. The competition between bacteria for Fe is reflected in the
chemical diversity of native siderophores: most bacteria produce a
unique siderophore that is recognisable only by its cognate cell surface
receptor. Molecules that thwart regular Fe(III)-siderophore mediated
uptake present a platform for the design of new narrow spectrum
antibiotics. In our group, we have modified the structure of a siderophore
native to a non-pathogenic gram positive bacterium to produce a suite
of potential antibiotic compounds. Of the four compounds we prepared,
one of these prevented growth of our target bacterium at an MIC of 60
nmol/cm2 lawn. The chemical differences in the family of compounds
we prepared is subtle, which presents some intrigue with regard to fully
understanding the mechanism of action. These results will shed new
light on the potential of tackling antibiotic design via iron deprivation
and the possibilities of designing siderophore-based compounds
against mammalian pathogens, including Pseudomonas aeruginosa,
Bordetella pertussis and Mycobacterium tuberculosis. In other work, we
have prepared new siderophore conjugates that show promise as new
therapeutics for the treatment of iron-overload disease in humans. In
this paper, details of each of these programs will be discussed.
POSTERS MONDAY & TUESDAY
POS-MON-65
STRUCTURAL REARRANGEMENTS OF THE
P. FALCIPARUM GAMETOCYTE: AN ESSENTIAL
PROCESS FOR PARASITE TRANSMISSION
Dearnley M.K.1, 2 , Dixon M.W.A.1, 2, Yeoman J.1, 2, Hanssen E.3 and
Tilley L.1, 2
1
La Trobe Institute for Molecular Science, La Trobe University, VIC
3086, Australia. 2ARC Centre of Excellence for Coherent X-ray
Science, La Trobe University, VIC 3086, Australia. 3Bio21 Institute,
University of Melbourne, VIC 3000, Australia.
Transmission of the malaria parasite Plasmodium falciparum depends
on the production of the specialised sexual blood stage parasites called
gametocytes. Despite the fact that the first forms of the malaria parasite
identified were the sexual stages or gametocytes, many questions
remain about the fundamental biology of this lifecycle stage. Maturation
of the gametocyte to the transmissible stage V form is dependent on
the parasites ability to remodel its host RBC. During this remodelling,
parasite derived structures are formed within the RBC cytoplasm
called Maurer’s clefts which function as protein-sorting organelles
for parasite proteins en route to the RBC membrane. In this study we
assess the genesis and maintenance of the Maurer’s clefts and the
resident Maurer’s cleft protein the Ring Exported Protein 1 (REX1),
across gametocyte development. We also investigate the formation and
composition of the inner membrane complex (IMC) of the gametocyte
across several developmental stages. Previous studies have identified
this complex within the gametocyte; however few have drawn parallels
to the IMC found in the merozoite. We aim to identify novel constituents
of the IMC and investigate its resemblance to the IMC in the asexual
merozoite. Purified gametocytes from differing developmental stages
were examined by electron and fluorescence microscopy to map the
morphology of the developing gametocyte with particular emphasis
on the Maurer’s cleft using resident protein REX1 and the IMC protein
GAP50. An understanding of the structures and remodelling mediating
gametocyte maturation will prove invaluable in the development of novel
transmission blocking approaches.
POS-MON-67
DETERMINING THE INTRACELLULAR LOCALISATION
OF ATNHX5 AND ATNHX6 AND THEIR ROLE IN
CELLULAR TRAFFICKING AND SALT TOLERANCE
Ford B.A. and Gendall A.R.
Depratment of Botany, La trobe University, Victoria, Australia.
AtNHX5 and AtNHX6, like AtSOS1 and AtNHX1-4 are part of the
large CPA1 monovalent cation/proton antiporter sub group of the
sodium hydrogen exchanger super family. AtNHX5 has been shown
to suppress the Na+ sensitive phenotype of the nhx1 yeast mutant
and is up-regulated in response to NaCl. Arabidopsis plants over
expressing the tomato AtNHX5/6 ortholog LeNHX2 showed increased
tolerance to high Na+ concentrations. The intracellular localisation of
AtNHX5 and AtNHX6 in Arabidopsis has been determined by studying
the expression of a 35S::NHX5ORF:CFP, a 35S::NHX6ORF:YFP and
a 35S::YFP:NHX6ORF. Our results show that AtNHX5 and AtNHX6
co-localise to the same intracellular location and are not localised to
either the tonoplast or the plasma membrane, and may be localized to
endosomes. Endosomal compartments are the main transport vesicles
in the secretory and endocytic pathways. It has been proposed that the
endosomal localized NHXs regulate endosomal pH and are essential
for protein sorting in the endocytic and secretory pathways. Preliminary
analysis of root tip cells from the nhx5 nhx6 double mutant shows a
disruption to the normal development of lateral roots. It may be the case
that in the nhx5 nhx6 double mutant the endosomal traffic required to
deliver auxin influx carriers AUX1 and LAX3 to their ultimate destination
is disrupted preventing the normal auxin signaling required for lateral
root development.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 117
POS-TUE-66
RAB2A PARTICIPATES IN THE TRAFFICKING AND
SECRETION OF APOLIPOPROTEIN E
Dinnes D.L.1, Kockx M.1, Jessup W.1 and Kritharides L.1, 2
1
Macrophage Biology Group, Centre for Vascular Research, UNSW,
Sydney, Australia. 2Concord Repatriation General Hospital, University
of Sydney, Sydney, Australia.
Apolipoprotein E (apoE) is a ~34kDa glycoprotein that is secreted from
several cells, including macrophages, hepatocytes and astrocytes, with
known roles in immunoregulation, protection from atherosclerosis and
Alzheimer’s disease. We have previously shown that apoE is transported
in vesicles along the microtubular network and is regulated by PKA,
PP2B (calcineurin) and intracellular Ca2+ (Kockx M et al, Circ Res (2007)
101:608; Kockx M et al, J Biol Chem (2009) 284:24144). However, the
transport proteins regulating its vesicular movement are unknown. The
aim of this study was to identify if any proteins of the Rab GTPase family
play a role in this process. HEK293 cells stably transfected with human
apoE or apoE tagged with GFP (apoE-GFP) were subjected to Rab1A,
2A, 6A, 8A, 10 or 27B siRNA using non-silencing sequences as a
control. Silencing of Rab2A inhibited the rate of secretion 5-fold relative
to control, whereas silencing of other Rab family members had no
effect. Rab2A silencing had no effect on apoE mRNA levels, increased
apoE protein levels within cells and pulse chase metabolic labeling
confirmed a direct inhibition of apoE secretion. Confocal microscopy
demonstrated accumulation of apoE in a perinuclear compartment after
Rab2A silencing, and this was supported with a loss of highly sialylated
apoE isoforms (determined by 2-dimensional electrophoresis) implying
a block of transport through the Golgi network. We have demonstrated
for the first time a major role for the GTPase Rab2A in regulating apoE
transport and secretion, the manipulation of which may provide new
avenues for modulating apoE secretion.
POS-TUE-68
BINDING OF P110 RETINOBLASTOMA PROTEIN
INHIBITS NUCLEAR IMPORT OF SV40 LARGE TUMOUR
ANTIGEN THROUGH A PHOSPHOREGULATED
MECHANISM
Fulcher A.J.1, Dias M.M.1 and Jans D.A.1, 2
1
Monash University, Clayton, Victoria, Australia. 2ARC centre of
Excellent for Biotechnology and Development.
Nuclear import of the simian virus SV40 large tumour antigen (T-ag) is
dependent on its nuclear localisation signal (NLS) within amino acids
126-132 that is recognised by the importin α/β1 heterodimer, as well
as a protein kinase CK2 site at serine 112 upstream of the NLS, which
enhances the interaction c. 50-fold. Here we show for the first time
that T-ag nuclear import is negatively regulated by further N-terminal
sequences (amino acids 102-110) which represent the binding site (BS)
for the retinoblastoma (Rb) tumour suppressor protein. Quantitative
confocal laser scanning microscopic analysis of the transport
properties of T-ag constructs with or without Rb binding site mutations
in living transfected cells or in a reconstituted nuclear transport system
indicate that the presence of the RbBS significantly reduces nuclear
accumulation of T-ag. A number of approaches, including the analysis
of T-ag nuclear import in an isogenic cell pair with and without functional
p110Rb implicate p110Rb binding as being responsible for the reduced
nuclear accumulation, with the serine106 phosphorylation site within the
RbBS appearing to enhance the inhibitory effect. The involvement of
p110Rb in modulating T-ag nuclear transport has implications for the
regulation of nuclear import of the other proteins from the various other
viruses of medical significance that interact with p110Rb, and how this
may relate to transformation.
POSTERS MONDAY & TUESDAY
POS-MON-69
REGULATORY ROLE OF PRIP IN EXOCYTOSIS
THROUGH THE INTERACTION WITH PROTEIN
PHOSPHATASE
Gao J., Takeuchi H., Zhang Z. and Hirata M.
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental
Science, Kyushu University.
Exocytosis is one of the most basal and important cellular events. The
minimal machinery for the final step, membrane fusion, is SNARE
complex consists of syntaxin, SNAP-25 and VAMP. Phospho-state of
these proteins are known to be involved in the regulation of exocytosis.
We have shown that a protein phosphatase-1 anchoring protein, PRIP
(phospholipase C-related but catalytically inactive protein), has some
inhibitory role in regulating exocytosis. In the present study, we examine
the role of PRIP in phospho-dependent regulation of exocytosis using
pheochromocytoma cell line, PC12 cells that secrete noradrenalin
(NA). Pretreatment of the cells with forskolin enhanced NA secretion
and the enhancement was gradually diminished by removing forskolin.
Exogenous expression of PRIP accelerated the decreasing process of
NA secretion. Correlatively with the NA secretion, SNAP-25 was strongly
phosphorylated by forskolin treatment of the cells and dephosphorylated
gradually after removing forskolin. In addition, exogenous expression
of PRIP accelerated the dephosphorylation process. We also showed
that dephosphorylation of SNAP-25 at Thr138, phosphorylated by cAMP-
dependent protein kinase, was mainly catalyzed by PP-1. These results
suggest that PRIP is involved in phospho-dependent regulation of
SNARE proteins through modulating the activity of protein phosphates-1,
thus regulating exocytosis. .
POS-MON-71
TOXICITY OF AMYLOID TRANSTHYRETINS TO DOWN
SYNDROME FIBROBLAST
Annanon S., Kaewmeechai S. and Prapunpoj P.
Department of Biochemistry, Faculty of Science, Prince of Songkla
University, Hat Yai, Songkhla 90112, Thailand
Amyloidosis is a group of human diseases characterized by the intra-
or extracellular deposition of aggregated amyloid proteins, leading to
progressive disruption of the normal tissue architecture and consequently
impairing organ function. Among of these proteins, amyloid β (Aβ) which
is the major constituent of amyloid plaques in brain cortex of Alzheimer
‘s disease (AD) and Down syndrome (DS), and transthyretin (TTR) are
of interest. Inter-relationship in particular the protective function of TTR
on cytotoxicity of Aβ has been demonstrated. Among peripheral tissues,
fibroblasts from DS patients were shown carrying Aβ protein precursor
(APP) and having endocytic dysfunction similar to neurons in AD. In
this study, the cytotoxicity and the protective effect on Aβ of TTR were
explored in greater details in normal and DS fibroblasts. Human wild-
type TTR and TTR variants including V30M and L55P were synthesized
using the heterologous gene expression system of Pichia pastoris. Cell
viability and apoptosis were examined and compared. The preliminary
results showed that not only Aβ but also amyloidogenic TTR V30M
and L55P were toxic to both normal and DS fibroblasts, however with
different sensitivity.
Page 118 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-70
ARL5B IS A GOLGI-LOCALISED SMALL G PROTEIN
INVOLVED IN REGULATION OF RETROGRADE
TRANSPORT
Houghton F.J. and Gleeson P.A.
Bio21 Molecular Science and Biotechnology Institute, University of
Melbourne.
Small G proteins play an essential role in the regulation of membrane
traffic. Small proteins (~20kD) include members of the Rab, Arf and Arl
families. The Arf/Arl family members were identified initially by sequence
homology (Kahn et al., 2006). In the GTP-bound form, the Arfs/Arls
are able to bind specific effectors. Although some Arf-like or Arl family
members have been well characterised, such as Arl1 (Lu et al., 2001)
and ARFRP1/Arl3 (Panic et al., 2003; Setty et al., 2003), the function of
many Arls remains undefined. An important role of small G proteins is
in the regulation of membrane traffic at the trans-Golgi network (TGN)
(Derby et al, 2004; Lu and Hong, 2003). The cDNA encoding a number
of Arls were amplified by RT-PCR from HeLa cells, mutated to the GTP
or GDP bound forms by PCR mutagenesis and cloned into GFP vectors.
Using confocal microscopy analysis of transfected HeLa cells, we
identified Arls that localise to the Golgi. Arl5bWT and Arl5bQ71L localise
to the Golgi in close proximity to TGN golgins, and Arl5bQ71L-GFP is
observed on tubules associated with the TGN. Functional analysis was
conducted using siRNA depletion and membrane transport was studied
using antibody internalisation assays. Depletion of Arl5b by RNAi in
cultured cells results in disruption of the intracellular localisation of
mannose 6-phosphate receptor, and alters the transport dynamics of
the membrane cargo TGN38 and the shiga toxin B fragment between
endosomes and TGN. Therefore, Arl5b is a trans-Golgi small G protein
which regulates endosome-TGN transport. Kahn, R.A., et al. J Cell Biol
(2006), Lu,L. and Hong,W. Mol Biol Cell (2003), Derby, M C. et al. J
Cell Sci.(2004)., Lu,L., et al. J Cell Sci (2001), Panic,B., et al. Curr Biol
(2003), Setty, S.R., et al Curr Biol (2003).
POS-TUE-72
LONG-TERM MEMORY AND LEARNING IMPAIRMENT
IN NEDD4 HETEROZYGOUS MICE
Bongiorno D.1, 2 , Boase N.3, Kumar S.3 and Poronnik P.1, 2
1
School of Medical Science. 2Health Innovations Research Insitute
(HIRi), RMIT University, Bundoora. 3Centre for Cancer Biology, SA
Pathology, Frome Road, Adelaide.
Nedd4 (Neural precursor cell Expressed Developmentally Down-
regulated 4) a ubiquitin ligase (E3) has been implicated in neuronal
development. Although numerous potential substrates of Nedd4 have
been uncovered, the exact physiological function(s) of Nedd4 remain
unclear (Yang and Kumar 2010). As Nedd4 knockout mice die at birth
due to growth retardation and vascular defects, we investigated the
impact of Nedd4 in memory and learning using Nedd4 heterozygous
mice. Nedd4 heterozygous (n=13) and wild-type (n=8) littermate controls
were assessed for short-term memory using Y-maze. Long-term spatial
memory and learning was also assessed using Morris Water Maze
(MWM). Time taken to locate a hidden platform corresponds to the
learning phase. On the final day of testing, the platform is removed and
duration to enter the platform quadrant corresponds to the memory
phase. We found that Nedd4 heterozygote mice took significantly longer
to find the hidden platform than wild-type mice in MWM test (Day 6,
65.3±9.5 and 27.0±4.6 sec). Furthermore, when the hidden platform
was removed, Nedd4 heterozygote mice also took significantly longer
to enter the platform quadrant compared to wild-type mice (23.0±5.4
and 6.4±1.3 sec). However, there was no significant difference in time
spent in the novel arm of the Y-maze between Nedd4 heterozygote
and wild-type mice (150.4±5.4 and 158.3±8.7 sec). These data show
that in Nedd4 heterozygous mice, where ~50% of the ubiquitin ligase is
removed is sufficient to produce long-term spatial memory and learning
deficits, with spared short-term memory. Yang B, Kumar S. 2010. Nedd4
and Nedd4-2: closely related ubiquitin-protein ligases with distinct
physiological functions. Cell Death Differ 17:68-77.
POSTERS MONDAY & TUESDAY
POS-MON-73
THE INVOLVEMENT OF EXOSOMES IN
INTERCELLULAR PRION TRANSMISSION
Coleman B.M.1, 2, 3, Hanssen E.3, 5, Masters C.L.4, Lawson V.A.2, 4 and
Hill A.F.1, 3, 4
1
Department of Biochemistry and Molecular Biology, The University of
Melbourne. 2Department of Pathology, The University of Melbourne.
3
Bio21 Molecular Science and Biotechnology Institute, The University
of Melbourne. 4Mental Health Research Institute, The University of
Melbourne. 5Electron Microscopy Unit, Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne.
Prion diseases are invariable fatal neurodegenerative disorders, which
are associated with an abnormal isoform (PrPSc) of the host encoded
cellular prion protein (PrPC). The diseases are also transmissible, with the
infectious agent postulated to be composed principally of PrPSc. However,
the conditions, cofactors, conformations and aggregates necessary for
transmission remain to be defined. PrPSc and prion infectivity are present
within exosome preparations. Exosomes are 40-100 nm membrane
vesicles of endocytic origin released by most cell types in vitro and
recent studies have also identified them in vivo in body fluids. However,
due to limitations associated with standard sucrose density gradients it
is not clear whether prion infectivity localises within exosomes or with
an alternate, potentially unique vesicle. Recent developments in virus
purification techniques have enabled the separation of virus particles
(such as HIV-1 and influenza) from exosomes and other membrane
vesicles. This was previously unobtainable using standard sucrose
density gradients. We have demonstrated the application of a novel,
rate-zonal gradient separation technique to show that prion infectivity is
indeed associated with exosomes; a finding that is further validated with
biochemical techniques. A benefit of this technique is that it produces
highly purified exosomes without disturbing their native structure, making
them amenable to a variety of downstream analytical techniques. Using
this technique coupled with transmission electron microscopy we
have found that the ultra-structure of exosomes is far more intricate
than previously thought. This could potentially provide insight into the
currently unknown mechanism by which exosomes interact with cells.
POS-MON-75
DYNEIN/COPII INTERACTION WITH MUTANT SOD1 IN
SOD1G93A TRANSGENIC MICE AND NSC34 CELLS
Farg M.1, Walker A.1, Turner B.2, Horne M.2 and Atkin J.1, 2
1
1 Department of Biochemistry, La Trobe University | Bundoora,
Victoria. 2Howard Florey Institute, University of Melbourne, Australia.
Recent evidence suggests that disruption of the dynein/dynactin
transport machinery occurs in ALS. Mutation in the dynactin subunit
p150 Glued causes human ALS and mice overexpressing mutant dynein
heavy chain or Tgdynamitin mouse display slowly progressive motor
neuron degeneration. Also, mutant but not wildtype SOD1 physically
interacts with dynein and dynein co-localises with mutant SOD1
inclusions. Impairment of dynein-dynactin function by p50 dynamitin over-
expression interferes with endosome trafficking and results in Golgi
fragmentation. Fragmentation of the neuronal Golgi apparatus has
been observed in both sporadic and SOD-mediated familial ALS as
well as in pre-symptomatic transgenic SOD1G93Aanimals. Bidirectional
transport between the endoplasmic reticulum (ER) and Golgi apparatus
is mediated by the dynein transport machinery. Proteins exiting the
ER are transported in vesicles coated by coat protein complex II
(COPII). Recently there has been a surge of publications describing the
importance of ER stress in ALS. We described induction of the whole
unfolded protein response in both transgenic SOD1G93A animals and in
human sporadic ALS patients. We hypothesised that disruption in ER to
Golgi trafficking may contribute to ER stress in ALS. Anterograde and
retrograde transport proteins were examined in transgenic SOD1G93A
mice and in NSC34 cell lines transfected with mutant and wt SOD1. We
demonstrated a physical interaction between mutant and not wildtype
SOD1 and both COPII and dynein in transgenic SOD1G93A animals at
p10, 60 days before the onset of symptoms and before the onset of
ER stress. This was confirmed; in NSC34 stable cell lines express
human SOD1 A4V/ G85R/ G37R and G93A. Also, COPII/ COPI found
to co-localise with mutant SOD1 inclusions. These data suggest that
alteration in dynein mediated transport is a very early event in disease
and preceeds ER stress in ALS.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 119
POS-TUE-74
EVALUATION OF SRU BIND LABEL-FREE
TECHNOLOGY FOR ASSESSMENT OF KAPPA OPIOID
RECEPTOR LIGANDS IN CELL-BASED ASSAYS
Deuis J.1, Vetter I.2, Lewis R.2 and Cabot P.1
1
School of Pharmacy, University of Queensland, Australia. 2Institute for
Molecular Bioscience, University of Queensland, Australia.
Opioid receptors have long been known to be effective targets for the
production of analgesia. Recent interest has developed in targeting the
kappa opioid receptor (KOR) subtype, which couples to inhibition of
adenylate cyclase through Gαi. Label-free technology is now available
to assess ligand-receptor interactions in cell-based assays, replacing
more conventional measurement of second messenger signalling. The
aim of this study was to assess the ability of BIND label-free technology
(SRU Biosystems) to detect the activation of the KOR. HEK-293 cells
transfected with the rat KOR were exposed to varying concentrations
of U-50488, a KOR agonist, with or without naloxone, an opioid
receptor antagonist. Addition of U-50488 caused a naloxone-sensitive,
concentration-dependent increase in peak wavelength shift with an
EC50 of 2.7 nM, consistent with literature values. Therefore BIND label-
free technology appears to be an effective technique to assess activity
of KOR agonists in cell-based assays, and may be a useful tool in the
future to identify new KOR agonists.
POS-TUE-76
ASCENDING GABAERGIC/PEPTIDERGIC CONTROL
OF AROUSAL, STRESS AND MOTIVATED BEHAVIOUR:
FOCUS ON NUCLEUS INCERTUS AND RELAXIN-3
SIGNALLING
Gundlach A.L.1, Ma S.1, 2, Smith C.M.1, Ryan P.J.1, Hossain M.A.1,
Wade J.D.1, Bathgate R.A.D.1, Verberne A.J.M.2, Blasiak A.3 and
Olucha-Bordonau F.E.4
1
Florey Neuroscience Institutes, The University of Melbourne,
Australia. 2Department of Medicine, Austin Health, The University of
Melbourne, Australia. 3Institute of Zoology, Jagiellonian University,
Krakow, Poland. 4Department of Anatomy, University of València,
València, Spain.
The nucleus incertus (NI) in the ventromedial pontine grey is a major
source of ascending GABAergic projections that innervate limbic and
hypothalamic regions involved in arousal, sleep/wakefulness and
related autonomic and neuroendocrine functions. NI neurons and other
small midbrain populations express the neuropeptide, relaxin-3; and
existing anatomical and functional studies suggest relaxin-3 receptor
(RXFP3) signalling should modulate ‘behavioural state’, arousal and
responses to stress, and related behaviours. The precise nature of
these actions and the circuits and transmitters/peptides involved are
not known, however. We have conducted studies in rats and mice that
reveal: (i) NI neurons in the rat are activated by behavioural activity
and neurogenic stressors, and by CRF and orexin; (ii) relaxin-3 neurons
in rat and mouse innervate networks controlling circadian activity and
metabolic balance, and the emotional and cognitive circuits of the
amygdala and septohippocampal system; (iii) central RXFP3 activation
increases feeding in satiated rats; (iv) RXFP3 activation/inhibition in
medial septum modulates spatial working memory and hippocampal
theta rhythm in the rat; (v) RXFP3 activation in central amygdala can
block fear expression and may enhance fear extinction in rats; and (vi)
relaxin-3 knockout mice are hypoactive and have altered sleep patterns
during the dark/active phase of the circadian cycle. These studies
have increased our knowledge of the role of the ascending NI GABA/
relaxin-3 network in homeostatic and complex behaviours and identified
its potential as a therapeutic target in neuropsychiatric disease.
POSTERS MONDAY & TUESDAY
POS-MON-77
OXIDATIVE STRESS INDUCES MULTIPLE CASPASE-
INDEPENDENT CELL DEATH PATHWAYS IN PRIMARY
CORTICAL NEURONS
Higgins G.C.1, Devenish R.J.1, Beart P.M.2 and Nagley P.1
1
Department of Biochemistry and Molecular Biology, Monash
University, Wellington Rd, Clayton 3800, Victoria, Australia. 2Florey
Neuroscience Institutes, University of Melbourne, Parkville, Victoria
3010, Australia.
Neuronal cells can undergo a diverse range of death responses. These
death outcomes were previously thought to be limited to either apoptosis
(involving caspases and energy-dependent) or unregulated necrosis
(independent of both caspases and the need for ATP). We have recently
shown that primary cortical neurons from mice exposed to an acute insult
of hydrogen peroxide (H2O2) undergo caspase-independent cell death
that is highly regulated, predominantly manifesting autophagic cell death
and programmed necrosis. In this current work we show these neurons
undergo caspase-independent cell death when exposed to a chronic
dose of superoxide (provided by exogenous xanthine oxidase in the
presence of catalase, which acts as a sink for H2O2). We found that while
key mitochondrial intermembrane space proteins (cytochrome c, Smac,
AIF and Endonuclease G) are redistributed to cytosol, downstream
caspases (-3, -7, -9) were not activated. While this outcome was similar
to that of our previous work done with H2O2, we found that knockdown
of either Endo G (programmed necrosis) or Atg7 (autophagic cell death)
using siRNA was much less potent in blocking cell death. We conclude
that superoxide invokes a diverse death response that involves some
autophagic cell death and programmed necrosis, but is predominantly
unregulated necrosis. This work highlights the significance of the type
of oxidative stress exposure, in relation to the specificity of the neuronal
cell death outcomes.
POS-MON-79
INVESTIGATING THE ROLE OF COPPER IN THE
NERVOUS SYSTEM
Hwang E.C.J. and Burke R.
School of Biological Sciences, Monash University, Wellington Rd,
Clayton, VIC, 3800, Australia.
Copper is an important trace metal required in balanced amounts
for proper cellular function and development. Strict control of copper
levels is required as copper dyshomeostasis results in multiple adverse
effects, such as cellular damage due to the generation of reactive
oxygen species. Proper copper homeostasis is achieved via the action
of copper transport proteins which serve to either import, sequester or
export copper. While the overall role of copper in the body has been
investigated quite well, less is known about the specific role of copper in
the nervous system. Previous studies have shown that copper is likely to
play a role in both the function and development of the nervous system,
however, further study is require to elucidate the specific mechanisms
by which this occurs. Using Drosophila Melanogaster as a model, I have
created a system within which to study the effects of both copper excess
and scarcity on the nervous system. This has been achieved through
the targeted misexpression of copper transport proteins in the nervous
system via use of the GAL4-UAS system, thereby generating an in vivo
model within which to study the effects of copper dyshomeostasis. I
have shown that both copper excess and scarcity result in the disruption
of nervous system function, and have investigated some of the
mechanisms by which this occurs.
Page 120 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-78
THE C-SRC PROTEIN TYROSINE KINASE IS
REGULATED BY CALPAIN MEDIATED CLEAVAGE
UPON OVER STIMULATION OF GLUTAMATE
RECEPTOR IN CULTURED PRIMARY NEURONS
Hossain M.I. and Cheng H.C.
Department of Biochemistry and Molecular Biology, Bio21 Institute,
University of Melbourne.
Glutamate-induced excitotoxicity is one of the main features of ischemic
stroke and neurodegenerative diseases. Excess glutamate causes over-
stimulation of NMDA receptor leading to excessive influx of extracellular
calcium ion into neurons. The excessive influx of calcium ion activates
calpains which cleave many functional proteins and in turns enhances
neuronal death. c-Src, an important member of non-receptor Src family
protein kinases (SFKs), binds to scaffolding proteins in the NMDA
receptor to form a complex to regulate activity of the receptor. In this way
c-Src plays crucial role in glutamate-induced neuronal death. Herein we
report that under glutamate-induced excitotoxicity, c-Src is cleaved by
calpain in cultured primary cortical neurons. The same phenomenon
was also found when recombinant c-Src was digested with calpain
in vitro. Using an N-terminal directed c-Src antibody we mapped the
cleavage site to the N-terminal region of c-Src. We also monitored
kinase activity of the protein under excitotixic condition using specific
Src optimal peptide and phosphospecific antibodies. Kinase activity
assay demonstrated that c-Src activity undergoes a biphasic change
– it increases shortly after glutamate treatment but decreases once the
calpain-mediated cleavage starts. In summary results of our studies
demonstrate the involvement of c-Src in excitotoxic neuronal death. We
demonstrate for the first time that calpain mediated cleavage of c-Src
as regulatory mechanism in neurons. Further investigation on the effect
of calpain mediated cleavage of c-Src on its activity and regulation may
provide insights into the mechanism of excitotoxic neuronal death.
POS-TUE-80
THE EXPRESSION OF TNF AND ITS RECEPTORS IN
SCHIZOPHRENIA AND MOOD DISORDERS
Jeon W.J.1, 2 , Gibbons A.S.1, 2 and Dean B.1, 2
1
Rebecca L. Cooper Laboratories, the Mental Health Research
Institute, Parkville, Victoria, Australia. 2Department of Psychiatry, the
University of Melbourne, Parkville, Victoria, Australia.
Abberant expression of cytokines involved in pro-inflammatory pathways
have been proposed to underlie the pathology of schizophrenia and
mood disorders. We have recently reported an increase in the level of
tumor necrosis factor (TNF) protein in the dorsolateral prefrontal cortex
(Brodmann’s area (BA) 46), but not the anterior cingulate cortex (BA 24),
from subjects with major depression. We sought to determine whether
the mRNA expression of TNF and its receptors is altered in the cortex
of post-mortem subjects with schizophrenia and mood disorders. Real-
time PCR was used to measure the levels of TNF, TNFR1 and TNFR2
mRNA in BA 24 and BA 46 from subjects with schizophrenia (n=20),
major depression (n=10), bipolar disorder (n=10) and matched control
subjects. The level of TNF mRNA was significantly increased in BA 24,
but not BA 46, from subjects with schizophrenia compared to controls
(p<0.05). The level of TNFR1 mRNA was increased in BA 24 (p<0.05)
and BA 46 (p<0.0001) in subjects with schizophrenia. TNFR2 mRNA
expression was not altered in schizophrenia (p>0.05). Furthermore, there
was no change in the mRNA levels of TNF and either receptor in BA 24
and BA 46 from subjects with major depression and bipolar disorder
compared to controls (p>0.05). Our findings suggest that abnormal
TNF signalling may be involved in the pathology of schizophrenia.
Furthermore, the increased TNF protein expression in BA 46 that we
previously reported in subjects with major depression does not appear
to result from increased gene expression.
POSTERS MONDAY & TUESDAY
POS-MON-81
TARGETED GENE DELIVERY TO MICROGLIA VIA THE
SCAVENGER RECEPTOR CLASS B, TYPE I
Malmevik J.M.K.1, Rogers M.-L.1, Nilsson M.2, Nakanishi Y.3, Rush R.A.1,
Sims N.R.1 and Muyderman H.1
1
Centre for Neuroscience, Flinders Medical Science and Technology,
Flinders University, Australia. 2Institute for Neuroscience, Goteborg
University, Sweden. 3Faculty of Pharmaceutical Sciences, Kanazawa
University, Japan.
Microglia are the major contributors to inflammatory responses in the
brain. Investigations of microglia are limited by the difficulty of selectively
targeting these cells within the complex environment of the mature brain.
Receptor-mediated gene delivery constitutes a non-viral approach
which also avoids immune responses that can be produced even by
highly modified viral vectors. In this study, we tested the possibility
of selectively targeting microglia in vivo utilising receptor-mediated
gene delivery via the scavenger receptor class B type I (SR-BI). SR-
BI expression in mixed glial primary cultures and in purified microglial
cultures was confined to microglia. Incubations of an antibody targeted at
the extra-cellular domain of SR-BI resulted in microglia-specific uptake.
This SR-BI antibody was linked to the polycation polyethylenimine
(PEI) and bound to a plasmid encoding green fluorescent protein
(GFP) to form an ‘immunogene’. GFP expression was seen in microglia
following incubations of this construct in mixed glial primary cultures.
Intrahippocampal infusions of the ‘immunogene’ resulted in a substantial
GFP expression in CD11b- and ED1-positive microglia around the
infusion site (n=5). GFP-positive microglia were found up to 4.2 mm
from the site of infusion. No colocalisation with astrocytic or neuronal
markers was found. Control infusions with PEI and the GFP plasmid
alone produced no expression of the reporter gene (n=3). This research
demonstrates for the first time the use of a non-viral transfection system
to selectively target the microglial cell population in vitro and in vivo.
POS-MON-83
CLINICAL SIGNIFICANCE OF URINE SURVIVIN MRNA
IN PROSTATE CANCER DETECTION
Almaghrebi M., Kehinde E.O. and Kapila K.
Kuwait University - Faculty of Medicine.
Survivin is one of the most tumor-specific molecules, which antagonizes
apoptosis and promotes tumor associated angiogenesis. Thus, it
comes to no surprise that it is overexpressed in many types of cancers.
In prostate cancer, PSA levels alone have a low overall efficiency of
accurate diagnosis. Survivin was evaluated as an alternative molecular
marker in patient urine samples. Expression levels were measured by
quantitative reverse transcription-real time polymerase chain reaction
(RT-PCR) and correlated with clinicopathological data in urine samples
from patients with BPH, BPH & Prostatitis, and prostate cancer. Survivin
levels were significantly elevated in urine from prostate cancer patients
compared with healthy controls (P <.001) and with noncancerous
prostate disorders: BPH (P <.05) and BPH with Prostatitis (p <0.05).
An optimal cutoff value of 25 pg was determined. Accordingly, 21% of
patient samples had survivin levels above 25 pg, of which only 3% were
healthy individuals. In contrast, 55% of individuals with survivin levels
above 25 pg were prostate cancer patients. The results indicate that
urine survivin levels are elevated during prostate cancer developmnt,
demonstrating its strength as a potential marker for discriminating
benign from malignant disease.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 121
POS-TUE-82
TOWARDS ISOFORM-SELECTIVE DYNAMIN
INHIBITORS
Mariana A.1, Hill T.A.2, McGeachie A.B.1, Chau N.1, Daniel J.1, Gorgani N.N.1,
McCluskey A.2 and Robinson P.J.1
1
Cell Signalling Unit, Children’s Medical Research Institute, The
University of Sydney, Westmead, NSW 2145, Australia. 2Department
of Chemistry, University of Newcastle, NSW 2308, Australia.
The GTPase enzyme dynamin I is predominant in brain; dynamin II is
ubiquitously expressed; and dynamin III is found in brain and testes.
Dynamin I and dynamin II are required for synaptic vesicle endocytosis
(SVE) and receptor-mediated endocytosis (RME), respectively. Dynamin
II has a second function in the abscission that completes mitosis.
Therefore, dynamin I-specific inhibitors might be useful in diseases
that involve SVE (epilepsy), while dynamin II-specific inhibitors might
be effective as anticancer drugs with minimal side effects. Our team
has developed various classes of dynamin inhibitors which have been
discovered using a malachite green dynamin I-based GTPase assay
(dynamin I being activated by liposomes) and dynamin II-mediated
RME cell-based assay. We have now developed a dynamin II-based
GTPase assay, using dynamin II produced by transient expression
in Sf9 insect cells, which retains high specific activity comparable to
endogenous brain dynamin I, as well as dynamin I cell-based assay
using synaptosome. When we tested our classes of dynamin inhibitor
that have been published (MiTMAB, pyrimidynes) in our 4 assays, they
appear to be non-selective (PAN inhibitors). We recently published new
class of dynamin inhibitors (iminodyns). These drugs inhibit dynamin
GTPase activity, as well as endocytosis. By utilising our 4 type of assays,
the iminodyns do not show any isoform-selectivity for dynamin I or II
GTPase activity. Despite this, the iminodyns include three nanomolar
potent dynamin I and II inhibitors, which also block endocytosis.
Nevertheless, our screening system allows further development
towards isoform-selective dynamin inhibitors from the iminodyns as well
as other classes.
POS-TUE-84
IDENTIFYING THE ROLE OF SPECIFIC NON-
CONSERVED RESIDUES ON PI3K IN THE DISCOVERY
OF ISOFORM-SELECTIVE PI3K INHIBITORS
Amran S.I.
Monash Institute of Pharmaceutical Science, 381 Royal Parade, Vic
3052 Australia.
Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that catalyzes the
biosynthesis of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3 - second messengers
that trigger a wide range of downstream signalling cascades involved in
cell survival, growth, adhesion and proliferation. The heterodimeric class
1 PI3K proteins are composed of a regulatory subunit (p85) complexed
with either one of 4 different isoforms of catalytic subunit (p110α, p110β,
p110δ and p110γ). The PI3KCA gene encoding the α-isoform has been
found to be frequently mutated in cancers such as breast, prostate,
colon, liver and brain. The overall aim of this program is to elucidate the
molecular mechanism of binding of isoform selective small molecule
PI3K inhibitors. Previous studies have shown that specific regions within
the catalytic subunit contain non-conserved residues that contribute
to isoform selectivity. In this study mutant P13Kα isoforms were
generated by swapping the residues of the α-isoform for corresponding
residues of other isoforms using site-directed mutagenesis followed
by recombination of the mutant sequences into the baculovirus vector
system. The resultant proteins were expressed in insect cells and
purified. The purified enzymes have been characterised using western
blot analysis and kinase assays. With retained enzyme activity these
mutant isoforms can be used to identify the role of non-conserved
residues in inhibitor binding and contribute to rational design of novel
PI3K inhibitors.
POSTERS MONDAY & TUESDAY
POS-MON-85
EVASION STRATEGIES OF ECHINOCOCCUS
GRANULOSUS TO TH1 HOST PROTECTIVE RESPONSE
DURING HUMAN INFECTION
Amri M. and Touil-Boukoffa C.
Team: “Cytokines and NOSynthases”, Laboratory of Cellular and
Molecular Biology, Faculty of Biological Science, USTHB, PB 32, El-
Alia, 16111, Algiers, Algeria.
Background: Human echinococcosis is one of the world’s major
zoonotic infections. It usually manifests as unilocular cyst(s) mainly
located in the liver. More recently, we have highlighted an evident role
of IFN-gamma (Th1 cytokine) in parasite killing by NOS2 (Nitric oxide
Synthase2) pathway. Moreover, IL-10 (Treg cytokine) production seems
to be an evasive mechanism taken by the parasite to establish in the
host by Arginase pathway (Amri et al., 2007 & 2009). Of note, NOS2 and
Arginase are known to compete for the common substrate, L-Arginine.
Moreover, IL-10 downregulates IFN-gamma production. Indeed, more
researches are required to identify factors present in parasite cyst
which affect protective Th1 response in Echinococcus granulosus
human infection. Method: We investigate the effect of laminated-
layer (accelullar layer of hydatic cyst) extract (LLs) on Th1/Treg and
NOS2/Arginase balance in culture performed with mononuclear cells
(PBMC) of hydatid patients and healthy donors. Furthermore, we have
investigated the effect of LLs on parasite viability in PBMC-parasite
cocultures. Results: Our results demonstrated that LLs reduced IFN-
gamma/NO production and enhanced IL-10 production and Arginase
activity. In addition, LLs enhanced parasite survival in vitro. Similar
findings are observed in cultures and cocultures performed with PBMC
of patients and healthy donors. Moreover, the major antigenic fraction
in LLs: the fraction 4 (12kDa, purified by chromatography) has the same
effect as LLs. Conclusion: Collectively, the present study provides
evidence that Echinococcus granulosus laminated layer impairs Th1
protective response and allow the parasite to survive. Inhibition of these
mechanisms seems to be important issue to address during the design
of anti-hydatic treatment.
POS-MON-87
ATRX IS A SERTOLI CELL SURVIVAL FACTOR
AND REGULATOR OF SPERMATOGENESIS VIA
INTERACTION WITH ANDROGEN RECEPTOR
Bagheri-Fam S.1, Argentaro A.1, Svingen T.2, Combes A.2, Koopman P.2
and Harley V.1
1
Prince Henry’s Institute of Medical Research, Melbourne, Victoria,
Australia. 2Institute of Molecular Bioscience, Brisbane, Queensland,
Australia.
The ATR-X syndrome, (α-thalassemia mental retardation, X-linked) is
a developmental disorder affecting males. The testicular abnormalities
of ATR-X patients comprise small testes, few seminiferous tubules, and
a lack of germ cells. ATRX protein remodels chromatin in vitro though
its bona fide target genes are unidentified. To understand the gonadal
role of ATRX, we inactivated Atrx specifically in Sertoli cells (ScAtrxKO
mice) from embryonic day 14.5. Fluorescence-based three-dimensional
modeling at E17.5 revealed that testis cord volume in ScAtrxKO mice is
~30% of wildtype, with discontinuous or isolated testis cords apparent.
While Sertoli cell apoptosis is increased by 10-fold when compared to
wildtype, no difference in Sertoli cell proliferation was observed. These
data suggest that ATRX is required for fetal Sertoli cell survival and, in
turn, for elongation of fetal testis cords. Adult ScAtrxKO testes weigh
~25% of wildtype suggesting that seminiferous tubule hypoplasia is
primarily established during fetal life. Histological examination showed
that a third of tubules contain germ cells arrested in late meiosis or
at the round spermatid stage. We found that the Androgen Receptor
(AR)-dependent genes, Rhox5 and Claudin3, were significantly down-
regulated in Sertoli cells of ScAtrxKO testes. Moreover, we show that
ATRX and AR proteins interact in the TM4 Sertoli cell line and co-
operatively activate the Rhox5 promoter. In summary, ATRX plays an
important role in Sertoli cell survival during fetal testis development and
in adult testis function, where ATRX protein interacts with AR to regulate
the transcription of AR-dependent genes that control spermatogenesis.
Page 122 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-86
ETHYLENE MEDIATED DEFENCE AGAINST THE ROOT
INFECTING FUNGAL PATHOGEN RHIZOCTONIA
SOLANI IN MEDICAGO TRUNCATULA
Anderson J.P.1, Lichtenzveig J.1, 2, Oliver R.P.2 and Singh K.1
1
CSIRO Plant Industry, Floreat, Western Australia. 2Australian Centre
for Necrotrophic Fungal Pathogens, Murdoch, Western Australia.
The fungal necrotrophic pathogen Rhizoctonia solani (Kühn) is a
significant constraint to the production of a range of crops as diverse
as cereals, canola and legumes, including causing the second largest
disease constraint to rice production in China and India. No strong
genetic resistance has been identified in these crops despite wide
ranging primary and secondary germplasm screens, suggesting
alternative strategies to improve resistance are required. In this study
we characterise moderate resistance to R. solani AG8 identified in
the model legume Medicago truncatula. The activity of an ethylene
and jasmonate responsive promoter element was associated with
the moderate resistance as was the induction of specific ethylene/
jasmonate response transcription factors. Over-expression of some
of these transcription factors in transgenic Medicago “hairy roots”
increased resistance to R. solani as well as to another destructive root
pathogen, the oomycete Phytophthora medicaginis. Over-expression of
these genes had no apparent impact on symbiosis with nitrogen fixing
bacteria as the transgenic roots formed root nodules at comparable rates
to wild type. This suggests that enhanced resistance to root diseases
can be uncoupled from symbiotic plant-microbe interactions in the same
tissue and ethylene dependent control of nodule number is distinct from
ethylene dependent defenses.
POS-TUE-88
POLYMORPHISM OF SOD2 GENE IN PATIENTS OF
BRONCHIAL ASTHMA
Bhadoria D.P.2 , Bhadoria K.3, Dutta K.2, Kumar M.1, Singh B.1, Singh
S.1, Kumar R.1, Bhadoria P.2, Anand R.2 and Sharma G.L.1
1
Institute of Genomics and Integrative Biology, University Campus Mall
Road, Delhi- 110007, India. 2Maulana Azad Medical College and Lok
Nayak (Irwin) Hospital, New Delhi 110002, India. 3Graduate School of
Science, University of Melbourne, Victoria 3010 Australia.
Superoxide dismutase (SOD2) is an antioxidant protein and
polymorphism of its gene may contribute to susceptibility to bronchial
asthma. There are no studies on polymorphism in SOD2 gene from the
Indian subcontinent. The present study was conducted to investigate
association between SOD2 gene polymorphisms and pathogenesis of
bronchial asthma in Indian patients. Fifty patients of asthma diagnosed
as per ATS guideline 1987 and 50 normal controls were included in this
study for drawing blood. Genomic DNA was isolated from peripheral
blood leucocytes and used for the amplification of SOD2 gene. PCR
product was subjected to sequencing by Applied Biosystems 3730 DNA
sequence analyzer. The sequencing data thus obtained was analyzed
using SISA and Epi Info statistical softwares. Screening of sequencing
data of the subjects in either group identified an intronic mutation at
base number 14788 (rs2842980. Twenty eight subjects of asthma (56%)
and 9 from the control group (18%) were heterozygous (A/T) for this
SNP. One patient as well as one control was found to be homozygous
mutant. Wild type allele T was present in 70% of asthmatics and 89%
of controls showing the role of A/T genotype susceptibility of asthmatic
patients to the disease.The occurrence of T to A mutation at 14788 base
position was higher in asthmatics than in controls (p<0.001) indicating
its involvement in asthma and it could be an important risk factor the
disease.
POSTERS MONDAY & TUESDAY
POS-MON-89
STATUS OF ANTIOXIDANTS IN BRONCHIAL ASTHMA
Bhadoria P.2 , Bhadoria K.3, Dutta K.2, Kumar M.1, Singh B.1, Singh S.1,
Kumar R.1, Bhadoria D.P.2, Anand R.2 and Sharma G.L.1
1
Institute of Genomics and Integrative Biology, University Campus Mall
Road, Delhi- 110007, India. 2Maulana Azad Medical College and Lok
Nayak (Irwin) Hospital, New Delhi 110002, India. 3Graduate School of
Science, University of Melbourne, Victoria 3010 Australia.
There is an increased production of reactive oxygen species (ROS)
by inflammatory cells in the airways of patients of bronchial asthma,
resulting into oxidative stress. In order to combat and neutralize the
deleterious effects of ROS, various endogenous antioxidant processes
occur in the hosts involving enzymatic and non-enzymatic mechanisms.
The present study was undertaken to investigate the status of
antioxidant markers in Indian asthmatic patients. Fifty asthmatics
diagnosed as per ATS guidelines (1987), and 50 normal controls were
recruited for drawing blood. Sera separated from whole blood was used
for estimation of antioxidant markers namely, Superoxide dismutase
(SOD), Catalase, Ascorbic acid and lipid peroxidation. Total SOD was
estimated by the method of Kono et al. (1978). The activity of catalase
enzyme was assayed by the method of Beers and Sizer (1952). Ascorbic
acid assay was performed by the method of Natelson el al. (1971). The
assay for microsomal lipid peroxidation byproduct, Malondialdehyde,
was performed by the method of Wright et al 1981. Results revealed that
there was marked decrease in total SOD (p<.01) and Catalase (p<.01) in
asthmatics as compared to controls. However, there was no significant
difference in ascorbic acid levels between patients and controls (p>.05).
There was increased lipid peroxidation and antioxidant levels were found
to be reduced in asthmatics. It seems that their level was not sufficient
to scavenge free radicals produced during oxidative stress and tissue
damage.
POS-MON-91
TARGETING THE TROPOMYOSIN ISOFORM TM5NM1
IMPAIRS TUMOUR SURVIVAL, PROLIFERATION AND
MIGRATION
Bonello T.1, Stehn J.1, Schevzov G.1, Coombes J.1, McCluskey A.2,
Haass N.3, Dixon I.4 and Gunning P.1
1
Department of Pharmacology, School of Medical Sciences, University
of New South Wales, NSW, Australia. 2School of Chemistry, University
of Newcastle, NSW, Australia. 3Centenary Institute of Cancer Medicine
and Cell Biology, University of Sydney, NSW, Australia. 4Genscreen
Pty Ltd, Melbourne, VIC, Australia.
The actin cytoskeleton is a fundamental regulator of key cellular
functions including proliferation, motility and apoptosis. Aberrations
in these processes are hallmarks of tumorigenesis, making the actin
cytoskeleton an attractive chemotherapeutic target. The function of
actin filament populations is regulated by their association with distinct
tropomyosin isoforms. As cells transform they demonstrate an increased
reliance on a subset of low molecular weight (LMW) tropomyosins. The
LMW isoform, TM5NM1, has been shown in over-expression and knock-
down models to impart a proliferative advantage on the cell and regulate
directed cell movement. Objective: We have developed a novel class
of compounds designed to target TM5NM1 containing filaments, and
aim to characterise their effect on cellular function. Summary of
Results: The lead compound, TR100, reduced viability in a panel of
neuroblastoma and melanoma cell lines (average LC50 ~2-3μM) and
inhibited survival and growth in a 3D melanoma model which simulates
the tumour microenvironment. To dissect the role of TR100 in cell
death, FACS analysis was performed with the human melanoma cell
line SK-N-MEL28. Cells treated with 5μM TR100 underwent G0/G1
cell cycle arrest, while programmed cell death was induced at higher
concentrations of TR100. Finally, migration towards a directional cue,
measured by in vitro scratch wound assay, was significantly impaired at
non-lethal concentrations of TR100. Conclusions: We have described
a novel class of chemotherapeutic compounds which specifically target
an actin filament population required for tumour survival, proliferation
and migration.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 123
POS-TUE-90
EFFECT OF ANTIOXIDANTS SUPPLEMENTATIONS IN
SALT-INDUCED DYSLIPIDAEMIA IN ALBINO RATS
Bilbis L.S., Saidu Y., Ahmad K., Abbas A.Y., Baba A.U. and Shuaib A.M.
Usmanu Danfodiyo University Sokoto, Nigeria PMB 2346 Sokoto,
Nigeria.
Cardiovascular disease (CVD) is associated with many risk factors
including oxidative stress and dyslipidaemia. The current work
evaluated the effects of antioxidants supplementation on salt-induced
dyslipidaemia in albino rats. Rats were grouped into 11 groups of 7 rats
each. Groups 2-11 were fed 8% salt diets for 4 weeks while group 1
served as control and were fed normal rat feed. Water was provided
to all the groups ad libitum. The animals in groups 3-11 were then
supplemented with vitamin A; vitamin C; vitamin E; Cu; Mn; Zn; vitamins
A,C and E combined; Cu, Mn and Zn combined; and all the vitamins and
minerals combined respectively for additional 4 weeks simultaneously
with salt loading. Group 2 was not supplemented and served as the
negative control. The body weight changes, pulse rate of the rats were
monitored. Serum levels of Total cholesterol, triacylglyceride, low
density lipoprotein cholesterol, very low density lipoprotein cholesterol,
high density lipoprotein cholesterol and glucose were estimated. The
results indicated that the vitamins reduced significantly serum lipid
profiles and the atherogenic index by up to 80 %. The serum glucose
levels of the rats supplemented with antioxidant vitamins and minerals
were also significantly (P<0.05) lowered compared with the negative
control group. These results suggest that the reduction of serum lipid
profile and glucose level may be due to regulation of cholesterol and
lipoprotein metabolism and increased insulin sensitivity as a result of
the supplementations. It may thus suggest that the antioxidants may
provide protection against CVDs and metabolic syndrome in salt
induced dyslipidaemia in rats.
POS-TUE-92
INVESTIGATION OF MITOCHONDRIAL FUNCTION IN A
METHYLMALONIC ACIDURIA MOUSE MODEL
Buck N.E.1, Siddiqui T.1, Laskowski A.2, Frazier A.E.2, Pitt J.J.3,
Thorburn D.R.2, 3 and Peters H.L.1
1
Cell & Gene Therapy Group, Murdoch Childrens Research Institute
and Department of Paediatrics, University of Melbourne, Royal
Children’s Hospital, Parkville, Victoria, Australia. 2Mitochondrial
Research, Murdoch Childrens Research Institute and Department
of Paediatrics, University of Melbourne, Royal Children’s Hospital,
Parkville, Victoria, Australia. 3VCGS Pathology, Murdoch Childrens
Research Institute, Royal Children’s Hospital, Parkville, Victoria,
Australia.
Methylmalonic aciduria (MMA) is an organic acid disorder resulting from
a defect in the mitochondrial enzyme methylmalonyl-CoA mutase. We
have developed an MMA mouse model that recapitulates key aspects
of the intermediate form of MMA: elevated methylmalonic acid levels
in urine, blood and various organs and survival into adulthood. We
have investigated the impact of deficient methylmalonyl-CoA mutase
on mitochondrial enzyme activities. Respiratory chain complexes (I, II,
III, IV), citrate synthase and aconitase activities were assayed. There
was no difference between Co II, aconitase and citrate synthase activity
between MMA and control mice, suggesting that the tissues were not
affected by significant oxidative stress. Comparison of MMA to control
mouse samples show reduced levels (up to 50%) of the complexes with
subunits encoded by mitochondrial DNA (I, III and IV) in the liver and
muscle, and Co III, IV in the brain (kidney enzyme activity unchanged).
Fluorescent microscopy showed no difference in the number and
distribution of mitochondria in kidney, muscle, lung or skin from control
and MMA mice. Mitochondrial DNA levels were determined using
real-time PCR by comparing Mtco1 to β-actin. The amount of mtDNA
present in MMA mouse kidney, heart and muscle were 60±20% the
amount present in controls. There was little change in the amount of
mitochondrial DNA in MMA mouse liver and brain (85±23% of controls).
These data support the concept that pathogenesis of MMA may be
mediated by an affect on expression of respiratory chain complexes
with subunits encoded by mitochondrial DNA.
POSTERS MONDAY & TUESDAY
POS-MON-93
THE ROLE OF UBIQUITIN IN THE REPLICATION OF
HEPATITIS C VIRUS
Cardeira S.S. and Bishop N.E.
Microbiology Department, La Trobe University, Bundoora, Vic,
Australia.
Hepatitis C Virus (HCV) infects 170 million people worldwide. Its high
prevalence, rate of chronic infection and the major risk of developing
liver cirrhosis and hepatocellular carcinoma make hepatitis C a
prominent global problem. The HCV genome encodes 10 proteins,
four structural proteins -core, E1, E2 and p7- and six non-structural
proteins -NS2, NS3, NS4a, NS4b, NS5a and NS5b. Current treatment
for hepatitis C is effective in only approximately 50% of cases. By
increasing our knowledge regarding the non-structural proteins of HCV,
and their role in replication, new targets for antiviral treatments may
be revealed. Ubiquitin is a 76 residue protein that becomes covalently
bound to specific lysine residues of target proteins. The ubiquitination of
proteins is a regulatory post-translational modification, much like protein
phosphorylation. Ubiquitin is known to be important for the budding of
many enveloped viruses from cellular membranes, a crucial step in viral
replication. My work is focused on characterizing the role of ubiquitin
in the replication of HCV. The NS5a protein has been implicated in
establishment of persistent HCV infections by its ability to modulate
intracellular signalling pathways. For example NS5a expression inhibits
the degradation of epidermal growth factor receptor, a process highly
dependent on ubiquitination, but the exact mechanism is unknown.
HCV core protein is also known to be ubiquitinated, which leads to its
subsequent degradation. I am screening HCV proteins for interactions
with proteins involved in cellular ubiquitination processes. I have
preliminary evidence for the direct interaction of ubiquitin with one of
the HCV non-structural proteins, and am investigating the wider role for
ubiquitin in HCV replication.
POS-MON-95
DETECTION OF PROGRP-DERIVED PEPTIDES
IN HUMAN COLORECTAL CANCER CELLS AND
ASSESSMENT OF THEIR ROLE IN CELLULAR
PROLIFERATION.
Patel O.1, Chang M.1, Nordlund M.2, Shulkes A.1 and Baldwin G.1
1
Department of Surgery, University of Melbourne, Austin Health,
Melbourne, Australia. 2Department of Medical Biochemistry,
Radiumhospitalet, Rikshospitalet University Hospital, Oslo.
Oneel Patel1, Mike Chang1, Marianne S. Nordlund2, Arthur Shulkes1 and
Graham Baldwin1. Background and aim: Amidated gastrin-releasing
peptide (GRP) is the prototypical autocrine growth factor. We have
previously demonstrated that non-amidated peptides derived from the
C-terminus of proGRP are also biologically active in colorectal cancer
(CRC) cell lines in vitro, via a receptor distinct from the GRP receptor
[1]
. This study investigates the quantities of proGRP-derived peptides
Methods: proGRP-derived peptides obtained from boiling water
extracts of the human CRC cell lines DLD-1, SW1222, HCT 15, HT-29
and HCT 116 were quantitated by region-specific ELISA. Proliferation of
DLD-1 cells after reduction of proGRP-derived peptide concentrations
by transfection with proGRP shRNA was measured by [3H]-thymidine
incorporation. Results: In CRC cell extracts ELISA assays for proGRP-
derived peptides containing residues 48-61, 56-88 or 48-88 detected
3-15, 10-60 and 20-152 fmol/106 cells, respectively. Little or no GRP18-
27amide or GRP1-27amide was detected. Stable transfection of DLD-1
cells with proGRP shRNA significantly reduced propresent in a panel of
5 CRC cell lines as well as the effect of such peptides on proliferation in
vitro. liferation. Conclusions: These results indicate that non-amidated
peptides derived from the C-terminus of proGRP are present in CRC
cells and stimulate the proliferation of CRC cells in vitro. Such peptides
are attractive targets for novel CRC therapies. Reference: 1. Patel O.,
Dumesny C., Shulkes A. and Baldwin G.S. (2007) C-terminal fragments
of the gastrin-releasing peptide precursor stimulate cell proliferation via
a novel receptor. Endocrinology. 148: 1330-1339.
Page 124 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-94
THE MITOCHONDRIAL CITRATE CARRIER IS
REQUIRED FOR CHROMOSOME INTEGRITY IN
DROSOPHILA AND HUMAN CELLS
Carrisi C.1, Morciano P.2, Cestra G.3, De Benedetto G.E.1, Corona
D.F.V.4, Musio A.5, Cenci G.2 and Capobianco L.1
1
University of Salento. 2University of L’Aquila. 3Sapienza University.
4
University of Palermo. 5ITB Pisa.
Chromosomal aberrations are key events in the initiation and progression
of cancer. We isolated a P-element induced mutation in Drosophila that
causes chromosome fragmentation. We named sea the gene specified
by this mutation. The P-element is inserted into the 5’UTR of CG6782
which encodes a protein orthologous to the mammalian citrate carrier
SLC25A1. It exports citrate from mitochondria supplying the cytosol
with acetyl units. Sea shows biochemical properties similar to those of
SLC25A1 indicating a functional conservation of this carrier. We found
that Sea is reduced in mitochondria of sea mutants which also exhibit
a reduced citrate transport activity and low levels of citrate in cytosolic
extracts as revealed by LC/MS. Interestingly, western-blot of nuclear
protein extracts and immunostaining of polytene chromosomes with
anti-acetylated histone antibodies revealed a reduction of AcH3 and
AcH4 in sea mutants. Therefore, the chromosome breakage phenotype
observed in sea mutants seems to be a consequence of reduced levels
of histone-acetylation. Notably, SLC25A1 siRNA-treated human primary
fibroblasts exhibited a phenotype similar to Drosophila sea mutants and
low levels of AcH4. These results suggest an unexpected evolutionary
conserved role for Sea/SLC25A1 in the chromosome integrity providing
an intriguing link between cellular metabolism and epigenetics.
POS-TUE-96
β-ARRESTIN1 PROMOTE CHRONIC MYELOCYTIC
LEUKEMIA PROGRESS DEPENDENT ON JNK PATHWAYS †
Zhang P.1, Long J.2, Li K.1, Liu H.2, Tan J.1, Tu Z.2 and Zou L.1
1
Center for Clinical Molecular Medicine, Children’s Hospital, Chongqing Medical
University, Chongqing 400014, China;. 2Department of Clinical Biochemistry
and the Key Laboratory of Laboratory Medical Diagnostics in the Ministry of
Education, Chongqing Medical University, Chongqing 400016, China.
Arrestins (Arrs) are scaffold proteins consisting of four members: β-arrestin1
(β-Arr1), β-arrestin2 (β-Arr2), x-arrestin, and s-arrestin. Only β-Arr1 and
β-Arr2 are ubiquitously expressed. The traditional functions of β-arrestins are
to mediate desensitization, sequestration, and recycling of G protein-coupled
receptors (GPCRs). Mounting evidence suggests that, in addition to regulation of
GPCR signals, β-arrestins also serve as modulators in a number of intracellular
signaling pathways, including ERK, JNK and ASK1, which play important roles
in the regulation of various cellular functions in both normal and malignant cells.
Some reports have showed that β-Arrs participate in tumor-related signaling
pathways such as p53/MDM2, TGF-β1, IGF1R pathways, which function vitally
as cell antiapoptosis, cell growth, and proliferation in tumor cells. We have
previously disclosed that rapid xenograft tumor progression in β-Arr1 transgenic
mice by enhancing tumor angiogenesis and PI3K inhibitors suppressed the
β-Arr1-elevated MMP9 activity and VEGF. However, little is known about β-Arrs
function in leukemia, especially in chronic myelocytic leukemia (CML). Here
we demonstrated that the expression of β-Arrs apparently increased in the
bone marrow (BM) and periphery blood (PB) samples from leukemia samples
compared with benign hematological disease patients. However, there was
no significant difference of β-Arr expressions among the diverse subtypes of
leukemia (AML, ALL and CML) samples (P>0.05). But the relative expression
of β-Arr1 was always higher than β-Arr2 in the same patient of leukemia. We
further found that over-expression of β-Arr1 could promote leukemia cells to
proliferate, and vice versa in CML K562 cells in vitro. Moreover, K562 cells
knocking out β-arrestin1could activate JNK signal pathways, and JNK inhibitors
could block the JNK activation by β-arrestin1 in cells. The results from CML
xenograft mice further showed that the survival rate was much higher in K562-
siRNA-β-arrestin1 mice than in K562-β-arrestin1 mice, which was closely
related to JNK activation. Furthermore, JNK inhibitors could affect the tumor
growth of K562-siRNA-β-arrestin1 mice. In conclusion, β-arrestin1 could
promote chronic myelocytic leukemia progression dependent on JNK signal
pathways, which disclosed the pathogenesis mechanism and signal pathways
of β-arrestin1 regulating CML. [† This wok was financial supported by Natural
Science Fund of China (30871103, 90919013), Chongqing Natural Science
Fund CQCBST 20082207 and 2008 New Century Excellent Talent Program
from Education Ministry of China.].
POSTERS MONDAY & TUESDAY
POS-MON-97
THE SEROTONIN 5-HT4, RECEPTOR SPLICE VARIANTS
INTERACTS WITH SPECIFIC PDZ DOMAIN PROTEINS
VELI 1-3/LIN7A, B, C HOMOLOGUES: MECHANISMS IN
RECEPTOR TARGETING
Chinkwo K.A., Coupar I.M. and Irving H.R.
Medicinal Chemistry and Drug Action, Monash Institute of
Pharmaceutical Sciences, Monash University, Parkville VIC 3052,
Australia.
Several disorders of the gastrointestinal tract are associated with
abnormal serotonin metabolism and / or signalling such as irritable bowel
syndrome (IBS). The largest quantity of 5-HT4 receptors is present in
the intestine of mammals and have modulatory and prokinetic functions
and form a clinical target for IBS. Alternative splicing results in 11 splice
variants of the 5-HT4 receptor, most differing in their C-terminal (Coupar
et al., 2007) and the 5-HT4 (a, d, e, f and g) receptor splice variants
possess canonical type 1 or type 2 PDZ domains. Mouse 5-HT4a
receptor interacts at the C-terminus with Veli3 (Joubert et al, 2004). The
Veli or Lin7A, B or C homologue proteins are involved in post synaptic
vesicle function and localization (Jo et al., 1999). We hypothesise that
the human 5-HT4 receptors containing canonical PDZ domains may
interact with Lin7A, B or C homologues and that this interaction may be
important in contributing to receptor function (including down-regulation)
in the gastrointestinal tract. We have shown that the 5-HT4 a, b, c and d
transcripts are expressed relatively strongly in the human sigmoid colon
and that the Lin7 A, B and C homologue transcripts are also expressed
in the colon (Chetty et al, 2009). To follow up these observations, we are
investigating the interaction between 5-HT4a and 5-HT4d receptor splice
variants and Lin 7 homologues in COS-7 and colonic cell lines. Here we
report on the generation of N-terminal FLAG tagged 5-HT4 receptor and
Lin 7 homologues with C-terminal V5, c-Myc and HA constructs and
their expression in COS-7 cells. Protein interactions will be detected
using immunoprecipitation and immunofluorescence techniques will be
used to visualise cellular co-localisation and receptor recycling. Chetty
et al., 2009 Neurogastroenterol Motil 21:551–558.e15; Coupar et al.,
2007 Curr Neuropharmacol 5: 224-31; Jo et al., 1999 J Neuroscience
19:4186-4199; Joubert et al, 2004 J Cell Sci 117:536-5379.
POS-MON-99
A MOLECULAR BASIS FOR THE DIFFERENTIAL
EXTRACELLULAR FUNCTION OF PAI-1 AND PAI-2 IN
CANCER
Cochran B.J.1, Croucher D.R.2 and Ranson M.1
1
Illawarra Health and Medical Research Institute, University of
Wollongong. 2Cancer Research Program, Garvan Institute of Medical
Research.
Plasminogen activator inhibitors type-1 (PAI-1, SERPINE1) and type-2
(PAI-2, SERPINB2) are potent irreversible inhibitors of the urokinase-
type plasminogen activator (uPA). The strong prognostic value of tumour
expression of uPA and PAI-1 in metastasis and poor patient outcome is
well established. Conversely, tumour expression of PAI-2 is associated
with favourable outcome and increased relapse free survival. We
have previously demonstrated that the interaction between uPA:PAI-2
and receptors of the low density lipoprotein receptor (LDLR) family is
markedly different to that of uPA:PAI-1, offering a molecular basis for
the contrasting prognostic data. Herein, we validate this hypothesis by
replacing residues of PAI-2 with those corresponding to LDLR binding
sites in PAI-1, thereby introducing LDLR binding. This has profound
impacts on the physiological functionality of PAI-2; not only increasing
the clearance rate of uPA from the cell surface, but altering non-
proteolytic functions via the activation of pro-mitogenic cell signalling
pathways. This work provides the first definitive explanation of what, on
the surface, appears to be paradoxical biological functionalities.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 125
POS-TUE-98
EFFECTS OF HPV ONCOPROTEINS ON HLA-A2
PROMOTER IN CERVICAL CARCINOMA CELL LINE
Mohd Habib S.H.1, 3, Abdullah A.1, 3, Othman S.1, 3, Karsani S.A.2, 3 and
Yusof R.1, 3
1
Department of Molecular Medicine, Faculty of Medicine, University
of Malaya, 50603 Kuala Lumpur Malaysia. 2Biochemistry Division,
Institute of Biological science, Faculty of Science, University
of Malaya, 50603 Kuala Lumpur, Malaysia. 3Drug Design and
Development Research Group, University of Malaya, 50603 Kuala
Lumpur, Malaysia.
Human papillomavirus (HPV) infection, particularly type 16, is
causally associated with the development of cervical cancer. The key
transforming proteins of high risk HPV are E6 and E7, which block
cell cycle exit in epithelial cells committed to differentiation, thereby
allowing viral replication. The major cellular target of E6 and E7 are
p53 and retinoblastoma cell proteins respectively, which play a pivotal
role in negative regulation of cell growth. E6 and E7 are constitutively
expressed in cervical cancer and their continuous expression is
required for maintenance of the transformed phenotype. The activities
of E6 and E7 are regulated by viral E2, a transcription regulator protein
that is characteristically non-functioning in cancer cells. In this study
we are prospecting the ability of E2 in mitigating human MHC class 1
gene, specifically on HLA-A2 promoter region using a dual luciferase
assay system. We demonstrate that introduction of viral E2 via transient
transfection can markedly up regulate the activity of HLA-A2 promoter
by as much as 40%. This can be taken as direct evidence that the E6/E7
are responsible for suppressing that activity of HLA-A2, as the ectopic
re-introduction of E2 would inhibit the expression of E6/E7.We are
currently investigating the underlying mechanisms behind this uptrend
and the possible role of E2 in up-regulating the effect of interferon.
POS-TUE-100
BIOCHEMICAL CHARACTERIZATION OF
ABNORMAL FIBRINOGENS CAUSING HEREDITARY
DYSFIBRINOGENEMIA
Kotlin R., Suttnar J. and Dyr J.E.
Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20
Praha 2, Czech Republic.
Fibrinogen, a 340 kDa glycoprotein, consists of three different pairs of
polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene
(FGA, FGB, and FGG), clustered on chromosome 4q32.1. The molecule
is posttranslationally modified - phosphorylated, glycosylated and
sulphated. Fibrinogen is synthesized by hepatocytes and is secreted
to the circulation, where it plays a crucial role in the hemostasis,
angiogenesis, platelet aggregation, cell migration and inflammation.
Inherited defects in fibrinogen are very rare and may cause life
threatening complications like thromboses or serious bleeding.
Dysfibrinogenemia is a disease characterized by inherited abnormality
in the fibrinogen molecule, resulting in functional defects. We have
biochemically characterized five new cases of dysfibrinogenemia with
abnormal coagulation test results. Genomic DNA was amplified by PCR
and dideoxysequencing was performed. Functional examinations were
carried out using fibrin polymerization, fibrinolysis, and measurement
of kinetics of fibrinopeptide release. Abnormal fibrinogens were studied
by SDS-PAGE and mass spectroscopy. We have found two unrelated
families with substitution Aα Arg16Cys and two unrelated families with
substitution Aα Arg16His. Mutant chains were found to be present in
the patient plasmas; abnormal fibrinopeptides release and impaired
fibrin polymerization were obtained. Mutations are situated at the site
of fibrinopeptide release catalyzed by thrombin. The last case is a novel
mutation γ Tyr363Asn impairing fibrin polymerization. Tyr 363 is situated
in the fibrinogen polymerization site and substitution γ 363Asn changes
the conformation of the interacting site. This work was supported by
a grant of the IGA of the Ministry of Health, nr. NS 9636-3/2008; by
a grant of The Ministry of Health, nr. 2373601; and by a grant of The
Academy of Sciences of the Czech Republic, nr. KAN200670701.
POSTERS MONDAY & TUESDAY
POS-MON-101
REDUCED FERROPORTIN EXPRESSION EXPLAINS
WHY INTESTINAL IRON ABSORPTION IN THE
NEONATAL RAT IS HYPORESPONSIVE TO STIMULI
THAT REDUCE ABSORPTION IN ADULTS
Darshan D., Wilkins S.J., Frazer D.M. and Anderson G.J.
Queensland Institute of Medical Research, Brisbane, Australia.
Neonatal mammals have a particularly high rate of intestinal iron
absorption to ensure an adequate supply of iron for growth. In adults,
iron absorption responds to a range of systemic signals, but whether the
neonatal gut is similarly responsive remains unclear. We thus examined
in rats whether elevated body iron (i.p. iron dextran; 0.3mg/g) and
inflammation (LPS; 100ng/g), treatments that reduce iron absorption
in adults, have similar effects in neonates. Gene expression was
measured by qPCR and protein levels by western blotting. Intestinal iron
absorption was determined by measuring the retention of an oral dose
of 59Fe. Neonatal rats (15 days old) absorbed 93.1±2.0% of an oral iron
dose compared to 39.1±2.6% in adults (7 weeks old). In response to iron
dextran and LPS, iron absorption in adults decreased by 53% and 72%
respectively, whereas the corresponding reductions in suckling animals
were only 14% and 9%. Despite this, expression of the iron-regulatory
hormone hepcidin was strongly induced by both iron dextran and LPS
at both ages. The hepcidin target, ferroportin (Fpn), was expressed at
the mRNA level but not at the protein level in the duodenum of suckling
animals up to 17 days of age. Expression was robust in adults. The
Fpn1A splice variant, which is under iron-dependent translational
control, predominated in neonates. In conclusion, iron absorption in
the neonate is largely refractory to systemic signals. This reflects the
limited expression of Fpn protein in the small intestine at this time and
the dominance of an iron-dependent Fpn splice variant. This adaptation
helps to ensure maximal iron supply to the growing pup.
POS-MON-103
APOE POLYMORPHISM IN CORONARY HEART
DISEASE PATIENTS FROM PUNJAB, INDIA
Dhiman S.R.1, Khanna D.2 and Balgir P.P.2
1
Department of Human Biology, Punjabi University, Patiala-
147002,Punjab, India. 2Department of Biotechnology, Punjabi
University, Patiala-147002, India.
Although there is a decline in several forms of infectious diseases with
a general improvement in the health and socio-economic status, there
is an increase in various diseases like, hypertension, stroke, diabetes
and coronary heart disease due to the changes in life style and dietary
habits. Coronary Heart Disease (CHD) contributes significantly to the
morbidity and mortality in many countries and is becoming the leading
cause of death in developing countries too. It has been shown that there
is a high prevalence of CHD, in both, urban and rural populations of
India.In the present study, Apolipoprotein E genotypes were determined
by Polymerase chain reaction at SNPs rs7412 and rs 429358 amongst
152 Coronary Heart Disease (CHD) patients and compared with
300 normal healthy individuals (Controls) from North Western Indian
population of Punjab, India. All the three ApoE alleles - ApoE2, ApoE3
and ApoE4 were observed in the CHD patients and normal controls.
The frequency of ApoE3 allele was found to be the highest, followed by
ApoE4 and ApoE2 alleles, both, in the CHD patient, as well as, in the
Control series. Chi-square analysis reveals statistically non-significant
differences between the patient and control series (Chi-square =1.435,
d.f. = 2, P = 0.4879). ApoE being a prominent mediator in cholesterol
homeostasis pathway has a significant influence on the plasma lipid
levels. The influence of ApoE genotypes on plasma lipid and lipoprotein
levels will be discussed.
Page 126 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-102
CONTRIBUTION OF METABOLIC PERTURBATIONS
TO THE NEURODEGENERATIVE EVOLUTION OF
ALZHEIMER’S DISEASE
Daulatzai M.A.
University of Melbourne, Parkville, Vic 3010, Australia.
Mak Adam Daulatzai Sleep Disorders Group/EEE, University of
Melbourne, Victoria, Australia Alzheimer’s disease (AD) is relentlessly
progressive with significant burden on medical and financial resources.
Our longevity is increasing and aging is the most important risk factor.
AD is diagnosed only when considerable brain damage is done. The goal
of this paper is to address important senescent stigmata, and highlight
metabolic disturbances that exacerbate AD neuropathogenesis. The
latter is correlated with cholinergic hypofunction and neuronal losses in
several key neocortical areas including hippocampus, basal forebrain
(BFB), entorhinal cortex (ERC), posterior cingulate cortex (PCC), and
retrosplenial parietal cortex (RPC) to name a few. These affected
regions possess amyloid plaques and neurofibrillary tangles (NFT).
Neuroimaging has shown significant hypoperfusion of BFB and several
key brain regions in old rats and elderly humans. These perfusion
decreases occur prior to substantial brain atrophy in AD. Furthermore,
there is significant evidence of decreased cerebral glucose metabolic
rate (CMRglc) in parietal and temporal cortices also years prior to clinical
AD diagnosis. Most consistently documented CMRglc hypometabolism
occurs in parietal cortex, particularly the most vulnerable RPC.
Respiration is essential to life. Upper airway (UA) patency is due to
motor activity of dilator muscles, particularly genioglossus innervated
by hypoglossal nerve. Nocturnal hypofunction of genioglossus results
in variable collapse of UA. Almost all elderly suffer from some kind of
sleep-disordered breathing condition including obstructive sleep apnoea
which has reached an epidemic proportion. There is well documented
involvement of brain stem nuclei including reticular, solitarius, ambiguous,
motor nucleus of vagus, and hypoglossal, in ventilatory regulation,
genioglossus function and UA patency. Hypofunction/atrophy of the
above nuclei in elderly, results in UA collapse, hypoxia, hypoxemia,
depressed CMRglc, and enhancement of AD neuropathology. Significant
data would be presented to substantiate this thesis.
POS-TUE-104
EFFECTS OF RHO GTPASES IN METASTASIS
Dinsdale B., Li S.F. and Parish R.W.
LaTrobe University.
Transition from a primary carcinoma to secondary tumours result in
higher mortality rates. The spread of cancer, is primarily dependant on
cell migration, cell invasion and metastasis. DNA arrays have defined
a pattern of gene expression that correlates with the progression to
metastasis, and among the genes found to play a role is a member
of the RAS-superfamily of small guanosine triphosphates (GTPases),
RhoC. RhoC is a known regulator of cellular migration through its effects
on the cytoskeleton and cellular adhesion. Changes in expression
and activation of RhoC can result in a metastatic phenotype. RhoC is
over-expressed in metastatic cells of many cancers, and as such we
are interested in varying expression in three sublines from a murine
mammary model (derived from spontaneously arising tumour in a
Balbc/cfC3 mouse) that demonstrate varied metastatic potential. By
ectopically expressing RhoC in different sublines, we have observed
distinct morphological changes that are indicative of a significant shift
in metastatic potential. We have carried out Matrigel invasion assays
to further characterise these transgenic lines, in conjunction with
creating RhoC loss-of-function mutants to further investigate its role in
metastasis.
POSTERS MONDAY & TUESDAY
POS-MON-105
SEC14-DEPENDENT SECRETION OF THE FUNGAL
INVASIN, PHOSPHOLIPASE B1, IS ESSENTIAL FOR
PATHOGENICITY OF CRYPTOCOCCUS NEOFORMANS
Djordjevic J.T.1, 2, Chayakulkeeree M.1, 2, 3, Johnston S.4, Bijosono Oei J.1, 2,
May R.4, Williamson P.R.5 and Sorrell T.C.1, 2
1
Faculty of Medicine, University of Sydney. 2Centre for Infectious Diseases
and Microbiology, Westmead Millennium Institute, Westmead Hospital,
Sydney. 3Faculty of Medicine, Siriraj Hospital, Mahidol University,
Bangkok, Thailand. 4School of Biosciences, University of Birmingham,
Edgbaston, Birmingham, United Kingdom. 5National Institute of Allergy
and Infectious Diseases, NIH Bethesda, MD, USA.
The secretory pathway of the AIDS-related pathogen, Cryptococcus
neoformans, exports phospholipase B1 (Plb1), an enzyme essential
for fungal invasion and dissemination throughout the host. Using gene
deletion analysis, secretion of Plb1, but not other major virulence
determinants, is shown to be dependent on CnSEC14-1, which encodes
a putative phosphatidylinositol transfer protein (PITP). Similar to the
Plb1 deletion mutant (CnΔplb1), the Plb1 secretion-deficient CnSEC14-1
deletion mutant (CnΔsec14-1) displayed reduced lung and brain
burdens and attenuated pathogenicity in an animal model. Furthermore,
expulsion of live C. neoformans from macrophages was attenuated
in both Plb1 secretion-deficient strains. CnSEC14-1 restored high-
temperature growth to an S. cerevisiae SEC14TS mutant confirming
it to be the orthologue of the secretion-essential Golgi PITP-encoding
gene, ScSEC14. Deletion of CnSEC14-2, a closely-related homologue
of CnSEC14-1, and CnSFH5, the more distantly-related SEC fourteen-
like homologue, singly or in combination (CnΔsec14-2, CnΔsfh5 and
CnΔsec14-2/CnΔsfh5, respectively), abrogated neither Plb1 secretion nor
pathogenicity. However, Plb1 secretion and pathogenicity were restored
in CnΔsec14-1 following genetic reconstitution with either CnSEC14-1
or CnSEC14-2, despite CnΔSEC14-2 retaining a wildtype phenotype,
confirming that CnSEC14-2 over-expression functionally compensates
for CnSEC14-1. C. neoformans therefore uses distinct mechanisms to
export virulence determinants, with CnSEC14-1 constituting part of a
Plb1-specific secretion pathway essential for pathogenicity.
POS-MON-107
DOES DYSFERLIN DO MORE THAN JUST REPAIR
MEMBRANES?
Evesson F.J.1, 2 , Peat R.A.1, Lek A.1, 2, Lemckert F.A.1, 2, North K.N.1, 2
and Cooper S.T.1, 2
1
Institute for Neuroscience and Muscle Research, The Children’s
Hospital at Westmead, Westmead, 2145, NSW, Australia. 2Discipline of
Pediatrics and Child Health, The University of Sydney, Sydney, 2000,
NSW, Australia.
Dysferlin is a muscle membrane protein which is mutated in a form of
late onset muscular dystrophy. Dysferlin belongs to the ferlin family of
proteins which have ancient origins in eukaryotic biology, and emerging
roles in a variety of vital cellular processes. Deficiency of dysferlin is
characterised by an impairment of rapid calcium-activated resealing of
damaged muscle fibres, although its precise role in this process remains
poorly understood. We have created a panel of dysferlin expression
constructs bearing domain deletions or targeted patient mutations for
use in cell biology assays studying protein lifetime, plasma membrane
expression and trafficking. We have used co-expression models and co-
immunoprecipitation to study associations and interactions of dysferlin
with other proteins involved in cellular trafficking and membrane repair.
Our studies demonstrate that dysferlin is a dynamic transmembrane
protein, showing relatively transient expression at the plasma membrane
of cultured muscle cells (half-life of ~ 3 hours). This plasma membrane
expression is attenuated in domain deletion mutants, and in several
patient missense mutants, resulting in a significant acceleration of
endocytosis and protein degradation. Our efforts are now focused on
defining the constituents and cargo of dysferlin’s endocytic pathway.
Recent results demonstrate that syntaxin-4 demarks the dysferlin
endocytic route. We are now defining the relationship between dysferlin
and syntaxin-4, with potential relevance to the regulated exocytosis of
membrane resealing, or the altered immune function often observed in
patients with dysferlin muscular dystrophy.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 127
POS-TUE-106
STEAROYL-COA DESATURASE 1 DEFICIENCY
REDUCES APOPTOSIS IN THE HEART OF OBESE OB/
OB MICE
Dobrzyn P.1, Pyrkowska A.1, Miyazaki M.2, Ntambi J.M.3 and Dobrzyn A.1
1
Laboratory of Cell Signaling and Metabolic Disorders, Nencki
Institute of Experimental Biology, Warsaw, Poland. 2Divisions of
Renal Diseases and Hypertension & Endocrinology, Metabolism and
Diabetes, University of Colorado, Denver, CO, USA. 3Departments
of Biochemistry and Nutritional Sciences, University of Wisconsin-
Madison, Madison, WI, USA.
Rationale: Stearoyl-CoA desaturase (SCD), the rate limiting enzyme
in the synthesis of monounsaturated fatty acids, was recently shown
to be involved in regulation of cardiac substrate utilization. Objective:
In the present study, using ob/ob;SCD1-/- mouse model, we tested the
hypothesis that lack of SCD1 could improve steatosis and left ventricle
function in leptin deficiency. Methods and Results: We show that
disruption of SCD1 gene significantly improves cardiac function in ob/
ob mice by correcting systolic and diastolic dysfunction and decreasing
left ventricle mass and diameter. The improvement is associated with
reduced expression of genes involved in fatty acids (FA) transport and
lipid synthesis, and reduction in cardiac free FA (by 39%), diacylglycerol
(by 20%), TG (by 33%), and ceramide (by 34%) levels. The rate of FA
beta-oxidation is also significantly lower in the heart of ob/ob;SCD1-/-
mice compared to ob/ob controls. Moreover, SCD1 deficiency reduces
cardiac apoptosis in ob/ob mice due to increased expression of
antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase
and caspase-3 activities. Conclusions: Reduction in myocardial
lipid accumulation and inhibition of apoptosis appear to be the main
mechanism responsible for improved left ventricle function in ob/ob
mice caused by SCD1 deficiency. These results suggest that inhibition
of SCD could be a potential therapeutic strategy for the treatment of
obesity-related cardiomyopathies. This work was supported by Polish
Ministry of Science and Higher Education grant N N301 0129 33.
POS-TUE-108
ABILITY OF α-CASEIN IN PREVENTING OF AMYLOID
FIBRIL FORMATION OF STRESSED α-LACTALBUMIN
IN CROWDED SYSTEM
Ghahghaei A. and Faridi N.
Department of Biology, Faculty of Science, University of Sistan and
Baluchestan, Zahedan, Iran.
Amyloid fibrils arise from the slow aggregation of intermediately folded
protein states. In this study the kinetics of the protein fibril formation
of α-lactalbumin and its prevention by αs-casein in the presence and
absence of crowding agent, dextran (68 kDa) have been compared
by thioflavin T binding assay. It was found that αs-casein, a molecular
chaperone found in bovine milk, is a potent in vitro inhibitor of
α-lactalbumin fibrillization. The effect of αs-casein in preventing fibril
formation was significant, although reduced in comparison with the
absence of crowding agent, dextran. Interaction between chaperone
and α-lactalbumin and structural change in target protein are also
indicated by intrinsic fluorescence intensity, ANS binding assay, CD
spectroscopy and size-exclusion HPLC. In summary, α-casein interact
with α-lactalbumin and prevent amyloid formation but not as well as in
the presence of crowding agent, dextran.
POSTERS MONDAY & TUESDAY
POS-MON-109
A HIGH-THROUGHPUT METHOD FOR THE DETECTION
OF HOMOEOLOGOUS GENE DELETIONS IN
HEXAPLOID WHEAT
Fitzgerald T.L.1, Kazan K.1, Li Z.2, 3, Morell M.K.2, 3 and Manners J.M.1
1
CSIRO Plant Industry, 306 Carmody Road St Lucia QLD 4067
Australia. 2CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601,
Australia. 3CSIRO Food Futures National Research Flagship, PO Box
93, North Ryde 1670, NSW, Australia.
Plant lines featuring full or partial gene deletions provide a useful resource
for the study of gene function by reverse genetics and also have the
potential to be exploited for crop improvement. The use of deletion lines
in polyploid plant species has been impeded by the presence of multiple
copies of homoeologous genes corresponding to each of the ancestral
genomes. Such homoeologous gene copies usually show high DNA
sequence homology, and may have either redundant or homoeologue-
specific functions. Hexaploid (‘bread’) wheat (Triticum aestivum) is an
economically significant polyploid crop species, and is an allopolyploid
possessing three progenitor genomes designated as ‘A’, ‘B’, and ‘D’.
To facilitate the screening for specific homoeologous gene deletions in
hexaploid wheat, we have developed a TaqMan qPCR-based method
that allows high-throughput and large-scale detection of homoeologous
copies in any gene of interest. To test the system, we chose the
TaPFT1 gene, an orthologue of the disease susceptibility gene PFT1 in
Arabidopsis, where mutants lacking a functional PFT1 gene have been
shown to possess increased resistance to a Fusarium pathogen. We
applied this method to the screening of 4500 M2 mutant wheat lines
generated by Heavy Ion Irradiation in order to identify knockouts of each
homoeologous copy of TaPFT1. We detected multiple lines featuring
deletions of each TaPFT1 homoeologue, and confirmed these deletions
using a CAPS method. Mutants in TaPFT1 and other wheat disease
susceptibility genes are now being further developed for phenotypic
testing for potential changes in response to fungal pathogens.
POS-MON-111
INDUCED SPLICE-SWITCHING TO STUDY DYSTROPHIN
ISOFORM FUNCTION AND EXPRESSION
Fletcher S., Adams A., Greer K., Johnsen R.J. and Wilton S.D.
University of Western Australia, 35 Stirling Highway, Crawley WA
6009.
Duchenne muscular dystrophy (DMD) is an X-linked, relentlessly
progressive muscle wasting disorder with a predictable course and
fatal outcome. DMD is caused by mutations in the dystrophin gene that
prematurely terminate the protein, and affects 1 in 3500 male births.
Although treatment options have been limited, advances in clinical care
have doubled the life expectancy of affected boys over the last 2-3
decades, but do not address the primary etiology of DMD, the absence
of dystrophin. Biological therapeutics are now becoming available, and
antisense oligomer-mediated splice manipulation to exclude selected
exons can overcome disease-causing mutations and restore dystrophin
expression. We show that peptide-conjugated phosphorodiamidate
morpholino oligomer induced splice-switching can also be used to alter
isoform expression, or induce a frame-shift and prevent translation
of functional products. Exclusion of selected exon blocks to yield in-
frame transcripts can allow mapping of functional protein domains,
based upon exon boundaries, and permits physiological evaluation of
dystrophin isoforms to aid in the design of splice-manipulation therapies.
It is also possible to efficiently disrupt the normal dystrophin mRNA
reading frame and ablate dystrophin expression in wild-type mouse
muscle. Total suppression of dystrophin gene expression was induced
by selected exon removal and maintained for several weeks in vivo,
resulting in severe dystrophic pathology in diaphragm within 4 weeks
of commencing treatment in wild-type neonatal mice. Manipulating
expression through altered splicing patterns could be applied to many
different genes, offering the opportunity to induce transient mouse
models to study the consequences of gene suppression or isoform
selection in vivo.
Page 128 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-110
UNTANGLING THE WEB OF HEPATITIS C VIRUS NON-
STRUCTURAL PROTEIN 4B FUNCTION
Fitzsimmons N.M. and Bishop N.E.
La Trobe University, Bundoora.
Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and/or
hepatocellular carcinoma, in an estimated three percent of the human
population. HCV is a single-stranded, positive-sense RNA virus,
classified in its own genus Hepacivirus within the Flaviviridae family.
The virus encodes ten proteins: Core, E1, E2, p7, NS2, NS3, NS4A,
NS4B, NS5A, and NS5B. The Core, E1 and E2 proteins are structural
proteins, while the remainders are non-structural proteins. My project
focuses on the role of the non-structural protein NS4B, which has a
poorly-characterized role in viral replication. A number of positive-
sense RNA viruses replicate their genome on the surface of intracellular
membranes. Some viruses also rearrange the intracellular membranes.
The purpose of rearranging membranes is not fully understood however
some hypotheses are that they could be used as a physical support
for the viral replication complex, allow compartmentalization of viral
products, may permit viral RNA tethering, or perhaps protect the RNA
from host defenses. For HCV these rearranged membranes are termed
membranous webs or membrane-associated foci (MAFs). Evidence to
date suggests NS4B plays a role in formation of these MAFs. Current
data indicate the membranes rearranged by HCV are derived from both
the endoplasmic reticulum and early endosomes. However, both the
molecular mechanisms involved, and the impact on host cell function
due to the MAF formation, is poorly characterized. I will present my
latest data on the characteristics and formation of HCV-induced MAFs,
and data on the impact of the membrane rearrangements on normal
host cell function. My results are based on findings in cultured cells over-
expressing HCV NS4B.
POS-TUE-112
MODULATION OF MHC CLASS I GENE EXPRESSION
BY DENGUE VIRUS
Othman S., Abdul Rahman N. and Yusof R.
Drug Design and Development Research Group, University of Malaya,
50603 Kuala Lumpur, Malaysia.
Mosquito-borne dengue fever (DF) and dengue haemorrhagic fever
(DHF) are caused by dengue viruses belonging to the Flaviviridae family
that comprised four closely related but antigenically distinct serotypes,
DV1 to DV4. There are over 100 million cases of dengue infections, half
a million cases of DHF and 24000 deaths worldwide every year, with
more than 2.5 billion people at risk for endemic transmission. Despite
aggressive efforts in dengue research, the control of dengue diseases
awaits complete elucidation of its complex immunopathogenesis,
many theories of which showed participation of both vector and host
responses, involving both innate and adaptive components of the
human immune response. In the adaptive immune response, the
major histocompatibility complex (MHC) class I molecules are critical
for antigen-specific immune recognition, expressed on the surface
membrane of virtually all mammalian cells to display fragmented pieces
of foreign antigen. In contrast to many viruses that escape this host
response by suppressing the MHC Class I pathway, infection by other
Flaviviruses has been shown to up-regulate the cell surface expression
of MHC Class I complex. In dengue infection, the mechanisms by
which the virus regulates the MHC Class I remain elusive. Hence, this
study elucidates comprehensively and systematically the effect of all
serotypes of dengue virus infection on the expression and regulation of
transcriptional activities of the human MHC class I HLA-A2 gene. This
adds further understanding to the role of dengue virus in manipulating
the MHC antigen presentation pathway in this immune-pathogenic
disease that has been posing great economic and social burden on
affected many nations, Malaysians included.
POSTERS MONDAY & TUESDAY
POS-MON-113
D-AMINO ACID OXIDASE: PATHOPHYSIOLOGICAL
BASIS AND MOLECULAR TARGET FOR
SCHIZOPHRENIA
Fukui K., Ono K., Abou El-Magd R.M., Iwana S., Kawazoe T., Chung S.P.,
Song Y., Shishido Y., Yorita K. and Sakai T.
Division of Enzyme Pathophysiology, The Institute for Enzyme
Research, The University of Tokushima, Tokushima, JAPAN 770-8503.
D-Amino acid oxidase (DAO) has been proposed to be involved in
the decreased glutamatergic neurotransmission in schizophrenia and
the modulation of the enzyme activity is expected to be therapeutical.
Here we show the inhibitory effect of a classical antipsychotic drug,
chlorpromazine, and an atypical drug, risperidone, on human DAO
activity. As for chlorpromazine, human DAO was inhibited with Ki
value of 0.7 mM. We also found that the oligomerized chlorpromazine
produced by irradiation, showed an enhanced inhibition (Ki =7 μM).
Risperidone was also shown to have a hyperbolic mixed-type inhibition
with Ki value of 41 μM. The effect of risperidone was then tested using
rat C6 cells and stable C6 transformant over-expressing mouse DAO
(designated as C6/DAO) as well as pig kidney epithelial cell line (LLC-
PK1). Risperidone exhibited a protective effect from D-amino acid
and oxidative stress induced cell death in both C6/DAO and LLC-PK1
cells. In search of the in vivo pathophysiological role of DAO, we then
analyzed the distribution of DAO mRNA and protein in the brain. In rat,
the distribution of DAO mRNA was newly detected in choroid plexus
(CP) epithelial cells in addition to glial cells of pons, medulla oblongata,
and especially Bergmann glia of cerebellum. In agreement with the
results in rat, the immunoreactivity for DAO was detected in glial cells
of rhombencephalon and in CP in the human brain. Furthermore, higher
level of DAO expression was observed in schizophrenic CP epithelial
cells than that in non-schizophrenic cases. These results suggest that
DAO expression level is altered in schizophrenia and the increase in
DAO expression is involved in aberrant D-amino acid metabolism. In
particular, gene expression of DAO in CP suggests that DAO may regulate
D-amino acid concentration by modulating the cerebrospinal fluid, and
may be regarded as a potential therapeutic target for schizophrenia.
POS-MON-115
POLYMORPHISM DEPENDANT SIGNALING BIAS IN
THE HUMAN CALCITONIN RECEPTOR
Furness S.G.B., Khreish M., Chrisopoulos A. and Sexton P.M.
Drug Discovery Biology Laboratory, Monash Institute of
Pharmaceutical Sciences, Parkville Victoria 3052, Australia.
The calcitonin receptor is a Family-B G protein-coupled receptor that
mediates responses to the peptide hormone calcitonin. Calcitonin is
involved in calcium homeostasis by inhibition of osteoclast-mediated
bone resorbtion and potentiation of renal calcium secretion. Calcitonin
also exerts anorectic and analgesic effects in the central nervous
system as well as suppressing gastric secretion and intestinal mobility.
A number of single nucleotide polymorphisms have been reported
for the calcitonin receptor. Of these, a coding polymorphism resulting
in either Leucine or Proline at position 447 in the carboxy terminal
tail has been reported to be associated with osteoperotic risk. We
hypothesized that these polymorphisms may result in altered signaling
profiles providing a molecular basis for apparent calcitonin receptor
dependant clinical changes in calcium homeostasis. Since both salmon
and human calcitonin are used clinically, we also hypothesized that
these ligands may show different signaling profiles with respect to this
polymorphism. A single previous report using a transient transfection
model demonstrated no difference in calcitonin stimulated adenylate
cyclase activity at either polymorphic variant of the receptor (Wolfe et.
al. Mutat Res. 2003; 522:93-105.). We have used several model cell
backgrounds, COS-7, 3T3 and HEK-293, to analyse differences in
signaling arising from these polymorphs. We have shown that there are
a number of polymorphism-dependant changes in signaling efficacy
toward adenylate cyclase, extracellular signal-regulated kinase (ERK)
phosphorylation and intracellular calcium release. Further, we have
demonstrated that these signaling biases are dependent on cellular
background. We have also observed that there are no apparent
polymorphism dependent differences in signaling profiles in response
to salmon and human calcitonin in these model cell systems.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 129
POS-TUE-114
IRON HOMEOSTASIS IN WHOLE BODY AND
INTESTINE-SPECIFIC HEPHAESTIN KNOCKOUT MICE
Fuqua B.K.1, Wolkow N.2, Bell A.1, Hsu J.1, Nguyen M.P.1, Yu C.C.1,
Dunaief J.L.2, Anderson G.J.3 and Vulpe C.D.1
1
Department of Nutritional Science and Toxicology, University
of California, Berkeley, USA. 2FM Kirby Center for Molecular
Ophthalmology, Scheie Eye Institute, University of Pennsylvania, USA.
3
Queensland Institute of Medical Research, Brisbane, Australia.
Hephaestin (Heph) is a vertebrate multicopper ferroxidase (MCF)
important for the transfer of dietary iron from intestinal cells to the
blood. The gene is mutated in the sex-linked anaemia (sla) mouse and
results in severe iron deficiency. However, sla mice still retain some
hephaestin ferroxidase activity. They survive, breed and their anaemia
improves with age. Heph is also expressed at low levels in other tissues,
including the brain, pancreas, and placenta, but its role in these tissues
is less clear. To gain a better understanding of the role of Heph in iron
homeostasis, we used the cre-lox system to generate knockout mouse
models with whole body and intestine-specific (villin promoter) ablation
of Heph. Both types of mice are small, yet viable, indicating that Heph
is not essential and that other mechanisms exist, MCF-dependent or
not, to compensate for Heph deficiency. At 8 weeks of age the knockout
mice remain small, but their rate of growth is comparable to that of wild-
type littermates. Both strains also develop a microcytic, hypochromic
anaemia, a picture consistent with severe iron deficiency, confirming that
Heph plays an important role in iron acquisition. In addition, mice born
to knockout mothers, regardless of pup genotype, exhibit truncal hair
loss that improves after weaning, also consistent with iron deficiency.
These mouse models will serve as valuable tools to study the role of
Heph and associated proteins in iron transport in the small intestine and
other tissues.
POS-TUE-116
ANTI-HELICOBACTER PYLORI ACTIVITY OF EDIBLE
PLANT FROM NORTHEASTERN THAILAND
Gamnarai P.1, Rojpibulstit P.1, Kangsadalampai S.1, Boonsiri P.2,
Daduang J.3 and Vilaichone R.K.1
1
Faculty of Medicine, Thammasat University (Rangsit Campus),
Pathumthani, 12121. Thailand. 2Faculty of Medicine, Khon Kaen
University, Khon. Kaen, 40002, Thailand. 3Faculty of Associated
Medical Sciences, Khon Kaen University, Khon. Kaen, 40002,
Thailand.
ABSTRACT Helicobacter pylori is a common bacterial pathogen in
human. It is found in association with peptic ulcer diseases and gastric
cancer. In Thailand, the infection has been reported to be higher in
northeastern part than in any other regions of the country. Surprisingly,
the prevalence of peptic cancer among people in this region was very
low. The explanation for this finding was not clear. It was thought that
a variety of local plants in their daily foods may play an indirect role in
the cancer protection by reducing the severity of the H. pylori infection
and, consequently, lower the rate of peptic cancer. In this study, we
assessed the anti-H. pylori activity of 21 edible plants from northeastern
Thailand to look into the potential nutraceuticals against the H. pylori
ATCC 43504. Minimum inhibitory concentration (MIC) was determined
by agar dilution method for ethanol and ethyl acetate plant extracts. Of
all 21 plant extracts, ethanol extracts of Polygonum adoratum Lour.,
Limnocharis flava (L.) Buchen., Limnophila aromatica Merr, Tiliacora
triandra Diels, and Spilanthis acmella Murr. (MIC 125 microgram/ml) and
ethyl acetate extracts of Polygonum adoratum Lour., Justicia gangetica,
Limnophila aromatica Merr, Cratoxylum formosum subsp. pruniflorum
(Kurz) Gogel., Cratoxylum formosum (Jack) Dyer ssp, Tiliacora triandra
Diels, Ottelia alismoides (L.) Pers., Hydrocharis morsus-ranae Linn.,
Tacca chantrieri Andre and Spilanthis acmella Murr. (MIC 62.5 to
125 microgram/ml) exhibited the highest anti-H. pylori activity. This
information provides more insights into another role of edible plant as
an anti-bacterial neutraceutical food.
POSTERS MONDAY & TUESDAY
POS-MON-117
SUBCELLULAR TRAFFICKING OF VIRAL PROTEINS
IS CENTRAL TO VIRAL IMMUNE EVASION AND
PATHOGENICITY
Moseley G.W.1, Wiltzer L.1, Oksayan S.1, Rowe C.L.1, Marsh G.2, Wang L.F.2,
Blondel D.3, Ito N.4 and Jans D.A.1
1
Monash University, Clayton, Victoria, Australia. 2AAHL, Geelong,
Victoria, Australia. 3Gif-sur-Yvette, France. 4Gifu Univeristy, Gifu,
Japan; CNRS.
The principal host response to viral infection is the production of
interferon (IFN), which induces a potent antiviral state in host cells.
Productive infection therefore requires evasion/subversion of the IFN
system, which is achieved by viral proteins with IFN-antagonist activity.
These IFN-antagonists often undergo regulated subcellular trafficking via
interaction with host cellular transport machinery (nuclear import/export
proteins and cytoskeletal factors), enabling the targeting of multiple
stages of intracellular IFN signaling pathways in specific subcellular sites.
IFN-antagonist trafficking thus represents a potential target for antiviral
therapy. To demonstrate the importance of IFN-antagonist trafficking to
viral pathogenicity, we have examined the archetypal IFN-antagonist of
rabies (P-protein), finding that it undergoes intricately regulated targeting
via several nuclear export (NESs) and nuclear localization (NLSs)
sequences, as well as sequences for association with microtubules
(MTs) and the MT motor, dynein. This appears to be central to P-protein’s
targeting and inactivation of the vital IFN signaling factors IRF3 and
STAT1 within different cellular compartments. Our recent data indicate
that NES-mediated P-protein nuclear export is essential to its inhibition
of IFN-dependent STAT1 signaling, and is a key pathogenicity factor
for infectious rabies virus, the first such demonstration for any virus.
Our data further indicate that P-protein nuclear import makes similar
crucial contributions to IFN-antagonist function, and that P-protein-MT
interaction provides additional, novel mechanisms of STAT1 inhibition.
These data indicate that efficient viral immune evasion requires viral
protein interaction with diverse cellular factors in multiple subcellular
sites, with implications for the development of therapies for incurable
human-pathogenic viruses.
POS-MON-119
THE EXPRESSION OF TRANSMEMBRANE TUMOUR
NECROSIS FACTOR IS INCREASED IN THE ANTERIOR
CINGULATE OF SUBJECTS WITH BIPOLAR DISORDER
Gibbons A.S., Tawadros N., Scarr E. and Dean B.
Rebecca L. Cooper Laboratories, Mental Health Research Institute of
Victoria, Parkville, Victoria 3052, Australia.
Altered pro-inflammatory cytokine signalling has been implicated in the
pathologies of mood disorders and schizophrenia. We have previously
reported an increase in the expression of the transmembrane isoform of
tumour necrosis factor (tmTNF) in Brodmann’s area (BA) 46 but not BA
24 from subjects with major depression, while the expression of soluble
isoform of TNF (sTNF) was not altered in either region. To determine
whether TNF is involved in other psychiatric disorders, we examined
TNF expression in post-mortem subjects with bipolar disorder (n=10)
and schizophrenia (n=20) and matched control subjects. tmTNF
and sTNF protein expression was measured using Western blotting.
Contrasting our previous observations in major depression, the level
of tmTNF was significantly increased in BA 24 (mean ratio of internal
standard ± SE: CON = 2.78 ± 1.03; BD = 7.15 ± 1.75; P<0.05) but not
BA46 (CON = 0.88 ± 0.10; BD = 1.84 ± 0.47; P>0.05) from subjects
with bipolar disorder. The level of sTNF protein was not changed in
subjects with bipolar disorder in either region examined (BA 24: CON
= 1.31 ± 0.17; BD = 1.49 ± 0.26; P>0.05; BA 46: CON = 1.35 ± 0.15; BD
= 1.44 ± 0.17; P>0.05). The expression of tmTNF and sTNF protein was
not altered in subjects with schizophrenia in BA 24 or BA 46 (P>0.05).
While our findings suggest increased tmTNF expression is associated
with mood disorders, the affected brain regions differ between bipolar
disorder and major depression. Furthermore, aberrant TNF signalling
in mood disorders may not involve the classical pro-inflammatory
signalling pathways mediated by sTNF.
Page 130 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-118
COMPARING THE CHAPERONE ACTIVITY OF
GLYCEROL AND β-CASEIN IN PREVENTING
AGGREGATION OF PROTEINS IN CROWDED SYSTEM
Ghahghaei A. and Rahmany Asgarabad F.
Department of Biology, Faculty of Science, University of Sistan and
Baluchestan, Zahedan, Iran.
Aggregation of proteins is a frequent occurrence during their transition
from random coil to native structure. The influence of glycerol and
β-casein in the refolding of ovotrnasferrin, insulin, α-lactalbumin and
catalase under aggregating conditions was studied by visible absorbtion
spectroscopy, intrinsic fluorescence, ANS binding assay and HPLC.
Both chaperone effectively prevented aggregation of ovotransferrin, and
α-lactalbumin during renaturation of proteins in the absence of dextran.
As dextran increased the rate of aggregation, β-cacein and glycerol was
found less efficient in preventing them. It was observed that dextran
accelerated insulin aggregation but it had little effect on the time to the
onset of insulin aggregation. β-Casein and glycerol were found equally
effective in preventing aggregation of insulin both in the presence and
absence of dextran. Glycerol effectively suppressed thermally induced
aggregation of catalase whereas β-casein co precipitated of proteins.
In conclusion glycerol is a more effective chaperone than β-casein to
protect protein from aggregation and precipitation. The more enhanced
protein reactivation by glycerol may be due to their more ability to bind
to hydrophobic sites in protein folding intermediate(s) followed by their
subsequent removal as the protein refolds. In addition structural change
in β-casein at higher temperature may explain the different effects of
glycerol and β-casein in protecting catalase.
POS-TUE-120
MITOCHONDRIAL ABNORMALITIES IN THE
MECP2TM1TAM MOUSE MODEL OF RETT SYNDROME
Gold W.A.1, Williamson S.L.1, Pelka G.J.2, Tam P.P.L.2, Gibson J.1 and
Christodoulou J.1, 3
1
NSW Centre for Rett Syndrome Research, Western Sydney Genetics
Program, Children’s Hospital at Westmead, Sydney, Australia.
2
Embryology Unit, Children’s Medical Research Institute, Sydney,
Australia. 3Disciplines of Paediatrics & Child Health and Genetic
Medicine, University of Sydney, Australia.
Rett Syndrome (RTT) is a severe childhood neurodevelopmental
disorder, primarily caused by mutations in the X-linked gene methyl-
CpG-binding protein 2 (MECP2). RTT occurs predominately in females
with an incidence of 1 in 8-10,000. There is considerable phenotypic
variability, with ‘classical’ RTT patients exhibiting developmental arrest
in infancy, stereotypic hand movements, loss of communicative skills,
progressive loss of intellectual functioning, and the deceleration of head
growth. Phenotypic similarities between RTT and patients with primary
mitochondrial respiratory chain disorders prompted investigations into
abnormalities of the respiratory chain function as well as mitochondrial
ultrastructure in RTT patients as well as in mouse models. The aim of this
study was to investigate the mitochondrial function of the brain and other
organs in the Mecp2tm1Tam mouse model, by investigating the individual
respiratory chain complexes (COI, COII, COIII, COII+III and COIV)
using spectrophotometric techniques. When we compared symptomatic
mice to their wild type litter mates we observed an increase in COIII
activity (wt=171.3±58.9; null=256.6±75.8, p=0.0302) in the heart, and
a decrease in COII+III (wt=111.2±46.59; null=61.19±20.89, p=0.003)
and COIV (wt=151.6±53.75;null=79.56±32.49, p=0.0007) activity in
skeletal muscle. Interestingly, we found no changes in activity in the
brain of symptomatic mice. Furthermore, no differences were observed
in mitochondrial activity in pre-symptomatic mice. This data imply that
perturbations in mitochondrial function appear to occur only at disease
onset and may contribute to the clinical phenotype in RTT patients.
POSTERS MONDAY & TUESDAY

POS-MON-121 POS-TUE-122
THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN SHARK ANTIBODIES AND NCAMS AS NOVEL
TYPE 1 DIABETES DIAGNOSTICS
Gorasia D.G., Dudek N.L., Wee S., Mangum J.E., Hubbard M.J. and Griffiths K., Casey J., Parisi K. and Foley M.
Purcell A.W. Biochemistry Department, Latrobe University, Australia.
The University of Melbourne.
Variable domains from the shark immunoglobulin new antigen receptor
Type I Diabetes (T1D) is an autoimmune disease which results (IgNARs) and homologous domains from human neural cell adhesion
from the specific destruction of pancreatic beta cells. Proinsulin is molecules (NCAMs) are small, robust molecules which can be engineered
a target of infiltrating T cells but it’s unclear why it’s trageted. A role to bind antigens of interest with high affinity and specificity. IgNARs
of misfolded proinsulin in diabetes is seen Akita mice which harbors have superior physicochemical properties to conventional mammalian
a mutated proinsulin II (Cys96Tyr) gene and develops diabetes due to antibodies, showing greater stability to extreme temperature, urea
endoplasmic reticulum (ER) stress. Recently, protein misfolding and concentration, and pH. There is significant potential for their application
associated cellular stress is emerging as a key factor in beta cell death as diagnostic reagents, particularly in point of care situations with high
mediated by apoptosis. Proinsulin folding takes place in the ER but ambient temperature and humidity. Similarly, NCAMs have widespread
little is known about how ER chaperones guide proinsulin production, potential as therapeutic and diagnostic reagents. We used a phage
particularly under conditions of ER oxidative stress. In this study we display approach to select high affinity binders from IgNAR and NCAM
have characterized a number of chaperones that assist in the folding libraries, and detail and compare here their various properties.
of proinsulin. The tools to study proinsulin folding were generated by
expressing flag tagged proinsulin in a pancreatic beta cell line (NIT-1).
Immunoprecipitation studies were performed using flag antibody. We
have identified a number of chaperones such as BiP (GRP 78), PDI and
DnaJ homolog subfamily B member 11 (DnaJ11B or HSP 40), that interact
with proinsulin. It has been shown that BiP interact with proinsulin, to
our knowledge this will be the first study to show that BiP together with
Dnaj 11 B interact with proinsulin. Our study on proteomic analysis of
islets from NOD mice showed that islets undergo oxidative stress during
development of diabetes as chaperones like BiP were upregulated.
Further understanding of the role, each identified chaperone plays in
proinsulin folding may provide insights into how proinsulin is targeted
during development of T1D and how oxidative stress affects folding of
proinsulin.

POS-MON-123
PROPAGATION OF THE PRION PROTEIN VIA THE
EXOSOMAL PATHWAY
Guo B.B.1, 2 , Vella L.J.2, 3, Cappai R.2, 3, 4, Bellingham S.A.1, 2, 4 and
Hill A.F.1, 2, 4
1
Department of Biochemistry and Molecular Biology, The University
of Melbourne, Victoria 3010, Australia. 2Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Victoria 3010,
Australia. 3Department of Pathology, The University of Melbourne,
Victoria 3010, Australia. 4Mental Health Research Institute of Victoria,
Melbourne, Victoria, Australia.
Prion diseases are a group of fatal neurodegenerative diseases
characterised by conversion of the host encoded prion protein, PrPC,
into the infectious isoform, PrPSc. Unlike other neurodegenerative
diseases, prion diseases are transmissible. According to the protein
only hypothesis, the misfolded variant acts as an infectious agent,
instigating the conversion of PrPC to PrPSc. Despite extensive research,
the pathway by which this infection is transmitted between cells and
tissues remains elusive. We have identified a role for exosomes in
transmitting prion infectivity between different cell types. Exosomes
are small membranous vesicles released by cells, and are derived
from the endosomal system. Both PrPC and PrPSc has been detected
in exosomes isolated from two cell models: the rabbit epithelial (RK13)
cell line overexpressing mouse PrP; and the mouse hypothalamic GT1-7
cell line expressing endogenous levels of PrP. Exosomes allow efficient
intercellular spread of prion infectivity, however the factors that govern
the sorting of PrP into exosomes is unknown. A difference between the
N-terminal immunoreactivity and glycosylation patterns of exosomal and
cellular PrP was observed, prompting the hypothesis that PrP isoforms
undergo preferential packaging into exosomes. Previous studies have
identified mono-ubiquitination as a tag for exosomal sorting and we
aimed to investigate if exosomal PrP is ubiquitinated. This modification
could have important implications for intercellular transmission of prion
infectivity as it could be a pre-requisite or consequence of exosomal
sorting.
POS-TUE-124
FIELD TRIALS OF TRANSGENIC COTTON
EXPRESSING A DEFENSIN GENE FOR FUNGAL
CONTROL
Hinch J.M.1, Gaspar Y.M.1, McGinness B.S.1, McKenna J.A.1, Anderson
M.A.2 and Heath R.L.1
1
Hexima Limited c/o School of Botany, The University of Melbourne Vic
3010. 2Dept of Biochemistry, La Trobe University Vic 3086, Australia.
Cotton plants (Gossypium hirsutum) expressing a gene encoding the
defensin NaD1 from the female sexual tissues of Nicotiana alata have
been tested for enhanced resistance to fungal pathogens in the field.
Transgenic cotton plants were tested in Queensland and in New South
Wales, Australia in fields naturally infested with Fusarium wilt fungus
(Fusarium oxysporum f. sp. vasinfectum) or Verticillium wilt fungus
(Verticillium dahliae). Three trials against Fusarium wilt in the growing
seasons of 2006-2007, 2007-2008 and 2008-2009 demonstrated that
the defensin technology gave very clear advantages to cotton plants,
with almost three times the survival rate in a high disease year compared
with plants that lack the defensin gene. The surviving plants had higher
numbers of bolls per plant and consequently a higher yield per plant.
Two trials against Verticillium wilt in 2007-2008 and 2008-2009 also
demonstrated a yield increase associated with defensin technology. No
adverse phenotypic effects or phytotoxicity were observed in transgenic
defensin cotton throughout the trials.

OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 131

POSTERS MONDAY & TUESDAY
POS-MON-125
PROTEOMIC STUDY ON RAPAMYCIN TREATED
HUMAN CANCER CELL LINE
Rajagopalan S.1, Guha N.1, Reddy Y.1, Joseph S.1, Lateef S.1 and
Hennessy T.2
1
Agilent Technologies, India Pvt.Ltd, Bangalore, India. 2Agilent
Technologies, Singapore Pte Ltd, Yishun, Singapore.
Rapamycin is an immunosuppressant drug that specifically inhibits
mTOR activity and cellular hyper-proliferation in many cell types
resulting in G1 growth arrest. In the current study, we aim to understand
the effect of rapamycin treatment on various biological pathways using
genomic, proteomic and metabolomic analysis of rapamycin treated
human cancer cell line HEK293. Human cell line HEK293 was treated
with rapamycin and cells were harvested at 15min, 45min, 2hrs and
16hrs after rapamycin treatment. Control samples without rapamycin
treatment were likewise harvested at similar time points. Proteins
were extracted from the cells using PPS silent extraction kit, reduced,
alkylated and digested using trypsin. Peptides were analyzed in five
replicates on a Q-TOF coupled to an HPLC chip system. Differentially
expressed features between the rapamycin treated and non-treated
samples were identified. Concentration of rapamycin was optimized
with respect to cell toxicity and the optimum concentration was found to
be 40nM. Proteins were extracted from one million cells of treated and
control samples respectively at the specified time points. Experimental
procedures for protein extraction, digestion and LC-MS analysis were
optimized. Approximately 3000 features were obtained in each LC-MS
analysis. The reproducibility in the replicate LC-MS analyses of the
digested protein mixture was tested and found to be good. Statistically
significant differentially expressed features were analysed for control
and treated samples of corresponding time points and in a subsequent
experiment identified using targeted MS/MS analysis and database
search.
POS-MON-127
EFFECTS OF MISSENSE MUTATIONS IN PAH ON
PROTEIN QUATERNARY STRUCTURE AND ENZYME
FUNCTION: IMPLICATIONS FOR DISEASE SEVERITY IN
PHENYLKETONURIA
Ho G.1, 2 , Reichardt J.3 and Christodoulou J.1, 2
1
Genetic Metabolic Disorders Research Unit, Children’s Hospital at
Westmead, Sydney, Australia. 2Disciplines of Paediatrics & Child
Health and Genetic Medicine, University of Sydney, Sydney, Australia.
3
School of Medical Sciences, University of Sydney, Sydney, Australia.
Phenylketonuria (PKU) is a recessive inborn error of phenylalanine
metabolism, predominantly caused by mutations in the phenylalanine
hydroxylase (PAH) gene. There is a high level of genetic heterogeneity,
with over 500 mutations reported to date. The majority of mutations
are missense or non-synonymous amino acid changes, which are
postulated to destabilise the structure of the folded protein. In PAH, a
homotetrameric protein, slight local changes can reduce the efficiency
of catalytic function by decreasing the amount of properly-folded
tetramers, the sole functional form. To study the effects of different
missense mutations we transfected COS-7 cells with PAH of wild-
type human sequence or containing a missense mutation previously
identified in patients. Protein lysates were separated by native PAGE
and analysed by Western blot. Quaternary structure (tetramer, dimer
or monomer) was deduced by size. The amount of intact tetrameric
protein roughly correlated with the predicted severity of the missense
mutations based on patient phenotype. Missense mutations known to
cause the most severe form of PKU (e.g. p.F299C and p.R408W) led to
very low or undetectable levels of tetrameric PAH, whereas mutations
(p.V245A, p.E390G) causing the mild form of PKU had similar levels
of tetrameric PAH compared to wild-type. Mutations of the latter group
showed decreased levels of enzyme activity, suggesting that mutations
of amino acids involved in catalytic activity in PAH are likely to cause
a less severe form of the disease compared to mutations causing
structural instability.
Page 132 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-126
MINCLE IS A NOVEL REGULATOR OF THE
INNATE IMMUNE RESPONSE TO TREHALOSE-6,6-
DIMYCOLATE
Hitchens K.J., Manzanero S. and Wells C.A.
Eskitis Institute for Cell and Molecular Therapies, Griffith University.
Macrophage inducible C-type lectin, Mincle, plays a non-redundant
role in the innate immune systems recognition and response to the
opportunistic pathogen Candida albicans. Recently, Mincle has been
demonstrated to be a key receptor for Trehalose-6,6-dimycolate
(TDM). TDM is a highly inflammatory component of the Mycobacterium
tuberculosis cell wall. TDM alone can induce granuloma formation and
is a component of the tuberculosis subunit vaccine. Absence of Mincle
results in poor induction of T cell immunity to the vaccine, making it
important for generating a memory immune response. In this study
significant differences were found in the inflammatory profile of Mincle
knockout (in comparison to wild type) macrophages responding to TDM.
The absence of Mincle resulted in the ablation of some inflammatory
responses (i.e. TNFα) or in the partial reduction of others (ie. IL-1β).
Surprisingly, the response to TDM’s building blocks, the molecules
Trehalose or Mycolate alone, appeared to be independent of Mincle.
Our results establish that Mincle works both independently of and in
collaboration with other receptors in the macrophage response to TDM,
and is only involved the response to the whole TDM molecule. Therefore
Mincle plays a key role directing part of the inflammatory response to
TDM.
POS-TUE-128
G-QUADRUPLEX STABILIZER BMVC4 INDUCES
SENESCENCE THROUGH BOTH INHIBITING
TELOMERASE ACTIVITY AND REPRESSING OF HTERT
EXPRESSION IN CANCER CELLS
Huang F.-C.1, Chang T.-C.2 and Lin J.-J.1
1
Institute of Biopharmaceutical Sciences, National Yang-Ming
University, Taipei, Taiwan. 2Institute of Atomic and Molecular Sciences,
Academia Sinica, Taipei, Taiwan.
Telomeres consist of tandem repeats of guanine-rich sequences that
can fold into a variety of four-stranded structures, G-quadruplexes.
Telomerase is the enzyme responsible for telomere replication and
represents a promising neoplasia therapeutic target. Inhibition of
telomere replication can be achieved by stabilization of G-quadruplex
DNA structures. Here we designed and synthesized carbazole
derivatives that stabilized G-quadruplex DNA structure. We found 3,6-
bis(4 -methyl-2-vinylpyrazinium iodine) carbazole (BMVC4) increased
the melting temperature of G-quadruplex and inhibited telomerase
activity. The BMVC4-treated cells ceased to divide after a lag period.
Hallmarks of senescence including morphological changes, detection
of senescence-associated β-galactosidase activity were detected
in BMVC4-treated cancer cells. The BMVC4-induced senescent
phenotype is accompanied by progressive telomere shortening and
partial co-localization of the DNA damage foci and telomere association
protein TRF2, indicating that BMVC4 caused telomere uncapping
after long-term treatments. Interestingly, we also found that BMVC4
repressed the expression of hTERT, the catalytic subunit of telomerase.
Repression of hTERT appeared to be caused by the reduction of c-myc,
a major transcription factor, upon BMVC4 treatments. Since disruption
of the G-quadruplex-forming sequence located at the promoter of c-myc
eliminated the repressing effects of BMVC4, the results indicated that
BMVC4 repressed c-myc through stabilization of the G-quadruplex
structure located at the promoter of c-myc. All together, our results
indicated that BMVC4 induced senescence through both inhibiting
telomerase and repressing telomerase expression.
POSTERS MONDAY & TUESDAY
POS-MON-129
CHARACTERIZATION OF TOXICITY OF ALS-LINKED
SOD1 MUTANT PROTEINS
Jang J.Y.1, Yoon E.J.1, Cho H.M.1, Kim Y.M.1, Rhim H.S.2 and Kang S.M.1
1
School of Life Sciences and Biotechnology, Korea University,
Seoul 136-701, Korea. 2Research Institute of Molecular Genetics,
Department of Biomedical Sciences, College of Medicine, the Catholic
University of Korea, Seoul 137-701, Korea.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative
disease caused by the degeneration of motor neurons. The disorder
causes muscle weakness and atrophy throughout the body. In many
cases there is a family history of the disease, in which case it is referred
to as familial ALS (FALS). Of these, 20% are mapped to the Cu/Zn
superoxide dismutase (SOD1) gene on chromosome 21. SOD1 is an
enzymatic antioxidant found in the cytosol, nucleus, peroxisomes, as
well as the mitochondrial intermembrane space of eukaryotic cells
and in the periplasmic space of bacteria. The mechanism for mutant
SOD1 toxicity remains unknown. However, two main hypotheses are
the impairment of the functions of important proteins by interaction of
misfolded mutant SOD1 proteins and the toxic effect of mutant SOD1
aggregates. Therefore, an attractive therapeutic approach could
be to reduce levels of mutant SOD1 expression in neuronal cells.
Previously, we found that intracellular Aβ directly interacted with SOD1.
Furthermore, we mapped the SOD1 binding region to Aβ amino acids
26-42. Interestingly, intracellular Aβ bound to the SOD1 G93A mutant
with greater affinity than to wild-type SOD1. Using a fragment (a.a. 26-
42) of Aβ as a competitive inhibitor, we reduced the interaction between
mutant SOD1 and proteins that were reported to bind mutant SOD1.
Furthermore, we mapped a domain in SOD1 that interacts with the
proteins. We also developed ways to disperse SOD1 mutant aggregates.
These studies will contribute to therapeutic approach of ALS.
POS-MON-131
THE ROLE OF GLUTAREDOXIN1 IN CELLULAR
COPPER HOMEOSTASIS
Ackland S.1, Singleton W.1, McInnes K.1, McKirdy R.1, Winnall W.2,
Mercer J.1 and La Fontaine S.1
1
Centre for Cellular and Molecular Biology, School of Life and
Environmental Sciences, Deakin University, Burwood, VIC. 3125.
2
Centre for Reproduction and Development, Monash Institute of
Medical Research, Clayton, VIC. 3125.
Copper (Cu) is an essential heavy metal that is required by all
organisms for a wide range of critical enzymes that harness the CuII/
CuI redox couple. Hence, it is important in many physiological and
disease processes. Uncontrolled intracellular Cu levels can lead to the
generation of excessive reactive oxygen species (ROS) and oxidative
stress. The dire consequences of Cu dysregulation are evident in the
inherited Cu transport disorders, Menkes and Wilson diseases, and in
neurodegenerative diseases. Cells have developed systems to regulate
levels of metal ions and ROS to prevent their toxic accumulation. The
mammalian Cu-transporting P-type ATPases, ATP7A and ATP7B
regulate the Cu status of the body by transporting Cu into the secretory
pathway for incorporation into essential enzymes, and by relocating to the
cell periphery to efflux excess Cu. We have identified new key roles for
glutathione (GSH) and the thiol oxidoreductase, glutaredoxin 1 (GRX1),
in regulating the activity of ATP7A and ATP7B. GSH is a major redox
buffer in cells and is the most abundant intracellular antioxidant. GRX1
has a protective role in oxidative stress-related conditions and plays
a pivotal role in redox regulation of protein function. The Cu-ATPases
are glutathionylated. That is, they are postranslationally modified at
their Cu-binding cysteines to form protein-SSG mixed disulfides. When
Cu levels are elevated, GRX1 interacts with ATP7A and ATP7B to
deglutathionylate them, thus allowing them to bind Cu for subsequent
transport. The effects of GRX1 overexpression and knockdown on Cu-
ATPase activity, cellular Cu homeostasis and overall cell growth and
viability will be discussed.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 133
POS-TUE-130
HEPATOMA-DERIVED GROWTH FACTOR (HDGF)
IS CORRELATED WITH MALIGNANCY AND
INVASIVENESS OF BREAST CANCER
Chen S.C.1, Yeh M.H.4, Chen H.Y.3 and Tai M.H.1, 2, 3
1
Institute of Biomedical Sciences, National Sun Yat-Sen University,
Kaohsiung 804, Taiwan. 2Department of Medical Education and
Research, Kaohsiung Veterans General Hospital, Kaohsiung 813,
Taiwan. 3Department of Biological Sciences, National Sun Yat-
Sen University, Kaohsiung 804, Taiwan. 4Department of Surgery,
Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.
Hepatoma-derived growth factor (HDGF) is a novel growth factor that
participates in the pathogenesis of a variety of cancers. However, the
role of HDGF in breast cancer progression remains unclear. This study
aimed to elucidate the association of HDGF expression with prognosis
of breast cancer. A total 93 surgically resected breast cancer specimens
were collected for immunohistochemical analysis. It was found that
HDGF was significantly elevated in breast cancer tissues compared with
non-tumor ones. Moreover, elevated HDGF expression was correlated
with tumor grades, stages, Ki-67 proliferation index, and lymph node
metastasis. By analysis of HDGF expression in various human breast
cancer cell lines, it was found that HDGF was significantly up-regulated
at the protein level in the most invasive human breast cancer cell line,
MDA-MB-231. Moreover, exogenous supply of recombinant HDGF
augmented the invasiveness of MDA-MB-231 cells in a dose-dependent
manner. Thus, it is concluded that HDGF over-expression is correlated
with malignancy and invasiveness of breast cancer. Besides, HDGF
may represent a novel diagnostic and therapeutic target for breast
cancer.
POS-TUE-132
TWO BAM COMPLEXES IN KLEBSIELLA PNEUMONIAE
Alcock F.H., Clements A. and Lithgow T.J.
Host-Pathogen Molecular Biology Unit, Department of Biochemistry
and Molecular Biology, Monash University, Clayton, 3800, Australia.
The cell envelope of Gram negative bacteria consists of the cytoplasmic
membrane, a peptidoglycan layer and an outer membrane, which
contains lipopolysaccharide in the outermost leaflet. The majority of
clinical K. pneumoniae isolates also produce a mucoid polysaccharide
capsule. These two polysaccharide layers are primarily responsible
for the barrier quality of the outer membrane, which provides the cells’
primary defence against fluctuating environmental conditions and
toxic small molecules. Different classes of outer membrane proteins
are required for uptake of sugars and cofactors essential for growth.
Correct assembly of outer membrane proteins, LPS and capsular
polysaccharide, and its regulation, is a complex task, mediated by
a number of multi-component machines and pathways. The BAM
complex is a multimeric machine found in the outer membrane of all
Gram-negative bacteria, which functions to assemble β-barrel proteins
in the membrane. The central component of this complex is BamA,
an essential, 90 kDa β-barrel protein, with homologues in the outer
membranes of mitochondria and chloroplasts. We analysed the genome
of Klebsiella pneumoniae, a Gram-negative bacterial pathogen, and
found that it encodes two BamA-like proteins: the canonical BamA,
identified as such by gene synteny, and a second isoform, which
we have called BamK. We are using a combination of microbiology,
microscopy, cell biology and biochemical techniques to investigate the
localisation and protein partners of BamK, and its role in assembly of
the K. pneumoniae cell envelope.
POSTERS MONDAY & TUESDAY
POS-MON-133
INVESTIGATING THE ROLES OF KRüPPEL-LIKE
FACTORS IN MEGAKARYOCYTE DIFFERENTIATION
Artuz C.M.1, Mak K.S.1, Funnell A.P.W.1, 2, Pearson R.C.M.1, 2 and
Crossley M.1, 2
1
School of Biotechnology and Biomolecular Sciences, University
of New South Wales, NSW 2052. 2School of Molecular Bioscience,
University of Sydney, NSW 2006.
The Krüppel-like factors (Klfs) are a family of transcription factors
that bind GC rich sites and CACCC-boxes present in the promoters
and enhancers of many genes. They have been shown to participate
in a diverse range of biological processes including erythropoiesis,
adipogenesis and stem cell biology. One of the family’s transcriptional
activators, erythroid Klf (Eklf or Klf1) is best known for its roles in red
blood cell differentiation. Recently, it has also risen to prominence
as an inhibitor of the megakaryocyte program. Erythrocytes and
megakaryocytes differentiate from a common progenitor, termed the
Megakaryocyte/Erythroid Progenitor (MEP). In order to elucidate the
mechanisms by which Klf1 influences differentiation decisions in the
MEP, it is important to consider two highly related repressor Klfs, basic
Klf (Bklf or Klf3) and Klf8. The three Klfs have been shown to function
in a tight regulatory circuit. Klf1 activates both Klf3 and Klf8, and Klf3
represses Klf8. Since these Klfs recognise similar DNA motifs, they may
compete or act redundantly. Our aim is to understand the molecular
network of gene control through which Klf1, 3 and 8 influence cell fate
during differentiation in the MEP, particularly in megakaryopoiesis. Given
that Klf1 acts primarily as a transcriptional activator, we are investigating
whether it indirectly represses megakaryopoiesis through driving either
or both of the repressors Klf3 and Klf8. Our strategy is to establish the
molecular contributions that Klf1, Klf3 and Klf8 make to the regulation of
megakaryopoiesis, using both cell lines and knockout mouse models.
POS-MON-135
CELL BIOLOGY OF AUTISM: CHARACTERISATION OF
THE DELETED IN AUTISM-1 GENE FAMILY
Aziz A. and Bishop N.
Department of Microbiology, La Trobe University, Bundoora.
Autism spectrum disorder (ASD), first described by Leo Kanner in
1943, is among the most heritable of all neurological conditions, with
a prevalence of at least 1:100 being commonly accepted. Variation in,
or deletion of, many different genes appears causative of ASD, and an
emerging common theme is a defect in the secretory pathway, secreted
proteins and/or regulators of these. The Deleted In Autism 1 (DIA1)
gene was recently identified in a study designed to identify recessive
autism genes. The normal cellular function of the DIA1 gene product is
unknown. We have carried out detailed bioinformatics-based analyses
of DIA1 which has revealed a closely related human gene. We have
named this gene DIA1R, for DIA1-related. DIA1R was generated by a
gene duplication event preceding the divergence of vertebrates. While
deletion of DIA1 is implicated in development of ASD, DIA1R localizes
to a region of the X chromosome implicated in mental retardation and
syndromes with ASD-like features. We found both gene products to
be ubiquitously expressed, including in brain tissue. At the cellular
level, both proteins have predicted signal peptides, indicating a role in
the secretory pathway. We are currently investigating the subcellular
localisation and cellular role of both DIA1 and DIA1R. Characterising the
role of genes involved in ASD will lead to a better understanding of the
biological basis of ASD, leading to opportunities for improved diagnosis
and therapy for those with ASD.
Page 134 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-134
EZRIN AND NHERF ARE REQUIRED FOR THE
RECOMBINANT PODXL ENHANCING ADHESION,
MIGRATION AND CELL-CELL INTERACTIONS
Fernandez D.2, Nowakowski A.2, Vilar-Egea M.P.2, Parrilla R.1, 2 and
Ayuso M.S.1, 2
1
Centre of Biological Research, CSIC, Calle Ramiro de Maeztu 9,
28040 Madrid, Spain. 2CIBER de Enfermedades Raras, ISCII.
Podocalyxin (PODXL) is a type I transmembrane glycoprotein initially
described in the epithelial cells of the kidney glomeruli, postulated to
exert antiadhesive properties; however, we have previously observed
that heterologous expression of recombinant PODXL enhanced the
adherence of cells to immobilized ligands, spreading, migration, as well
as the interaction with other cells. The enhancing effect of PODXL on
cell adhesion and migration decreased significantly in deletion mutants
of the cytosolic domain or the DTHL domain. Thus, the integrity of the
cytosolic carboxylic domain of PODXL, included the terminal DTHL
domain, is required for PODXL to exert its effect on cell adhesion and
cell-cell interactions. In the podocytes of kidney glomeruli the DTHL
domain of PODXL interacts with ezrin and NHERF to establish a link
with the actin cytoskeleton, essential for the function of podocytes. To
determine whether the recombinant PODXL expressed in CHO cells was
also linked to the cytoskeleton through ezrin and NHERF we performed
immunoprecipitation studies of cell extracts. We detected ezrin in anti-
PODXL immunoprecipitates and PODXL when the immunoprecipitation
was performed with anti-ezrin or with anti-NHERF. Thus, recombinant
PODXL expressed in CHO cells enhance the cell adhesion properties
even though the signaling to the cytoskeleton are similar to the one
reported in cells like podocytes that show anti-adherent properties.
These observations suggest the cellular environment might be a crucial
factor in defining the functional properties of PODXL.
POS-TUE-136
MOLECULAR SIZES OF ACID-INSOLUBLE AND ACID-
SOLUBLE GLYCOGEN AND THEIR RESPONSES TO
EXERCISE AND RE-FEEDING
Barnes P.D.1, Clode P.2 and Fournier P.A.1
1
School of Sport Science, Exercise and Health, The University of
Western Australia. 2Centre for Microscopy, Characterisation and
Analysis, The University of Western Australia.
Muscle glycogen exists as acid soluble (ASG) and acid insoluble (AIG)
forms, with AIG reported to be the most responsive fraction to exercise
and re-feeding when glycogen is extracted using homogenisation-
free protocols, whereas ASG is the most responsive fraction using
homogenisation-dependent protocols. It has been proposed that AIG
corresponds to a population of lower molecular weight glycogen species
that act as precursors for ASG synthesis. However, the molecular size
distributions of AIG and ASG in skeletal muscles and their responses
to changes in glycogen levels have never been examined. Using
transmission electron microscopy analyses of AIG and ASG from
muscles extracted using a homogenisation-free and homogenisation-
dependent protocols, we compared the molecular size distributions of
AIG and ASG from fed Wistar rats as well as the responses of these
fractions to 3-minutes of intense exhausting swimming with or without
a subsequent 24-hours re-feeding period in rats previously fasted
for 24 hours. Contrary to the interpretations made in the literature,
the molecular size distribution of both AIG and ASG were normally
distributed, with similar ranges and average molecular sizes irrespective
of extraction protocol. Immediately after exercise, the ASG molecular
size distribution shifted markedly towards smaller glycogen molecules,
and AIG distribution changed relatively little. After 24 hours of recovery,
both the AIG and ASG molecular size distributions returned to those
found in fed rats. All changes in total glycogen concentrations were
accounted for by the ASG fraction. In conclusion, the different responses
of AIG and ASG suggest that these are physiologically distinct glycogen
populations.
POSTERS MONDAY & TUESDAY
POS-MON-137
FUNCTIONAL STUDIES OF HOMEDOMAIN-
INTERACTING PROTEIN KINASE USING C. ELEGANS
AS A MODEL
Berber S. and Nicholas H.R.
School of Molecular Bioscience, The University of Sydney, NSW 2006.
The Homeodomain-Interacting Protein Kinases (HIPK) are a family
of serine/threonine protein kinases shown to be critical in regulation
of many cellular processes including cell survival, proliferation and
apoptosis. Since there are four HIPK family members in mammals that
show some redundancy, functional studies of this protein family can be
simplified with the nematode Caenorhabditis elegans since it expresses
a single HIPK protein (HPK-1). Both HIPKs and HPK-1 localise to nuclear
speckles suggesting that they are likely be involved in analogous cellular
processes. To expand on this knowledge we performed phenotypic
analyses on a worm strain carrying a deletion mutation within hpk-1 with
the global aim of discovering some of the in vivo functions of worm HPK-
1. Additionally, to gain insights into molecular mechanisms of HPK-1
function, microarray analysis was performed on the hpk-1 mutant strain.
These analyses have revealed that various cellular processes may be
regulated by HPK-1 including ageing, metabolism and expression of
collagens.
POS-MON-139
REGULATION OF COPPER UPTAKE AND EFFLUX IN
DROSOPHILA INTESTINAL ENTEROCYTES
Binks T. and Burke R.
School of Biological Sciences, Monash University. Wellington Rd
Clayton 3800 Victoria, Australia.
All animals require the essential biometal copper as an enzymatic
cofactor for processes as diverse as energy production, free radical
detoxification and pigmentation. Most copper is absorbed via the
polarized enterocytes that line the intestinal lumen. Active transport
is required to transfer copper across the apical enterocyte membrane
from the lumen into the cells, then again across the opposite basolateral
enterocyte membrane and out into the bloodstream for distribution
throughout the body. Therefore the intestinal enterocytes are an
excellent system to study directional transport of copper through a
polarized cell layer. We are studying this process in the Drosophila
larval midgut, using targeted over expression and suppression of known
copper transport and copper chaperone genes together with two Green
Fluorescent Protein (GFP) reporter genes that respond to copper
depletion and copper accumulation respectively. We will also present
protein localization studies in these midgut cells using GFP fusion
proteins created for the major fly copper uptake and efflux proteins.
And using our Drosophila enterocytes model, we will demonstrate novel
roles in copper homeostasis for genes previously implicated in cellular
vesicle trafficking / transport pathways.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 135
POS-TUE-138
SUMO MODIFICATION OF THE ZINC-FINGER PROTEIN
CTCFL/BORIS
Bernier-Latmani J. and Shaw P.
Experimental Pathology Division, Institute of Pathology, University of
Lausanne.
Post-translational modification by the small ubiquitin-like SUMO
proteins (SUMO-1 and SUMO-2/3) is recognized to play an important
role in target protein function by affecting DNA binding, sub-cellular
localization, protein stability and co-factor association. CTCFL is a
paralog of the ubiquitous genome regulator CTCF, but is expressed solely
in the testis and some cancers. Due to high homology in their respective
zinc-finger regions it has been proposed that CTCF and CTCFL bind
similar DNA sequences but perform different functions. CTCFL has
been implicated in the establishment of methylation at the Igf2/H19
imprinting control region and transcriptional regulation of a number of
genes including BRCA1, c-myc, and BAG1. Recently CTCF has been
shown to be modified by SUMO, whereby enhancing repression of a
c-myc promoter reporter construct. In the present work we show that
CTCFL is efficiently SUMOylated in vitro and when over-expressed
in 293T cells. Endogenous CTCFL is SUMOylated, as determined by
immunoprecipitation of SUMO-1 and SUMO-2/3 modified proteins in
K562 cells. Both SUMO-1 and SUMO-2/3 are attached at two residues,
K181 and K645, as shown by point mutation analysis. Characterization
of the functional consequences of CTCFL SUMOylation is ongoing.
Alteration of CTCFL sub-cellular localization by SUMOylation is being
analyzed by immunofluorescence confocal microscopy. Furthermore,
the effect of SUMOylation on CTCFL DNA-binding and gene regulation
activity is being assessed using EMSA and luciferase reporter assays.
Also, the consequence of SUMOylation on the stability of CTCFL is
being investigated. This work will help elucidate basic CTCFL function
and potentially provide a basis to understand its role in both testis and
cancer.
POS-TUE-140
CHARACTERIZATION OF MAVI-P35, A BACULOVIRAL
MULTIPLE CASPASE INHIBITOR
Brand I.L.1, George C.1, Lovric M.1, Pantaki D.1, Kitevska T.1, Clem
R.J.2 and Hawkins C.J.1
1
La Trobe University, Department of Biochemistry, Melbourne 3086,
Australia . 2Molecular, Cellular and Developmental Biology Program,
Arthropod Genomics Center, Division of Biology, Kansas State
University, Manhattan, KS 66506, USA.
P35 from Autographa californica (Ac) MNPV was the first identified
multiple caspase inhibitor expressed by a baculovirus to prevent the
host cell from undergoing infection-induced apoptosis. A number of P35
relatives have since been identified, including some less closely related
homologues belonging to the P49 sub-family. Ac-P35 has been studied
extensively. It protects against mammalian, nematode and insect cell
death and inhibits all caspases tested, with the exception of the initiator
caspases DRONC, Sf-caspase-X and caspase-9. In contrast, the best
studied P49 sub-family member SlNPV-P49 seems to be a generally
less potent caspase inhibitor, but it can inhibit Ac-P35-resistant initiator
caspases. A new member of the P35-family has been recently identified
by sequencing the genome of the baculovirus Maruca vitrata (Mavi)
MNPV. This P35-family member (Mavi-P35) shares 85% sequence
homology to Ac-P35, but its active loop possesses a different caspase
cleavage sequence, reminiscent of sequences efficiently cleaved by the
insect initiator caspase DRONC. This suggested to us that, unlike other
members of the P35 sub-family, Mavi-P35 may inhibit initiator caspases
such as DRONC. In this study, Mavi-P35 was characterized by testing
its ability to inhibit caspase-induced yeast death and apoptosis of
mammalian and insect cells. Recombinant Mavi-P35 was tested in
vitro against a range of caspases and their susceptibility to inhibition
was quantitated. Our results show that Mavi-P35 is able to inhibit an
even broader range of caspases than Ac-P35, including the Drosophila
melanogaster caspase DRONC.
POSTERS MONDAY & TUESDAY
POS-MON-141
THE GENETICS AND CELL BIOLOGY OF COPPER
HOMEOSTASIS IN DROSOPHILA
Lye J.1, Binks T.1, Hwang J.1, Camakaris J.2 and Burke R.1
1
School of Biological Sciences, Monash University. Wellington Rd
Clayton, Victoria 3800. Australia. 2Department of Genetics, University
of Melbourne. Parkville, Victoria 3010. Australia.
Copper is an essential micronutrient required as a cofactor for enzymes
such as Cytochrome C oxidase, Cu, Zn Superoxide dismutase and
Tyrosinase. Disruption of copper homeostasis can result in serious
human conditions such as Menkes disease (copper deficiency) and
Wilson disease (copper toxicity). The major copper uptake (Ctr1)
and efflux (ATP7) genes are highly conserved from yeast through to
vertebrates. Drosophila has three Ctr1 orthologues and a single ATP7
orthologue, DmATP7. We are using a combination of targeted gene
over expression and gene suppression to examine the in vivo role of
the key copper transport genes in various Drosophila tissues. We find
that reduction of copper uptake or increase in copper efflux in cuticle-
producing cells results in a striking hypopigmentation phenotype due
to lack of activity of the Laccase cuproenzyme. Genetically-induced
copper deficiency in the Drosophila adult eye results in a severely
flattened eye phenotype while increased copper uptake in the eye
causes a rough eye phenotype typical of increased cell death. We will
present genetic interaction studies showing that the effects of increased
copper uptake can be compensated for by increasing copper efflux and
are exacerbated by blocking copper efflux. We will also present data
showing how the cellular localization of these copper transport proteins
fits with their proposed cellular functions, and exciting recent data from
the Australian Synchrotron where we have used X ray Fluorescence
Microscopy to produce elemental maps of Drosophila tissues and prove
directly for the first time, that our genetic manipulations do indeed alter
copper levels in a targeted manner in vivo.
POS-MON143
PURINE BIOSYNTHESIS AND ITS REGULATION IN
NODULES OF SUB-TROPICAL LEGUMES
Chen C.1, Bussell J.2, 3, Atkins C.2 and Smith P.1
1
School of Biological Sciences, The University of Sydney, NSW
2006, Australia. 2School of Plant Biology, The University of Western
Australia, WA 6009, Australia. 3Umea Plant Sciences Center,
Department of Forest Genetics and Plant Physiology, Swedish
University of Agricultural Sciences (SLU), 901 83 Umea, Sweden.
Sub-tropical legumes such as cowpea and soybean (mainly of the
tribe Phaseoleae) export fixed nitrogen as ureides. De novo synthesis
of purine nucleotide is the major pathway for assimilation of fixed
nitrogen in these legumes and the product, IMP, is oxidised to form
ureides that are exported from the nodules. There are 10 enzymatic
steps in the purine biosynthetic pathway and the enzymes involved
are encoded by nine genes (the pur genes). The role of the products
of nitrogen fixation in regulation of these genes was investigated in
nodules of cowpea by growing plants with their root systems in an
atmosphere devoid of nitrogen (80% Ar: 20% O2). In the absence
of N2 (plants grown in Ar:O2) expression of Vupur1, 4, 5, 6, 7 and 8
is at a level required to maintain basic cellular processes only. In air
the level of these transcripts increases dramatically after N2 fixation
begins, suggesting transcription is regulated by the products of fixation.
Other transcripts studied (Vupur2, 3, 9, IMPDH and uricase) showed no
change in the level of expression over time, however, the expression of
Vupur2 and 9 and IMPDH was reduced in Ar:O2 compared to air while
that of Vupur3 and uricase was the same in both treatments. We have
investigated promoters of the two genes encoding the fifth enzyme in
the pathway, AIR synthetase (Gmpur5-1 and Gmpur5-2), in soybean
using GUS/GFP fusions. The Gmpur5-1 promoter drives expression in
all cells but the infected cells of the nodule while the Gmpur5-2 promoter
drives expression almost exclusively in infected cells. We are currently
investigating the regulation of these promoters in response to nitrogen
using these constructs. We are also investigating the targeting of the
enzymes to organelles in nodules.
Page 136 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-142
PEPTIDASE EXPRESSION IN LYMPHOCYTES
Chen Y., Yao T.W., Chowdhury S., Gorrell M.D. and Yu D.M.T.
Centenary Institute, Sydney Medical School, University of Sydney,
NSW, Australia.
The dipeptidyl peptidase (DP) IV gene family is specialized in hydrolyzing
the prolyl bond, which is useful for degrading many peptide hormones.
DPIV (CD26) has roles in lymphocyte activation and proliferation. DPIV
and its closest relative, fibroblast activation protein, also have roles
in liver disease. We have associated DP8 and DP9 expression with
disease pathogenesis [1] and observed DP8 and DP9 expression by
leukocytes and B and T cell lines while others have shown a role for
DP9 in processing antigenic peptides [Geiss-Friedlander R, et al., JBC
2009]. A greater understanding of DP8 and DP9 in the immune system is
needed. This study investigated DP8 and DP9 expression in stimulated
lymphocytes and diseased human liver. DP8 and DP9 expression levels
were measured by Western blot and quantitative PCR in Jurkat T cells
and Raji B cells treated with mitogen, hydrogen peroxide, dithiothreitol,
Mitomycin C or serum starvation. DP8 and DP9 protein levels were
upregulated in mitogen stimulated Jurkat T cells but unaffected by the
other treatments. DP9 protein expression was similar in human primary
biliary cirrhosis liver compared with normal liver. The novel finding that
DP8 and DP9 expression was upregulated in activated T cells suggests
that, like DPIV/CD26, DP8 and DP9 might have roles in T cell activation.
Reference: [1] Yu, D.M.T., et al., The in vivo expression of dipeptidyl
peptidases 8 and 9. J Histochem Cytochem 2009. 57:1025-1040.
POS-TUE-144
AUTOPHOSPHORYLATION OF ATM AT SERINE 1981
MEDIATES THE INTERACTION WITH MDC1 AND
STABILIZES ATM AT SITES OF DNA DOUBLE-STRAND
BREAKS
Davis A., Zheng C. and Chen D.J.
Division of Molecular Biology, Department of Radiation Oncology,
University of Texas Southwestern Medical Center, Dallas Texas, USA.
Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular
response to DNA damage. In response to DNA double strand breaks
(DSBs), ATM is autophosphorylated at serine 1981. Although this
autophosphorylation is widely considered a sign of ATM activation, it
is still not clear if autophosphorylation is required for ATM functions
including localization to DSBs and activation of ATM kinase activity. In
this study, we show that localization of ATM to DSBs is differentially
regulated with the initial localization requiring the MRE11-RAD50-NBS1
complex and sustained retention requiring autophosphorylation of ATM
at serine 1981. Ablation of the autophosphorylation site affects the ability
of ATM to phosphorylate its downstream targets after DNA damage
especially at later time point after irradiation and confer radioresistance.
Biochemical evidence shows that autophosphorylated ATM directly
interacts with the FHA domain of MDC1. Knock-down of MDC1 protein
recapitulates the effects of S1981A mutation on the retention of ATM
at DSBs and phosphorylation of downstream substrates. Moreover,
ablation of the ATM autophosphorylation site and MDC1 depletion did
not show any additive effect on radiosensitivity. Together, these data
illustrate the importance of autophosphorylation at serine 1981 for the
interaction of ATM with MDC1 which serves to stabilize ATM at DSBs
and thereby promote a full-scale response to DNA damage in human
cells.
POSTERS MONDAY & TUESDAY
POS-MON-145
SAME SIGNAL TRIGGERS DIFFERENT CELL
RESPONSES IN VARIOUS CELL LINES
Cheung T.W. and Lam Y.W.
City University of Hong Kong.
Andrographolide (Andro), a diterpenoid lactone isolated from a traditional
herbal medicine. Andrographis paniculata, is an abundant component of
the plant Andrographis that has been commonly used as a folk remedy
for alleviation of inflammatory disorders in Asia for millennia. Also, it is
known to possess potent anti-cancer activities. According to previous
studies, it can trigger most of cell lines to go through G1/S arrest
followed by apoptosis. Cell cycle checkpoints are regulatory pathways
that control the order and timing of cell cycle transitions and ensure that
critical events are completed with high fidelity. In addition, checkpoints
respond to damage by arresting the cell cycle to provide time for repair
and by inducing transcription of genes that facilitate repair. However,
a feature of tumor cells is the alteration of appropriate cell-cycle
progressions which are closely related apoptotic process. Currently,
people mainly uses anti-mitotic drugs which work by perturbing spindle
assembly through the activation of the spindle assembly checkpoint for
treating cancers. The drugs cause mitotic arrest, and triggers apoptosis.
Based on past study, the response of HepG2 cells is different from the
other cell lines under Andro treatment. To clarify what the differences
are, we use live cell imaging and western blotting approaches in the
study. We showed that Andro induces both G2/M and G1 arrest in two
tested cell lines. Then, in HeLa cells, it quickly goes into apoptosis. But,
HepG2 cells mainly stay long in cell cycle arrests until necrosis occurred.
The differences may not relate to DNA damaging or increasing ROS
production. And, many M-phase related proteins show some regulation
to reason the longer G2/M arrest observation in HepG2 cells, not in
HeLa cells.
POS-MON-147
PROTEOMIC PROFILE OF THE SOYBEAN
SYMBIOSOME MEMBRANE
Clarke V.C.1, Loughlin P.C.1, Jacoby R.P.2, Taylor N.L.2, Millar A.H.2,
Day D.A.3 and Smith P.M.C.1
1
School of Biological Sciences, The University of Sydney, NSW,
Australia. 2ARC Center of Excellence in Plant Energy Biology, The
University of Western Australia, WA, Australia. 3Flinders University,
SA, Australia.
Soybeans are able to form a symbiotic association with soil bacteria,
Bradyrhizobium japonicum, whereby atmospheric nitrogen is fixed by the
bacteria and made available to the plant in exchange for nutrients. This
symbiotic relationship occurs within specialised root structures termed
nodules. Free-living Bradyrhizobium bacteria invade the soybean roots
and become engulfed within the plant cell, surrounded by a membrane
of plant origin known as the symbiosome membrane (SM). It is this
membrane that regulates the movement of solutes from plant to bacteroid
(the symbiotic form of the rhizobium) and vice versa. The SM is a unique
structure containing an array of plant-derived proteins through which
the plant can regulate the symbiosis. Previous attempts to characterise
the protein complement of this membrane have been hindered by its
hydrophobic nature and the absence of a complete soybean genome
for reference. In this study, SM was isolated from mature nitrogen-fixing
soybean root nodules and analysed using shotgun proteomic techniques.
The recent release of the complete soybean genome has allowed for
the identification of peptide sequences. Our initial proteomic analysis of
soybean SM has identified forty putative SM-localised proteins, including
ten previously localised to the SM such as nodulin-26, an aquaporin.
Bioinformatic analysis of novel matches has suggested that half may
contain membrane-spanning domains, making them candidates for SM
transporters. Further in-depth proteomic analysis of the membrane is
currently being undertaken and the function and localisation of putative
SM proteins will be investigated.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 137
POS-TUE-146
FUNCTIONAL CHARACTERIZATION OF A NOVEL
AURORA-A BINDING PROTEIN, AIBP IN CENTROSOME
STRUCTURE AND SPINDLE FORMATION
Chou C.H1, 2 , Wu C.H. 2, Huang C.Y. 3, Hsu C.M. 1, Howng S.L.4 and
Hong Y.R.1, 2
1
Department of Biological Sciences, National Sun Yat-Sen University,
Kaohsiung,Taiwan. 2Department of Biochemistry, Faculty of Medicine,
College of Medicine, Kaohsiung Medical University, Kaohsiung,
Taiwan. 3Institute of Clinical Medicine, National Yang-Ming University,
Taipei, Taiwan. 4Department of Neurosurgery, Kaohsiung Medical
University Hospital, Kaohsiung, Taiwan.
Aurora-A is involved in chromosome alignment, centrosome maturation,
mitotic spindle assembly and regards to an oncogene. Aurora-A is also
known to bind to several other proteins affecting its up-regulation or
down-regulation and localization. However, how these different binding
signals work together to regulate Aurora-A is not properly known. To
explore more Aurora-A interacting proteins, the low-copy yeast two-
hybrid screening using Aurora A as bait protein was performed. One
novel gene, AIBp, was demonstrated to associate with Aurora-A in
yeast two-hybrid method and in vitro GST pull-down assay. Molecular
characterization showed that AIBp possessed a binding site at the
C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also
interacts with hNinein at the N-terminal, which overlaps a previously
reported hNinein and GSK3β binding site. In kinase assay, AIBp interacts
with the Aurora-A kinase domain functions as a positive regulator,
whereas AIBp binding to hNinein appears to block the phosphorylation
of hNinein by both Aurora-A and GSK3β. siRNA-mediated elimination
of AIBp from HeLa cells, results in a donut-like shape, asymmetrical
spindle pole and multiple spindle pole formation. We also demonstrated
that both AIBp and Aurora-A are co-overexpressed in various brain
tumors. These studies demonstrate that AIBp may not only be required
for the dynamic movement of Aurora-A at the centrosomes and spindle
apparatus during the cell cycle, and may also be important during brain
tumorigenesis.
POS-TUE-148
TROPOMYOSIN TM5NM1 MEDIATES ACTIN-
DEPENDENT CELLULAR PROLIFERATION
Coombes J.D.1, 2 , Schevzov G.1, Stehn J.1, Creed S.3, Desouza M.1,
Bach C.T.2, 3, O’Neill G.2, 3, Musgrove E.4 and Gunning P.1
1
Oncology Research Unit, School of Medical Sciences, University of
New South Wales. 2Sydney Medical School, University of Sydney.
3
Kids Research Institute, Children’s Hospital at Westmead. 4Cancer
Research Program, Garvan Institute of Medical Research.
Progression through the cell cycle is associated with profound changes
in the actin cytoskeleton. Disruption of the actin cytoskeleton with
pharmacological agents causes arrest in G1 phase and failure to enter
S-phase, indicating that an intact actin cytoskeleton is required for cell
cycle progression. The lack of specificity of these agents has hindered
the analysis of the mechanisms responsible for this regulation. We have
previously identified functionally distinct populations of actin filaments,
each containing different isoforms of the actin filament-associated
protein, tropomyosin (Gunning, O’Neill, Hardeman, Physiol Revs 2008).
In the current study, we hypothesised that a specific population of Tm
isoform-containing actin filaments regulates cell proliferation. The role
of Tm5NM1-containing actin filaments was investigated in knock-out,
knock-down, and overexpression model systems. Our data show that
embryonic fibroblasts (MEFs) isolated from Tm5NM1/2-knockout mice
have an impaired rate of proliferation in response to growth factor
stimulation. This is accompanied by a dysregulation of MAPK signalling
(phospho-ERK1/2) and decreased expression of the key G1-phase
mediator, Cyclin D1. Neuroblastoma SHEP cells treated with siRNA
against Tm5NM1/2 also have reduced proliferation rates and decreased
Cyclin D1 expression. In comparison, Tm5NM1-overexpressing
neuroblastoma-derived B35 cells display accelerated proliferation,
and reduced propensity to withdraw from the cell cycle when treated
with differentiation factors. These data suggest that actin-filaments
containing Tm5NM1 mediate G1 phase progression, potentially via
controlling the fidelity of signalling through the ERK/MAPK pathway.
This indicates that Tm5NM1 has potential as a novel anti-proliferative
therapeutic target.
POSTERS MONDAY & TUESDAY
POS-MON-149
DEXAMETHASONE INHIBITS INTESTINAL SECRETORY
CELL ER STRESS BY UPREGULATING DEGRADATION
OF MISFOLDED PROTEIN ER STRESS (ERAD)
Das I.1, 2 , Png C.W.1, Tran T.1, 3, Eri R.1, Lourie R.1, 3, Adams R.1, Oancea
I.1, 3, Crane D.1, 2, Florin T.1, 3 and McGuckin M.1, 3
1
Mater Medical Research Institute. 2Griffith University. 3University Of
Queensland.
Endoplasmic reticulum (ER) stress in intestinal secretory cells has been
linked with the pathogenesis of Inflammatory Bowel Diseases (IBD)
which introduces ER stress, the unfolded protein response (UPR) and
ER-associated degradation (ERAD) of misfolded proteins as pathways
for therapeutic targeting in IBD. Glucocorticosteroids are an effective
treatment for IBD, but little is known of their effect on ER stress. This
study determined the effect of a glucocorticosteroid (dexamethasone,
Dex) on ER stress in cultured LS174T colonic cells and in Winnie
mice with intestinal secretory cell ER stress caused by Muc2 mucin
misfolding. mRNA expression of ER stress markers GRP78, spliced-
XBP1, ATF6 and CHOP; glycoprotein chaperones CNX and CRT; and
ERAD genes EDEM1, VCP and SEC61 were measured by qRT-PCR.
Histological analysis was used to assess intestinal inflammation and
mucin biosynthesis. Upregulation of GRP78 (a key indicator of ER stress,
15.1±1.3 and 14.3±1.0 fold increased by tunicamycin and thapsigargin in
LS174T cells, respectively) was completely inhibited by Dex (P<0.001).
All other ER stress markers followed the same pattern. Winnie mice
showed accumulation of misfolded Muc2 in secretory cells and a 40±3
fold increase in intestinal Grp78 compared to wild-type mice, and
elevated expression of all other ER stress/UPR markers. Four weeks
treatment with Dex significantly inhibited the increase in ER stress
genes (P<0.001) and substantially increased production of correctly-
folded Muc2 (P<0.001). The ERAD genes Edem1 and Vcp were the
only ER stress-related genes that increased in Dex-treated mice. These
results suggest that Dex ameliorates ER stress by enhancing removal
and degradation of the mis-folded Muc2 from the secretory cells by up-
regulation of proteins involved in the ERAD pathway.
POS-MON-151
KRüPPEL-LIKE FACTOR 3 (KLF3) AND THE UV-
RESPONSE PATHWAY
Dewi V.A., Pearson R.C.M. and Crossley M.
School of Biotechnology and Biomolecular Sciences, University of
New South Wales, NSW, 2052.
Following DNA damage, cells enter either cell cycle arrest or apoptosis
depending on the severity of the damage. Upon extensive DNA damage
by ultra violet (UV) light, a master regulator of cell cycle and apoptosis,
p53, is phosphorylated by the kinase, Homeodomain-interacting protein
kinase 2 (Hipk2). This phosphorylation of p53 stabilises the protein
and is recognised as a hallmark of apoptosis. The transcriptional co-
repressor C-terminal binding protein 2 (Ctbp2) is also phosphorylated by
Hipk2 upon DNA damage. In contrast to p53, phosphorylation of Ctbp2
marks it for degradation. Krüppel-like factor 3 (Klf3) is a transcriptional
repressor known to recruit Ctbp2 to silence target genes. Using in vitro
kinase assays we have shown that Hipk2 can also phosphorylate Klf3.
This led us to hypothesise that Klf3 plays a role in the UV-response
pathway. Using NIH-3T3 as a model cell line, we conducted time course
experiments to study the effect of UV irradiation on the expression of
Klf3 and its target genes. We observed downregulation of both Klf3 and
its co-repressor Ctbp2 following irradiation. We are currently exploring
the hypothesis that changes in expression of Klf3 target genes play an
important role in the response to DNA damage.
Page 138 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-150
AGED-RELATED INCREASE IN XANTHINE OXIDASE
EXPRESSION AND ACTIVITY IN SPLEEN FROM MICE
Vida C.1, Gonzalez E.1, Hernandez O.1, Rodriguez-Teres S.1, Hernanz
A.2, Corpas I.1 and De la Fuente M.1
1
Department of Physiology. Faculty of Biology. Complutense University
of Madrid (Spain). 2Department of Biochemistry. La Paz Hospital.
Madrid. Spain.
In mammalian cells, one of the major sources of reactive oxygen
species (ROS) is the enzyme xanthine oxidase (XO), which uses
hypoxanthine and xanthine as reducing substrates producing ROS,
such as superoxide anion and hydrogen peroxide. XO has been
implicated in a variety of pathophysiological states and oxidation stress-
related diseases. In addition, ageing is based on chronic stress, with an
excessive production of ROS and a loss of the antioxidant defences, in
which the immune system has an important role. The aim of the present
work was to determine the age-related changes in XO expression and
activity in mouse spleen. ICR-CD1 adult (6 month-old), mature (13
month-old), old (18 month-old) and long-lived (31 month-old) female
mice were used. Animals were sacrificed and spleen was obtained,
which was homogeneized in order to determine XO activity using a
comercial kit (Invitrogen). The XO protein expression was assayed by
western-blot analysis. The results showed that both XO expression
and activity increase with ageing. XO activity levels were significantly
increased in old (p≤0,001) and long-living (p≤0,001) mice with respect to
adult mice. Finally, we analyzed the XO protein expression in this tissue,
which showed a significant increase in old (p≤0,05) and long-living mice
(p≤0,01). Nevertheless, mature mice show similar XO activity levels
and XO protein expression as compared to the adults. In conclusion,
the increase in the XO expression and activity in spleen from older
mice may have a crucial role in the pathophysiological changes of
the ageing process. MICINN(BFU2008-04336); Research-Group-
UCM(910379ENEROINN); RETICEF (RD06/0013/0003).
POS-TUE-152
THE SECRETIN PROTEIN SUPER-FAMILY: NEW
STRUCTURES FOR BACTERIAL OUTER MEMBRANE
PROTEINS
Dunstan R., Alcock F., Webb C. and Lithgow T.
Department of Biochemistry & Molecular Biology, Monash University,
Clayton, 3800 Australia.
It was considered for some time that all integral membrane proteins in
bacterial outer membranes had an architecture referred to as a β-barrel,
and recently it has become clear that these proteins are assembled by
the BAM complex. Very recently a new architecture of bacterial outer
membrane proteins was discovered: large, multimeric complexes
containing amphipathic helical transmembrane domains rather than
the conventional β-barrel structures. These “secretins” form pores for
the secretion of proteins of the type II and type III secretion systems,
formation of type IV pili and the assembly of filamentous bacteriophage.
They also form the outer membrane collar of bacterial flagella. The
precise mechanism and kinetics of secretin assembly and insertion
into the OM is unclear. Using a Hidden Markov Model (HMM) strategy,
based on sequences of known secretins, novel candidate secretins
have been identified from the enteropathogenic Escherichia coli strain
E2348/68. We are tailoring an assay in which the kinetics of assembly of
these secretins can be determined and aim to determine the machinery
responsible for secretin assembly in bacterial outer membranes.
POSTERS MONDAY & TUESDAY
POS-MON-153
POLLEN-SPECIFIC DEPOLL GENE EFFECTS THE
EXPRESSION OF LTP PROTEINS IN ARABIDOPSIS
THALIANA
Duplakova N.1, 2 and Honys D.1, 2
1
Laboratory of Pollen Biology, Institute of Experimental Botany ASCR,
Rozvojová 263, 165 02 Prague 6, Czech Republic. 2Department of
Plant Experimental Biology, Faculty of Science, Charles University,
Viničná 5, 128 44 Prague 2, Czech Republic.
Plant cell wall is highly organized and complex structure consisting of
carbohydrates, proteins and aromatic compounds surrounding and
separating cells from outer environment. It has been assigned many
different functions like regulation of cell volume, protoplast protection,
cell shape determination and many others including its key role in
morphogenesis. The pollen wall represents its special type as it is
composed by two distinct layers - exine and intine. Moreover, pollen wall
is covered with unique and specialized lipid pollen coat with functions
in pollen dispersal and pollen stigma recognition. The list of cell wall
proteins is very extensive and one of these, lipid-transfer proteins
(LTPs), are present in pollen wall separable fraction. LTP proteins are
basic, soluble, 9-kDa proteins representing up to 4% of total soluble
proteins in higher plants. They were found to be secreted and located
extracellularly, in the cell wall, although earlier hypotheses considered
their function in intracellular lipid dynamics. Several roles for LTPs
were suggested in plant growth and development, defense reactions
against phytopathogens, symbiosis and the adaptation of plants to
various environmental conditions. Here we show that T-DNA insertion
in pollen-specific gene depoll encoding 21 kDa (185 aa) C2 protein
caused increased expression of numerous LTPs in male gametophyte
accompanied by defects in pollen cell wall formation or cell death.
Acknowledgment: Authors gratefully acknowledge the financial support
from the Czech Science Foundation (grant 522/09/0858) and Ministry
of Education, Youth and Sports of the Czech Republic (grants LC06004
and OC10054).
POS-MON-155
TARGETS OF RNA-BINDING PROTEIN MUSASHI-1 IN
MOUSE GERM CELL DEVELOPMENT
Fraser B.A.1, 2 , Sobinoff A.2, Pye V.J.2, Roman S.D.1, 2, Hime G.R.1, 3,
Siddall N.A.1, 3, Koopman P.1, 4 and McLaughlin E.A.1, 2
1
ARC Centre of Excellence in Biotechnology & Development.
2
Reproductive Science Group School of Environmental & Life Science,
University of Newcastle, Callaghan NSW 2308. 3Dpt of Anatomy &
Cell Biology, University of Melbourne, Parkville VIC 3010. 4Institute for
Molecular Biosciences, University of Queensland, St Lucia QLD 4072.
RNA-binding proteins, such as Musashi-1 (Msi-1), can contribute to
posttranscriptional control and are believed to have an important role
in the maintenance of germline stem cells and germ cell differentiation.
We have previously confirmed that Msi-1 is predominately expressed
in gonocytes and mitotic spermatogonia. Protein-RNA pulldown and
microarray and bioinformatic analysis has provided us with a number
of possible targets of Msi-1 in the testes. Further to this, we aimed: i)
to generate mice that over-express Msi-1 in differentiating male germ
cells and examine the resultant phenotype; (ii) to use RNA interference
to knockdown the expression of Msi-1 in cultured spermatogonial cells;
and (iii) to use a Musashi-1 specific antibody to immunoprecipitate target
RNA from a spermatogonial cell lysate. Initial phenotypic analysis of the
transgenic mice indicated that, whilst these mice produced spermatozoa,
they have reduced fertility or are infertile. They have anomalous sperm
formation and the sperm have a decreased ability to bind to mature
oocytes. Increasing transgene expression resulted in male sterility
due to spermatogenic arrest and a total absence of mature sperm.
Quantitative gene expression of the putative targets demonstrated
significantly increased transcript levels with concomitant increase in
Msi-1 transgene expression. The relative expression of the target genes
was down-regulated in cultured spermatogonia following shRNA Msi-1
knockdown. Immunoprecipitated mRNA from spermatogonial lysate was
significantly enriched with Msi-2 mRNA. In conclusion, our preliminary
results indicate that Msi-1 is required for normal spermatogenesis and
that it may target a number of mRNAs, particularly Musashi-2, to achieve
posttranscriptional control of mouse spermatogonial differentiation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 139
POS-TUE-154
PROLIFERATIVE AND SURVIVAL PATHWAYS IN
OESOPHAGEAL CANCER
Esau L.E. and Hendricks D.
University of Cape Town, Cape Town, Western Province, South Africa.
Oesophageal cancer is the 7th most common cancer worldwide and
available treatment does not significantly enhance patient survival. The
epidermal growth factor receptor EGFR is commonly over-expressed
in oesophageal cancer and its over-expression correlates with tumour
aggression and survival. Reports exists that the insulin-like growth factor
receptor 1 IGF-1R plays a role in survival and that interactions exist
between EGFR and IGF-1R. The objective of this study is to determine
the role of EGFR and IGF-1R in proliferation and survival of oesophageal
cancer. EGFR and IGF-1R function in cultured oesophageal cancer
cell biology was determined by using western blot analysis, inhibitor to
EGFR or IGF-1R shRNA. Receptors were activated with specific ligand.
EGFR kinase domain phosphorylation activated IGF-1R as receptors
were shown to immunoprecicpitate with each other. Targeting EGFR
resulted in decreased proliferation and an IC50 of +/-10uM however
increased Akt activity was observed. IGF-1R knockdown resulted in
increased pEGFR and pERK1/2 with diminished Akt activity which lead
to a 3 fold increased sensitivity to EGFR inhibitor compared to EGFR
inhibition alone. EGFR and IGF-1R show potential as drug targets in
Oesophageal cancer as combined targeting of these two receptors
show increased response than targeting EGF receptor alone.
POS-TUE-156
THE GOLGI-LOCALISED CALCIUM ATPASE IN BREAST
CANCER
Grice D.M.1, Vetter I.2, Faddy H.M.1, Kenny P.A.3, Monteith G.R.1 and
Roberts-Thomson S.J.1
1
School of Pharmacy, The University of Queensland, Brisbane, QLD,
4072, Australia. 2Institute for Molecular Biosciences, The University
of Queensland, Brisbane, QLD, 4072, Australia. 3Department of
Developmental and Molecular Biology, Albert Einstein College of
Medicine, Bronx, New York, USA.
Golgi-localised calcium ATPases maintain the calcium concentration
of the Golgi apparatus and as such are associated with cell signalling,
apoptotic signalling and post-translational modification of proteins.
We assessed the mRNA expression of one of these Golgi-localized
calcium ATPases in 295 breast tumour samples classified into five
transcriptional subtypes each associated with a different prognosis and
response to therapeutic treatment. The Golgi-localised calcium ATPase
was enriched in particular breast cancer subtypes. To characterize
the role of this calcium ATPase in breast cancer, its expression was
silenced in MDA-MB-231 MCF7 and T-47D breast cancer cell lines
using Dharmacon OnTarget plus Smartpool siRNA. We assessed
the consequences of ATPase silencing on cell viability, response to
induction of endoplasmic reticulum stress and affects on G-protein
coupled receptor cytosolic calcium signaling stimulated by trypsin,
thrombin and ATP using a high throughput FLIPRTETRA imaging system
and the potential regulation of Golgi-resident enzymes. Our results
suggest that alterations in the expression of a Golgi-localised calcium
ATPase may be a feature of specific breast cancer subtypes, and that
inhibition of the expression of these calcium pumps differentially affects
pathways important in breast cancer progression.
POSTERS MONDAY & TUESDAY
POS-MON-157
CONTROL OF CELL GROWTH AND SURVIVAL VIA
14-3-3 (ZETA) AND TYPE 1 INSULIN-LIKE GROWTH
FACTOR RECEPTOR
Hadzir S.1, Ramshaw H.2, Krake R.2, Delaine C.1, Guthridge M.2,
Wallace J.C.1, Lopez A.F.2 and Forbes B.E.1
1
School of Molecular and Biomedical Sciences, University of Adelaide,
Adelaide SA,5005. 2Division of Human Immunology, Centre for Cancer
Biology, Adelaide, SA, 5000.
We have developed the first isoform specific 14-3-3ζ-/- mouse. 14-3-3ζ-
/-
mice exhibit post-natal growth deficiency (P7 onwards) with 20-30%
reduction in size compared to wild type littermates. 14-3-3 proteins are
intracellular phosphoserine/phosphothreonine binding proteins involved
in modulation of crucial biological processes such as proliferation, signal
transduction, metabolism, cell cycle and apoptosis. Importantly, 14-3-3ζ
binds the type 1 insulin-like growth factor receptor (IGF-1R) and several
proteins involved in IGF-1R downstream signalling pathways including
IRS-1 and 2, p85, Akt and Raf. These interactions modulate IGF-1R
survival, proliferation and transformation activities. Initial observations
suggest a perturbation of the GH/IGF-I axis in 14-3-3ζ-/- mice. Preliminary
analyses have revealed low pituitary growth hormone (GH) levels and
low serum IGF-I (~ 2.7 fold), IGF binding protein-3 (IGFBP-3) and acid
labile subunit (ALS) levels. 14-3-3ζ-/- mice growth deficiency is evident
before weaning (P7 onwards) whereas GH deficiency models show
growth deficiency only at puberty and IGF-I knockout mice are pre and
postnatally growth deficient The phenotype suggests it is different to
most mouse models of IGF-I deficiency, although it is reminiscent of the
hypothalamic IGF-1R knockout phenotype. Interestingly, we have shown
a perturbation in the IGF-1R signalling via Akt and Erk1/2 in embryonic
fibroblasts derived from the 14-3-3ζ-/- mice. Therefore, our evidence so
far suggests that the growth deficiency is driven by abnormalities in GH/
IGF-I axis and perturbation in 14-3-3ζ action involving IGF-1R signalling
(via Akt and Erk). The mechanism underlying the GH deficiency is still
not clear.
POS-MON-159
ASSEMBLY OF MITOCHONDRIAL PORINS:
ADAPTATION TO AN ENDOSYMBIOTIC LIFESTYLE
Hewitt V.L., Gabriel K., Traven A. and Lithgow T.J.
Department of Biochemistry and Molecular Biology, Monash
University, Melbourne.
The outer membrane of mitochondria is the interface between the
organelle and its host. The SAM (sorting and assembly machinery) is
embedded in this membrane where it helps insert and assemble vital
membrane proteins and protein complexes. The core components of
the SAM complex are conserved across eukaryotes but it appears to be
able to co-opt additional subunits for some tasks. While previous studies
have identified some of these subunits there is still very little known
about how the core of the sorting and assembly process really works
even for the most abundant proteins, such as porin (VDAC). Though
extensively studied in Saccharomyces cerevisiae, understanding the
details of the assembly and insertion of porin has been hampered by
the instability of the porin complex. Preliminary studies in Candida
albicans show porin assembles more quickly and into a more robust
complex than in Saccharomyces. Import of radiolabeled proteins into
purified mitochondria is used to elucidate the role of the SAM complex
in this process. This more stable model system enables a more
thorough comparison of the core SAM subunit, Sam50 and its bacterial
homologue BamA (Omp85). Understanding the origins, interactions and
roles of these components can help reveal how free-living bacteria were
assimilated into the cell to become mitochondria.
Page 140 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-158
OXIDATION AND INFLAMMATION INCREASE WITH
AGEING IN IMMUNE CELLS FROM MEN AND WOMEN
Mate I.1, Arranz L.1, Garcia-San Frutos M.V.1, Vida C.1, Hernanz A.2 and
De La Fuente M.1
1
Department of Physiology. Faculty of Biology. Complutense University
of Madrid. 2Biochemistry Department. La Paz Hospital. Madrid.
(Spain).
A direct relationship exists between ageing and increasing oxidation
and inflammation. The immune cell functions change with ageing
and this immunosenescence seems to be a consequence of this
age-related oxidative and inflammatory stress. We have proposed a
relevant role of the immune cells on oxi-inflamm-ageing. The aim of
the present work was to determine the age-related changes in several
oxidative and inflammatory parameters as well as in some oxidative
stress-related functions in leukocytes from men and women. A total
of 334 healthy human volunteers were studied (171 women and 163
men), and divided into three age groups: adult (30-49 years of age),
adult-mature (50-59 years) and mature (60-79 years). Blood samples
were extracted and the following parameters analyzed in neutrophils
and lymphocytes: adherence to tissues, spontaneous mobility (SM),
chemotaxis, phagocytosis, proliferation of lymphocytes in response to
mitogens, superoxide anion (SA) levels, TNF-α secretion in response to
LPS, as well as plasma C-reactive protein (CRP). The results showed
statistically significant increases of CRP, SA, TNF-α, adherence and SM
in mature groups with respect to adults, whereas the other functions
were decreased. These changes were more significant in women than
in men. Thus, oxi-inflamm-ageing seems to increase in postmenopausal
women as a possible consequence of the loss of the protective role of
oestrogens. Financial support: MICINN (BFU2008-04336); Research-
Group-UCM (910379ENEROINN); RETICEF (RD06/0013/0003).
POS-TUE-160
MARSUPIAL INTERFERON INDUCIBLE
TRANSMEMBRANE PROTEINS
Hickford D.E., Shaw G. and Renfree M.B.
Zoology Department, The University of Melbourne.
The Interferon Inducible Transmembrane (IFITM) family is a group of
cell surface proteins with roles in diverse cellular processes, including
promoting homotypic cell adhesion, mediating cell differentiation
and acting as a transducer of anti-proliferative signals. In humans,
IFITM genes are up-regulated in some cancers and IFITM2 acts as
a pro-apoptotic factor. In the mouse and human the IFITM genes are
clustered; five Ifitm genes within a 68 kb locus on chromosome 7 in
the mouse and four genes within a 26.5 kb locus on chromosome 11 in
humans. Sequence comparison suggests that gene duplications at the
mouse and human IFITM locus have occurred independently in both
species. Despite the broad range of processes the IFITM genes have
been implicated in, nothing is known about marsupial IFITMs. We have
identified five IFITM homologs in the tammar wallaby Macropus eugenii,
four of which are clustered in a 74.5 kb locus. There are four homologs
in the opossum Monodelphis domestica, three of which are clustered on
chromosome 3. Amino acid identity between the various IFITM proteins
is 27-76% in the tammar and 35-64% in the opossum. Sequence
analysis suggests that similar to the mouse and human, IFITM genes
have been duplicated independently in marsupials. Using RT-PCR, we
analysed the expression of IFITM genes in adult organs, embryos and
fetuses of the tammar wallaby. Expression of the tammar IFITM genes
differs to that in the mouse, suggesting that the roles of the IFITM genes
may not be conserved between marsupials and eutherians.
POSTERS MONDAY & TUESDAY
POS-MON-161
DIFFERENTIAL EXPRESSION OF HEDGEHOG
SIGNALING COMPONENTS AND SNAIL/E-CADHERIN
IN HUMAN BRAIN TUMORS
Hong Y.R.1, 2 and Chou C.H.1, 2
1
Department of Biochemistry, Faculty of Medicine, College of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan. 2Department of
Biological Sciences, National Sun Yat-Sen University, Kaohsiung,
Taiwan.
The Hedgehog (Hh) transcription factor Gli induces transformation
of epithelial cells via induction of Snail, a repressor of E-cadherin.
Epithelial-mesenchymal transition is also a determinants of the
progression of tumorigenesis, following downregulation of E-cadherin.
However, the role of Hh signaling components and Snail/E-cadherin in
brain tumors is not yet fully understood. We analyzed the expression of
Hh signaling components and Snail/E-cadherin in 69 brain tumors by
reverse transcription–polymerase chain reaction (RT-PCR). The data
showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69),
Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis.
Our results also indicated that Snail and E-cadherin showed opposing
expression in malignant tumors (high grade astrocytoma and metastasis).
Snail and E-cadherin showed less correlation in benign brain tumors. We
further investigated mutations of Gli2 and Snail by RT-PCR and direct
sequencing. No mutation was observed on Gli2 but several sporadic
mutations on Snail were found, including S96G, S111L, S111L/S119Y
and one nonsense mutation at codon 158 (Y158*). An in vitro E-cadherin
promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants
were decreased 15%, 25% and 50%, respectively, whereas Y158* was
increased 40% compared to wild type. Furthermore, our data showed
that wild type Snail and S96G, S111L, S111L/S119Y translocated into the
nucleus, while the Y158* mutant failed to translocate into the nucleus.
Taken together, our results demonstrate that Hh signaling components,
the expression and mutations of Snail, and the expression of E-cadherin
may play an important role in human brain tumorigenesis.
POS-MON-163
IDENTIFICATION OF PROTEIN-PROTEIN
INTERACTIONS REGULATING SODIUM-PROTON
ANTIPORTER ACTIVITY
Huynh D.1, 2 and Gendall T.1
1
Department of Botany, La Trobe University, Bundoora, Victoria 3086,
Australia. 2Cuulong Delta Rice Research Institute, Cantho, Vietnam.
In plants, the concentration and ratio of Na+ and K+ is crucial for
homeostasis, particularly in stressful conditions, such as salinity, cold
and drought. Ion homeostasis in plants is regulated by a large number
of ion exchangers that control the movement of ions across the plasma
and intracellular membranes. The class of sodium-proton exchangers
designated as NHEs in animals or NHXs in plants and yeast, is highly
conserved throughout eukaryotes. NHX5 and NHX6 are intracellular-
localized proteins that are widely conserved in diverse plants. The
C-terminal tail of NHX5 and NHX6 is also well conserved in many plant
species suggesting that it may mediate a protein-protein interaction and
act as a part of pH sensing mechanism. To investigate the functional
significance of this well conserved C-terminal tail in NHX5 and NHX6,
a yeast 2-hybrid screen has been performed to identify interacting
proteins. The predicted C-terminal tail and the highly conserved regions
of both NHX5 and NHX6 have been used as baits to screen existing
2-hybrid libraries. Positive colonies will be identified, and the interaction
further will be analyzed using by deletion analysis of both the C-terminal
region of NHX5 and NXH6, and the interacting protein(s). These results
will be validated by in vitro pull down assays using recombinant proteins.
To determine if NHX5 and NHX6 function as homo- or hetero-dimers,
interactions between NHX5 and NHX6 have also been investigated by
yeast two-hybrid system, bimolecular fluorescence complementation
and co-immuno-precipitation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 141
POS-TUE-162
CYTOTOXICITY AND OPTICAL-PHYSICAL STUDIES OF
6-ALKYNYL-COUMARIN DERIVATIVES
Hisao C. and Hong F.
Department of Chemistry, National Chung Hsing University, Taiwan.
Many coumarin derivatives are biologically important molecules that
possess antifungal, antibacterial, antimicrobial insecticidal, anti HIV and
antibiotic activites. The coumarin moiety as chromophore of fluorescent
ion indicator is a useful physical property for the bio-imaging technique
in biological systems. It was expected that modification on coumarine
with conjugated multiple bonds might bring about the desired change
in either red-shift of wavelength or intensity of the emission. It is our
intention to apply these compounds to biological systems by combining
both the cytotoxicity and fluorescence property. Several attempts in
the modification of 6-Br-coumarine with substituted alkynes by the
Sonogashira reaction were pursued. Indeed, it led to the formation of
several coumarine derivatives with expected red-shift of wavelength
in fluorescence. It was also found that optical property was affected
greatly by slight change the substitutents on the phenyl moiety which
is linked to coumarin mainframe through a triple bond. Moreover, we
applied these coumarine derivatives to biological systems, especially,
on the study of cytotoxicity towards HeLa cells. These newly-made
compounds indeed showed higher cytotoxicity towards cells compared
with the unmodified one.
POS-TUE-164
THE HIJACKING OF MITOCHONDRIAL IMPORT
MECHANISMS BY PORB FROM NEISSERIA
MENINGITIDIS
Jiang J.-H.1, 2 , Davies J.3, Strugnell R.A.2, Lithgow T.1 and Gabriel K.1
1
Department of Biochemistry and Molecular Biology, Monash
University, Victoria, Australia. 2Department of Microbiology and
Immunology, the University of Melbourne, Victoria, Australia.
3
Department of Microbiology, Monash University, Victoria, Australia.
PorB is a β-barrel protein from the pathogen Neisseria meningitidis
that is targeted to host cell mitochondria and modulates apoptosis
during bacterial invasion. The sorting and assembly machinery (SAM)
is required for eukaryotic β-barrel protein import into mitochondria.
To investigate the mechanisms behind PorB import, S35 -labeled PorB
was in vitro translated and incubated with mitochondria isolated from
mouse tissue or import machinery deficient yeast strains. Folded
PorB was distinguished from unfolded protein using semi-native gel
electrophoresis. The rate of PorB folding in mammalian mitochondria
is decreased if the mitochondrial outer membrane is ruptured by hypo-
osmotic shock. This treatment releases soluble inter-membrane space
(IMS) proteins such as the small Tim(s) and hence this suggests a role for
IMS proteins in PorB folding. Mitochondria isolated from Sam50 mutant
which are deficient in import of native mitochondrial β-barrel proteins
also fold PorB at a slower rate. Taken together, the import of PorB into
mitochondria is SAM pathway dependent. The exact localization of
PorB in mitochondria and the roles of other SAM components will also
be assessed in the future.
POSTERS MONDAY & TUESDAY
POS-MON-165
EXPRESSION AND REGULATION OF THIOREDOXIN IN
CANCER CELLS DURING HYPOXIA
Karlenius T.C.1, 2 , Shah F.L.1, 2, Clarke F.M.1, 2 and Tonissen K.F.1, 2
1
School of Biomolecular and Physical Sciences, Griffith University,
Nathan QLD 4111, Australia. 2Eskitis Institute for Cell and Molecular
Therapies, Griffith University.
Redox homeostasis is crucial for cell survival. Too much oxygen in the
cell leads to oxidative stress through the production of ROS, which
reacts with the cells macromolecules causing cell damage and finally
cell death. The cells defend themselves against oxidative stress through
the production of antioxidants, which either neutralise ROS or reverse
ROS-induced damage. In contrast, low oxygen levels in the cell lead
to hypoxia. Under hypoxic conditions a signalling pathway involving a
key regulator termed hypoxia-inducible factor (HIF) is switched on. HIF
drives the induction of many genes controlling multiple cell functions
such as angiogenesis, metabolism and apoptosis/survival. Thus, the
level of oxygen in a cell dictates the molecular response of cells through
modulation of gene expression. Furthermore, both oxidative stress and
hypoxia are common features of solid tumours. Both oxidative stress and
hypoxia lead to changes in the cellular redox balance within cancer cells.
High levels of antioxidants and redox control systems, especially the
Thioredoxin system, are often observed in cancer cells and are believed
to play a major role in cancer progression. We are currently investigating
the expression and regulation of Thioredoxin in breast cancer cell lines
cultured in hypoxic conditions and after re-oxygenation.
POS-MON-167
CIAP ANTAGONISTS AND IFNγ ACTIVATE CASPASE
AND RIPK1 DEPENDANT DEATH PATHWAYS IN
CANCER CELLS
Khan N.1, Miasari M.1, Chau D.1, Wong W.W.-L.1, Mckinlay M.2,
Chunduru S.K.2, Benetatos C.A.2, Condon S.M.2, Vaux D.L.1 and Silke
J.1
1
La Trobe University, Melbourne, VIC 3086, Australia. 2TetraLogic
Pharmaceuticals 343 Phoenixville Pike Malvern, PA 19355.
Synthetic IAP antagonists have been shown to kill tumour cells as
single agents or sensitise them to existing anti-cancer treatments,
validating Inhibitor of apoptosis (IAPs) proteins as targets for anti-
cancer therapeutics. We have previously shown that a peptide-mimetic
IAP antagonist compound has similar effects on cells as the TNF family
cytokine, TWEAK. Because some cancer lines can be sensitised to
TWEAK cytotoxicity by co-treatment with IFNγ, we hypothesised that
IFNγ might synergise with IAP antagonists to kill tumor cells. Consistent
with our hypothesis, tumor cells that are sensitive to TWEAK/IFNγ
were killed when treated with IFNγ and IAP antagonist while primary
untransformed lines were unaffected. JAK/STAT signaling was
required for cell death because synergistic killing could be blocked
by SOCS1 overexpression, but the TNFR1 pathway was not. Another
distinguishing feature of this IAP antagonist/IFNγ death was that it could
not be blocked by caspase inhibition alone even though cells displayed
classic apoptotic features. Surprisingly, caspase inhibition together with
a RIPK1 inhibitor (Nec1) significantly attenuated synergistic killing by
Compound A and IFNγ, as did RIPK1 knockdown. Our results suggest
that IAP antagonists can activate both apoptotic and non-apoptotic cell
death which may extend their clinical use.
Page 142 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-166
PHYSIOLOGICAL SIGNIFICANCE OF D-AMINO
ACID DEGRADATIVE ENZYMES IN NEMATODE
CAENORHABDITIS ELEGANS
Katane M.1, Saitoh Y.1, Seida Y.1, Kawata T.1, Maeda K.1, Sekine M.1,
Furuchi T.1, Kobuna H.2, Sakamoto T.1, Inoue T.3, Arai H. 3, Nakagawa Y.
1
and Homma H. 1
1
Department of Pharmaceutical Life Sciences, Kitasato University,
Tokyo, Japan. 2School of Medicine, Tokyo Women’s Medical University,
Tokyo, Japan. 3Graduate School of Pharmaceutical Sciences, The
University of Tokyo, Tokyo, Japan.
Among free D-amino acids existing in living organisms, D-serine
(D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser
has been proposed as a neuromodulator that regulates L-glutamate-
mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting
as a co-agonist. On the other hand, D-Asp has been proposed to
play important roles in regulating developmental processes, hormone
secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp
oxidase (DDO) are known as stereospecific degradative enzymes that
catalyze the oxidative deamination of D-amino acids. DAO and DDO
reportedly regulate endogenous D-Ser and D-Asp levels, respectively,
as well as mediate the elimination of accumulated exogenous D-amino
acids in various organs. Previously, we demonstrated that nematode
Caenorhabditis elegans has at least one active DAO gene and
three active DDO genes, and that these enzymes exhibit different
and distinctive enzymatic properties. In this study, to elucidate the
physiological roles of the C. elegans DAO and DDOs, we examined the
localization of these enzymes within the whole body of C. elegans using
green fluorescent protein-based gene expression analysis, revealing
that the spatiotemporal distributions of these enzymes differ from one
another. We also examined several phenotypes of four C. elegans
mutants in which each gene for these enzymes is partially deleted and
inactivated. We will report the phenotypes of these C. elegans mutants
in comparison with those of wild-type C. elegans, as well as alterations
in D-amino acid levels within the body.
POS-TUE-168
INVESTIGATION OF HOW RASACT COOPERATES
WITH RHOGEF2 OVER-EXPRESSION AND SCRIBBLE
MUTANTS IN TUMOURIGENESIS
Khoo P., Brumby A.M., Humbert P.O. and Richardson H.E.
Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne,
Victoria.
scribble (scrib) is a tumour suppressor gene which regulates cell polarity
in Drosophila epithelial cells. Expression of activated Ras (RasACT) in
scrib - clones in the Drosophila eye disc results in the development of
tumours, characterised by decreased cell death, loss of cell polarity,
hyper-proliferation and reduced differentiation. RhoGEF2, a gene
involved in cell shape changes, also cooperates with RasACT in eye
disc clones to result in tumourigenesis. Jun kinase (jnk) activity is both
up-regulated and required for tumourigenesis in RhoGEF2 and RasACT
tumours and scrib - + RasACT tumours. We compare the development
of tumours arising from the co-expression of RhoGEF2 and RasACT in
eye disc with scrib - + RasACT tumours and investigate the pathways
downstream of RhoGEF2 in its cooperation with RasACT in clones.
We show that RhoGEF2 + RasACT clonal tissue over-grows but not to
the same extent as in scrib - + RasACT tumours and is not as invasive.
Preliminary data indicate that when Rho 1, Rho kinase or Zipper levels
are reduced in RhoGEF2 + RafGOF (a downstream effector of Ras) clones
using UAS-RNAi transgenes, morphological defects are suppressed and
differentiation is restored. These results suggest that Rho1, Rho kinase
and Zipper are required for tumourigenesis arising from RhoGEF2 and
RafGOF co-expression in clones.
POSTERS MONDAY & TUESDAY
POS-MON-169
PHYSIOLOGICAL FUNCTION OF A NOVEL NF-κB-
REGULATING MOLECULE, NUCLING, IN IMMUNE
SYSTEM
Kim S.M., Sakai T., Tran N.H. and Fukui K.
Division of Enzyme Pathophysiology, The Institute for Enzyme
research, The University of Tokushima.
Nucling is a novel stress-induced protein isolated as a marker for
cardiac development. Recent our studies have revealed that Nucling is a
regulator of nuclear factor-kappa B (NF-κB) operated by its cytoplasmic
retention through the physical interaction with Nucling. Notably, Nucling
is considered to be important for the regulation of NF-κB signals in liver.
Nucling-KO mice showed several liver dysfunctions including hepatitis
and cancer with some defects of NF-κB signal in liver. In addition, a
number of kupffer cells, residential macrophages in liver, and hepatic
dendritic cells apoptotically decreased in the Nucling-KO mice. Thus we
focused on the function of Nucling in immune system. Further analyses
revealed that macrophages of the Nucling-KO mice were decreased in
lung. On the other hand, Nucling-KO mice were resistant to endotoxin
(Lipopolysaccharide) similarly to TNFα-KO mice. It is correspond to our
previous report showing the defect of NF-κB signal in Nucling-KO mice.
Our recent studies indicated that the spleen of Nucling-KO mice showed
abnormally activated germinal centers without immunologic stimulation.
Besides, we observed dissimilarity of B cell subset distribution between
Nucling-KO mice and wild-type mice. Therefore, we speculate that there
are some differences between Nucling-KO mice and wild-type mice
concerning the functions of macrophages in the aspect of its quantity or
activity. Nucling is considered to play an important role in the immune
system with the regulation of NF-κB signal pathway.
POS-MON-171
A ROLE FOR SODIUM/PROTON EXCHANGERS AND
INTRACELLULAR HYDROGEN ION CONCENTRATION
IN REGULATING VITAMIN C-DRIVEN ELECTRON
TRANSPORT ACROSS THE PLASMA MEMBRANE
Lane D.J.R. , Robinson S.R. , Czerwinska H. and Lawen A.
1 2 2 1
1
Department of Biochemistry and Molecular Biology, School of Biomedical
Sciences, Monash University. 2School of Biomedical Sciences and
Psychology & Psychiatry, Monash University.
Ascorbate is the major electron donor to a transplasma membrane
electron transport (tPMET) system that was originally identified in human
erythrocytes [1]. This plasma membrane redox system appears to transfer
electrons from intracellular ascorbate to extracellular oxidants (e.g., non-
transferrin-bound iron). Though this phenomenon has been observed in
nucleated cells, its mechanism and regulation are not well understood.
Here we have examined both facets of this phenomenon in K562 cells and
primary astrocyte cultures. Using ferricyanide as the analytical oxidant
we demonstrate that tPMET is enhanced by dehydroascorbate uptake
via facilitative glucose-transporters, and subsequent accumulation of
intracellular ascorbate. Additionally, we demonstrate that this stimulation
is not due to ascorbate that is released from the cells, but is dependent
only on a restricted intracellular pool of the vitamin. Substrate-saturation
kinetics suggest an enzyme-catalysed reaction across the plasma
membrane by an as-yet-unidentified reductase that relies on extensive
recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated
tPMET by the Na+/H+-exchange inhibitors amiloride and 5-(N-ethyl-N-
isopropyl)amiloride, which is diminished by bicarbonate, suggests that
tPMET activity may be regulated by intracellular pH. In support of this
hypothesis, tPMET in astrocytes was significantly inhibited by ammonium
chloride-pulse-induced intracellular acidification, while it was significantly
stimulated by bicarbonate-induced intracellular alkalinisation. These
results suggest that ascorbate-dependent tPMET is enzyme-catalysed and
is modulated by NHE activity and intracellular pH [2]. Though the identity of
the putative enzyme(s) responsible for electron transfer across the plasma
membrane has yet to be identified, it has been suggested that members of
the ubiquitous cytochrome b561 family (e.g., Dcytb) are likely to be involved.
[1] Lane and Lawen (2009) Free Radic. Biol. Med. 47, 485-495. [2] Lane et
al. (2010) Biochem. J. 428, in press.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 143
POS-TUE-170
REACTIVE OXYGEN SPECIES GENERATED THROUGH
NADPH OXIDASE MEDIATE UV-INDUCED GSK-3BETA
ACTIVATION
Kim H.S., Kwon Y.H., Kim J.Y. and Sohn J.
Department of Biochemistry & Molecular Biology, Korea University
College of Medicine, Seoul 136-705, Korea.
Previously, we have reported that UV induced ATR-dependent GSK-3β
activation and subsequent proteasomal degradation of p21 protein in an
ubiquitination-independent manner. Here, we show that reactive oxygen
species (ROS) are generated by UV irradiation and GSK-3β activation.
ROS generation was detected in the cytosol and mitochondria at 5 min
after UV exposure. NADPH oxidase contributed to the ROS generation
since chemical inhibitors of NADPH oxidase, DPI and AEBSF, abrogated
it. NOX4 mRNA was expressed in MCF-7 cells, and transfection of the
cells with NOX4 siRNA prevented UV-induced ROS generation. DPI
and apocynin also inhibited UV-induced GSK-3β activation indicating
that ROS generated by NADPH oxidase mediated GSK-3β activation.
Further supporting redox-sensitive activation of GSK-3β, treatment of
cells with hydrogen peroxide activated GSK-3β. ROS scavengers and
NOX4 siRNA abolished p21 protein degradation after UV-irradiation,
confirming the previous finding that p21 phosphorylation by GSK-3β
was required for p21 degradation. To investigate whether changes in
the [Ca2+]i mediated NADPH oxidase activation after UV irradiation,
the effect of Ca2+ chelators on ROS production was assessed. Indeed,
pretreatment with EGTA or BAPTA-AM blocked ROS generation
indicating Ca2+-dependent activation of NADPH oxidase. In MCF-7
cells, increase in [Ca2+]i was detected < 5 min after UV irradiation and
continued up to 2 h. Since GSK-3β activation by UV was shown to be
dependent on ATR, it was next studied whether an increase in [Ca2+]i
and ROS generation were upstream to ATR activation. Phosphorylation
of ATM/ATR substrates after UV irradiation was observed 15 min after UV
irradiation. Pretreatment of cells with BAPTA-AM, Tiron or NAC prevented
their phosphorylation indicating that ATR activation was dependent on
intracellular Ca2+ and ROS. Taken together, UV mobilizes intracellular
Ca2+ and thereby activates NADPH oxidase. ROS generated through
NADPH oxidase then activate ATM/ATR and that, in turn, induces GSK-
3β activation and p21 degradation.
POS-TUE-172
GSK3β INTERACTS WITH AND PHOSPHORYLATES
BCL2L12 IN BRAIN CANCER CELLS
Lin C.C., Chen W.J, Lin R.C., Chou C.H., Wu C. and Hong Y.R.
Department of Biochemistry, Faculty of Medicine, College of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan.
A novel GBM oncoprotein, Bcl2-Like 12 (Bcl2L12, a BH2 containing
protein), was recently identified which is significantly expressed in the
majority of primary GBM tumor specimens and distantly related to
canonical Bcl-2 proteins. By using large scale yeast 2-hybrid screening,
Bcl2L12 was found as a GSK3β binding partner in testis cDNA library.
Our data showed that Bcl2L12 middle fragment (118-240 residues)
which locates outside of Bcl2L12 C-terminal BH2 motif is responsible
for the binding to GSK3β and also confirmed by far-western blotting
experiment. In vitro kinase assay showed that GSK3β phosphorylates
Bcl2L12 at S156 but not Bcl2L12A (Bcl2L12 splicing transcript variant),
LiCl (GSK3 inhibitors) and phosphatase were used to confirm that
GSK3β indeed phosphorylates Bcl2L12 at S156. Interestingly, the S156
phosphorylation site on Bcl2L12 does not fit the GSK3β consensus
sequence (S/T-X-X-X-S/Tp or S/T-P). To explore the significance of
the interaction between GSK3β with Bcl2L12 and Bcl2L12 S156A
for assessing the morphological changes and caspases 2, 3 and 7of
apoptosis, we transfected Bcl2L12 and Bcl2L12 S156A into the HeLa
and Glioma cell lines (U87MG and GBM 8401).The data showed that
Bcl2L12 induced apoptosis in Hela cell, whereas in GBM and U87MG
may function as anti-apoptosis. Ongoing studies will combine TMZ (an
alkylaing agent against recurrent glioma) to examine how Bcl2L12 gets
involved and which may function as a chemotherapy biomarker in brain
tumorgenesis.
POSTERS MONDAY & TUESDAY
POS-MON-173
PLASTIDAL STARCH PHOSPHORYLASE IN SWEET
POTATO ROOTS IS PROTEOLYTIDY REGULATED BY
THE 20S PROTEASOME
Lin Y.C., Chou I.M., Chen A.N., Cheng C.H., Chang S.C. and Juang R.H.
Department of Biochemical Science and Technology, Institute of
Microbiology and Biochemistry, National Taiwan University, Taipei 106,
Taiwan.
Starch phosphorylase (SP, EC 2.4.1.1) catalyzes the reversible
phosphorolysis of starch and produces glucose 1-phosphate (Glc-1-P)
as one of its products. Plants express two isoforms of SP, which are
classified as low-affinity (L-form SP, L-SP or Pho1) and high-affinity
(H-form SP, H-SP or Pho2) types according to their binding affinities
to starch. Although the role of L-SP in starch metabolism is unclear,
several studies have found that the gene expression and catalytic
activity of L-SP correlate with starch content in plants, indicating that
L-SP might be involved in starch biosynthesis. In addition, L-SP in
Arabidopsis leaves might play a role in the tolerance of abiotic stress
(Zeeman et al., 2004). Previous studies with sweet potato roots have
shown that the activity of L-SP may be regulated by proteolysis of the
central 78-amino acid peptide (L78). Removal of L78 increased the
catalytic activity of L-SP in a phosphorolytic direction (Chen et al.,
2002). During the purification of L-SP from sweet potato roots, Chang
(1999) observed an unknown high molecular weight complex (HX),
whose mobility is significantly slower than the typical L-SP on non-
denaturing polyacrylamide gel electrophoresis, also performed L-SP
activity. Following this observation, we utilize mass spectrometry,
coimmunoprecipitation, agarose-gel based double diffusion, two-
dimensional gel electrophoresis, and confocal microscopy as the
tools and demonstrate that HX was composed of L-SP and the 20S
proteasome. Furthermore, we found that HX was reduced immediately
after 45°C heat treatment, and then a stepwise degradation of L-SP
was observed in a time-dependent mode which was strongly inhibited
by MG132, suggesting that the 20S proteasome was involved in L-SP
turnover. In addition, kinetic studies indicated that the proteolytic
modification of L-SP might increase the binding affinity of L-SP against
soluble starch and subsequently enhance its phosphorolytic activity.
This work demonstrates the role of 20S proteasome as a regulator of
L-SP activity which might be controlled by environmental heat stress.
POS-MON-175
PSPC1 IS AN IMPORTIN ALPHA
NUCLEOCYTOPLASMIC TRANSPORT CARGO
Major A.T.1, 3, Hogarth C.A.4, Miyamoto Y.2, 3, Young J.C.2, 3, Jans D.A.2,
3
and Loveland K.L.1, 2, 3
1
Department of Anatomy and Developmental Biology, Monash
University, Australia. 2Department of Biochemistry and Molecular
Biology, Monash University, Australia. 3The ARC Centre of Excellence
in Biotechnology and Development, Australia. 4School of Molecular
Biosciences, Washington State University, USA.
Importin proteins facilitate classical nuclear import by binding nuclear
localisation sequences (NLSs) on cargo proteins and translocating
them into the nucleus. Through a Y2H screen of an E12.5 mouse
testis cDNA library, we identified paraspeckle protein 1 (PSPC1) as
an importin α2 cargo. The long isoform of PSPC1 is 523aa, contains
two putative NLSs and is highly expressed in the testis. PSPC1 is
a marker for paraspeckles, a distinct subnuclear domain formed
around the non-coding RNA transcript, NEAT1, and present only in
differentiated cells. Paraspeckles are rich in RNA transcripts and two
other core paraspeckle proteins, PSF and NONO. Recent research
suggests that paraspeckles form a structural base by which A to I edited
RNA transcripts may be retained within the nucleus. We addressed
the hypothesis that trafficking of PSPC1 into the nucleus and its
subsequent localisation into paraspeckles is mediated specifically by
importin α2. Binding between PSPC1 and both importin α2 and α6
was confirmed using recombinant proteins in an ELISA based binding
assay. The extent of PSPC1 nuclear import was quantitated following
transient transfection of HeLa cells with constructs that over-express
GFP-tagged full length and truncated (dominant negative) constructs
encoding several importins. Data are collected following co-transfection
of DsRed2-tagged PSPC1 or assessed following endogenous PSPC1
detection via immunostaining. Dominant negative importin α2 appears
able to reduce the number of detectable paraspeckles, indicating the
importance of regulated importin synthesis to cell function.
Page 144 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-174
P21-ACTIVATED KINASE 1 IS IMPORTANT FOR
SURVIVAL OF COLORECTAL CANCER CELLS
Liu H., Huynh N., Baldwin G. and He H.
Department of Surgery, Austin Health, The University of Melbourne,
Heidelberg, VIC, Australia 3084.
Background and Aim: P21-activated kinase 1 (PAK1) functions
as a key node in various signalling pathways leading to cell survival,
migration and growth. PAK1 is overexpressed and activated in several
human tumors including colorectal cancer (CRC), which is the second
most common cause of cancer death. PAK1 is also required for vascular
endothelial growth factor (VEGF) expression and consequently for
angiogenesis, which in turn promotes tumor growth and metastasis.
VEGF is a downstream target gene of hypoxia-inducible factor 1α (HIF-
1α) which is involved in cellular adaptation to hypoxia and important for
cell survival. The aim of this study was to investigate the importance of
PAK1 in CRC cell survival. Methods: PAK1 knock-down (KD) clones of
the human CRC cell line DLD1 were obtained by stable transfection with
shRNA targeting PAK1. Hypoxia was mimicked by treatment with CoCl2
and cell survival was measured by [3H]-Thymidine incorporation. Protein
expression was determined by Western Blot and VEGF production was
measured by ELISA. Results: After treatment with 150μM CoCl2, cell
survival was significantly reduced by 40% in PAK1 KD cells (P<0.05). The
expression of HIF-1α and the production of VEGF were both significant
lower in PAK1 KD cells compared to control cells. Conclusion: This
study demonstrates that PAK1 is required for cell survival and VEGF
production of CRC cells. PAK1 may regulate these cellular processes
through a HIF-1α-dependent pathway.
POS-TUE-176
REGULATION OF EMBRYONIC GLOBINS BY THE
KRüPPEL-LIKE FACTORS
Mak K.S.1, Funnell A.P.W.1, Pelka G.J.2, Radziewic T.2, Power M.2, Tam
P.P.2, Pearson R.C.M.2 and Crossley M.1
1
School of Biotechnology and Biomolecular Sciences, University of
New South Wales, NSW 2052. 2Embryology Research Unit, Children’s
Medical Research Institute, Westmead, NSW, Australia.
Basic Krüppel-like Factor (Bklf/Klf3) is one of 17 members of the Krüppel-
like family of transcription factors that regulate biological processes
such as cell proliferation, differentiation and apoptosis. Bklf is highly
expressed in erythroid tissues, consistent with its direct activation by
Erythorid Krüppel-like Factor (Eklf), an erythroid-specific transcriptional
activator. Bklf knockout mice exhibit a mild deficiency in the erythroid
population Chromatin immunoprecipitation experiments have shown that
Bklf directly binds to the Klf8 gene which is up-regulated in the Bklf-null
erythroid tissues. Klf8, like Bklf, functions primarily as a transcriptional
repressor, recognising similar DNA sequences and repressing genes by
recruiting the same co-repressor, Ctbp. Given the similarities between
Bklf and Klf8, we hypothesize that Klf8 might functionally compensate
for the absence of Bklf and that this may account for the mild phenotype
of the Bklf knockout mouse. We have intercrossed Bklf and Klf8 mutant
mice and purified erythroid cells from compound mutants of various
genotypes. Affymetrix arrays have been performed to study the
differences in gene expression resulting from the disruption of Bklf or
Klf8 or both. Eliminating Klf8 activity in erythroid cells should reveal not
only the full biological function of Bklf, but also distinguish target genes
that are shared between or specific to Bklf and Klf8.
POSTERS MONDAY & TUESDAY
POS-MON-177
HEPATOCYTE GROWTH FACTOR (HGF) ACUTELY
REGULATES THE EPITHELIAL JUNCTIONAL
CYTOSKELETON AND ZONULA ADHERENS BY
CONTROLLING JUNCTIONAL MYOSIN VI
Mangold S.
Institute for Molecular Bioscience, 306 Carmody Rd., Brisbane 4072,
QLD.
HGF plays an important role in various morphogenetic events during
embryonic development, as well as being implicated in tumor invasion
and metastasis. Many studies have investigated the long-term effect of
HGF treatment on epithelial cells. HGF binds with high affinity to the
receptor tyrosine kinase c-Met. Activation of c-Met leads to recruitment
of adaptor proteins and in turn to activation of a multitude of down stream
signalling molecules, including PI3-Kinase, Src and Ras/MAPK. The
resulting signalling cascades initiate a broad range of cellular responses
such as proliferation, migration, invasion and even complex events like
branching morphogenesis in 3D matrices. All of these effects occur
after long-term treatment (several hours to days) with HGF. Our lab is
interested in the formation and function of E-cadherin based cell-cell
adhesions and their significance both under physiological conditions
and in disease. To allow morphogenetic changes or processes like cell
migration, the zonula adherens and the cytoskeleton must be able to
respond quickly and in an orchestrated fashion to the initiating HGF signal.
In this study we investigate the acute effects of early HGF treatment on
the epithelial junctional cytoskeleton and the zonula adherens in CaCo2
cells. We report a dramatic re-organization of the apical actin ring within
the first 15min of HGF treatment, which is accompanied by a loss of
Myosin VI from the adherens junctions. Myosin VI is an unconventional
actin binding motor protein that has previously been shown to play a role
in the stabilization of adherens junctions and the regulation of F-actin
organization. We identify Myosin VI as a novel down stream target of
HGF signalling and elucidate that Calcium signalling plays a crucial role
in the HGF induced regulation of junctional Myosin VI.
POS-MON-179
GENETIC TRANSFORMATION IN COLLETOTRICHUM
TRUNCATUM ASSOCIATED WITH ANTHRACNOSE
DISEASE OF CHILI PEPPER
Auyong A.S.M., Ford R. and Taylor P.W.J.
BioMarka/Plant Health Centre, Department of Agriculture and Food
Systems, 3010 Parkville, The University of Melbourne, Australia.
Colletotrichum truncatum is the causal agent of chili pepper
anthracnose causing severe economic loss through reduced yield and
marketability of infected fruit. To this extent an efficient and reliable
transformation system of the pathogen is required to prove the function
of isolated pathogenicity genes through gene silencing or mutagenesis.
Agrobacterium tumefaciens-mediated transformation (ATMT) system
was developed for C. truncatum. A. tumefaciens carrying a hygromycin
phosphotransferase gene (hph) and a green fluorescent protein (GFP)
gene was used to transform the conidiospores of two C. truncatum
pathotypes (F8-3B and BRIP26974). Optimum transformation efficiency
was obtained when equal volume of A. tumefaciens strain AGL1 carrying
either pJF1 or pPK2 binary vector was transformed with 1000000
C. truncatum conidiospores/ml and co-cultivated at 24°C for three
days. Southern blot analysis and TAIL-PCR indicated that most of the
transformants contained randomly inserted, single copies of the T-DNA.
Infection and colonization of chili pepper fruit at the mature red stage
with F8-3B-GFP and BRIP26974-GFP confirmed the virulence of these
transformed pathotypes. Analysis by fluorescent microscopy showed
that colonization of parenchyma cells of fruit pericarp tissue occurred by
subcuticular intramural infection. In the susceptible Capsicum annuum
genotype, fungal hyphae was detected between the cell walls of
parenchyma cells up to 1 cm in advance of the visible lesion, indicating
that C. truncatum entered a short endophytic stage in its disease cycle
before becoming necrotrophic.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 145
POS-TUE-178
ACTIVATION OF INSULIN-LIKE GROWTH
FACTOR BINDING PROTEIN-3 (IGFBP-3) BY DNA-
DEPENDENT PROTEIN KINASE (DNA-PK) OR
ATAXIA TELANGIECTASIA MUTATED (ATM) IS NOT
NECESSARY FOR ITS PROAPOPTOTIC FUNCTIONS
Marzec K.A., Martin J.L. and Baxter R.C.
Kolling Institute of Medical Research, University of Sydney, Royal
North Shore Hospital, St Leonards, New South Wales 2065, Australia.
Chemotherapeutic drugs such as doxorubicin, which induce apoptosis
by causing DNA double strand breaks (DSB), are opposed by the
phosphoinositide-3-kinase-related protein kinases, DNA-PK and ATM,
which promote DSB repair. Our laboratory has previously described the
phosphorylation by DNA-PK of IGFBP-3, which was reported by others
to be required for the pro-apoptotic function of IGFBP-3. The aim of
this study was to investigate the dichotomy where DNA-PK inhibition
enhances apoptosis by preventing DSB repair, yet opposes apoptosis
by inhibiting IGFBP-3 phosphorylation. ATM phosphorylates similar
sites to DNA-PK and may also have a role in activating IGFBP-3. We
assessed the response of phenotypically normal breast cells (MCF-10A)
and breast cancer cells (Hs578T, MDA-MB-231 and MDA-MB-468) to
doxorubicin, alone or with the ATM inhibitor KU55933 or the DNA-PK
inhibitor NU7441 (KuDOS Pharmaceuticals), monitoring caspase-3
activity in cell lysates as a marker of apoptosis. Apoptosis induced over
24-72 h by 0.3-1 μM doxorubicin was significantly enhanced in all cells
treated with 1 μM NU7441 or 10 μM KU55933. To investigate its role
in this response, endogenous IGFBP-3 was downregulated in Hs578T
cells ≥80% with small interfering RNA. Doxorubicin-induced apoptosis
was significantly reduced in IGFBP-3-silenced cells compared to control
cells, confirmed by decreased cleavage of the caspase-3 substrate,
poly(ADP-ribose) polymerase. These data suggest that endogenous
IGFBP-3 potentiates doxorubicin-induced apoptosis, and this activity is
not prevented by DNA-PK or ATM inhibition.
POS-TUE-180
PROTEOMIC STUDIES ON A HIGH AMYLOLYTIC
NIGERIAN MAIZE CULTIVAR
Awoyinka O.A.1, 2 , Adebawo O.O. 1, 2 , Daini O.A.2 and Dipanker C.3
1
Babcock University, Ilisan-Remo, Nigeria. 2Olabisi Onabanjo
University, Ago-Iwoye, Nigeria. 3Indian Institute of Science, Bangalore
India.
This study sought a high amylolytic Nigerian maize cultivar. It also
established the amino acid sequence that forms the primary structure of
the β amylase found in the maize cultivar. Purification steps comprising
of fractional precipitation by ammonium sulphate, gel filtration and
anion exchange chromatography was used respectively to purify β
amylase from the malt of TZEE*TZEE-W*DEMARSCUS*TZEE-W
before determination of the primary structure of the β-amylase with the
aid of Matrix assisted laser desorption ionization time- of- flight (MALDI-
ToF) mass spectrometry. Results obtained showed that An apparent
60 KDa monomeric protein was detected on a single dimensional 10%
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE). Well complete primary structure deduced showed a signature
of a highly conserved ubiquitous not yet reported β-amylase comprising
of 505 amino acid sequence. However, mass spectrometry analysis
further carried out on concomitant proteins eluted with the β-amylase
during partial purification gives a hypothetical insight into the metabolic
activities of the high amylolytic maize cultivar during malting. This
could be explored in quest of improving the diastatic power of the
Nigerian maize cultivar. Keywords: β-amylase, Malting, Maize, Mass
spectrometry and Proteomics.
POSTERS MONDAY & TUESDAY
POS-MON-181
NON-DESTRUCTIVE HIGH-THROUGHPUT
PHENOTYPING IN THE PLANT ACCELERATOR TO
STUDY PLANTS UNDER SALT AND WATER STRESS
CONDITIONS
Berger B.1, 2, 3 and Tester M.1, 2, 3
1
The Plant Accelerator. 2Australian Center for Plant Functional
Genomics. 3University of Adelaide, Waite Campus, Urrbrae SA, 5064.
Salinity and drought have a major impact on agricultural productivity,
likely to increase with climate change. To secure global food production,
crop plants with increased yields under these stress conditions are
urgently needed. Whereas gene and marker discovery have become
faster than ever, it is now the phenotyping process that is increasingly
limiting the generation of more tolerant crop plants (Finkel, 2009; Furbank,
2009; Tester and Langridge, 2010). Both salinity and drought trigger
dynamic physiological responses that require continuous phenotypic
measurements to be able to dissect overall tolerance mechanisms into
individual traits. The Plant AcceleratorTM combines automated plant
handling and high-throughput imaging to alleviate the phenotyping
bottleneck. Water stress regimes are controlled and recorded with
automated watering and weighing stations. At the same time, various
phenotypic traits can be assessed non-destructively by digital imaging
in different wavebands. Colour images can be used to estimate biomass
production and to measure growth rates and the degree of senescence.
Temperature differences between control and stressed plants, commonly
used as a surrogate for stomatal conductance, are determined with
infrared cameras. Furthermore, changes in leaf water content over time
are monitored using near infrared imaging. The Plant AcceleratorTM,
therefore, enables researchers to monitor important aspects of the
physiological changes occurring under salt and water stress conditions.
The high-throughput capacity provides the opportunity to screen large
populations, making possible forward genetics approaches to the
study of many relevant aspects of plant responses to salt and drought
stress. Finkel, E. (2009) Science 325, 380-381. Furbank, R.T. (2009)
Functional Plant Biology, 36, v-vi. Tester, M. and Langridge, P. (2010)
Science 327, 818-822.
POS-MON-183
GENE EXPRESSION OF PONCIRUS TRIFOLIATA,
CITRUS SUNKI AND THEIR HYBRIDS UNDER
INFECTION OF PHYTOPHTHORA PARASITICA USING
REAL-TIME QUANTITATIVE PCR
Boava L.P., Cristofani-Yaly M., Mafra V.S., Kubo K.S., Stuart R.M. and
Machado M.A.
Centro APTA Citros Sylvio Moreira, CP4, 13490-970, Cordeiropolis-
SP, Brazil.
Phytophthora nicotianae Breda de Haan (Phytophthora parasitica
Dastur) is an important oomycete causing damage in Citrus nurseries
and orchards and around the world. To integrate breeding and genomics
programs to provide resistance against P. parasitica, the identification
of genes involved in disease response is required. In this study, we
proposed that genes differentially expressed between resistant and
susceptible hybrids of P. trifoliata and C. sunki (resistant and susceptible,
respectively) may provide key candidates to identify transcripts involved
in disease resistance. We investigated gene expression in pools of four
resistant and four susceptible hybrids in comparison to their parents 48
hours after P. parasitica inoculation using real-time quantitative PCR.
Our analysis searched up regulated genes (fold ≥ 2 and p-value ≤ 0.05)
in the resistant genotypes relative to the susceptible, found in previous
microarray study. These genes were selected due to the biological
interest based on their function according to CitEST database, among
them, genes that encode enzymes participating in defense-related
metabolic pathways such as the biosynthesis of phenylpropanoids and
antimicrobial compounds such as phytoalexins, flavonoid biosynthesis
and resistance genes such as CC-NBS-LRR and TIR-NBS-LRR.
Our results showed that all analyzed transcripts were up regulated in
P. trifoliata relative to C. sunki and in the resistant pool relative to C.
sunki and to susceptible pool. This suggests that a number of defense
strategies are activated following the recognition event encoded by
Phytophthora resistance genes. The genes that we have identified as
up regulated across the resistant genotypes will be valuable for ongoing
work in eQTL mapping. Financial support: FAPESP-INCT/CNPq.
Page 146 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-182
MOLECULAR ANALYSIS OF PHOSPHITE INDUCED
RESPONSES IN ARABIDOPSIS
Berkowitz O.1, Jost R.2, Hardy G.S.E.J.1 and O’Brien P.A.1
1
Murdoch University, School of Biological Sciences and Biotechnology.
2
The University of Western Australia, School of Plant Biology.
Phosphite (H2PO3-) is a phosphate analog widely used to protect
plants from oomycete pathogens such as Phytophthora and Phytium.
Phytopthora species are prominent pathogens in agriculture, e.g.
Phytopthora infestans being the causing agent of potato blight (Irish
potato femine). Phytopthora cinnamomi has devastating effects
(“dieback disease”) on native ecosystems with over 2000 plant
species at risk in Western Australia alone. Phosphite is the only known
protectant of plants and exhibits a complex mode of action. At elevated
concentrations it directly inhibits the pathogen’s growth by interference
with its phosphate-dependent metabolism which is paralleled in plants
grown on high phosphite concentrations. At the same time it also inhibits
the plant’s phosphate starvation response, e.g. the up-regulation of
high-affinity phosphate transporters, and thus has constrictive effects
on plant growth under low phosphate supply. In addition to these direct
effects phosphite also induces some of the plant’s defence responses,
e.g. treatment of plants leads to increased expression of defence
genes. However, the underlying mechanism of this indirect effect is not
understood. We have started to characterise the impact of phosphite on
plant defence responses by analyses of gene expression and metabolic
pathways. Transgenic plants have been generated that express a
microbial phosphite dehydrogenase which converts phosphite into
phosphate. These plants are a valuable tool to dissect direct from
indirect phosphite effects.
POS-TUE-184
AUXINS: CRUCIAL TO GRAPE BERRY RIPENING
Bottcher C.1, Harvey K.1, Forde C.2, Boss P.K.1 and Davies C.1
1
CSIRO Plant Industry, PO Box 350, Glen Osmond SA 5064, Australia.
2
Nestle S.A., 1800 Vevey, Switzerland.
Fruit ripening is a complex process which seems to be largely regulated
by plant hormones. In contrast to climacteric fruit, such as bananas and
tomatoes, the ripening of non-climacteric fruit, e.g. strawberries and
grapes, seems to be less dependent on ethylene and might be controlled
by several other hormones. We are investigating the role of hormones
during grape berry development and are testing their ability to manipulate
ripening. While the application of some hormones, e.g. absicic acid and
brassinosteroids, can advance the onset of berry ripening (veraison)
others, e.g. auxins, delay it. The work presented here focuses on the
ripening-delaying effects of auxins that have been reported for a number
of climacteric and non-climacteric fruit. Of particular interest is the ability
of synthetic auxins like 1-naphthaleneacetic acid (NAA) to retard grape
berry ripening. This ability to delay ripening, and therefore harvest,
has potential advantages for the wine industry. NAA applied to berries
approximately two weeks prior to veraison delayed ripening by about
ten days as measured by reduced sugar levels and skin colouration.
A decrease in levels of indole-3-acetic acid (IAA), the most abundant
auxin in plants, occurs during the development of a number of different
fruit. This decrease also occurs in grapes and the potential for IAA to act
as an endogenous inhibitor of grape berry ripening is a current research
focus. In this context the role of IAA-conjugation in the initiation of berry
ripening is examined.
POSTERS MONDAY & TUESDAY
POS-MON-185
IN VIVO ROLE FOR FLAVONOIDS IN AUXIN
TRANSPORT AND GRAVITY RESPONSES
Buer C.S. and Djordjevic M.A.
ARC Centre of Excellence for Integrative Legume Research; Research
School of Biology; College of Medicine, Biology, and Environment; The
Australian National University; Canberra ACT; Australia.
Flavonoids are bioactive plant secondary metabolites with a myriad
of important functions (Buer et al., 2007, 2009, 2010). The well-
characterised Arabidopsis thaliana transparent testa (tt) mutants are
compromised in various flavonoid pathway structural enzymes and
regulatory genes (Buer et al., 2007, 2008). Several lesions result in
differential root flavonoid accumulation when compared to wild-type
roots (Buer et al., 2010). This differential accumulation allowed testing
whether certain flavonoids controlled auxin transport and subsequent
root gravity responses. The greatest changes in gravity response,
root elongation, and auxin transport were in the mutants accumulating
quercetin (tt3, tt8, tt10, and ttg1). Increased quercetin accumulation
caused rapid gravitropic curvature, faster root elongation rates, and
higher auxin transport inhibition compared to the wild type. Those
mutants with no or low quercetin accumulation (tt4, tt5, and tt6) were
inhibited in gravity responses and had increased auxin transport rates
confirming earlier experiments with tt4 (Buer and Muday, 2004). These
in vivo assays extend earlier in vitro assays (Jacobs and Rubery, 1988)
showing that flavonoids inhibit auxin transport with quercetin showing
the greatest effects and that flavonoids are important for normal auxin
flux and plant development. Buer C, Muday G, Djordjevic M (2007) Plant
Physiology 145: 478-490. Buer C, Muday G, Djordjevic M (2008) Plant
Signalling and Behavior 3: 415-417. Buer C, Djordjevic M (2009) Journal
Experimental Botany 60: 751-763. Buer C, Imin N, Djordjevic M (2010)
Journal of Integrative Plant Biology 52: 98-111. Buer C and Muday G
(2004) Plant Cell 16: 1191-1205. Jacobs M and Rubery P (1988). Science
241: 346-349.
POS-MON-187
RNAI-MEDIATED MANIPULATION OF NITROGEN
DEFENCES IN PLANTS USING PYRIDINE ALKALOID
BIOSYNTHESIS IN NICOTIANA AS AN EXPERIMENTAL
SYSTEM
Dalton H.L., DeBoer K.D., Neale A.D. and Hamill J.D.
School of Biological Sciences, Monash University, VIC, Australia.
Alkaloid synthesis in plants usually involves the diversion of amino
acid precursors from primary into to secondary metabolism. A good
example is the production of toxic pyridine alkaloids, which are a
feature of all species in the genus Nicotiana and some other genera
in the Solanaceae family. Alkaloids are recognised as being important
defensive compounds, the synthesis of which can increase in response
to wounding in order to provide protection from predators in native
habitats. Wound- or jasmonate-induced production of nicotine in
Nicotiana tabacum is correlated with elevated transcript and enhanced
activity of a number of key alkaloid biosynthetic enzymes such as
ornithine decarboxylase (ODC) and arginine decarboxylase (ADC).
Our previous research has indicated that antisense-mediated down
regulation of ADC transcript levels has very little effect upon capacity of
transgenic N. tabacum to produce nicotine. In contrast, RNAi-mediated
down regulation of ODC transcript levels in N. tabacum produced a
substantial decrease in nicotine levels which was accompanied by a
marked increase in concentrations of anatabine. Treatment of ODC-
RNAi hairy root cultures and regenerated plants with methyl-jasmonate
did not restore the plants capacity to produce normal amounts of nicotine
and anatabine. These results suggest that ODC has an important role
in providing the putrescine used in alkaloid biosynthesis in N. tabacum,
particularly in the wound response. Ongoing work is examining the
wider effects of these RNAi manipulations upon activity of related genes
and enzymes in primary and secondary metabolism and the capacity of
these transgenic Nicotiana tissues to redirect nitrogen from secondary
metabolism back into primary metabolism and growth – particularly in
response to abiotic and biotic stresses.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 147
POS-TUE-186
SPECTRAL EXTENSION OF PHOTOSYNTHESIS
Chen M.
University of Sydney.
Chlorophyll a is typically the major photosynthetic pigment in all oxygenic
photosynthetic organisms (plants, algae and Cyanobacteria) with Chl b
and c playing only an accessory light-harvesting function. The newly
discovered cyanobacterium Acaryochloris marina contains Chl d as its
major pigment. Chl d in Acaryochloris is involved in both the accessory
light-harvesting processes and as the primary donor pair in Photosystem
I and Photosystem II. This is the first known example where a chlorophyll
other than Chl a is involved in the primary chemical reaction in a reaction
center as is the case in all other oxygenic photosynthetic organisms.
The unique optical properties of chlorophyll d fill in the absorption gap
between chlorophylls (Chl a/b) and bacteriochlorophylls which has
shattered the old scheme that Chl a is the only major chlorophyll of
photosynthesis in oxygenic organisms, and has broadened the potential
to extend the range of photosynthesis into a new spectral region. Chl
d is the only Chl known so far that can replace all roles of Chl a in
oxygenic photosynthesis. Here, I wish to report the discovery of the
biochemical steps, at gene and protein levels, leading to the formation
of the unique pigment chlorophyll d, which will lead us to the possibility
for genetic engineering of these important chlorophylls. A potential
new biotechnology application has been developed. We are seeking
the application in improving the crop spectral properties, by extending
their light absorption properties into a new spectral region. Recently,
we have discovered an unknown chlorophyll from stromatolites pigment
extractions. Using various spectral analyses, we confirmed that it is a
new type of chlorophyll molecule, which showed a even further red-shift
absorption Qy peak. This discovery challenges the limitation of spectral
wavelength region for oxygenic photosynthesis, which may open new
bioenergy applications.
POS-TUE-188
CHARACTERISATION OF TWO NECROSIS AND
ETHYLENE INDUCING PROTEIN-LIKE (NLP) GENES
FROM SCLEROTINIA SCLEROTIORUM
Dinsdale A.J.1, Van Den Elsen F.2, Barnett R.1 and Plummer K.1
1
La Trobe University, Bundoora, Victoria, Australia. 2Wageningen UR
Plant Breeding, AJ Wageningen, The Netherlands.
The necrotrophic phytopathogen, Sclerotinia sclerotiorum, induces
plant cell death in order to colonise host plants and release nutrients.
This pathogen is known to secrete various compounds, including oxalic
acid and lytic enzymes during infection. Infection is often facilitated in
other necrotrophs (such as Fusarium oxysporum and Botrytis cinerea)
by the secretion of small, phytotoxic effector molecules (necrosis and
ethylene inducing peptides, NEPs). We have cloned two genes from
Sclerotinia sclerotiorum with significant similarity to NEPs called NEP-
like1 (Nlp1) and Nlp2. Both NLPs appear to induce necrosis when
transiently expressed in Nicotiana tobaccum and N. benthamiana.
Multiple 35S fusion constructs, designed to investigate the effect of in
planta NLP protein expression (with or without signal peptides) have
also been completed and transformations are currently underway. Both
genes have also been expressed in Pichia pastoris resulting in purified
NLP1 and NLP2 that will be used for further transient assays to assess
the specific response induced in host plants when presented with one
or both proteins. Multiple Nlp1RNAi mutant lines have been generated
which display inhibited growth rates both in vitro and in planta; further
characterisation of these will be presented. Expression localisation
studies are also underway using Nlp promoter:GFP fusion constructs.
POSTERS MONDAY & TUESDAY
POS-MON-189
CHLAMYDOMONAS REINHARDTII - A MODEL
ALGA FOR THE STUDY OF TRIACYLGLYCEROL
BIOSYNTHESIS AND LIPID BODY BIOGENESIS
Djordjevic M.A., Chen H.-C., Hillier W., Hocart C. and James G.
Plant Science Division, Research School of Biology, The Australian
National University, Canberra, Australia.
C. reinhardtii is an emerging model algal system to study triacylglycerol
biosynthesis and storage in lipid bodies (Wang et al., Eukaryot. Cell,
2009; 8: 1856-1868; Moellering et al., Eukaryot. Cell, 2010; 9: 97-106;
Yanto et al., Metabolic Engineering, doi:10.1016/j.ymben 2010.02.002;
Dean et al., Bioresource Technology, 2010; 12: 4499-4507). Normally,
C. reinhardtii, when cultivated under nutrient limited conditions, stores
carbon predominantly as starch with only low levels as triacylglycerols.
Mutations in ADP-glucose pyrophosphorylase inhibit starch synthesis
and concomitantly redirect carbon into lipid biosynthesis and storage of
triacylglycerols in lipid bodies when nitrogen deprived. We have found
C. reinhardtii can store 40% of dry cell weight as lipid under nitrogen
starvation at low light (100 µmol m-2 s-1). Fourier transform infrared
(FTIR) spectroscopy was used to develop high throughput methods
for semi-quantitative measurements of cellular protein, carbohydrate
and lipid content. Targeted transcriptional profiling by quantitative real-
time PCR was performed to investigate the temporal relationships of
key genes involved in the induction of lipid biosynthesis and those
associated with lipid body formation. Proteomic analysis on isolated
lipid bodies identified a key lipid body associated protein. We conclude
that C. reinhardtii is an alga that can be manipulated to produce high
levels of triacylglycerols making it a novel model to study triacylglycerol
biosynthesis and lipid body biogenesis.
POS-MON-191
THE IMPACT OF CHLOROPLAST NUMBER ON
PHOTOSYNTHETIC PROPERTIES OF TOBACCO
Evans J.R.1, Tazoe Y.1, Pengelly J.J.L.1, Nugent G.D.2 and Von
Caemmerer S.1
1
Research School of Biology, Australian National University. 2RMIT.
The mesophyll conductance to CO2 diffusion between intercellular
airspaces and chloroplast stroma depends on the surface area of
chloroplasts exposed to intercellular airspace. We characterised
chloroplast division mutants in tobacco to investigate the effects of
chloroplast number on mesophyll conductance to CO2 diffusion.
We analysed four different lines: two lines which had only one to two
chloroplasts per cell, one line which had more chloroplasts compared
to control and control plants. Tobacco plants were grown under full
sunlight in a greenhouse. Gas exchange was measured concurrently
with carbon isotope discrimination to assess photosynthetic properties
and the drawdown in CO2 concentration between intercellular airspaces
and chloroplasts. Leaves were subsequently sampled for Rubisco
and chlorophyll content and quantitative anatomy. Mesophyll cells in
leaves from plants with 1-2 chloroplasts per cell were distorted. The
large chloroplast covered most of the cell wall area, but was not always
in close contact with the cell wall, buckling away in places. Despite
large differences in chloroplast numbers between different lines, CO2
assimilation rates, mesophyll conductance, Rubisco content and leaf
anatomy were similar. The relationship between Rubisco content and
CO2 assimilation rate at a given CO2 concentration in the chloroplast
will be assessed.
Page 148 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-190
Evaluation of growth, nodule development
and the presence of SAT1 homologs in the
Australian wild soybean relative Glycine
canescens
Ebrahimie E., Mazurkiewicz D., Mohammadidehcheshmeh M.,
Chiasson D. and Kaiser B.
School of Agriculture Food and Wine, The University of Adelaide,
Australia.
SAT1 is a basic helix-loop-helix transcription factor located on the
peribacteroid membrane in soybean nodules (Kaiser et al 1998). Loss of
SAT1 activity in soybean limits nodule development and nitrogen fixation
producing a typical nod plus, fix minus nodule phenotype (Loughlin et
al unpublished results). We are interested in the role of this protein in
symbiotic nitrogen fixation and the signaling cascades it participates in.
We have identified SAT1 homologs in all sequenced plants (legumes
and non-legumes) often present in multiple gene families. As part of this
examination we have looked for the presence of SAT1 in other Glycine
species. The perennial soybean, Glycine canescens F.J.Herm. is widely
distributed throughout inland regions across Australia. G. canesscens is
a wild relative of G. max however it is often found growing in extreme
environments where both water and nutrients are often limiting. Very
little is known about the genetic and physiological properties of G.
canesscens. In this study we examined the physiological properties of
G. canescens growth and nodule development and evaluated the level
of conservation in nodule expressed SAT1 genes. G. canescens was
found to contain both determinate (young) and indeterminate nodules
when inoculated with Bradyrhizobium japonicum (CC1601a). The
nodules were capable of nitrogen fixation as plants grew adequately in
the absence of external nitrogen. We identified four SAT1 homologs in
G. canescens, which are related to those found in G. max. A summation
of our findings and a bioinformatic analysis of the SAT1 clones will
presented.
POS-TUE-192
THE MODIFICATION OF FLOWER COLOUR IN
DIANTHUS CARYOPHYLLUS
Fiorito S.D.1, Parish R.W.1, Brugliera F.2 and Gendall A.R.1
1
Department of Botany, La Trobe University, Bundoora, Victoria,
Australia, 3086. 2Florigene PTY LTD, 1 Park Drive, Bundoora, Victoria,
Australia, 3086.
Flower colour is an important evolutionary aspect for many plants,
attracting a variety of pollinators such as insects, birds and small
mammals. The interaction of pigments, co-pigments, metal ions, inter-
and intra- molecular staking and vacuolar pH can contribute to an
array of colours in petals, which is valuable to the floricultural industry.
Anthocyanin compounds, synthesized via the flavonoid biosynthetic
pathway, are the most abundant group of pigments associated with
flower colours, occurring in the majority of plant species. Recombinant
DNA technology has been utilised by the floricultural company
Florigene, to create novel flower colours, with a particular emphasis on
the generation of blue petal colour. Florigene were the first company to
release the purple coloured carnation (Dianthus caryophyllus) varieties
known as the FLORIGENE Moon series TM. Various transcription factors
and putative pH regulators genes will be identified in carnation using a
degenerate primer approach. A rapid transient system has been applied
to carnation floral tissues to generate transient down regulation of
known genes within the flavonoid biosynthetic pathway and putative pH
regulators. Constructs have been generated and the transient system
tested; with GUS expression optimised. A selection of constructs will
be stably transformed into carnation to confirm if the technology is
functional in carnation. Once these genes have been identified amiRNA
or RNAi constructs will be generated and transient and/or stable down
regulation applied.
POSTERS MONDAY & TUESDAY
POS-MON-193
PROTEOMIC ANALYSIS OF THE RESPONSE OF
WHEAT CULTIVARS THAT DIFFER IN THEIR CAPACITY
TO WITHSTAND DROUGHT
Ford K.L., Cassin A. and Bacic A.
Australian Centre for Plant Functional Genomics, School of Botany,
The University of Melbourne.
Using a series of multiplexed (iTRAQ) experiments we have studied
the changes in protein abundance of three Australian bread wheat
cultivars (Triticum aestivum). The three cultivars differ in their ability
to maintain grain yield under drought conditions. Plants were grown in
the glasshouse with cyclic drought treatment that mimics conditions in
Southern Australia’s wheat growing regions. Buffer soluble proteins were
isolated from the leaves of the wheat plants and the iTRAQ system with
multiple rounds of chromatography was used to follow the changes in
protein abundance, a pooled internal standard was used to allow cross
experiment comparison. The study contributed to a wheat database of
2,183 proteins, as well as identifying 110 leaf proteins that changed in
abundance in response to drought. The number of significant changes in
the cultivars at the different time points reflected their differing response
to drought. In general we observed an increase in proteins involved
in oxidative stress metabolism and a down regulation of proteins
involved in photosynthesis and the Calvin cycle. Drought responsive
proteins, including dehydrins and LEA proteins were also significantly
upregulated.
POS-MON-195
A MODEL DESICCATION-TOLERANT GRASS,
SPOROBOLUS STAPFIANUS GANDOGER
Gaff D.F. , Blomstedt C.K. , Neale A.D. , Hamill J.D. , Ghasempour
1 1 1 1
H.R.2 and Sutaryono Y.A.3
1
School of Biological Sciences, Monash University, Clayton 3800,
Australia. 2Department of Biology, Razi University, Taghbostan,
Kermanshah, Iran 6. 3Lembaga Penelitian Universitas Mataram,
Jl.Pendidikan No. 37, Mataram, 83125 NTB, Indonesia.
Of the ~40 grasses that can recover from dehydration to air-dryness,
Sporobolus stapfianus is a most versatile tool for research into
desiccation tolerance in vegetative grass tissue. This African tropical-
subtropical perennial grass is easily propagated by subdivision or from
seed. A number of comparisons of desiccation-tolerant and desiccation-
sensitive leaves can be made, eg. leaves become desiccation-tolerant
while they dry attached to intact plants in the light, whereas leaves
detached before drying remain desiccation-sensitive, - this permits
comparison of leaves with identical genotypes. Many desiccation-
sensitive Sporobolus species and 6 desiccation-tolerant ones are
available for comparison with S. stapfianus. S. stapfianus is salt tolerant
and its roots adapt to withstand waterlogging if they grow into water. Of
7 desiccation-tolerant grass species tested in preliminary field trials in
the Northern Territory, S. stapfianus, a C4 grass, produced the greatest
dry matter increment per unit basal area. S. stapfianus has favorable
digestibility, and satisfactory protein and phosphate levels. S. stapfianus
exemplifies an advanced stage of an evolutionary trend in desiccation
tolerant plants toward increased importance of the dehydration phase
for induction of desiccation tolerance accompanied by synthesis of
protectants and extensive proteomic changes. In the resurrection
monocot Borya contracta and dicot Craterostigma plantagineum, the
induction of desiccation tolerance during drying is controlled by the
phytohormone abscisic acid (ABA), the induction in S. stapfianus is ABA-
independent. S. stapfianus offers a valuable resource for future research
on desiccation tolerance in a most important family economically.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 149
POS-TUE-194
THE AFFECTS OF LIGHT ON CYANOGENESIS IN
FORAGE SORGHUM
Fromhold S.M.1, O’Donnell N.H.1, Blomstedt C.K.1, Stuart P.N.2,
Gleadow R.M.1, Hamill J.D.1 and Neale A.D.1
1
School of Biological Sciences, Monash University, VIC, AUS. 2Pacific
Seeds, QLD, Australia
Sorghum is a C4, drought resistant plant species, which is also
cyanogenic in all tissues except the mature seeds. The cyanogenesis
pathway in sorghum produces the stable cyanogenic glucoside,
dhurrin. When the plant tissue is disrupted the dhurrin is hydrolysed
to release hydrogen cyanide (HCN). At low concentrations HCN can
be metabolised safely by mammals but at higher concentrations it is
toxic as it causes metabolic asphyxiation. The HCN potential (HCNp) of
sorghum is important in the cattle industry as forage sorghum is used
as an alternate feed crop over summer. Environmental factors, such as
drought, are known to increase the HCNp of sorghum but little is known
regarding the affects of other conditions, including light levels. Analysis
of the putative promoter regions of the three biosynthetic genes in the
cyanogenesis pathway; CYP79A1, CYP71E1 and UGT85B1, showed
that in all three sequences there are potential light and circadian
response elements. It has been postulated that sorghum may also go
through a circadian or diurnal rhythm in relation to its cyanogenesis
pathway. An experiment has been conducted to compare different light
treatments on forage sorghum that were hydroponically grown and
placed in cabinets with controlled temperature and nutrients. The plants
subjected to three light treatments; total light, total darkness and 14/10
light/dark cycle, and plants were harvested every four hours for 60
hours. This experiment was designed to determine if there is a circadian
or diurnal effect on the cyanogenesis pathway in forage sorghum and
the results will be discussed.
POS-TUE-196
SCLEROTIAL FORMATION IN SCLEROTINIA
Greenhill A.1, Gendall A.1, Benny U.2, Rollins J.2, Porter I.3 and
Plummer K.1
1
La Trobe University, Victoria, Australia. 2University of Florida, Florida,
USA. 3Victorian Department of Primary Industries, Victoria, Australia.
Sclerotinia diseases cause over $100M loss to vegetable crops annually
in Australia, with significant losses worldwide. Control of these diseases
is severely impeded by the pathogen’s ability to produce sclerotia;
highly melanized structures crucial to the pathogen’s propagation,
reproduction and survival. T-DNA mutants have been created via
Agrobacterium-mediated transformation. In this process a short DNA
sequence (T-DNA) is inserted randomly into the fungal genome. As the
sequence of this T-DNA tag is known, the gene or region into which
it has been inserted can be identified. Approximately 1200 T- DNA
mutants were created and screened for altered sclerotium phenotypes,
with approximately 40 of these presenting a sclerotia-minus, or aberrant
sclerotia phenotype. T-DNA insertion points have been identified in a
number of these mutants and work is underway to characterise the
interrupted genes. In addition to this genetic study the role of melanin
in sclerotial formation is also being examined. Vectors to knock out or
silence melanin biosynthesis genes have been constructed. Additionally,
chemical melanin inhibitors have been tested against Sclerotinia in vitro.
This work is supported by funding from Horticulture Australia Limited
and an Australian Postgraduate Award.
POSTERS MONDAY & TUESDAY
POS-MON-197
EXPRESSING RED RUBISCOS IN GREEN PLANTS
Gunn L.H. and Whitney S.M.
Plant Sciences Division, Research School of Biology, The Australian
National University, Canberra A.C.T. 0200, Australia.
The CO2-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco) has a pervasive influence on photosynthetic
carbon assimilation in plants. In higher plants Rubisco is comprised
of eight catalytic large subunits (L, rbcL gene, plastome encoded)
and eight small subunits, essential for maximal activity (S, rbcS gene,
nuclear encoded). Despite millions of years of evolution, Rubisco is an
inefficient enzyme as O2 competes with CO2 as a substrate producing
a product (2-phosphoglycolate) whose dissipation via photorespiration
consumes energy. In nature, Rubisco shows considerable catalytic
diversity with some variants in red algae having properties that surpass
those in higher plants. Replacement of higher plant Rubisco with that of
some red-algae (in theory) has the potential to improve photosynthetic
C-assimilation, however such endeavours have been thwarted by the
finding that the folding and assembly properties of Galdieria sulphuraria
Rubisco (a red algae) could not be met by tobacco plastids. Here
we expand on this work to introduce L- and S-subunits from the red
algae Griffithsia monilis and a “red-like” Rubisco from the bacterium
Rhodobacter sphaeroides into inverted repeat regions of a plastome
transforming tobacco-master line (cmtrL1). To evaluate whether the
putative molecular chaperone CbbX (coded by the cbbX gene located 3’
to the rbcL-S genes in red algae) influences assembly of G.sulphuraria
Rubisco in tobacco, the rbcL-S-X operon have also been introduced into
cm
trL1. The current status of this research will be presented.
POS-MON-199
IDENTIFICATION OF FLAGELLAR MASTIGONEME
PROTEINS FROM PHYTOPHTHORA
Blackman L.M.1, Arikawa M.2, 3, Yamada S.2, Suzaki T.2 and Hardham A.R.1
1
Plant Sciences Division, Research School of Biology, Australian
National University, Canberra, ACT 2601, Australia. 2Department
of Biology, Graduate School of Sciences, Kobe Univesity, Nada-ku,
Kobe 657-8501, Japan. 3Department of Cardiovascular Control, Kochi
Medical School, Nankoku, Kochi 783-8505, Japan.
Motile, flagellated zoospores of Phytophthora and Pythium species play
a key role in pathogen dissemination and the initiation of infection of host
plants. The diseases these pathogens cause are highly destructive and
result in extensive losses in agriculture and natural ecosystems worldwide.
Tripartite tubular hairs called mastigonemes on the anterior flagellum of
Phytophthora and Pythium and other protists in the Stramenopile taxon
are responsible for reversing the thrust of flagellar beat and for cell motility.
Immunoprecipitation experiments using antibodies directed towards
mastigonemes on the flagella of zoospores of Phytophthora nicotianae
have facilitated the cloning of a gene encoding a mastigoneme shaft
protein in this pathogen. Expression of the gene, designated PnMas2,
is up-regulated during asexual sporulation, a period during which many
zoospore components are synthesized. Analysis of the sequence of the
PnMas2 protein has revealed that, like other Stramenopile mastigoneme
proteins, PnMas2 has an N-terminal secretion signal and contains four
cysteine-rich epidermal growth factor (EGF)-like domains. Evidence
from non-denaturing gels indicates that PnMas2 forms large oligomeric
complexes, most likely through disulphide bridging. Bioinformatic
analysis has revealed that Phytophthora species typically have three
or four putative mastigoneme proteins containing the four EGF-like
domains. These proteins are similar in sequence to mastigoneme
proteins in other Stramenopile protists including the algae Ochromonas
danica, Aureococcus anophagefferens and Scytosiphon lomentaria and
the diatoms Thalassiosira pseudonana and T. weissflogii.
Page 150 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-198
MANIPULATION OF TOXIC NITROGEN SECONDARY
METABOLISM IN PLANTS AND OPPORTUNITIES IN
PLANT BIOTECHNOLOGY
Hamill J.D., Blomstedt C.K., Dalton H.L., DeBoer K.D., DeGuzman G.,
Gleadow R.M., Ling H.Y., O’Donnell N.H., Neale A.D. and Walmsley A.M.
School of Biological Sciences, Monash University, Melbourne, Victoria
3800.
Many plants produce high levels of toxic nitrogen-containing specialized
metabolites, such as alkaloids and cyanogenic glycosides, which
generally are regarded as offering environmental protection against
predators. Using Nicotiana (dicot - alkaloids) and Sorghum (monocot
- cyanogenic glycosides) as experimental species, we are studying the
interplay between primary and secondary metabolism and the extent to
which movement of nitrogen can be experimentally redirected in vivo.
A screening program to identify mutations in key genes necessary for
cyanide production in Sorghum is well underway. We have also produced
transgenic lines of Nicotiana with markedly altered patterns of pyridine
alkaloids, both qualitatively and quantitatively. These studies have
relevance to biotechnology, in relation to the redirection of metabolites to
produce plants with altered growth potential. They also have relevance
to other ongoing research interests involving the production of plant-
made vaccines in plants without concomitant negative connotations
that may be associated with the co-production of toxic nitrogen-based
secondary metabolites.
POS-TUE-200
MOLECULAR ANALYSIS OF THE EARLY STAGES OF
SOYBEAN NODULATION
Hayashi S.1, Reid D.1, Lorenc M.2, Stiller J.2, Edwards D.2, Ferguson B.J.1
and Gresshoff P.M.1
1
ARC Centre of Excellence for Integrative Legume Research, The
University of Queensland, St. Lucia, QLD 4072, Australia. 2Australian
Centre for Plant Functional Genomics, School of Land, Crop and
Food Sciences, The University of Queensland, Brisbane, Qld 4072,
Australia.
Nitrogen is typically a limiting factor for plant growth. Legumes can form
‘nitrogen-fixing root nodules’ de novo through a symbiotic association
with soil-living bacteria called rhizobia. This provides them with nitrogen,
thus enabling them to grow in nitrogen-poor environments. Nodule
formation is initiated via a chemical exchange between the plant and
the bacteria. This is followed by a series of signalling cascades in the
root, which induces cell proliferations leading to the formation of nodule
primordia. In recent decades, mutagenesis programs using several
legume species have identified numerous components required for
nodule development. These studies also indicated that root epidermal
and cortical/pericycle cells must tightly coordinate their development
during the early stages of nodulation in order to establish functional
nodules. To further identify genes that are activated during the early
stages of nodulation, we conducted a root transcriptome analysis using
high-throughput deep-sequencing technology (Illumina’s GAIIx) in
soybean (Glycine max). Soybean root tissue was harvested from the
zone of nodulation to specifically focus on nodulation-expressed genes.
The tissue was harvested at various times following rhizobia inoculation.
Over 6,000 differentially expressed genes were identified, including
known, and many previously unknown, nodulation-dependent genes.
In addition, we identified a number of novel molecular pathways (i.e.,
hormone biosynthesis and signalling) that are differentially regulated
during nodulation. Findings from these studies will be presented.
POSTERS MONDAY & TUESDAY
POS-MON-201
SILENCING OF THE PHOSPHOLIPASE C GENE
IN PROTOPLASTS FROM COFFEA ARABICA L.
SUSPENSION CELLS
Poot-Poot W.A., De Los Santos-Briones C., Munoz- Sanchez J.A. and
Hernandez-Sotomayor S.M.T.
Centro de investigacion Cientifica de Yucatan.
Phospholipase C (PLC) is an enzyme involved in signal transduction
pathways. PLC catalizes the hydrolysis of phosphatidylinositol
4,5 bisphosphate generating two second messengers: inositol
1,4,5-triphosphate (IP3) and diacylglycerol (DAG) which is immediately
phosphorylated to phosphatidic acid (PA) by diacylglycerol kinase
(DGK) in plant cells. Likewise, PLC has been observed that is involved in
the response to different types of stress such as drought, salinity, cold,
pathogens and metals such as Al3+. RNA interference (RNAi) technology
is a widely used tool to analyze gene function in different organisms. In
order to obtain a protocol for silencing PLC from protoplasts of C. arabica
suspension-cell, cells were treated with a mixture of hydrolytic enzymes.
To determine the concentration of polyethylene glycol (PEG) required for
the transformation, we made a curve to different concentrations of PEG
and viable protoplasts were quantified and the optimal concentration of
PEG to use for transformation was 20%. Protoplasts were exposure to 20
% (PEG) and the plasmid for RNAi which contains the catalytic fragment
of PLC from C. arabica plus the red fluorescent protein. Protoplasts
were observed with fluorescence microscopy at 12, 24, 48, 72 hours.
Our results showed greater transformation with of 20% PEG and the
suitable time to analyze the RNAi-PLC of 72 hours. These results allow
us to obtain information in a short time on the role of the PLC to different
stimuli. Acknowledgments: Scholarship to WPP CONACYT (157993)
and a CONACYT grant (98352) to SMTHS.
POS-MON-203
THE ARABIDOPSIS PHYTOSULFOKINE RECEPTOR
CONTAINS A FUNCTIONAL GUANYLYL CYCLASE
DOMAIN ENABLING CGMP-DEPENDENT
DOWNSTREAM SIGNALLING IN VIVO
Kwezi L.1, 2, Ruzvidzo O.2, Wheeler J.I.1, Govender K.2, Iacuonne S.1,
Thompson P.E.1, Gehring C.2, 3 and Irving H.R.1
1
Monash Institute of Pharmaceutical Sciences, Monash University,
381 Royal Parade, Parkville VIC 3052, Australia. 2University of the
Western Cape, Bellville 7535, South Africa. 3Computational Bioscience
Research Centre, 4700 King Abdullah University of Science and
Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.
Phytosulfokines (PSKs) are sulphated pentapeptides that stimulate cell
growth and differentiation in plants. The PSK receptor (PSKR1) is a
leucine rich repeat receptor like kinase but its role in mediating PSK
signals is undefined at a biochemical level. We identified a putative
guanylate cyclase (GC) catalytic centre in PSKR1 that is encapsulated
within the kinase domain. This is a different architecture to known animal
receptor GCs where a linker domain separates the GC catalytic centre
from the kinase homology domain. Hence we tested the hypothesis that
the GC worked in conjunction with the kinase to signal downstream
events in PSK signalling. We expressed the recombinant complete kinase
domain of AtPSKR1 and show that it has serine/threonine kinase activity
using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5
μM and Vmax of 1800 nmol min-1 mg-1 protein. This same recombinant
protein also has GC activity in vitro that is significantly enhanced when
calcium is present. Overexpression of the full length AtPSKR1 receptor
in freshly isolated protoplasts from Arabidopsis thaliana leaves results in
the endogenous basal cGMP levels being raised over 20-fold indicating
that the receptor has GC activity in vivo. In addition, we demonstrate
that PSK α itself induces small and rapid increases in cGMP levels
and that the relatively inactive non-sulphated peptide backbone n-PSK
does not. Together these results indicate that the AtPSKR1 contains
dual functioning GC and kinase catalytic activity and represents a novel
enzymatic class of kinases with overlapping catalytic domains.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 151
POS-TUE-202
SEQUENCE DYNAMICS OF AN ARABIDOPSIS
THALIANA GENOMIC LOCUS UNDER HIGHLIGHT (HL)
STRESS
Ichim M.C.1, Milcamps A.2, Papazova N.3, Depicker A.4, De Loose M.3
and Van Den Eede G.2
1
Stejarul Research Centre for Biological Sciences. 2Joint Research
Centre. 3Institute for Agricultural and Fisheries Research. 4Flanders
Interuniversity Institute for Biotechnology.
The integration of the T-DNA into the plant genome is reported to
be often accompanied by simple or complex rearrangements at the
integration genomic locus. It is suspected that transgenic events,
instability can arise upon plant exposure to abiotic stresses. The
stability of the transgene and the sequences flanking the T-DNA is
an important aspect for the approval of GMPs. The structural stability
of transgenes and their integration locuses is a concern for risk
assessment, food labeling, traceability and post-release monitoring. We
have performed sequence analysis of a genomic locus, corresponding
to a T-DNA insertion point from an Arabidopsis thaliana transgenic line.
The analyses of the target locus will allow defining if and what kinds
of rearrangements are specifically induced under highlight (HL) stress
(PPFD of 600 μmole/m2/s), known to be a mutagenic oxidative stress due
to the generation of ROS molecules. In order to be able to detect even
the smallest mutations possible (SNPs) we have tested and optimized
the Single Strand Conformational Polymorphism analysis coupled with
Capillary Electrophoresis (SSCP-CE). The results indicate that in wt
plants, both those grown in standard conditions and those exposed to
highlight stress, the sequence considered does not undergo any major
or minor re-arrangement. Moreover, as method for mutation detection,
the SSCP-CE has proved to have the potential to be used in the field
of GMO research for stability analyses and post-release monitoring
purposes.
POS-TUE-204
A COMPARATIVE PROTEOMICS APPROACH FOR
IDENTIFICATION OF EFFECTORS IN VENTURIA
INAEQUALIS
Jones D.1, 2, Mesarich C.3, 4, Hill G.3, 4, Shiller J.2, Bowen J.3, Templeton M.3
and Plummer K.1, 2
1
Cooperative Research Center for National Plant Biosecurity. 2La
Trobe University. 3Plant and Food Research, New Zealand. 4The
University of Auckland, New Zealand.
Venturia inaequalis and V. pirina are the causative agents of apple and
pear scab (respectively). This project aims to identify and characterise
effectors in these species. Effectors are pathogen proteins involved
in infection. Effectors can also be recognised as foreign by plant
receptors, which then initiate a signal transduction cascade that results
in plant resistance. Breaking of resistance can occur if either the plant
receptor gene or the effector gene is lost, or if the receptor is unable
to recognise the effector due to a mutation. Due to this evolutionary
pressure, effectors often vary across species and races. Effectors
are therefore of interest for their direct role in infection, for their role in
activating plant resistance, and for use in developing molecular tests
that can differentiate strains of V. inaequalis and/or species of Venturia.
We are developing a PCR-based test using a putative effector, a fungal
hydrophobin; this test would be of use in surveillance, particularly in
Western Australia, which is currently free of V. inaequalis but requires
ongoing surveillance to verify area freedom. Using 2-D DIGE, we intend
to compare proteins from Venturia sp. grown on cellophane/PDA culture
against proteins on PDA culture, since V. inaequalis grows infection
structures (stroma) on cellophane but not PDA. Therefore, proteins
present only in cellophane culture may be involved in infection structures.
Isolated proteins can be identified either by de novo sequencing, or by
Peptide Mass Fingerprinting. To this end, the genomes of V. inaequalis
and V. pirina are being sequenced using pyrosequencing.
POSTERS MONDAY & TUESDAY
POS-MON-205
DOES PHOSPHITE MIMIC PHOSPHATE IN
REGULATING THE PHOSPHATE-STARVATION
RESPONSE IN PLANTS?
Jost R.1, Pharmawati M.2, Berkowitz O.3, Lambers H.1 and Finnegan P.M.1
1
School of Plant Biology, The University of Western Australia, Crawley
WA 6009, Australia. 2Biology Department, Faculty of Mathematics and
Natural Sciences, Udayana University Campus Bukit, Jimbaran, Bali.
3
School of Biological Sciences and Biotechnology, Murdoch University,
Murdoch WA 6150, Australia.
Phosphate is one of the most important macronutrients for plant
growth and elaborate mechanisms have evolved to sense and maintain
phosphorus homeostasis. Phosphite, a reduced form of phosphate, is
considered to be metabolically inert when taken up by plants, and is in
wide use as an effective fungicide against a wide range of devastating
plant diseases caused by oomycetes, particularly Phytophthora
species, e.g. P. infestans, the causative agent of the potato famine in
18th century Europe, and P. cinnamomi, the dieback pathogen that
currently threatens the long-term survival of many Australian native
ecosystems. The effects on plant productivity upon phosphite treatment
in the field vary and depend on the presence of phosphite-oxidizing
microorganisms in the soil, as well as the internal phosphorus status
of the plant. Phosphite has been demonstrated to suppress phosphate
starvation responses in plants with low phosphorus status, but to date
there is very little understanding of how phosphite interferes with the
plant phosphorus signalling networks and how/if this is linked to the
long-term resistance to P. cinnamomi observed in plants in the field
after aerial spraying of the host canopy. Our project aims to characterize
the molecular effects of phosphite on the phosphate-sensing machinery
within an Arabidopsis thaliana model.
POS-MON-207
EVOLUTION OF A CIS-ELEMENT THAT REGULATES
PETAL LOSS EXPRESSION IN THE PERIANTH OF THE
ARABIDOPSIS FLOWER
Kilinc A. and Smyth D.R.
School of Biological Sciences, Monash University, Melbourne, Vic
3800.
An outstanding issue in biology is the evolution of complex traits, and
how changes in regulatory networks drive this process. Flower structure
in the Brassicaceae is remarkably conserved. The outermost whorl
consists of four sepals, and the second whorl contains four petals
arising between them, giving a distinctive tetrameric appearance. The
PETAL LOSS (PTL) gene is a transcription factor of the trihelix family
with 29 members in Arabidopsis, a member of the Brassicaceae. The ptl
mutant phenotype is characterised by a loss of petals and aberrant sepal
development. PTL is expressed between developing sepal primordia
(termed the Inter Sepal Zone, ISZ) where it functions to suppress sepal
enlargement and to promote petal initiation in nearby internal spaces.
We have identified a specific 16 bp regulatory enhancer (RE) within the
sole intron of PTL which is required to activate its expression in the ISZ.
This element is highly conserved within orthologous genes in tetrameric
species of the related Brassicaceae, Cleomaceae and Capparaceae
families tested, but surprisingly, was not detected in more distantly
related pentameric families within the order Brassicales. The ISZ RE
shows close homology to a sequence within the LTRs of some gypsy-
like retrotransposons, although the latter sequences are 2 bp longer.
Deletion of these 2 nucleotides is sufficient to activate ISZ expression.
Evolution of the distinct tetrameric perianth pattern of Brassicaceae may
have arisen through insertion of a retroelement into the intron of the
PTL ancestral gene, followed by simple mutational change in the LTR
sequence to create an active ISZ RE and thus recruit PTL function to
this region.
Page 152 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-206
MODIFICATION OF THE INTERCELLULAR CONTACTS
IN PLANT SUSPENSION CELLS THROUGH
ADAPTATION TO ENVIRONMENTAL STRESS
Kasprowicz A.K.1, Michalak M.J.1, Baluska F.2 and Wojtaszek P.1
1
Department of Molecular and Cellular Biology, Faculty of Biology,
Adam Mickiewicz University, Umultowska 89, 61-614 Poznan. 2Institute
of Cellular and Molecular Botany, University of Bonn, Kirschallee 1,
53115 Bonn, Germany.
The cell wall-plasma membrane-cytoskeleton continuum is responsible
for mechanical integration of plant cells. Long-term water deprivation
leading to osmotic stress activates adaptory mechanisms which enable
cell to function more or less normally. During stepwise adaptation of
tobacco BY-2 suspension cells to growth in the presence of various
osmotically active agents, cell lines diverged into independent,
osmoticum type-specific lines. The changes in growth patterns were
accompanied by the alterations in the composition of cell walls. It
was determined that cell walls of cells grown in the presence of ionic
agents were homogenous, while longitudinal walls and cross-walls in
cells adapted to nonionic agents were significantly different. In plants,
cross-walls within cell files of axial organs exhibit specific properties
that allow them to act as domains of contact and intense intercellular
communication e.g. via endocytosis, and the sites of anchorage of
cytoskeleton. Similar properties were also observed in cross-walls
connecting cells grown in the presence of nonionic osmoticum. The
presented results were revealed with immunolocalization, live confocal
microscopy and proteomics approaches. This research was founded
by the Polish Ministry of Science and Higher Education grants: PBZ-
KBN-110/P04/2004 and 2944/B/P01/2008/34 to PW and grant of Dean
of Biology Faculty for PhD students and scholarship from European
Social Fund Programme for best PhD students from Wielkopolska to
AK.
POS-TUE-208
NOVEL TRANSIENT GENE-REPLACEMENT SYSTEM
TO INVESTIGATE RNA PROCESSING ACTIVITIES OF
DICER-LIKE ENZYMES IN PLANTS
Kim K.W.1, Eamens A.L.1, 2, Fusaro A.1, 2 and Waterhouse P.M.1, 2
1
University of Sydney NSW 2006. 2CSIRO Enquiries Locked Bag 10
Clayton South VIC 3169 Australia.
Since their discovery, RNA silencing pathways have been the focus of
intense research in both plants and animals. The use of HELA cells and
Drosophila embryo lysates have allowed animal RNA silencing pathways
to be studied rapidly in vitro, whereas in plants, RNA silencing pathways
have been studied mostly in vivo, within different stable mutants of
Arabidopsis thaliana. A.thaliana, has been used as the model plant for
studying plant RNA silencing pathways because of its short generation
time, complete annotated genome and its capacity to be transformed by
a non-tissue culture, floral dip method with stable transgenes. A rapid
alternative to stable transformation is the use of transient Agrobacterium-
mediated transformation (agro-infiltration). However, this technique
is only compatible with an Australian native, Nicotiana benthamiana,
whoes genome remains to be fully sequenced. In an attempt to combine
the benefits of both systems, we developed a novel transient gene-
replacement strategy to investigate the RNA processing activities of
Arabidopsis Dicer-like proteins in N. benthamiana. This involved the
transient knock-down of endogenous N. benthamiana DCL1 protein
expression using an artificial miRNA (amiRNA) that is specific to the
endogenous NbDcl1 gene, followed by the transient over-expression
of functional exogenous Arabidopsis DCL1 protein via agro-infiltration.
This resulted in the successful complementation of DCL1-dependent
processing of miRNA-precursors, and therefore demonstrated that the
concept of transient gene-replacement was achievable for studying
specific components of the miRNA silencing pathway.
POSTERS MONDAY & TUESDAY
POS-MON-209
STRUCTURAL BASIS OF DISEASE RESISTANCE IN
FLAX AGAINST FLAX RUST
Ve T.1, Williams S.1, 2, Valkov E.1, Stamp A.1, Sornaraj P.2, De Courcy-
Ireland E.2, Ellis J.G.3, Dodds P.N.3, Anderson P.A.2 and Kobe B.1
1
School of Chemistry and Molecular Biosciences, University of
Queensland, Brisbane 4072, Australia. 2School of Biological Sciences,
Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia.
3
CSIRO Plant Industry, Canberra, Australia.
Plant diseases significantly affect economically important crops. Plant
immunity is triggered by the recognition of a pathogen effector protein
by a plant resistance (R) protein, leading to the activation of plant
defences and a localized cell death response. The effectors usually
have roles in virulence and are structurally diverse, while R proteins
generally fall into a few conserved families. The effector-R recognition
event is poorly understood at molecular and structural levels. We have
used the fungal pathogen flax rust interaction with flax as a model
system to characterize this process. The flax R proteins consist of a core
nucleotide-binding, N-terminal Toll-interleukin-like (TIR), and C-terminal
leucine-rich repeat (LRR) domain. Previously, we have shown the direct
interaction of the effector proteins AvrL567 and AvrM with R proteins L6
and M, respectively (1,2). We also determined the crystal structure of
AvrL567 (3). Here, we report the first crystal structure of a TIR domain
from a plant R protein (L6) at 2.3 Å resolution. The structure reveals
important differences from the structures of mammalian TIR domains,
and highlights three separate functionally important protein surfaces,
involved in dimerisation, interaction with a downstream signalling
partner, and regulatory intramolecular interactions, respectively. We also
determined the crystal structure of flax rust effector protein AvrM, which
has no significant sequence similarity with proteins of known structure.
The 2.7 Å resolution structure reveals a novel L-shaped helical fold,
with two chains forming a dimer with an unusual non-globular shape.
Our results bring us a step closer to understanding the molecular basis
for the disease resistance process and the ability to engineer novel
resistance specificities. 1. Dodds et al (2006) Proc Natl Acad Sci USA
103: 8888. 2. Catanzariti et al (2010) Mol Plant Microbe Interact 23: 49.
3. Wang et al (2007) Plant Cell 19: 2898.
POS-MON-211
DE NOVO ASSEMBLY OF THE NICOTIANA ALATA
POLLEN TRANSCRIPTOME USING NEXT-GENERATION
SEQUENCING
Koh P.1, Cassin A.M.2, Edwards D.3, Bacic A.1, 2 and Newbigin E.1
1
Plant Cell Biology Research Centre, School of Botany, University of
Melbourne, VIC 3010, Australia. 2Australian Centre for Plant Functional
Genomics, School of Botany, University of Melbourne, VIC 3010,
Australia. 3Australian Centre for Plant Functional Genomics and School
of Land Crop and Food Sciences, University of Queensland, Brisbane,
Australia.
We report the sequencing of the Nicotiana alata pollen grain transcriptome
using a Next-Generation or deep-sequencing approach followed by de
novo sequence assembly. Next-Generation sequencing is increasingly
being used to profile transcriptomes although to date this approach has
mainly been used with model organisms where a sequenced genome is
used to align the transcriptome sequence reads. De novo transcriptome
assembly offers a way of using this powerful technology in non-model
organisms such as N. alata, for which there are few genomic resources. In
de novo assembly, long RNA transcripts are first converted into a library
of short cDNA fragments that are then sequenced in a high-throughput
manner to obtain small lengths of sequence information from one end of
each cDNA fragment. Computer programs then reconstruct full-length
and partial transcripts from this vast amount of sequence information.
Here we describe the workflow used to de novo assemble the N. alata
pollen transcriptome, some of the features of this transcriptome, and
RT-PCR validation of selected cDNAs.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 153
POS-TUE-210
IDENTIFICATION OF PLANT HOST PROTEINS
TARGETED BY FLAX RUST EFFECTOR PROTEINS
Koeck M.1, 2 , Dodds P.N.2, Jones D.A.1, Ellis J.G.2 and Hardham A.R.1
1
Australian National University, CMBE, RSB, PS. 2CSIRO Plant
Industry, Black Mountain, Canberra.
Secreted rust fungal proteins found inside the plant cell during infection
are thought to manipulate host metabolism and defence responses.
Flax (Linum usitatissimum) and its rust pathogen (Melampsora lini) is
a useful model system for this infectious disease, and several secreted
effectors have been identified from flax rust, including AvrL567. The
aim of this study is to identify proteins that interact with AvrL567, which
may represent such host targets. Using a Yeast-2-Hybrid screen with
cDNA from infected flax leaves, three interactors of AvrL567 could
be identified, namely LuCKX1, C2MP and SIA2. Interaction between
AvrL567 and these three targets was confirmed by a Bimolecular
Fluorescence Complementation assay in planta. LuCKX1 (Linum
usitatissimum cytokinin oxidase 1) is a cytosolic plant gene closely related
to AtCKX7, one of seven genes in Arabidobsis thaliana responsible
for irreversible degradation of cytokinin. These plant hormones are
involved in cell development such as lateral root formation and their
cellular concentration changes upon pathogen infection. Homologues
of the plant gene C2MP (C2-domain containing membrane-targeting
protein) have been reported to be involved in pathogen responses in
rice. A.thaliana lines expressing these rice genes were more resistant
to Pseudomonas syringae infection compared to the wild type plants.
The third interactor, SIA2 (Secreted Interactor of AvrL567a), is another
secreted fungal protein that may be a co-factor for AvrL567. SIA2 also
shows binding to two other rust effector proteins (AvrM and AvrP),
suggesting it may have an important general role in protein translocation
and infection. Additionally, homologous of SIA2 are associated with
pathogenicity in powdery mildew and rice blast fungi.
POS-TUE-212
CHARACTERISATION OF LEGUME ANGIOGENIC
ACTIVE METABOLITES
Kordbacheh F.1, Hocart C.1, Du Fall L.1, Oakes M.1, Bezos A.2, Parish C.1, 2
and Djordjevic M.1
1
ARC Centre of Excellence for Integrative Legume Research,
Plant Science Division, Research School of Biology, GPO Box
475, Australian National University, Canberra ACT 2601, Australia.
2
Department of Immunology and Genetics, The John Curtin School of
Medical Research, The Australian National University, PO Box 334,
Canberra, ACT, 2601 Australia.
Legumes have many beneficial health effects in humans and animals
due to their nutritional properties and they are a known source of
bioactive metabolites that affect plant, animal and bacterial cells (Buer
et al, Journal of Integrative Plant Biology, 2010, 52: 98-111; Dixon and
Sumner, Plant Physiology 2003; 131: 878-885; Banunas and Kinghorn
2005 Life Sciences 78: 431-441). We have isolated, identified and partially
characterised metabolites from legumes that modulate angiogenesis.
Angiogenesis is the formation of new blood capillaries, veins and
arteries from pre-existing blood vessels to supply blood and nutrients
and remove metabolic wastes from the tissues. Size filtration and high
performance liquid chromatography (HPLC) were used to purify and
isolate the bioactive metabolites from the crude extract. The bioactive
components were identified using activity-guided assays. An in vitro
angiogenesis bioassay based on in vivo blood vessel formation (Parish
et al, Cancer Research 1999; 59: 3433-3441) was used to discover at
least three different molecules that possess pro-angiogenic activity.
Mass spectrometry strategies including gas and liquid chromatography
(GC/MS, LC/MS) were used to determine the molecular masses of the
compounds, establish elemental formulas and determine structural
details of the bioactive compounds. These compounds may find utility
in cardiovascular disease and wound healing.
POSTERS MONDAY & TUESDAY
POS-MON-213
GENOME-WIDE TRANSCRIPTOME ANALYSIS
OF CITRUS HYBRIDS PLANTS RESISTANT OR
SUSCEPTIBLE TO CITRUS LEPROSIS C (CILV-C)
Kubo K.S.1, 2 , Cristofani-Yaly M.1, Ribeiro-Alves M.3, Boava L.P.1,
Costa F.M.1, 5, Kishi L.1, Mafra V.S.1, 2, Freitas-Astua J.1, Bastianel M.1
and Machado M.A.1
1
Centro de Citricultura Sylvio Moreira/IAC, CP4, 13490-970,
Cordeiropolis-SP, Brazil. 2Institute of Biology, CP 6109, State
University of Campinas – UNICAMP, 13083-970, Campinas, SP,
Brazil. 3Centro de Desenvolvimento Tecnologico em Saude (CDTS),
Fundacao Oswaldo Cruz (Fiocruz), 21040-900. Rio de Janeiro, RJ,
Brazil. 4Embrapa Cassava and Tropical Fruits, 44380-000, Cruz das
Almas, BA, Brazil. 5Department of Agronomy, Universidade Federal
Rural de Pernambuco, 52171-900, Recife, PE, Brazil.
The molecular response of citrus plants against Citrus leprosis C
(CiLV-C) is still unknown. To identify differentially expressed genes in
response to CiLV-C, pools of seven highly tolerant and seven highly
susceptible hybrids from Murcott tangor (Citrus sinensis x C. reticulata)
and Pera sweet orange (C. sinensis) were evaluated by microarray
analysis. In order to define susceptible and tolerant hybrids, they were
infested by viruliferous mites in 2002 and the symptoms were evaluated
during six years. Symptomless leaves of each hybrid were collected
for RNA extraction. The experiment was designed in ten experimental
blocks, the leaves were collected from four blocks, each being a
biological repetition and each repetition containing seven individuals.
The microarray chip contained 32,121, 18,873 and 12,873 unigenes
from Pera sweet orange, Ponkan mandarin (C. reticulata) and Poncirus
trifoliata, respectively. These unigenes were obtained from the Citrus
ESTs database (CitEST) of Centro de Citricultura Sylvio Moreira/IAC.
The microarrays were performed by Roche Nimblegen and Bayesian
moderated T-test yielded 466 diferentially expressed genes (p.val ≤ 0.05
and fold-change ≥ 2). From these, the majority was related to metabolism,
cell rescue, defense and virulence, and protein fate. Financial support
FAPESP 2007/00117-4/INCT/Cnpq.
POS-MON-215
TAPETUM AND POLLEN DEVELOPMENT REGULATED
BY ATMYB103 AND ITS APPLICATION IN HYBRID SEED
PRODUCTION
Li S.F., Phan H., Iacuone S., Avdic A., Xu Y., Pham H. and Parish R.W.
Botany Department, School of Life Sciences, La Trobe University,
Melbourne, Vic. 3086, Australia.
The Arabidopsis AtMYB103 gene codes for a R2R3 MYB domain protein
whose expression is restricted to the tapetum of developing anthers and
to trichomes. We have previously shown that blocking the function of the
AtMYB103 gene using either an insertion mutant or an AtMYB103EAR
chimeric repressor construct results in complete male sterility and failure to
set seed. These plants exhibit similar abnormalities in tapetum and pollen
development, with the tapetum and pollen degenerating prematurely. We
are now studying the molecular network regulated by AtMYB103 protein
and developing male sterility systems for hybrid seed production in crop
plants. AtMYB103 regulates the expression of genes that are involved in
tapetum and pollen development including the release of microspores
from tetrads. These putative target genes of AtMYB103 protein are being
characterized and they will provide clues to the AtMYB103 molecular
network. To develop a reversible male sterility system and an inducible
male sterility system for hybrid seed production, several AtMYB103
homologues were cloned from crop plants. They are highly conserved in
their deduced amino acid sequences and their promoters are only active
in developing anthers. These homologues are the functional equivalents
of AtMYB103 in regulating tapetum and pollen development as they were
able to restore male fertility of a male sterile atmyb103 mutant. The male
sterility systems using chimeric MYB103EAR repressors for hybrid seed
production are discussed.
Page 154 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-214
FORMATION OF SECOND WHORL ORGANS IN THE
ARABIDOPSIS FLOWER INVOLVES AUXIN INFLUX
AND IS SENSITIVE TO DISTORTION OF THE FLOWER
MERISTEM SHAPE
Lampugnani E.R., Kilinc A. and Smyth D.R.
School of Biological Sciences, Monash University, Melbourne, Vic
3800.
We are interested in how organ primordia are initiated within flowers, and
in identifying the genetic factors and pathways involved in this process.
Flowers of mutants of the PETAL LOSS (PTL) gene of Arabidopsis show
a progressive loss in the ability to generate petals, and the sepals are
wider, closer together and sometimes fused. PTL encodes a trihelix
transcription factor that is expressed between sepal primordia and in
sepal margins during early stages of flower development. Measurements
of the numbers of cells that express PTL in this region indicate that there
are more when PTL function is lost. Thus PTL may have a role in the
suppression of growth between sepals and in sepal margins. Disruption
of petal initiation in ptl mutants may be the consequence of disruption
of a signal required for nearby petal initiation. It seems likely that auxin
provides such a signal because disruption of auxin influx in aux1 ptl
double mutants further compromises petal initiation. This is supported
by observations on further disruptions to petal number in mutant
combinations of ptl, and aux1, with other genes, including cuc, and
eep1, which further disrupt flower meristem shape when their function is
lost. Changes in flower meristem shape apparently cause disruptions to
auxin signalling, and the initiation of second whorl primordia is especially
sensitive to variation in its strength.
POS-TUE-216
INVESTIGATING CELLULAR SIGNALLING PATHWAYS
IN CANCER CELL LINES TREATED WITH A NOVEL
COMPUTATIONALLY DESIGNED MYXOMA VIRUS T5
PEPTIDE ANALOGUE
Almansour N.M.1, Pirogova E.2 and Istivan T.1
1
School of Applied Sciences, Health Innovations Research Institute,
RMIT University, Bundoora West, Australia. 2School of Electrical and
Computer Engineering, Health Innovations Research Institute, RMIT
University, Melbourne, Australia.
M-T5 is a myxoma virus ankyrin-repeat host range protein that is shown
to be required for virus replication in rabbit lymphocytes and the majority
of human tumor cells. We investigated the cytotoxic effects of RRM-
MVT5, a short peptide analogue for this protein, on melanoma cell lines
as a future cancer therapeutic candidate. This novel computationally
designed bioactive peptide has significant apoptotic/ necrotic effects
on cancer cell lines and a negligible toxic effect on normal cells. Akt
activation is essential for normal cell processes such as programmed
cell death, proliferation and angiogenesis. Yet, the regulation of Akt
activation is impaired in cancer cells. It was previously shown that viral
M-T5 binds with phospho-Akt to regulate Akt signalling in some human
cancer cell lines. In this study we investigated the effect of RRM-MVT5
treatment on Akt activation in skin cancer cell lines to detect the possible
cellular pathway that is affected by this peptide. The cellular expressions
of total Akt and phspho-Akt in cell cultures of the malignant melanoma
cell line, MM96L, the human squamous carcinoma cell line COLO-16
and B16-F0 murine melanoma were detected using western blotting.
The levels of phospho-Akt expression were assessed in cell cultures
treated with RRM-MVT5 peptide analogue, in the presence or absence
of the PI3 kinase inhibitor (LY294002) Our preliminary results indicated
that the cytotoxic effects of RRM-MVT5 are not targeted towards this
pathway as the expression of both total and phospho-Akt in cancer cells
were not affected by this peptide analogue.
POSTERS MONDAY & TUESDAY
POS-MON-217
ANALYSIS OF SUBUNIT INTERACTIONS WITHIN THE
NUCLEOSOME REMODELLING AND DEACETYLASE
(NURD) COMPLEX
Alqarni S., Thong S., Josep F. and Mackay J.
School of Molecular Bioscience, University of Sydney, NSW, Australia.
Our lab is interested in the structure and function of the Nucleosome
Remodelling and Deacetylase (NuRD) complex, which is conserved
across a wide range of eukaryotes and expressed broadly in mammalian
tissues. This ~2-MDa complex appears to comprise at least ten
polypeptides, including Mi-2/CHD-family proteins, HDAC1/2, RbAp46,
RbAp48, MTA-family proteins (MTA1, MTA2 or MTA3), MBD2 or MBD3,
p66α and p66β. Despite its importance, almost nothing is known about
the structure, assembly or biochemical mechanism of action of this
complex. RbAp46/RbAp48 and MTA1/MTA2 are core members of the
complex and have been shown previously to interact with each other.
To identify the residues of MTA proteins that are critical for mediating
RbAp46/48 interactions, we generated a series of constructs encoding
truncations and mutants of MTA1 and MTA2. These constructs were
used to generate in vitro translated 35S-labeled proteins. Using pulldown
experiments, we tested the interactions between RbAp46/RbAp48 and
different truncations of MTA1 and MTA2. Our data indicate that RbAp46/
RbAp48 proteins interact with MTA1 constructs in the same manner and
we have identified a short motif in MTA1 that appears to be necessary for
the interaction with RbAp48. We have used a range of RbAp48 mutants
to delineate the MTA1-binding surface of RbAp48. Together, these data
provide the first detailed information on the inter-subunit interactions that
facilitate the assembly of the NuRD complex, and also provide insight
into the manner by which NuRD interacts with promoter-bound proteins
to regulate gene expression.
POS-MON-219
STRUCTURE OF DIHYDRODIPICOLINATE SYNTHASE
FROM BACTERIA AND PLANTS: INSIGHTS INTO
REGULATION AND INHIBITION OF A VALID DRUG
TARGET
Atkinson S.C., Dogovski C., Dobson R.C.J. and Perugini M.A.
Department of Biochemistry and Molecular Biology, Bio21 Molecular
Science and Biotechnology Institute, The University of Melbourne,
Victoria 3010, Australia.
Agrobacterium tumefaciens is a Gram-negative bacterium and the
causative agent of Crown Gall disease, which is characterised by the
formation of tumours at sites of environmental damage in plants. The
disease is common to stone fruit, including grape vines, causing millions
of dollars of damage each year to the wine industry worldwide. Despite
its widespread occurrence, there is currently no treatment for Crown Gall
disease. Accordingly, there is an urgent need to discover, characterise
and validate new antimicrobial targets in this organism. One such target
is dihydrodipicolinate synthase (DHDPS), which is an essential bacterial
enzyme that catalyses the condensation of aspartate semialdehyde
and pyruvate in the diaminopimelate (DAP) pathway, ultimately yielding
the amino acid lysine. Previous studies in Gram negative bacteria and
plants demonstrate that the downstream product of the DAP pathway,
lysine, can feedback inhibit DHDPS allosterically This project aims to
characterise the structure, regulation and inhibition of DHDPS from
both Agrobacterium tumefaciens (AgT) and the common grape vine,
Vitis Vinifera (Vv). Both AgT-DHDPS and Vv-DHDPS have been cloned,
expressed and purified to homogeneity. The recombinant enzymes are
enzymatically active, folded and form tetramers in solution. We have
solved the crystal structure of AgT-DHDPS in the apo, pyruvate-bound
and pyruvate & lysine bound forms and have used the apo structure to
conduct an in silico screen using a virtual library comprised of more than
4 million drug-like compounds. The screen has yielded 100 potential
inhibitors, targeting either the active or allosteric sites. 10 of these
compounds show greater than 10% inhibition in vitro at a concentration
of 25 μM. Inhibition studies using the Vitis vinifera form of the enzyme as
a control reveal that several of these hits show specificity towards AgT-
DHDPS and do not inhibit the plant enzyme in vitro. We are currently in
the process of further validating these hits both in vitro and in vivo.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 155
POS-TUE-218
A MODULAR BAM COMPLEX IN THE OUTER
MEMBRANE OF THE α-PROTEOBACTERIUM
CAULOBACTER CRESCENTUS
Anwari K.1, Poggio S.2, Jacobs-Wagner C.2 and Lithgow T.J.1
1
Department of Biochemistry and Molecular Biology, Monash
University, Melbourne, Australia. 2Department of Molecular, Cellular
and Developmental Biology and Microbial Pathogenesis Section, Yale
University, New Haven, Connecticut, United States of America.
The BAM (β-barrel assembly machinery) complex is a multi-subunit
protein complex present in the outer membrane of all Gram-negative
bacteria. It comprises of a core β-barrel protein, BamA and associated
lipoproteins that collectively participate in the folding and insertion
of β-barrel proteins. In this study, we demonstrate that the BAM
complex possesses modular characteristics and contains BamA and
three outer membrane lipoproteins (BamB, BamD and BamE) in the
α-proteobacterium, Caulobacter crescentus. In addition to the three
known lipoproteins, we identify an essential subunit of the BAM complex
referred to as peptidoglycan-associated lipoprotein (Pal). We propose
that Pal is a protein that anchors the BAM complex to the peptidoglycan
layer and promotes proximity to the inner membrane Sec machinery for
efficient outer membrane protein assembly.
POS-TUE-220
MECHANISTIC PERSPECTIVE FOR POLYMERIZATION
OF ADH AND ROLE OF α-CYCLODEXTRIN AS AN
ARTIFICIAL CHAPERONE FOR SOLUBILIZATION OF
LARGER AMORPHOUS NANOAGGREGATES
Barzegar A.1, 2 and Moosavi-Movahedi A.A.2
1
Research Institute for Fundamental Sciences (RIFS), University
of Tabriz, Tabriz, Iran. 2Institute of Biochemistry and Biophysics,
University of Tehran, Tehran, Iran.
A crucial problem of protein-based agents in biotechnology and
biopharmaceuticals is their long-term stability or so-called shelf-life.
The shelf-life of biotechnological potent enzymes is limited by thermal
stress because of self-assembly of proteins into nanoaggregates such
as nanoensembles or nanofilaments. Since biotechnological application
of mesophilic alcohol dehydrogenases (ADH) in the pharmaceutical
industry is limited because of easily polymerization during thermal
process. We have evaluated the mechanism of ADH aggregation
with a view to establish of thermal stabilization in order to improve the
shelf-life of the ADH enzymes. The fluorescence, circular dichroism,
UV-Vis spectrophotometry, dynamic light scattering (DLS) technique,
enzymatic activity assay, molecular dynamic and molecular docking
methods have been used for the mentioned aim. Based on the findings
we have proposed a new model of self-assembly for ADH enzymes that
construction of nuclei and growing to formless nanoaggregates without
enzymes denaturation and unfolding. Enzymes quaternary structural
changes delocalization of subunits lead to enzymes polymerization
without unfolding. Alpha cyclodextrin (α-CyD) caused two types thermal
stabilization of ADH enzymes including kinetic and thermodynamic
stabilities by masking delocalized regions of ADH chains. Delocalization
of ADH subunits, which causes the exposure of hydrophobic domains,
takes part in the enzyme polymerization and has proven to be beneficial
for aggregation inhibition and solubility enhancement within the host
α-CyD-nanocavity. The thermostabilization of ADH against aggregation
proposed for its biotechnological applications.
POSTERS MONDAY & TUESDAY
POS-MON-221
ANALYSIS OF THE CHROMATIN BINDING PROPERTIES
OF CHD4
Bendak K., Mansfield R.E., Burdach J., Crossley M. and Mackay J.
School of Molecular Bioscience, University of Sydney, NSW 2006,
Australia.
Chromosome helicase DNA–binding protein 4 (CHD4) is a major
component of the nucleosome remodeling and deacetylase (NuRD)
complex. The NuRD complex contains at least 10 subunits and
plays an important role in transcription regulation and development
throughout a wide range of eukaryotic species. Mammalian CHD4
contains two plant homeodomain (PHD) fingers. Recent results from
our laboratory show that these fingers are capable of binding in vitro
to the amino-terminus of histone H3. This interaction is enhanced by
acetylation or methylation of H3Lys9, whereas H3Lys4 methylation or
H3Ala1 acetylation inhibit the interaction. To examine the functional
significance of this interaction, we have sought to determine whether
CHD4 can bind to chromatin in vivo. Mutations have been introduced
into the PHD domains of full-length CHD4 that compromise binding to
H3 and chromatin immunoprecipitation (ChIP) experiments have been
used to assess the ability of the mutant CHD4 to colocalize in cells
with H3 bearing trimethylated Lys9. Additionally immunoprecipitation
experiments have been carried out, to establish recruitment of other
NuRD complex components through CHD4.
POS-MON-223
AP180 BINDING TO CLATHRIN IN ENDOCYTOSIS: A
NEW INSIGHT
Chan L.S., Robinson P. and Graham M.
Children’s Medical Research Institute, The University of Sydney.
The assembly protein AP180 is involved in membrane nucleation, which
is the first step of synaptic vesicle endocytosis (SVE). AP180 controls
the uniformity of synaptic vesicle size and shape. It has two known
binding partners – the adaptor protein AP-2, which recruits accessory
endocytic proteins to the budding membrane, and clathrin heavy chain,
which is the main coat component of the budding vesicle. Similar to other
major SVE proteins, AP180 is dephosphorylated when the synapse
is stimulated (depolarization). Our aim was to investigate the binding
of clathrin to different domains of AP180 and to determine if clathrin
binding is phopsho-regulated. We made domain-specific constructs for
AP180, and found a new clathrin binding site in AP180 that challenges
previous assumptions about clathrin binding. We have also completed a
phosphorylation map of AP180 and found that phosphorylation regulates
clathrin binding to AP180. The location of the new clathrin binding site
and the regulatory phosphorylation sites suggests a staged interaction
between clathrin and AP180. This has profound implications on the
mechanism of clathrin recruitment during stimulus-dependent SVE.
Page 156 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-222
UNRAVELLING THE ENZYMES OF THE TARTARIC ACID
BIOSYNTHETIC PATHWAY IN VITIS VINIFERA
Burbidge C.A.1, Soole K.L.1, Hayes M.A.2 and Ford C.M.2
1
School of Biological Science, Flinders University, GPO Box 2100, SA
5001, Australia. 2School of Agriculture, Food, and Wine, University of
Adelaide, Adelaide, SA 5005, Australia.
Tartaric (TA) and malic acid (MA) account for 90% of total acid in the
developing grape berry. While the majority of MA is metabolised before
harvest, TA levels remain constant throughout development. Little work
has been conducted into the synthesis of TA due to its rarity in higher
plants. Previous work has identified ascorbic acid (Asc, Vitamin C) as
the principle precursor of this acid, with additional evidence for a minor
synthetic route via D-gluconic acid. To date only one enzyme, L-idonate
dehydrogenase has been identified as having an active role in TA
production. Bacteria including Erwinia spp and Escherichia coli possess
enzymes capable of catalysing reactions identical to those involved in
synthesising TA in V. vinifera. Comparative analysis of the E. coli and
V. vinifera genomes has identified candidate genes that may encode
previously unidentified enzymes of the TA biosynthetic pathways.
Candidates were selected based on criteria including the presence of
cofactor binding sites and conserved bases. Cloning and expression
of enzymes pertaining to two separate steps of the biosynthetic
pathway has resulted in high amounts of soluble protein. Analysis of
these enzymes indicates activity towards their respective substrates.
Determination of TA levels, enzyme activity and gene expression over
berry development were used to confirm an enzymatic role for these
enzymes within the TA biosynthetic pathway.
POS-TUE-224
EXPRESSION AND PURIFICATION OF HUMAN G72 AND
DAO, TWO SCHIZOPHRENIA-RELATED PROTEINS
Chen S.Y.1, 2 , Chen Y.J.3, Wang C.M.1, Lane H.Y.4 and Chang H.T.1, 4
1
Graduate Institute of Molecular Systems Biomedicine, China Medical
University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C.
2
Master Program of Life Sciences, National Chung Hsin University,
No. 250, Kuo Kuang Road, Taichung 402, Taiwan R.O.C. 3Department
of Public Health, China Medical University, No. 91, Hsueh-Shih
Road, Taichung 404, Taiwan, R.O.C. 4Graduate Institute of Clinical
Medical Science, China Medical University, No. 91, Hsueh-Shih Road,
Taichung 404, Taiwan, R.O.C.
Schizophrenia, a mental disease, is caused by multigene dysfunction
or neurontransmitter dysregulation such as N-methyl D-aspartate
(NMDA) receptor agonist decreases and dopamine deficits. Previous
studies showed that patients with schizophrenia might have abnormal
regulations of D-amino acid oxidase (DAO) and G72. DAO is responsible
for D-amino acid oxidation to pyruvates and G72 is defined as a DAO
activator. However, the regulatory mechanisms of G72 and DAO are still
unclear. Thus, how G72 regulates DAO is the main theme of this study. In
order to characterize the interaction between G72 and DAO, we produed
and purified recombinant DAO-6H, G72-6H, MBP-DAO and GST-G72
from E. coli BL21(DE3)pLysS cells. The yield was obtained by growing
the cells overnight at 25 °C under induction with 0.5 mM IPTG during the
exponential phase of growth. We have succeeded to express DAO-6H,
G72-6H, and MBP-DAO. However, in which only MBP-DAO was purified
from soluble fraction. Initial testing showed that the enzymatic activity
of MBP-DAO is not high. It may be due to the MBP tag obstructing the
DAO activie sites. Thus the MBP tag will be removed to increase DAO
activities. Furthermore, for production of soluble G72-6H, BL21(DE3)
containing pG-KJE6 chaperone plasmids will be used. DAO is a key
enzyme for oxidizing D-amino acids in brain and its enzymatic activities
are thought highly correlated with schizophrenia. We expect to verify
DAO abnormally regulated by G72 in vitro and in vivo.
POSTERS MONDAY & TUESDAY
POS-MON-225
A POTASSIUM SWITCH OF ATP-INDUCED GROEL
CONFORMATIONAL CHANGES
Chen J.1, Makabe K.1, 2 and Kuwajima K.1, 2
1
Okazaki Institute for Integrative Bioscience and Institute for Molecular
Science. 2Department of Functional Molecular Science, the Graduate
University for Advanced Studies (Sokendai).
Bacterial chaperonin GroEL utilizes the co-chaperone GroES to assist
folding of cellular proteins in an ATP-dependent manner. An ATP-induced
allosteric communication through GroEL double rings is prerequisite
for the overall chaperonin cycle. It has been shown by Horovitz et.al.
(1995) that ATP-induced allosteric transitions of GroEL consist of two
phases with one at relatively low ATP concentrations (≤ 100 μM) and
the second at higher concentrations of ATP with a midpoint in the range
of 16 to 160 μM. Here we studied the effect of essential metal cofactor,
potassium ion, on the ATP-driven allosteric transitions of GroEL by
using a tryptophan mutated GroEL. Within the test range of potassium
concentration, two distinct patterns of ATP-induced allosteric transitions
were observed. We showed that at 50 mM potassium concentration,
a remarkable increasing second-phase of ATP-induced allosteric
transition was observed which is contrary to the decreasing phase at
10 mM potassium concentration. References [1] Yifrach O, Horovitz A.
Nested cooperativity in the ATPase activity of the oligomeric chaperonin
GroEL. Biochemistry. 1995, 34(16):5303-8. [2] Inobe T, Kuwajima
K. Phi value analysis of an allosteric transition of GroEL based on a
single-pathway model. J. Mol. Biol. 2004, 339(1): 199-205. [3] Poso D,
Clarke AR, Burston SG. A kinetic analysis of the nucleotide-induced
allosteric transitions in a single-ring mutant of GroEL. J. Mol. Biol. 2004,
338(5):969-77.
POS-MON-227
STRUCTURE AND FUNCTION OF THE TIMA PROTEIN
FROM CAULOBACTER CRESCENTUS
Civciristov S. , Clements A. , Purcell A. , Williamson N. and Lithgow T.
1, 2 1 2 2 1
1
Department of Biochemistry, Monash University, Clayton. 2Bio21
Institute, The University of Melbourne, Parkville.
Mitochondria have evolved from a bacterial endosymbiont which was
probably related to present day α-proteobacteria. They possess complex
molecular machines that are found in both mitochondrial membranes.
An example is the TIM23 protein translocase of the inner mitochondrial
membrane that is responsible for the final step of translocation of the
proteins destined to the mitochondrial matrix. The key components of
this complex are the Tim23 translocation channel, the mitochondrial
motor Hsp70 and the Tim44 protein which is required for docking to
the channel. C.crescentus is an α-proteobacterium which has a protein
which we named TimA and it’s a protein homologue of the yeast protein
Tim44. The aim of my study is to identify if the TimA protein interacts
with any other proteins as part of an inner membrane protein complex
in C.crescentus and to investigate its function. We found that TimA is a
part of a complex in the inner membrane of Caulobacter by BN-PAGE.
Immunoprecipitation with the TimA antibody followed by LC-MS/MS
revealed that TimA has two partner proteins: FtsH and HflC. These two
proteins are part of a complex in E.coli together with a third protein-
HflK. The function of this complex in E.coli is in inner membrane quality
control of misfolded proteins or jammed translocation machines such as
SecY and YidC. HflK/C were shown to negatively regulate the activity
of FtsH protease in E.coli. Having in mind that TimA proteins are only
found in α-proteobacteria, we are investigating the function of TimA in
Caulobacter as a novel component of the FtsH complex as well as its
involvement in inner and outer membrane remodeling.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 157
POS-TUE-226
STRUCTURE-ACTIVITY ANALYSIS OF CAROCIN S2
Chuang D.Y. and Wu J.L.
Department of Chemistry, National Chung-Hsing University.
Carocin S2, which contains CaroS2K and CaroS2I, is the low-molecular-
weight bacteriocin produced by Pecctobacterium carotovorium 3F-3.
CaroS2K is an 85kDa protein that consists of a membrane-tranlocation
domain, a receptor-binding domain, and a catalytic domain-in their
primary sequences. These phytopathogenic cells also produce
inhibitor protein CaroS2I (Mw=10kDa) located downstream of the
caroS2K gene in the carocin operon. Fruthermore, in order to minimize
potential activity of carocin within the producing cell, the association of
the immunity protein with its cognate CaroS2K is rapid, specific, and
high affinity. Our results demonstrate clearly that the minimal inhibitory
concentration of CaroS2K is closed to 4μg/ml with indicator strain SP33.
Here, we use several bioinformatics tools to predict the structure and
functional properties of CaroS2K. The analysis accompany with site-
directed mutagenesis reveal that the catalytic pocket centred around
His696, the general base of the cyclization step of the reaction. The
nucleophilic attack participates in the abstraction of a proton from the
2’-hydroxy group of the substrate ribose and in the protonation of the 5’-
oxy group of a leaving substrate. The negatively charged pentacovalent
transition state can than be stabilized by nearby positively charged
residues (Lys692 and Lys695). Other mutant bacteriocin affecting the
main residues forming the central groove (Phe760, Ser762, Trp764) or
its loop (Tyr734) for stacking on the RNA base are also not active.
POS-TUE-228
BIOSYNTHESIS OF CIRCULAR PROTEINS IN PLANTS
Conlan B.1, Gillon A.D.1, Barbeta B.L. 1, Colgrave M.3, Craik D.J.2, and
Anderson M.A.1
1
Department of Biochemistry, La Trobe University, Melbourne VIC
3086. 2Institute for Molecular Bioscience, University of Queensland,
Brisbane QLD 4072. 3CSIRO Livestock Industries, Brisbane, QLD
4067.
Plant cyclotides are a large family of naturally occurring circular proteins
that are produced from linear precursors and are thought to be processed
by asparaginyl endopeptidases to produce the circular peptide. The
production of the circular peptide through excision of the cyclotide
domains and ligation of the free N- and C-termini occurs through a little
studied mechanism. Within the plant Oldenlandia affinis, the prototypic
cyclotide producing plant, the linear precursor Oak1 is cleaved at two
sites releasing the kalata B1 domain that is then cyclised resulting in
the mature cyclotide. Previous work in our lab has shown that O. affinis
produces only the cyclic form of the kalata B1 peptide from the Oak1
precursor, whilst transgenic expression of the same precursor in plants
that do not normally produce cyclotides results in the formation of both
linear and circular forms of the peptide. We have studied the importance
of the residues surrounding the processing site of the cyclotide domain
by site-directed mutagenesis, which revealed a number of residues and
motifs essential for cyclisation. The substitution of a highly conserved
asparagine residue at the C-terminal processing site of the cyclotide
domain with an alanine completely inhibited cyclization. Similarly, when
the conserved C-terminal tri-peptide motif was truncated no cyclic
product was detected. The mutagenesis of a leucine in the C-terminal
tri-peptide motif produced a unique processing product with implications
for a novel mechanism of cyclisation. The observed processing may be
the result of enzyme activity never before documented in planta.
POSTERS MONDAY & TUESDAY
POS-MON-229
THE USE OF CELL PERMEABLE PEPTIDES TO INHIBIT
SPRY DOMAIN-CONTAINING SOCS BOX PROTEIN 2
(SPSB2) REGULATION OF INDUCIBLE NITRIC OXIDE
SYNTHASE (INOS)
D’Cruz A., Lewis R.S., Kuang Z., Norton R.S., Babon J.J. and
Nicholson S.E.
The Walter and Eliza Hall Institute of Medical Research.
The production of nitric oxide (NO) and related reactive nitrogen
species is important for the killing of intracellular pathogens. iNOS/
NOS2 (inducible nitric oxide synthase) is rapidly expressed in response
to infection and catalyses the high-output production of NO. We
have demonstrated that SPSB2 is a negative regulator of iNOS in
Poly(I:C) and LPS stimulated macrophages, targeting iNOS for poly-
ubiquitination and proteasomal degradation. The SPSB2 SPRY domain
interacts with high affinity with a short linear sequence in the N-terminal
region of iNOS. We were interested in investigating whether intracellular
delivery of an iNOS peptide could disrupt the endogenous SPSB2/
iNOS interaction resulting in enhanced NO production. The highly
cationic Arginine-8 (R8) and HIV-1 derived TAT sequences are known to
facilitate the entry of covalently attached cargo molecules into cells via
macropinocytosis. We utilized the R8 and TAT sequences to facilitate
entry of iNOS peptide into macrophages. While the R8-iNOS and TAT-
iNOS peptides bound the SPSB2 SPRY domain with high affinity in vitro,
much of the fluorescently labelled peptide was trapped in endosomes.
We then utilized the influenza virus-derived HA2 sequence to enhance
endosomal escape of fluorescently labelled R8-iNOS and TAT-iNOS.
Despite observing cytoplasmic localization of the labelled peptides, we
saw no differences in the level of iNOS protein expressed in response
to stimuli, highlighting the difficulties of using cell permeable peptides
to interfere with biological interactions. More experiments need to
be conducted to determine if the iNOS peptides remain biologically
available upon delivery to the cytoplasm.
POS-MON-231
ROLE OF THE BRCA1 BINDING PROTEIN BRAP2 IN
THE TESTIS
Fatima S.1, 3, Davies R.G.1, Fulcher A.J.1, Wagstaff K.M.1, Whiley P.2, 3,
Loveland K.L.2, 3 and Jans D.A.2, 3
1
Nuclear Signaling Laboratory,. 2Testis and Germ Cell Development
Laboratory, Department of Biochemistry and Molecular Biology.
3
Australian Research Council Centre of Excellence for Biotechnology
and Development, Monash University, Clayton, Victoria, Australia.
BRAP (BRCA1 binding protein) 2 is a phosphorylation-regulated negative
regulator of nuclear import (NRNI) of specific proteins. It is highly
expressed in testis, including a testis specific isoform, consistent with
a specific role in testicular development. In situ hybridization analysis
indicates BRAP2 expression in spermatogonia and spermatocytes, and
to a lesser extent in round spermatids, essentially at the same stages
when molecules responsible for nuclear protein import (Importins - IMPs)
are highly expressed. In cotransfection experiments, we determined
that BRAP2 amino acid 442-592 is sufficient to inhibit nuclear import of
IMPα/β-recognised cargoes from testis. In an attempt to determine the
interactome of BRAP2 in testis, we screened a yeast two-hybrid library
from human testis using a C-terminal fragment of BRAP2 (343-592) as
bait. Among various clones identified, several appeared to interact with
proteins binding to the actin cytoskeleton, or involved in mitotic spindle
function. Future work will involve documenting the apparent link between
nucleus and cytoskeleton in mammalian testicular development and the
role therein of BRAP2 and its interactors. Keywords: BRAP2, Testis,
Negative regulator of Nuclear Import (NRNI).
Page 158 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE230
MECHANISMS OF TRANSTHYRETIN AMYLOID
FORMATION
D’Souza D.G.1, Altland K.3, Richardson S.J.1, 2 and Pattenden L.K.1, 2
1
School of Medical Sciences, RMIT University, Bundoora, Australia.
2
Health Innovations Research institute, RMIT University, Bundoora,
Australia. 3Department of Human Genetics, University of Giessen,
Giessen, Germany.
Transthyretin (TTR) is a β-sheet rich homo-tetrameric protein that
transports retinol-binding protein and thyroid hormones. For unknown
reasons, TTR has the potential to dissociate to dimers, then monomers.
Through protein misfolding, the monomers can form insoluble fibrils
(amyloids) that disrupt normal cellular functions and result in disease.
Preliminary data has shown human TTR forms insoluble amyloid fibrils
under mild acidic conditions similar to those found in vivo, however
wallaby TTR under the same conditions remains as stable dimers. Each
human TTR monomer contains His31, Ser46, and His90, close to the
dimer interface where they help stabilise the complex. Wallaby TTR has
different amino acid residues in these key positions; Lys31, Ala46, Tyr90.
Our work seeks to understand why human TTR dimers are unstable
compared to wallaby TTR dimers. We hypothesise that protonation of
key Histidine residues in human TTR, absent in wallaby TTR, results
in destabilisation of the dimer, facilitating TTR amyloid formation. We
produced human, wallaby, and seven cross-species mutant TTRs.
SDS-PAGE and Western analysis proved the recombinant TTR subunits
were the correct size. The proteins were purified via ion-exchange
chromatography. Stability assays were then performed under mild acidic
conditions (pH 7.4-6.4). Fibril formation experiments of human TTR,
wallaby TTR and their variants were performed at pH 7.4 (physiological),
pH 6.5 (inflammation), pH 4.6 (standard non-physiological pH used to
make amyloid fibrils). The application of evolutionary comparative
biology to produce single amino acid variant TTRs provides a great
opportunity to study structure/function relationships and understand
molecular mechanisms underlying disease states.
POS-TUE-232
STRUCTURAL STUDIES WITH CROTOXIN B FROM
CROTALUS DURISSUS SUBSPECIES VENOM
Fernandes C.A.H.1, Salvador G.H.M.1, Colombi D.2, Soares A.M.3 and
Fontes M.R.M.1
1
Departamento de Física e Biofísica, Univ Estadual Paulista, UNESP,
Botucatu-SP, Brazil. 2Departamento de Parasitologia, Univ Estadual
Paulista, UNESP, Botucatu-SP, Brazil. 3Departamento de Análises
Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-
USP, Ribeirão Preto-SP, Brazil.
Crotoxin B is a basic phospholipase A2 found in the venom of several
Crotalus durissus ssp. rattlesnakes and is one of the subunits that
constitutes crotoxin, the mains component of the venom of these
snakes. This heterodimeric toxin is related to important envenomation
effects such as neurological disorders, myotoxicity and renal failure.
Although crotoxin was first crystallized in 1938, the first structural data
only become available two years ago by our research group, with the
crystallization of crotoxin B from Crotalus durissus terrificus venom.
However, this structure reveled an ambiguous result for the biological
assembly, which could be dimeric or tetrameric. In this work, we present
the crystal structure of crotoxin B from Crotalus durissus colillineatus
venom, an unambiguously dimeric complex formed by two crotoxin
B isoforms (CB1 and CB2). A structural comparison between these
crotoxin B structures reveals differences in salt bridges that are important
to oligomerization and significant Cα deviation of 54-72 region. These
structural data combined with SAXS and CD experiments with inhibitors
and site-directed mutagenesis studies, currently in progress, may give
relevant insights into the understanding of its neurotoxic mechanism of
action.
POSTERS MONDAY & TUESDAY
POS-MON-233
CHARACTERIZATION OF ANTI-STAPHYLOCOCCAL
PROTEIN (P128) DERIVED FROM BACTERIOPHAGE
FROM IN PROCESS TO PURIFICATION USING MICRO-
FLUIDIC BASED LC SYSTEM COUPLED TO AN
ADVANCED QTOF-MS
Gudihal R.1, Sundaram P.M.2, Madhavi H.N.2, Asrani J.2 and
Fouracre C.G.3
1
Agilent Technologies India Pvt. Ltd, Bangalore, India. 2GangaGen
Biotechnologies Pvt. Ltd, Bangalore, India. 3Agilent Technologies,
Sydney, Australia.
P128 is recombinantly expressed in E.coli. P128 has potent bactericidal
activity against methycillin resistant Staphylococcus aureus (MRSA)
and is currently under early development for use in humans. Liquid
chromatography/Mass spectrometry (LC/MS) technology is a powerful
and sensitive technique for characterization and identification of proteins.
Chemical modifications and degradation that can potentially occur during
manufacturing, formulation, and storage of proteins, necessitate reliable
and sensitive methods for the characterization of purity and structural
integrity. If suboptimal characteristics of biologics are discovered after
initiation of clinical trials can lead to serious and costly delays in time
to market. Here we present accurate molecular mass measurement,
impurity profiling and peptide mapping for P128 which are some of
the biophysical characterization for biopharmaceutical selection.
Micro-fluidic based HPLC-Chip which integrates sample enrichment,
chromatographic separation and nano-electrospray formation for
efficient and high sensitivity nanospray LC/MS was used for the study.
Pure P128 as well as in process samples were analyzed using C8
packed micro-fluidic HPLC Chip. Flow from the chip to the accurate-
mass QTOF MS based system was 600nL/min and occurred through
a nanospray tip etched into the polyamide material of the chip. While
for peptide mapping, purified P128 protein was subjected to trypsin
digestion followed by peptide separation and mass determination on
C18 HPLC Chip at flow rate of 600nL/min coupled to QTOF-MS system.
Further the MS/MS experiments were performed so as to confirm the
peptide sequence.
POS-MON-235
BEYOND PERMEABILISATION - THE ROLE OF
INTRACELLULAR TARGETS IN THE ACTION OF AN
ANTIFUNGAL PEPTIDE
Hayes B., Conlan B., Anderson M. and Van Der Weerden N.
La Trobe University, Department of Biochemistry, Plenty Road,
Bundoora 3086.
Fungal disease is an ever increasing burden in the fields of medicine and
agriculture. Fungal pathogens are responsible for damage and loss of
many important agricultural crops as well as human illnesses that are often
life-threatening, particularly in immunocompromised individuals. With
the appearance of resistance to current treatments, novel approaches
are required to treat and prevent fungal disease. Antimicrobial peptides
have been the focus of intense research in recent years for their use as
antibacterial agents and this focus is now turning to include their use as
antifungal agents. Plants naturally express a vast array of antimicrobial
peptides as part of their innate immune system. One such protein, the
plant defensin NaD1, from Nicotiana alata, has potent antifungal activity
and is being trialed in the protection of transgenic crops. Its mechanism
of action involves entry into the cytoplasm of fungal hyphae where it
potentially interacts with an intracellular target. We are using various
fluorescence based techniques to elucidate how NaD1 traverses the
cell wall and plasma membrane with the ultimate aim of identifying the
intracellular target.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 159
POS-TUE-234
EXPRESSION AND PURIFICATION OF RECOMBINANT
BENZALDEHYDE DEHYDROGENASE AND BENZOATE
DIOXYGENASE FROM RHODOCOCCUS RUBER UKMP-5M
Hamzah A., Rabu A. and Tvakoli A.
School of Biosciences and Biotechnology, Faculty Science and
Technology, University Kebangsaan Malaysia, Bangi 43600 Selangor,
Malaysia.
Rhodococcus ruber UKMP-5M strain utilizing aromatic compounds
such as toluene and biphenyl was isolated from oil contaminated soils
in Malaysia. The bacterium was able to growth in 0.5 and 1 mM toluene.
The enzyme activity for toluene degradation was determined with
Horseradish peroxidase (HRP) and Indole-indigo method. The catechol
production was determined spectrophotometrically by monitoring
the activity of catechol 1,2 dioxygenase (260 nm) and catechol 2,3
dioxygenase (375 nm), respectively. The enzyme activity for catechol
1,2 dioxygenase was 0.0142 U/ml and for catechol 2,3 dioxygenase
was 0.007 U/ml. These two enzymes, benzaldehyde dehydrogenase
(xylC) which oxidized benzaldehyde to benzoic acid and benzoate
dioxygenase (benA) which involved in the catechol production
were identified from upper (hydrocarbon-carboxylic acid) and lower
(carboxylic acid-tricarboxylic acid cycle) pathways. The selected genes
were amplified by polymerase chain reaction and cloned into E. coli
expression system. The proteins were expressed at 37°C and 22°C with
1mM and 0.5 mM IPTG (isopropyl β-D-thiogalactoside), respectively.
Benzaldehyde dehydrogenase (xylC) is a 24 KD protein with pI of 5.22
and benzoate dioxygenase is 25 KD with pI of 10.38. The recombinant
proteins were purified by ion exchange chromatography on DEAE-
Sephacel, and SP sepharose for xylC and benA.
POS-TUE-236
PAS-DOMAIN INTERACTIONS OF DROSOPHILA AND
MOUSE PERIOD CLOCK PROTEINS
Hennig S.1, Strauss H.2, Arens J.1, Schulze S.3, Yildiz Ö3, Theiss C.1
and Wolf E.1
1
Max-Planck Institut für molekulare Physiologie, Dortmund, Germany.
2
Max-Planck Institut für Biophysik, Frankfurt, Germany. 3Nanolytics
Gesellschaft für Kolloidanalytik mbH, Potsdam, Germany.
Most organisms exhibit a day-night activity cycle of approximately 24
hours due to a circadian pacemaker (lat. circa: about; dies: a day) which
is operated by autoregulatory translational and transcriptional feedback
loops. The function of PERIOD proteins, as central components of
the circadian clock, is controlled by synthesis, cellular localization,
phosphorylation, degradation as well as specific interactions with other
clock components. Furthermore PERIOD is able to form homodimers
via its tandemly organized PAS (PER-ARNT-SIM) domains. Here we
solved crystal structures of PAS (PER-ARNT-SIM) domain fragments
of Drosophila PERIOD (dPER) and mouse PERIOD2 (mPER2) to get
insights into the regulatory role in the circadian clock at an atomic level.
In the case of Drosophila PERIOD we were able to show the importance
of both the hydrophobic Trp482 and a C-terminal helix for homo dimer
stabilization. The mPER2 crystal structure represents the first three
dimensional structure of a mammalian clock protein. In contrast to
the Drosophila protein, the mouse homolog shows a homodimer
stabilized solely by interactions of the Trp419 (Trp482 in dPER) with
the PAS-B-β-sheet surface. By solving the PAS-domain structure of
both the Drosophila PERIOD and mammalian PERIOD2 and due to our
biochemical experiments, we were able to give the first insights into the
molecular mechanisms of their homo dimerization.
POSTERS MONDAY & TUESDAY
POS-MON-237
THE THREE-DIMENSIONAL STRUCTURE OF
THE BIFUNCTIONAL FAD SYNTHETASE FROM
CORYNEBACTERIUM AMMONIAGENES REVEALS
OLIGOMERIC ASSEMBLIES
Herguedas B.1, 2 , Martinez-Julvez M.1, Frago S.1, Medina M.1 and
Hermoso J.A.2
1
Deparmento de Bioquimica, Biologia Molecular y Celular / Institute
for Biocomputation and Physics of Complex Systems (BIFI).
University of Zaragoza. Pedro Cerbuna 12, 50009-Zaragoza
(Spain). 2Crystallography Deparment. Instituto de Quimica-Fisica
Rocasolano. Spanish National Research Council (CSIC). Serrano 119,
28006-Madrid (Spain).
FMN and FAD are essential cofactors in flavoproteins, a large group
of proteins involved in many vital processes including photosynthesis,
fatty acid metabolism, mitochondrial electron transfer chain, apoptosis
and DNA repair. FMN and FAD are syntesized in vivo from Riboflavin
(RF, Vitamin B2) in two sequential steps, the first one is related with
a Riboflavin Kinase (RFK) activity, and the second with an FMN
adenylyltransferase (FMNAT) one. In prokaryotes, both activities
reside in a bifunctional enzyme, FAD synthetase (FADS), whereas
in eukaryotes two independent enzymes catalyze each reaction.
Here we present the crystal structure of the bifunctional FADS from
Corynebacterium ammoniagenes (CaFADS) solved at 1.95Å. The
structure of the protomer reveals two independent modules with the
catalytic sites placed 40Å away. In the protomer, the product of the
first reaction –FMN- must be released from the protein surface and
subsequently bind in the catalytic site of the second reaction, where
it is a substrate. Crystal packing interactions also reveal an hexameric
assembly formed by the interaction of two trimers. In the oligomer, both
distance and orientation of the active sites from different protomers are
compatible with the direct transfer of FMN between modules during the
FAD synthesis. These results provide evidences, at molecular level, on
the mechanism of synthesis of FMN and FAD in prokaryotes.
POS-MON-239
THE STRUCTURE OF BOO/DIVA REVEALS A
DIVERGENT BCL-2 PROTEIN
Hinds M.G.1, Day C.L.2 and Rautureau G.J.P.1
1
Walter and Eliza Hall Institute of Medical Research, Parkville 3052,
Australia. 2Department of Biochemistry, University of Otago, Dunedin
9054, New Zealand.
Intrinsic cell death is mediated by interaction between pro-apoptotic
and pro-survival proteins of the B-cell lymphoma-2 (Bcl-2) family of
proteins. To initiate apoptosis the α-helix formed by the BH3 domain
of BH3-only proteins interacts with the hydrophobic groove of a pro-
survival protein. Binding events leading to pro-survival neutralization
depend upon a combination of conserved and variable contacts and
are typically of high affinity. We present the structure, binding properties
and biological implications of Bcl-2 protein Boo. Boo has a structure that
is highly homologous to other multi-domain Bcl-2 proteins where seven
amphipathic helices surround a central hydrophobic helix that forms the
base of a surface exposed hydrophobic groove. Although structurally
homologous to other Bcl-2 family proteins, Boo has significant sequence
differences in the groove, and it lacks affinity for the binding domain of
pro-apoptotic proteins. In addition the canonical BH3 domain present
in all pro-apoptotic proteins is absent. These observations suggest that
Boo may function in a distinct manner to modulate apoptotic signalling
pathways.
Page 160 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
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UNDERSTANDING HOW FLIPS INHIBIT DEATH
RECEPTOR-MEDIATED APOPTOSIS
Mirams R.E.1, Vajjhala P.R.1, Stojanovski S.1, Lambert L.K.2 and Hill J.M.1
1
School of Chemistry and Molecular Biosciences, The University of
Queensland, Brisbane QLD 4072. 2Centre for Advanced Imaging, The
University of Queensland, Brisbane QLD 4072.
Upon stimulation, death receptors such as Fas/CD95 recruit the adaptor
protein FADD and procaspase-8 into the death-inducing signalling
complex (DISC). Assembly of the DISC promotes the dimerisation and
activation of procaspase-8 via an induced proximity mechanism. This
process can be inhibited by a family of cellular and viral proteins known
as FLIPs. cFLIP exists as long (cFLIPL) and short (cFLIPS and cFLIPR)
splice variants, all capable of protecting cells from apoptosis by blocking
procaspase-8 activation at the DISC. Several herpesviruses and
poxviruses also express FLIPs to suppress apoptosis and promote their
survival in host cells. The hallmark of FLIPs is the presence of tandem
death effector domains (DEDs) that interact with the complementary DED
of FADD and prodomain of caspase-8 to hinder caspase recruitment
and activation. However, the underlying mechanisms remain unclear.
At present structural information on the assembly and regulation of the
DISC is relatively limited and DED complexes have remained elusive.
To further characterise the molecular basis of FLIP-mediated inhibition
of apoptosis, we have optimised the production of FADD for structural
studies and successfully formed a stable FADD-FLIP complex. Here
we present a detailed structural and biochemical analysis of the FADD-
FLIP complex. Our results offer new insights into the mechanism by
which FLIPs subvert death receptor signalling.
POS-TUE-240
BACTERIAL DIAMINOPIMELATE EPIMERASE FORMS
AN ACTIVE DIMER
Hor L.1, 2, 3, Dobson R.C.J.2, 3, Dogovski C.2, 3, Hutton C.A.1, 3 and
Perugini M.A.2, 3
1
School of Chemistry, University of Melbourne, Parkville, VIC,
Australia. 2Department of Biochemistry and Molecular Biology,
University of Melbourne, Parkville, VIC, Australia. 3Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne,
Parkville, VIC, Australia.
Given the rise in antibiotic resistance, it is has become necessary to identify
novel antibacterial drug targets. One potential candidate is diaminopimelate
(DAP) epimerase, which is an enzyme in the lysine biosynthesis pathway
of bacteria and plants. DAP epimerase catalyses the stereoinversion of
LL-DAP to meso-DAP, which is a crucial component of bacterial cell walls.
This study has cloned, expressed and purified DAP epimerase from
E. coli. The recombinant enzyme is folded, as determined by CD
spectroscopy, and is active in solution. Quaternary structure analysis
employing analytical ultracentrifugation indicates that DAP epimerase
is dimeric in solution. In addition, we have recently solved the crystal
structure of E. coli DAP epimerase (2.0 Å) in its open, active conformation.
The crystal structure crystallised as a dimer, which is consistent with
solution study results. We anticipate the crystal structure will aid in the
discovery of novel inhibitors of this enzyme.
POSTERS MONDAY & TUESDAY
POS-MON-241
SELF-INTERACTION OF THE SULPHATE
TRANSPORTER, SHST1
Leves F.P.1, Tierney M.L.2, Price G.D.1 and Howitt S.M.1
1
Research School of Biology, Australian National University, ACT
0200. 2John Curtin Sxhool of Medical Research, Australian National
University, ACT 0200.
Many transporter proteins form higher order structures, involving either
self interaction or interaction with other proteins. The SulP (SLC26)
family is a diverse anion transporter family found in all domains of life.
Genetic evidence suggests that members of this family may interact
with other members and therefore that these transporters may act
as oligomers, rather than monomers. We investigated the ability of
the sulphate transporter, SHST1, to interact with itself using the split-
ubiquitin yeast two hybrid system. We identified an interaction between
SHST1 molecules suggesting that in vivo, this transporter does form
oligomers. Transporters of the SulP family contain an intracellular,
C-terminal domain, the STAS domain, which is homologous to anti-
sigma factors These proteins have a role in protein/protein interaction
making the STAS domain a good candidate for an interaction domain in
SHST1. Deletion experiments were used to test whether the interaction
between SHST1 molecules was due to the STAS domain. It appears
that interaction is mediated at least partly by the STAS domain, but a
successful interaction requires that this domain be correctly oriented with
respect to the membrane. Mutagenesis of conserved residues within
the STAS domain has been used to investigate the role of particular
residues on both sulphate transporter function and on self-interaction.
Some of these mutations affect both transport function and interaction,
confirming a role for the STAS domain in interaction between SHST1
molecules.
POS-MON-243
DISSECTING THE MECHANISM OF BINDING OF
ISOFORM-SELECTIVE PI 3-KINASE SMALL MOLECULE
INHIBITORS
Jennings I.G.1, Zheng J.1, Imran S.I.1, Pinson J.1, Schmidt-Kittler O.2,
Kinzler K.W.2, Vogelstein B.2 and Thompson P.E.1
1
Medicinal Chemistry and Drug Action, Monash Institute of
Pharmaceutical Sciences, Parkville, Victoria, Australia. 2Johns Hopkins
University, Sidney Kimmel Cancer Centre, Baltimore, USA.
A number of small molecule inhibitors of Class 1 phosphatidyl 3-kinases
(PI3K) are now in Phase 1 clinical trials with potential to be used in the
treatment of a range of diseases. However there has been little progress
in the development of isoform-selective inhibitors against the highly
conserved class 1 PI3K isoforms, p110α, β, γ & δ. Selective inhibitors
have the potential to reduce or eliminate unwanted side effects of these
treatments. We are interested in developing an α-specific inhibitor as this
isoform as it has been shown to be mutated in some cancers resulting
in increased enzyme activity. In an attempt to elucidate the structural
mechanism of selectivity we have examined the 3-dimensional crystal
structure of the PI3K α and γ isoforms and identified 2 regions of non-
conserved amino acids within the PI3K catalytic site which we postulate
will confer isoform selectivity towards small molecule PI3K inhibitors
which bind at or near the ATP binding site. We have produced a range
of in vitro mutants of these amino acids in p110α. These mutants have
been co-expressed with the regulatory subunit, p85, in insect cells with
the resulting complex purified. Results will be presented which correlate
a change in inhibitor isoform selectivity with a mutation as a means to
identify amino acids critical for isoform selectivity. In addition several
chemical classes of inhibitors have been assessed for their isoform
selectivity against the four isoforms of PI3K. This has been followed by
tuning of the α-selectivity by the addition of substituents to the original
scaffold.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 161
POS-TUE-242
VISUALIZING DIFFERENCE OF CONFORMATIONS OF
C-SRC IN LIVE CELLS USING TETRACYSTEINE TAGS
AND REASH REACTIVITY
Irtegun S., Schweiggert J., Mulhern T. and Hatters D.
Bio21, The University of Melbourne.30 Flemington Road.The
University of Melbourne. Victoria 3010 Australia.
c-Src is the prototypic member of the Src family kinases and its activity
is upregulated in a wide variety of human cancers. While the structural
basis of c-Src catalytic regulation is well understood, the temporal and
spatial features of SFK regulation in cells remain unclear. The activity of
all SFKs depends on whether their conformation is “open” or “closed”. In
the open active conformation, the protein has an extended configuration;
while in the inactive closed conformation the protein is more globular
and compact. Because many cancers involve dysregulated Src
function, it remains critical to establish new approaches to monitor
where, within cells, the active and inactive forms accrue and change
conformation in a dynamic fashion. Here we have developed novel
reporters of c-Src conformation which enable us to distinguish between
the “open” active and “closed” inactive states of c-Src in live cells in
real time. The localization of c-Src has been determined using Green
Fluorescent Protein (GFP) fused to the C-terminus of the protein, while
the conformation of c-Src has been monitored by the presence of a
genetically encoded tetracysteine (TC) tag, which binds the biarsenical
dye ReAsH in a conformation-sensitive manner. We have recently
validated that our c-Src reporters work independently of intrinsic kinase
activity, subcellular localization and cell morphological differences
attributed to Src activation.
POS-TUE-244
DEVELOPMENT OF FLUORESCENT CHEMOSENSOR
SUBSTRATES FOR HIGH THROUGHPUT SCREENING
OF THERAPEUTIC INHIBITORS AND ASSAY OF
ONCOGENIC PROTEIN TYROSINE KINASE ACTIVITY
IN CHRONIC MYELOGENOUS LEUKEMIA CELLS
Kamaruddin M.A.1, 2 , Hossain M.I.1, Jarasrassamee B.1, Thompson P.2,
Scanlon D.1, Cheng H.C.1 and Graham B.2
1
Department of Biochemistry and Molecular Biology, Bio21 Institute,
University of Melbourne. 2Department of Medicinal Chemistry and
Drug Action, Monash Institute of Pharmaceutical Sciences, Monash
University.
The Src family of protein tyrosine kinases (SFKs) are the most
extensively studied protein tyrosine kinases involved in the formation
and progression of many types of cancers such as leukemia, colon and
breast carcinoma. Recently, aberrant activation and over-expression
of the SFK members of Lyn and Hck kinases were found to induce
development of drug resistant to chronic myelogenous leukemia (CML).
For this reason, development of small molecule compounds capable
of inhibition of SFKs are urgently needed for the treatment of patients
suffering from cancer. To facilitate the search and development of
these therapeutic small molecular SFK inhibitors, we have designed
and synthesized fluorescent chemosensor peptides suitable for high
throughput assay for SFKs activity. In our design, we combine state
of the art solid phase peptide technology and the flexibility of the click
chemistry to develop a library of fluorescent chemosensor peptide
substrates for SFKs. Among them, some can be used to monitor SFK
activity in CML cells with high selectivity and sensitivity. The availability
of these chemosensor peptides permit high throughput screening of
potential inhibitors of SFKs.
POSTERS MONDAY & TUESDAY
POS-MON-245
A PROTEOMIC APPROACH TO INVESTIGATE
THE MECHANISMS OF SODIUM EXCLUSION IN
PHYSCOMITRELLA PATENS
Kelly L., Ford K. and Bacic A.
Australian Centre For Plant Functional Genomics, School of Botany,
The University of Melbourne, VIC, Australia.
Reduced productivity from agricultural land caused by increasing
levels of soil salinity is a growing problem. Plants have evolved a
variety of protective mechanisms to adapt to these conditions. These
mechanisms in general involve exclusion from the most sensitive
tissue, compartmentalisation in the vacuole where elevated salinity
is less detrimental to cellular processes, and the production of
compatible osmolytes to compensate for the associated osmotic
effects. Physcomitrella patens has been shown to be tolerant to a
variety of abiotic stresses including salinity. This species is able to
survive in sodium chloride concentration up to 600mM, if exposure is
gradual. Uniquely, among plants, Physcomitrella patens and the related
liverwort Marchantia Polymorpha possess Na+-ATPase (ENA) pumps
capable of Na+ extrusion, in addition to the Na+/H+ antiporters utilised by
higher plants to transport sodium across the plasma membrane. Much
remains unknown about this addition mechanism for sodium exclusion,
absent from higher plants and how this process is regulated. Both the
Na+/H+ antiporter and the ENA proteins have been identified by tryptic
digest and mass spectrometry (MS) in the membrane fraction from
Physcomitrella patens treated with 100 mM NaCl. The current work
describes the application of subcellular and posttranslational specific
enrichment strategies combined with analysis by MS to Physcomitrella
patens grown under various salt conditions to shed light on this recently
discovered mechanism for salinity tolerance.
POS-MON-247
EXPLORING MOLECULAR DETERMINANTS OF
PROTEIN-CARBOHYDRATE INTERACTIONS IN THE
CARBOHYDRATE BINDING MODULE ISOFORMS OF
AMPK
Koay A.1, Bieri M.1, Petrie E.J.1, Bailey M.F.1, Park K.2, Gooley P.R.1 and
Stapleton D.1
1
Biochemistry and Molecular Biology, University of Melbourne,
Parkville, VIC, Australia. 2Biology, University of Incheon, Incheon,
Korea.
The AMP-activated protein kinase (AMPK) is an evolutionarily conserved
enzyme essential in sensing and regulating metabolic processes.
Two mammalian β-subunit isoforms exist, each containing a central
carbohydrate-binding module (CBM). Using NMR and fluorescence
spectroscopy, we show that the ubiquitous β1-CBM and muscle specific
β2-CBM possess different affinities and specificity toward glycogen
mimetics. We find that the β2-CBM binds carbohydrate up to ten-fold
more tightly than the β1-CBM counterpart with an 80% sequence
identity. Additionally, we observe that β2-CBM binds optimally to
α->1,6 glucosyl maltoheptaose, a mimetic of single-glucose branched
portions in glycogen that will be exposed during glycogen degradation
by glycogen debranching enzyme. A three-fold increase in affinity for
glucosyl-maltoheptaose was observed in β1-CBM when a threonine
is inserted into position 102. This threonine is naturally present in β2-
CBM (Thr101) but not in β1-CBM. Conversely, deletion of Thr101 in
β2-CBM saw a three-fold loss in affinity for glucosyl maltoheptaose.
Thermodynamic and NMR studies reveal more favourable enthalpy
change and diminished chemical shift perturbations in β2-CBM over β1-
CBM when titrated with ligand, suggesting an optimized carbohydrate-
binding site in β2-CBM. Using structural and biochemical approaches,
we aim to define the molecular determinants governing specificity and
affinity toward carbohydrates in the CBMs,ultimately contributing to our
knowledge on the biological significance of isoform-specific roles in
AMPK.
Page 162 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-246
CHARACTERISING A CASPASE-2 CLEAVAGE SITE
CONSENSUS AND IDENTIFYING ITS SUBSTRATES
Kitevska T.1, Roberts S.J.1, Scott F.L.2 and Hawkins C.J.1
1
Biochemistry, La Trobe University, Bundoora, VIC, Australia.
2
Burnham Institute for Medical Research, La Jolla, CA, United States.
Apoptosis is a vital pathway which maintains cellular homeostasis. This
pathway activates cysteine-aspartate proteases known as caspases. To
date, thirteen mammalian caspases have been identified and most have
been well characterised. However, the role of some remains enigmatic,
including that of caspase 2. Substrates of caspases appropriately
illustrate their function. This study hypothesises that the identification
of novel caspase 2 substrates may well explain the recently attributed
activity of caspase-2 in cell cycle control and tumour suppression.
The aim of this study is to determine the minimal substrate specificity
of caspase-2 and use that to screen for caspase-2 substrates using
a bioinformatics approach. The P5-P1’ minimal substrate specificity
of caspase 2 was determined using a unique transcriptional reporter
system in the yeast, Saccharomyces cerevisiae. Based on this hexa-
peptide profile, a screen for proteins complying with this consensus
was carried out using the program, Probability of Protease Specificity
(PoPS). The candidate substrates revealed by this screen are now being
tested for caspase 2-mediated proteolysis in vitro. Substrates shown to
be susceptible to cleavage in vitro will then be further characterised.
POS-TUE-248
MOLECULAR DESIGN AND TARGETED CELLULAR
DELIVERY OF ENGINEERED HUMAN DEOXYCYTIDINE
KINASE TO ENHANCE METABOLIC ACTIVATION OF
NUCLEOSIDE ANALOG PRODRUGS AND IMPROVE
ANTICANCER THERAPY
Konrad M.1, Ort S.1, Mcsorley T.1 and Lavie A.2
1
Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen,
Germany. 2University of Illinois at Chicago, Dept. of Biochemistry and
Molecular Biology, Chicago, IL 60607, USA.
Nucleoside analogs (NA) are widely used in chemotherapy of cancer
and viral infections. These compounds are commonly administered in
their unphosphorylated form (prodrug). NAs cross the cell membrane via
nucleoside transporters and are then converted to their 5’-triphosphorylated
states (NA-TP) by nucleoside and nucleotide kinases. The intracellular NA-
TPs can efficiently terminate synthesis of nucleic acids in viral replication
and switch on the apoptotic cascade in cancer cells. Poor activation and
off-target cytotoxicity often limit the efficacy of these prodrugs. NAs, such
as AZT (3’-azido-3’-deoxythymidine) for the treatment of HIV infection, the
guanosine analogs acyclovir (ACV) and ganciclovir (GCV) used against
Herpes virus, or the anticancer compounds AraC (cytosine arabinoside)
and gemcitabine (2’-deoxy-2’,2’-difluorocytidine), are phosphorylated by
different kinases. The rate-limiting reaction in metabolic activation often is
the first, and in some cases the second phosphorylation step. Our aim is to
improve enzyme-NA systems that are of potential use in the treatment of
certain cancers. We engineered the human enzyme deoxycytidine kinase
(dCK) such that it phosphorylates the prodrugs AraC and gemcitabine more
efficiently than does the wildtype enzyme. Remarkably, this enzyme is highly
active in phosphorylating non-physiological L-enantiomers of NAs that
are less toxic in vivo and biologically more potent than the corresponding
D-forms. The modified dCK is delivered to tumour cells via Epidermal
Growth Factor Receptor (EGFR1 and 2)-specific single chain antibodies
(scFv) or affibodies, and induces apoptosis upon administration of NA.
Thus, our work highlights the success of a novel Antibody-Directed Enzyme
Prodrug Therapy (ADEPT) concept to significantly improve NA-dependent
cancer chemotherapy.- Ref.: Sabini, E. et al. (2003) Nat.Struct.Biol. 10, 513-
9. Sabini,E. et al. (2007) J.Med.Chem. 50, 3004-14. Sabini, E. et al. (2008) J.
Mol. Biol. 378, 607-21. Hazra, S. et al. (2009) Biochemistry 48, 1256-63.
POSTERS MONDAY & TUESDAY
POS-MON-249
CHANNELLING SCENT: NEW APPROACHES TO
UNDERSTAND THE STRUCTURAL BASIS OF INSECT
ODORANT RECEPTION
Kralicek A.V.1, Law A.L.M.1, 2, Carraher C.1, 2, Christie D.L.2 and
Newcomb R.D.1, 2
1
The New Zealand Institute for Plant & Food Research Limited, 120
Mount Albert Rd, Auckland 1142, New Zealand. 2School of Biological
Sciences, University of Auckland, Auckland 1010, New Zealand.
In animals the sense of smell is mediated by large families of membrane
proteins known as odorant receptors (ORs). We are one of the first
groups to show that insect ORs are not G protein-coupled receptors,
as mammalian Ors are, but have a distinct membrane topology and
ability to function in the presence of G protein signalling inhibitors1.
Instead they comprise a novel family of seven transmembrane ligand-
gated ion channels2,3. As a major step towards understanding insect OR
structure/function we have overexpressed and reconstituted ORs into
artificial membranes. This offers a unique opportunity to investigate the
stoichiometry and structural nature of the interaction between the ligand-
binding and ion channel subunits of the receptor complex. Additionally we
are using a comparative approach to understand where odor recognition
is encoded. We have identified orthologues of the Drosophila receptor,
Or22a, that differ in their response to 2-heptanone using cell-based
assays4 (D. melanogaster EC50 = 1.8x10 -7 M, D. mauritiana EC50 = 5.4
x10 -10 M) and determined EC50 values for a series of chimeric receptors
to locate a region necessary for ligand binding. This work is contributing
to the design and development of real-time olfactory biosensors using
insect ORs and the design of compounds for pest control. 1) Smart et
al. (2008) Insect Biochem. and Molec. Biol. 38: 770-780, 2) Sato K et al.
(2008) Nature 452: 1002-1006, 3) Wicher et al. (2008) Nature 452: 1007-
1011, 4) Kiely et al. (2007) J. Neurosci. Methods 159: 180-194.
POS-MON-251
THE NATURAL HISTORY OF MITSUGUMIN-53 AND THE
MEMBRANE REPAIR COMPLEX
Lemckert F.1, Waddell L.1, Tran J.1, Zheng F.1, Evesson F.1, Chen A.1,
Clarke N.1, Ma J.2, North K.1 and Cooper S.1
1
Institute for Neuroscience and Muscle Research, the Children’s
Hospital at Westmead & the University of Sydney, Australia. 2Robert
Wood Johnson Medical School, the University of Medicine and
Dentistry of New Jersey, Piscataway, USA.
Defects in the muscle membrane repair pathway cause muscular
dystrophy. Dyferlin, responsible for limb-girdle muscular dystrophy 2B
and Miyoshi Myopathy, was the first muscle membrane repair protein
identified. Caveolin-3 and annexinA1 have since been implicated in this
pathway; mutations in caveolin-3 cause human muscle disease. A new
protein, mitsugumin-53 (MG53), interacts with dysferlin and is involved
in muscle membrane repair. MG53 accumulates at sites of membrane
damage in normal muscle fibres, and MG53 knockout mice display a
progressive muscular dystrophy and impaired resealing of isolated
muscle fibres. We sequenced MG53 in 50 patients with muscular
dystrophy of unknown genetic basis in which all other common causes of
muscular dystrophy had been excluded. Although no primary mutations
were identified, we still consider it a feasible disease gene candidate.
Little is known about MG53’s role in skeletal muscle and how this
and other membrane repair proteins may be intrinsically modulated in
patients with muscle disease. We characterised the expression of MG53
and other membrane repair proteins in control and diseased muscle
by Western blot and immunohistochemistry, to determine if levels and
localisation of these proteins (the membrane repair complex) change
with age or in response to disease, and if any correlation exists between
intrinsic modulation of membrane repair pathways and disease severity.
We show that dysferlin, MG53, caveolin-3 and annexinA1 are markedly
upregulated in muscular dystrophy, and by varying levels in different
forms of dystrophy. We assessed the degree of protein upregulation
through quantitative Western blot and examined the effects of primary
dysferlin, dystrophin and caveolin-3 protein deficiencies on modulation
of the other membrane repair components.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 163
POS-TUE-250
STRUCTURAL BASIS FOR THE INHIBITION OF
APOPTOSIS BY EPSTEIN-BARR VIRUS BHRF1
Kvansakul M.1, 2 , Wei A.1, Fletcher J.I.1, Willis S.N.1, Chen L.1,
Colman P.M.1 and Huang D.C.S.1
1
La Trobe Institute of Molecular Sciences, La Trobe University,
Kingsbury Drive, Bundoora, Victoria 3086, Australia. 2The Walter &
Eliza Hall Institute of Medical Research, 1 G Royal Parade, Parkville,
Victoria 3050, Australia.
Viruses must evade host apoptotic defences to ensure their own
survival. Despite the complexity of mammalian cell death processes,
viruses have evolved successful mechanisms for subverting the
apoptotic machinery, including homologs of the mammalian pro-survival
protein Bcl-2. The ubiquitous Epstein-Barr virus (EBV), a member
of the γ-herpesviruses, infects the epithelium of the oropharynx and
resting B cells. Acute infection manifests as infectious mononucleosis
or glandular fever, whereas chronic EBV-associated transformation
is associated with Burkitt’s lymphoma, Hodgkin’s disease and
nasopharyngeal carcinoma. EBV BHRF1 is a sequence, structural
and functional homologue of Bcl-2, however its mechanism of action
remained unclear. Previous structural studies indicated that BHRF1
lacks an accessible BH3 binding groove, and shows only weak affinity
for BH3 ligands. We show that BHRF1 is a potent inhibitor of apoptosis,
and confers chemoresistance in mouse lymphoma models similar to
mammalian Bcl-2. Next, we determined the crystal structures of BHRF1
in complex with Bim and Bak BH3 peptides and show that in contrast to
previous predictions, BHRF1 interacts with these proteins in a manner
similar to its mammalian counterparts. Structure-based mutagenesis
enabled us to address the molecular mechanisms underlying BHRF1
activity. We demonstrate that BHRF1 can prevent Bak activation by
direct interaction, but prevents Bax activation indirectly by sequestering
the BH3-only proteins Bim, Puma and tBid. Unlike mammalian pro-
survival proteins, BHRF1 does not interact with the selective/sensitizer
BH3-only proteins. These studies indicate that BHRF1 might be targeted
by small molecule mimetics of BH3-only proteins.
POS-TUE-252
ANALYSIS OF HOMOTYPIC AND HETEROTYPIC
INTERACTIONS OF ARENAVIRUS Z MATRIX PROTEIN
Loureiro M.E., Levingston MacLeod J.M. and Lopez N.
Instituto de Ciencia y Teconologia Dr Cesar Milstein (CEVAN),
CONICET. Saladilo 2468 (C1440FFX). Buenos Aires, Argentina.
Arenaviruses, such as Tacaribe virus (TacV) and its closely related
pathogenic Junin virus (JunV), are enveloped viruses with a bipartite
negative-sense RNA genome which encodes the nucleocapsid
protein (N), the precursor of the envelope glycoprotein complex
(GP), the polymerase (L) and a RING finger protein (Z). TacV-Z is a
multifunctional protein that has a key role on virus morphogenesis. In
addition, Z is able to self-associate, and exhibits an inhibitory effect
on viral RNA replication and transcription through its interaction with
the L polymerase (J.Virol. 77:10383-93, 2003). We have previously
shown that the region comprised between the residues G36 and R85
of Z is sufficient to maintain Z-L interactions and Z inhibitory functions.
To learn more about the roles of individual amino acids in the different
interactions of Z, a panel of point mutants of TacV-Z and JunV-Z was
created by in vitro mutagenesis. The interaction between Z mutants
and L protein was analyzed by a coimmunoprecipitation assay and a
minireplicon system was used to examine the effect of mutations on
viral RNA synthesis. The capacity of Z mutants to self-associate was
also evaluated by coinmunoprecipitation and crosslinking essays. Our
results show that single amino acid changes selectively interfere with
Z-Z or Z-L interactions.
POSTERS MONDAY & TUESDAY
POS-MON-253
DIFFERENT DOMAINS OF THE DROSOPHILA
INSULATOR PROTEINS INTERACT WITH CP190 AND
E(Y)2 PROTEINS MEDIATING VARIOUS FUNCTIONS OF
DROSOPHILA INSULATORS
Maksimenko O., Bonchuk A., Stakhov V. and Georgiev P.
Institute of Gene Biology RAS, Moscow, Russian Federation.
Insulators affect interactions between promoters and enhancers or
silencers and function as barriers for spreading of repressive chromatin.
A core of insulator’s protein part is highly specific DNA-binding protein
which binds DNA sequence of insulator. Such proteins responsible
for activity of the Drosophila insulators are dCTCF, Su(Hw) and Zw5
proteins. It contain multi-zinc-finger DNA-binding domain connected
with other poorly studied domains. Recently centrosomal protein CP190
has been identified as functional component of Su(Hw) and dCTCF-
dependent insulators. CP190 co-precipitates insulator proteins but
details of that interaction remain unknown. In this work we found that
CP190 is a major protein which precipitates with purified GST-tagged
C-terminal part of dCTCF, middle part of Zw5 from S2 cells crude extract.
Using GST pull-down assay we confirmed that CP190 and dCTCF, Zw5
directly interacts in vitro. We mapped region of CP190 sufficient for
interaction with dCTCF, Su(Hw) and Zw5 insulator proteins in GST pull-
down assay. Surprisingly such a region (308-470aa) overlaps with a
microtubule-interacting domain of CP190 and is independent of its zinc-
finger domain. Then we have studied interactions with another insulator
component - E(y)2/Sus1. Earlier we demonstrated the specific role of
the E(y)2 protein in the barrier activity of Su(Hw) insulators. In this work
we found that E(y)2 protein interacts with dCTCF in the yeast two-hybrid
assay. Using GST pull-down assay we confirmed that the dCTCF and
Su(Hw) zinc fingers were essential for interaction with E(y)2. E(y)2 co-
precipitates insulator proteins dCTCF and Su(Hw) from S2 cells. Thus
we suggest that insulator complexes containing different DNA-anchor
proteins have general functional activities owing to the same protein
partners responsible for enhancer-blocking and boundary activities of
Drosophila insulators.
POS-MON-255
THE N-TERMINAL EXTENSION OF
HOLOCARBOXYLASE SYNTHETASE IS REQUIRED
FOR RECOGNITION OF PROTEIN SUBSTRATES
Mayende L., Swift R.D., Bailey L.M., Wallace J.C., Booker G.W. and
Polyak S.W.
Discipline of Biochemistry, University of Adelaide, Adelaide, 5005.
Protein biotinylation is an example of a protein:protein interaction
with exquisite specificity. Biotin protein ligase (BPL) catalyses the
activation of biotin-dependent enzymes, through the attachment of the
prosthetic group biotin. The catalytic region of all BPLs is contained
in the conserved C-terminal region. Human BPL or holocarboxylase
synthetase (HCS), contains a long N-terminal extension that is not
present in bacterial BPLs. The structure and function of the N-terminal
region is poorly understood. In order to delineate the role of this extension
the domain structure of HCS was mapped using limited proteolysis. Two
protease-sensitive linker regions were identified, one between residues
151-153, the other at residue 314. The domain containing residues 159-
314 was implicated in the reaction mechanism despite being distal to
the C-terminally located active site. Mutations within this domain give
rise to the metabolic disorder multiple carboxylase deficiency (MCD),
which is poorly responsive to current therapies. Novel mutations in this
N-terminal region were generated using ‘error prone’ PCR and isolated
through a genetic screen in bacteria. Residues mutated in these isolates
were identified by DNA sequencing and found to be those normally
highly conserved between species. Using yeast-two hybrid analysis we
present the first evidence for an interaction between the N-terminal and
C-terminal halves of HCS. The region of N terminal domain required
for interaction was mapped to the domain encompassing residues 159-
314. We show that mutations in the N-terminal region compromise the
interaction of HCS with its protein substrate, but not the intramolecular
interaction between the two halves. This provides a new mechanism for
protein biotinylation as well as a molecular explaination for MCD.
Page 164 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-254
CATHEPSIN B AS A TARGET FOR CONTROL OF
BLOOD-FEEDING PARASITES
Jilkova A.1, Horn M.1, Rezacova P.1, Kopacek P.2, Caffrey C.R.3 and
Mares M.1
1
Institute of Organic Chemistry and Biochemistry, Academy of
Sciences of the Czech Republic, 16610 Praha, Czech Republic.
2
Institute of Parasitology, Biology Centre of the Academy of Sciences
of the Czech Republic, 37005 Ceske Budejovice, Czech Republic.
3
Sandler Center for Drug Discovery, QB3, University of California San
Francisco, San Francisco, CA 94158, USA.
Hemoglobin digestion is an essential process for blood-feeding
parasites. In blood flukes and ticks, hemoglobin degradation is
orchestrated by a network of aspartic and cysteine peptidases operating
at acidic pH. Our work focuses on cathepsin B, a cysteine peptidase
functioning as both an exo- and endopeptidase. In the hard tick, Ixodes
ricinus, a vector of Lyme disease and tick-borne encephalitis, we
applied functional proteomic approaches to demonstrate that cathepsin
B (IrCB) is the most abundant digestive enzyme critical for hydrolysis
of hemoglobin fragments. Accordingly, IrCB represents a candidate
antigen for developing novel anti-tick vaccines. In the human blood fluke,
Schistosoma mansoni, cathepsin B1 (SmCB1) is a therapeutic target for
the treatment of schistosomiasis. We have solved the crystal structure of
SmCB1 and analyzed the inhibitory regulation of the enzyme active site.
The identified structural relationships represent a potential tool for the
development of antischistosomal drugs. (This work was supported by
grants P207/10/2183 (GACR) and IAA600960910 (GAASCR), research
projects Z40550506 and Z60220518, and the Sandler Foundation.).
POS-TUE-256
CHARACTERIZATION OF ASSEMBLY FACTORS
INVOLVED IN COMPLEX I BIOGENESIS AND DISEASE
McKenzie M.1, Lazarou M.1, Thorburn D.R.2 and Ryan M.T.1
1
1Department of Biochemistry, La Trobe University, Melbourne,
AUSTRALIA. 22Murdoch Childrens Research Institute, Royal
Children’s Hospital, Melbourne, AUSTRALIA.
Mitochondrial Complex I (NADH: ubiquinone oxidoreductase) is an
~980 kDa multimeric enzyme composed of 45 subunits, with 7 subunits
encoded by mtDNA and the remainder by nuclear genes. The assembly
of human Complex I is not well understood, complicated by its large size
and its regulation by two genomes. In recent years a number of proteins
have been described which aid the assembly of Complex I, however the
exact functions of these ‘assembly factors’ remains unclear. We have
now defined the roles of various assembly factors which act at either
early, mid or late stages of Complex I assembly. C20orf7 and C8orf38
are important during early stages, acting as potential transcriptional
activators of the mtDNA-encoded Complex I subunit ND1. Pathogenic
mutations in the genes of either of these assembly factors result in a
translation defect and/or the rapid turnover of ND1, leading to Complex I
deficiency and patient death. The assembly factor C6orf66 (NDUFAF4)
also acts at early stages of assembly, however it does not interact
with any mtDNA-encoded subunits, instead aiding the assembly of
early matrix arm intermediates. During the middle stages of Complex
I biogenesis the assembly factors CIA30 (NDUFAF1) and ECSIT both
directly interact with the subunit ND2, suggesting roles for both as
molecular chaperones. At later stages, CIA30, ECSIT, and the assembly
factor B17.2L (NDUFAF2) are found in larger Complex I assembly
intermediates of ~830 kDa. However, B17.2L only co-precipitated with
ECSIT, suggesting the existence of different ~830 kDa species which
contain 1) CIA30 and ECSIT after assembling from smaller intermediates
or 2) B17.2L and ECSIT following the release of CIA30. Our studies
have helped to define the function of different assembly factors during
various stages of Complex I assembly, and have also provided insights
into how defects in these proteins disrupt Complex I biogenesis and lead
to mitochondria disease.
POSTERS MONDAY & TUESDAY
POS-MON-257
ELUCIDATING THE MOLECULAR DETAILS OF RSSB-
MEDIATED TURNOVER OF SIGMAS BY THE AAA+
PROTEASE, CLPXP
Micevski D., Spall S.K., Truscott K.N. and Dougan D.
La Trobe Institute for Molecular Sciences, La Trobe University,
Melbourne 3086, AUSTRALIA.
In bacteria, promoter recognition and transcription initiation requires
both the RNA polymerase core enzyme and a sigma factor. There are
seven different sigma factors in Escherichia coli, each of which are able
to recognise a specific subset of genes. The SigmaS (also known as
RpoS) regulon comprises ~10% of all E. coli genes, and is crucial for
the cells response to a variety of different stresses including glucose
starvation and DNA damage. As the master regulator of what is often
referred to as the “general” stress response it is a key factor in bacterial
adaptation. Given this crucial role in bacterial adaptation, the cellular
concentration of SigmaS is tightly controlled, not only at the transcriptional
and translational levels, but also most importantly by proteolysis. The
turnover of SigmaS is an ATP-dependent process, which is carried out
by the AAA+ protease, ClpXP. Interestingly an additional factor - RssB
(Regulator of SigmaS B) - is also required for SigmaS turnover, however
to date the mechanism by which the adaptor protein RssB prepares or
delivers SigmaS for ClpXP-mediated degradation remains unclear. One
model suggests that RssB delivers SigmaS to the unfoldase ClpX via a
direct interaction between the C-terminal tail of RssB and the N-terminal
domain of ClpX. To dissect the role of RssB, in ClpXP-mediated SigmaS
turnover, we have performed a range of different experiments including
site-directed mutagenesis, half-life and co-immunoprecipitation
experiments. Collectively our data favours a direct delivery model.
POS-MON-259
DIHYDRODIPICOLINATE SYNTHASE:
O-AMINOBENZALDEHYDE ASSAY FOR HIGH-
THROUGHPUT CHEMICAL SCREEN AND
CHARACTERISATION OF CHROMOPHORE
Mitsakos V.1, 2, 3, Hutton C.A.1, 2 and Perugini M.A.2, 3
1
School of Chemistry, The University of Melbourne, Parkville, VIC,
Australia. 2Bio21 Molecular Science and Biotechnology Institute, The
University of Melbourne, Parkville, VIC, Australia. 3Department of
Biochemistry and Molecular Biology, The University of Melbourne,
Parkville, VIC, Australia.
In search of a new class of antibacterial agents, the exploration of
compounds to target the enzyme dihydrodipicolinate synthase (DHDPS)
is described in this study. The colourmetric o-aminobenzaldehyde
(o-ABA) assay is an assay suitable for a high-throughput chemical
screen. Optimisation studies in cuvette format and subsequently 96-
well format have allowed the derivatisation of kinetic data using the
o-ABA assay, with values matching those shown from coupled assay
studies. In addition, the purple chromophore of the o-ABA reaction
has been analysed by UV/Vis-spectrophotometry, NMR and MS and
found to be a diazaanthracene. The characterisation of the product has
provided confidence that the assay is suitable for quantitative work. A
high-throughput screen (88,000 compounds) against DHDPS from the
pathogen B. anthracis has shown several hits in the micromolar range,
which will allow the design of analogues with potent inhibition (nano-
picomolar range) against DHDPS.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 165
POS-TUE-258
A MECHANISM OF DNA HOMOLOGY SEARCH, BASED
ON THE STRUCTURE OF DNA BOUND TO PROTEINS
THAT PROMOTE HOMOLOGOUS RECOMBINATION
Masuda T.1, Ito Y.2, Terada T.3, Shibata T.1 and Mikawa T.1, 4
1
Cllular & Molecular Biology Unit, RIKEN Advanced Science Institute.
2
Department of Chemistry, Tokyo Metropolitan University. 3Graduate
School of Agricultural and Life Sciences, The University of Tokyo.
4
Biometal Science Laboratory, RIKEN SPring-8 Center.
Homologous recombination (HR) plays a critical role in genetic diversity.
The RecA/Rad51 family of proteins is well known for promoting HR in
an ATP-dependent manner. Recently, the crystal structure of the RecA-
ssDNA complex has been determined. In addition, ATP-independent
HR proteins with significantly different structures to that of RecA/Rad51
proteins have been identified. However, the mechanism of HR remains
unclear. In this study, we demonstrate a base rotation mechanism
for HR based on the structure of ssDNA bound to HR proteins. We
determined the structure of ssDNA bound to five evolutionarily distinct
HR proteins and found that these proteins induced a common ssDNA
structure characterized by long inter-base distances and reduced DNA
base stacking. Furthermore, the molecular dynamics simulations of
the RecA-ssDNA complex indicated that the bases in RecA filament
rotate significantly, providing a mechanism for the homology search
process of HR. Interestingly, RNA was unable to adopt this extended
conformation. Our results suggest that the extended structure of
ssDNA is a critical determinant of HR, and have provided DNA with
an important evolutionary advantage over RNA for the development of
genomic diversity.
POS-TUE-260
EBITEIN1, A NOVEL ERK1/2 BINDING PROTEIN
Miura K. and Imaki J.
Department of Developmental Anatomy and Regenerative Biology,
National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama
359-8513, Japan.
EBITEIN1 is a novel protein that binds with ERK1/2 that is one of the
classical MAP kinases. EBITEIN1 was found abundantly in round
spermatids. During spermatogenesis, EBITEIN1 was first translated
after meiosis when cells became haploid. Then, the amount of
EBITEIN1 protein gradually increased, reaching a maximum at
Oakberg’s stage 9. The level of EBITEIN1 decreased such that it
was undetectable when the flagellum of the spermatozoon was
generated. EBITEIN1 was localized to the cytoplasm on a subcellular
level. Binding experiments using various deletion mutants identified
a 40-amino acid minimal sequence for binding ERK2. Furthermore,
binding experiments using substitution mutants indicated the crucial
role of arginine residues in this sequence. Based on empirical and
bioinformatic analyses, we proposed two domains in EBITEIN1. One
is EB domain, the minimal sequence for binding ERK2, and the other
is ECT domain, the EBITEIN1 C-terminal domain. EBITEIN1 bound to
nonphosphorylated and phosphorylated forms of ERK1 and ERK2,
respectively. Phosphorylation and dephosphorylation experiments
indicated that EBITEIN1 is usually phosphorylated in vivo and that it
is a substrate of ERK2. The ERK2-binding domain was required for
phosphorylation of EBITEIN1. Based on these results, it is suggested
that EBITEIN1 is a phosphoprotein and a downstream interactor of
ERK2 that participates in the intracellular signal transduction pathway,
especially in the morphogenetic development of round spermatids into
spermatozoa. Although EBITEIN1 cDNA was originally cloned from a
mouse brains cDNA library, the functions of EBITEIN1 in the brains
remain to be identified.
POSTERS MONDAY & TUESDAY
POS-MON-261
ANALYSIS OF THE CONSERVED CX3C MOTIF IN TIM9
AND TIM10 PROTEINS OF THE MITOCHONDRIAL
INTERMEMBRANE SPACE
Mooga V.P., Baker M.J., Stojanovski D. and Ryan M.T.
La Trobe University.
About ~99% of mitochondrial proteins are encoded by nuclear genes,
synthesised in the cytosol and are imported into the organelle. Specific
translocases have evolved in the outer and inner mitochondrial
membranes to sort nuclear encoded proteins to one of four sub
compartments; the outer membrane, the intermembrane space, the
inner membrane and the matrix. The Tim9-Tim10 complex of the
mitochondria acts to chaperone the movement of selected hydrophobic
precursor proteins through the intermembrane space. The crystal
structure of the yeast Tim9-Tim10 complex revealed that it is structurally
similar to its human counterpart. The conserved dual Cx3C motif in
Tim9 and Tim10 allows the formation of two intramolecular disulfide
bonds, which stabilise the helix loop helix topology. To analyse the
significance of the disulfide bonds in the biogenesis of Tim9 and Tim10
in vivo, yeast with Tim9 and Tim10 cysteine mutants were generated.
Removal of both disulfide bonds in either Tim9 or Tim10 resulted in a
lethal yeast phenotype. However, deletion of the inner disulfide bond
in Tim9 or Tim10 (tim9C39S/C55S, tim10C44S/C61S) caused only a
temperature sensitive phenotype, while removal of the outer disulfide
bond (tim9C39S/C55S, tim10C40S/C65S) resulted in minor growth
defects. Substrate import and assembly studies suggest that a single
disulfide bond is sufficient for the biogenesis and function of Tim9 and
Tim10, with the inner disulfide bond appearing to be more important
than the outer disulfide bond.
POS-MON-263
COLD ADAPTATION OF PROTEINS: A BIOPHYSICAL
STUDY OF A PSYCHROPHILIC ALPHA-AMYLASE AND
ITS STABILIZED MUTANTS
Cipolla, A.C., Damico, S.D. and Feller, F.G.
Laboratory of Biochemistry, Center of protein Engineering, Institute of
Chemistry B6a, University of Liege, B-4000 Liege, Belgium.
Permanently cold environments, like polar regions, have been colonized
by a great variety of psychrophilic organisms producing enzymes
adapted to function efficiently at low temperatures. We have investigated
the role of weak interactions in thermal adaptation of proteins by site-
directed mutagenesis of the psychrophilc alpha-amylase (AHA) from the
Antarctic bacterium Pseudoalteromonas haloplanktis. Two stabilized
multiple-mutants (Mut5 and Mut5CC) have been constructed. The
single mutations were selected by comparison of the presence of weak
interactions in a mesophilic homolog from pig pancreas, PPA. The three
enzymes AHA, Mut5 and Mut5CC have been analyzed by differential
scanning calorimetry, thermal and chemical denaturation. The flexibility
has been studied by acrylamide-induced fluorescence quenching. In
order to investigate the kinetic origin of the gain in stability, the kinetics
of unfolding and refolding in GdmCl have been monitored at 15°C. The
newly introduced weak interactions stabilized the mutants, protected
them against heat and chemical unfolding and also induced an effective
loss of flexibility. In addition, the two multiple-mutants exhibit an
increased optimum temperature for activity. The first results of kinetic
studies show a similar refolding phase but differences between the
three amylases in the unfolding phase. These results unambiguously
support the capital role of weak interactions in the balance between
activity, flexibility and stability and provide a better knowledge of the
adaptation of enzymes to cold temperatures.
Page 166 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-262
PROTEOMIC ANALYSIS OF RICE ROOT CULTURES
(ORYZA SATIVA L. SUB. INDICA) DURING SODIUM
CHLORIDE STRESS
Bhorncharoennop C.1, Phaonakrop N.2, Jaresitthikunchai J.2, Uyen U.1,
Atikankun S.1, Roytrakul S.2 and Wetprasit N.1
1
Department of Biotechnology, Faculty of Science, Ramkhamhaeng
University, Bangkok 10240, Thailand. 2Genome Institute, National
Center for Genetic Engineering and Biotechnology, Pathumthani
12120, Thailand.
Saline soil is one of the major problems that limit rice production in
Thailand. In salt stress response in rice, root is the first organ that
perceives salinity. In order to improve the salt-tolerant ability in rice
cultivars, the differentially expressed proteins in rice root during salt
stress are necessary to investigate. The roots of 2-month old salt-
sensitive (Pathumthani 1) and salt-tolerant (Hom-Jan) rice seedlings
were cultured in sugar-free liquid MS (Murashige & Skoog) media before
applying the salt stress (513 mM NaCl). The roots were then collected
at different exposure times as 0, 3, 6, 9 12, 24, and 48 h. Total proteins
were extracted and subjected to SDS-PAGE separation, proteins from
systematically sectioned gel lanes were identified by nano LC-MS/MS
of tryptic peptides. The LC-MS data were analyzed using DeCyderMS
2.0 followed by MASCOT software. Eight proteins were observed
as differentially expressed following sodium chloride stress in both
varieties and 4 proteins were identified to be the functional proteins.
These proteins involved in oxidative stress, photosynthesis system and
cell wall organization. Of these 8 proteins showed down-regulation in
root culture of the salt tolerant Homjan rice while 7 proteins exhibited
up-regulation in root culture of salt sensitive Pathumthani 1. Only 1
protein in Pathumthani 1 root performed an increasing at first and then
gradually decreasing expression pattern. These results indicated the
extent of the salinity response in rice root cells and suggested a number
of key regulatory proteins and pathways that are involved in modulating
the response of rice root cells to salinity. These will lead to gain more
understanding about salt stress response that could be useful for crop
selection and improvement of salt-tolerant in rice.
POS-TUE-264
COMPARATIVE STUDY OF HIV-1 TAT TRANSIENTLY
AND TRANSGENICALLY EXPRESSED IN TOMATO
Cueno M.E., Yamato H., Hibi Y. and Okamoto T.
Nagoya City University, Nagoya City, Aichi, Japan.
Plant-based protein production has grown to be an ideal method for
transient protein production since it is free from animal pathogens and
has lower cost of production. Current methods of protein production in
plants include transient expression and transgenic plant development.
In this study, we noted the advantages and disadvantages of Tat
protein transiently expressed in tomato calli with Tat protein expressed
in a transgenic tomato plant. Plant-optimized gene constructs was
synthesized and introduced into leaf calli through bombardment. One
set of bombarded calli was maintained in zeatin-containing medium
to allow Tat transient expression. Another set of bombarded calli was
maintained in growth medium to allow Tat-expressing transgenic
tomatoes. Immunological assays determined the presence of Tat in
both sets of tomatoes. Effects of Tat expression on tomato were visually
determined and confirmed by measuring cytokinin levels. Likewise,
production time and thermostability of Tat from both tomato sets were
compared. Ultimately, Tat produced from both sets were introduced
intradermally to Balb/c mice and immunogenic responses were observed
through ELISA and ELISPOT. Both tomato sets showed preferential
fusion protein expression and thermostable production in room
temperature. Transgenically expressed Tat impaired plant development
by differentially expressing cytokinin oxidase resulting to lower cytokinin
levels. Transiently expressed Tat was found to be secreted and absorbed
from bombarded and naive tomato calli, respectively. Both humoral
and cellular immune responses were induced using either tomato.
Interestingly, higher cellular immune response was induced using
transiently expressed Tat as compared to transgenically expressed Tat.
Our results show more advantages in transiently expressing Tat in plant
calli than developing transgenic tomatoes expressing Tat.
POSTERS MONDAY & TUESDAY
POS-MON-265
EVALUATION OF ALTERNATIVE MECHANISMS FOR
THE EFFICIENT PRODUCTION OF A PLANT-MADE
VACCINE
De Guzman G.M., Walmsley A.M., Webster D. and Hamill J.D.
Monash University, Clayton, VIC, Australia 3800.
Using plants as an alternative vaccine production system to current
systems has shown promise with a range of vaccines produced in a
range of plant systems. Plants provide possible advantages for oral
delivery, reduced cost, large scale production and storage. In this study,
hairy root cultures were utilized for their rapid growth, high genetic
stability, potential for rhizosecretion, and their ability to be regenerated
into whole plants. The B-subunit of the heat-labile toxin from E.coli
was expressed in hairy root cultures of Tobacco, Tomato and Petunia
to evaluate LTB production in and between each species. The hairy
root cultures of Tobacco and Petunia expressed LTB antigen at high
concentrations of LTB ~70ug/g FW tissue on average, while tomato
produced low amounts of antigen (~2ug/g FW). Throughout the root
growth cycle, LTB was shown to peak after 22 days however, a marked
growth impairment was observed correlating to high expression of LTB,
interestingly petunia growth was shown to be least affected by high
levels of LTB. The ability of hairy roots to secrete LTB into the supplied
media was also confirmed in each species; however improvement of
secretion methods is required for effectively testing secretion patterns.
The ability of hairy roots to regenerate into whole plants and retain their
high antigen production was also shown and the ability for cultures to
be re-established form these plants has been also shown This research
demonstrates hairy roots as a viable system for vaccine production, and
in particular roots derived from Petunia where high production coupled
with low growth inhibition, secretion potential and regeneration ability
allow for a very promising production system.
POS-MON-267
NOVEL BIOACTIVE SECONDARY METABOLITES FROM
AUSTRALIAN MEDICINAL PLANT ENDOPHYTES
Fazendin Y.K.1, Lim K.F.2, Cahill D.1, Rookes J.1, Conlan X.1 and
Barnett N.W.1
1
Deakin University, Life and Environmental Sciences, Pigdons
Road, Waurn Ponds, Vic, Australia 3217. 2Deakin University, Life
and Environmental Sciences, 221 Burwood Highway, Burwood, Vic,
Australia 3125.
Plant endophytes are producers of biologically active secondary
metabolites that have provided new antibiotics, antiparasitics,
antimalarial, and anticancer agents. There has been limited investigation
of endophytic microorganisms associated with Australian traditional
medicinal plants. A range of plant species, incorporating four diverse
families and seven genera, were collected from State and National
Parks around Victoria, Australia. Of the eight plant species selected,
endophytic fungi and bacteria were found in association with the
leaves and stems of Psilotum nudum, Ajuga australis, and Phebalium
squameum. The fungal endophytes were isolated on potato dextrose
agar (PDA), 10% V8 agar, and purified on malt agar. The ITS-5.8S-ITS2
rDNA region of each of the endophytic fungi was PCR amplified using
universal primers, and gene sequenced. A BLAST search was conducted
to identify the fungal isolates to genus. A preliminary co-culture screen
of the fungal endophytes against several fungal plant pathogens showed
antifungal activity. A large-scale culture of an endophytic fungus in
PDA broth (10 x 500 mL, 5 L) at room temperature (20–25 ° C) was
undertaken, followed by extraction of mycelia and growth media with
ethyl acetate. The extract is currently being screened for activity against
four bacterial strains, including an antibiotic resistant strain of E. coli),
and antioxidant activity using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging activity test and an acidic potassium permanganate
(KMnO4) chemiluminescence method. Anticancer activity will also
be tested on several cancer cell lines. Bioassay-guided fractionation,
and a range of spectroscopic techniques will be used to isolate and
characterise the bioactive constituent(s).
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 167
POS-TUE-266
DIFFERENTIAL ANALYSIS OF VITAMIN D TREATED
U937 MYELOID LEUKEMIC CELLS
Durand F.M.1, 2 , Levina V.1, 2, Cooke I.1, 2, Coley A.1, 2, Hoogenraad N.2
and Talbo G.H.1, 2
1
CRC for Biomarker Translation, La Trobe University, Melbourne,
Australia. 2:La Trobe Institute for Molecular Science, La Trobe
University, Melbourne, Australia.
Unlike normal progenitor cells, haemopoietic cells from patients with
acute myeloid leukemia have an apparent inability to differentiate
and terminally mature. U937 myeloid leukemic cells can be induced
to differentiate into macrophages by 1α,25-dihydroxyvitamin D3
(1α,25(OH)2D3). Growth arrest of these cells has been shown to
be blocked at G1 in the cell cycle, happening concomitantly with an
upregulation of the cell surface membrane proteinaceous monocyte/
macrophage-specific markers, CD14 and CD11b. Granulocyte-
macrophage colony-stimulating factor (GM-CSF), when used in
conjunction with 1α,25(OH)2D3 has been shown to have a synergistic
effect on U937 cells resulting in an increased upregulation of these
cell surface markers. Three batches of U937 cells were grown for
96 hours in media plus vehicle, media plus 1α,25(OH)2D3 and media
plus 1α,25(OH)2D3 and GM-CSF. Aliquots of cells were subjected to
our differential proteomics protocol. In short, the glyco moiety of the
outer membrane proteins were oxidised by NaIO4, the cells were lysed
and the oxidised carbohydrate covalently bound to hydrazide coupled
beads. The covalent binding of the proteins facilitates extensive washing
to remove membrane lipids and other debris. The bead bound proteins
were reduced, alkylated and digested by trypsin. The residual tryptic
glycopeptides were released by PNGase F and analysed by LC-ESI-
microTofQ-MS/MS as seen in the poster titled: “Analytical Prerequisites
for Differential and Quantitative Proteomics”. The known cell surface
proteinaceous marker CD11b was shown to be upregulated on the
1α,25(OH)2D3 as well as the 1α,25(OH)2D3 and GM-CSF treated cells,
while the marker CD14 upregulated on the 1α,25(OH)2D3 and GM-CSF
treated cells, only.
POS-TUE-268
OXIDATION OF TETRAHYDROPTERINES BY
QUINONES
Garcia Molina F.1, Muñoz Muñoz J.L.1, Martinez Ortiz F.2, Varon R.3,
Tudela J.1, García Canovas F.1 and Rodriguez-Lopez J.N.1
1
GENZ: Grupo Investigación Enzimologia.Deparatamento de
Bioquímica y Biologia Molecular A. Facultad de Veterinaria. Campus
de Espinardo. E-30100, Murcia (Spain). 2Grupo de Investigación
de Electroquímica Teórica y Aplicada.Departamento de Química
Física. Facultad de Química.Universidad de murcia. A. Correos
E-30100. Murcia, Spain. 3Departamento de Química-Física. Escuela
de Ingenieros Industriales de Albacete. Universidad de Castilla la
Mancha. Avda. España s/n. Campus Universitario, E-02071, Albacete,
Spain.
Tetrahydrobiopterine (6BH4) can diminish the oxidative stress of
keratinocytes and melanocytes by reducing the o-quinones generated
by the oxidation of the corresponding o-diphenols. In this work, we
demonstrate that 6BH4 and its analogues methyltetrahydropterine (MBH4)
and dimethyltetrahydropterine (DMBH4) can reduce all the o-quinones
studied, including 1,2 benzoquinone but not p-benzoquinone. The
formal potentials of different quinone/diphenol pairs obtained by square
wave voltammograms indicate that the o-quinones with withdrawing
groups are more potent oxidants than those with donating groups.
This work has been partially supported by grants from several Spanish
organizations. Ministerio de Educación y Ciencia (Madrid, Spain)
Project BIO2009-12956, from the Fundación Séneca (CARM, Murcia,
Spain) Projects 08856/PI/08 and 08595/PI/08, from the Consejería de
Educación (CARM, Murcia, Spain) BIO-BMC 06/01-0004 and from the
Consejería de Salud y Bienestar Social de la Junta de Comunidades
de Castilla La Mancha, Project FISCAM PI-2007/53. JLMM has a
fellowship from the Ministerio de Educación y Ciencia (Madrid, Spain)
Reference AP2005-4721. FGM has a fellowship from Fundación Caja
Murcia (Murcia, Spain).
POSTERS MONDAY & TUESDAY
POS-MON-269
CONSERVED BLOCK7 SEQUENCE OF BACILLUS
THURINGIENSIS CRY TOXINS. -THE INNOVATIONAL
PEPTIDE-TAG FOR EFFICIENT PROTEIN PRODUCTION-
Hayakawa T., Shimizu Y., Ishida T. and Sakai H.
Graduate School of Natural Science and Technology, Okayama
University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
We have developed a novel system to prepare proteins effectively
by using peptide-tag derived from the mosquitocidal Cry4Aa toxin of
a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag,
designated 4AaCter, facilitates the formation of crystal-like inclusion
bodies of glutathione S-transferase in Escherichia coli without loosing
the enzyme activity. In this study, we analyzed functional motif, in
particular Block7, which may work for formation of crystal-like inclusion
bodies. Block7 is a stretch of amino acid sequence conserved among
Cry toxins. We selected fourteen Cry toxins and synthesized the genes
encoding their Block7s. The Block7 peptides were expressed as GST
fusions and formation of crystal-like inclusion bodies in E. coli was
analyzed. As a result, among the 14 tested Block7 peptides, those from
Cry5Ba, Cry32Aa, and Cry48Aa formed crystal-like inclusion bodies as
well as that of Cry4Aa. But no inclusion observed for Cry47Aa Block7.
Formation efficiency of crystal-like inclusion body for the remaining 10
of the tested Block7 peptides was ranging from 39 to 66%. Thus, the
conserved Block7 may be one of factors responsible for crystallization
of Cry toxins, but the role of Block7 may vary with type of Cry toxin.
In addition, our results suggested the possibility to design a shorter
peptide-tag based on Block7 which form protein crystal efficiently.
Results of detailed mutational analyses will also be presented.
POS-MON-271
EFFICIENT PRODUCTION OF THE RECOMBINANT
CYSTATIN C USING INNOVATIONAL PEPTIDE-TAG
DERIVED FROM BACILLUS THURINGIENSIS CRY
TOXIN
Iwamoto S.1, Fan K.2, Sato S.1, 3, Hayakawa T.3, Sudo S.1 and Sakai H.3
1
Japan Lamb CO.,LTD, Department of Bioscience, Development
Division, Okayamadai-incubator 108, 1-1-1 Tsushima-Naka, Okayama
700-0082, Japan. 2JSR Corp., 25 Miyukigaoka, Tsukuba-shi,
Ibaraki, 305-0841, Japan. 3Graduate School of Natural Science and
Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku,
Okayama 700-8530, Japan.
Cystatin C is a potent inhibitor of lysosomal proteinases and produced by
all human cells with a nucleus. Since Cystatin C is a biomarker of kidney
function, the recombinant protein is highly demanded in the market of
diagnosis agent. In this study, we present an innovational production
system of the recombinant Cystatin C using a peptide-tag derived from
Bacillus thuringiensis Cry toxin. Fusion with this peptide tag, designated
4AaCter, facilitates the formation of alkali soluble protein inclusion in
Escherichia coli. The recombinant Cystatin C fused with 4AaCter at the
N-terminal end was successfully expressed and accumulated as protein
inclusion in E. coli. The yield of 4AaCter-Cystatin C was 40mg/L culture
and was five fold higher than that of Cystatin C without the tag. The
inclusion of 4AaCter-Cystatin C was solubilized in denaturing buffer and
purified by tag-based affinity chromatography. The purity of the final
product was more than 90% and the final yield was 8mg/L culture.
Western blotting revealed that the recombinant protein reacted with
anti-Cystatin C antibody as well as that of native Cystatin C. In addition,
latex agglutination assay showed that the reactivity of the recombinant
Cystatin C by our method was higher than that of the native one. The
novel system we have developed using 4AaCter is very simple and
enables efficient production of the recombinant Cystatin C with superior
quality in low cost.
Page 168 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-270
DEVELOPMENT OF ANALYTICAL METHODS FOR
SEQUENCING OLIGONUCLEOTIDES USING MALDI-
TOFMS
Minohata T.1, Hewetson J.W.2 , Yamazaki Y.1 and Yamada M.1
1
Applications Development Center, Analytical Applications
Department, Shimadzu Corporation, Kyoto, Japan. 2Shimadzu
Scientific instruments (Oceania) Pty Ltd., Rydalmere, Australia.
Studies of oligonucleotides containing both RNA and DNA are of great
interest in relation to the development of oligonucleotide therapeutics and
fundamental research. It has been recognized that various sequences
of oligonulcleotides indicate antisense effect or RNA interference, which
are responsible for effective and specific gene suppressions to regulate
expressions of unwanted proteins in living system. Although demand
for a precise sequencing method of short length oligos (20-30 mer) is
increasing to study biological function, only a few analytical methods
have been reported, such as acid-hydrolysis and nuclease treatment
in conjunction with mass spectrometry. On the other hand, MALDI-
TOFMS has been mainly applied to the detection of oligonucleotide
molecular weight. We will report studies of two sequencing methods
using MALDI-TOFMS, including on-plate acid-hydrolysis and in-source
decay (ISD) on MALDI-TOFMS. We modified a previous report of acid-
hydrolysis for DNA sequencing and investigated favorable conditions
for the reaction of sequencing 19 mer RNA. Hydrolysis was conducted
directly on the MALDI plate with a mixture of matrix, strong acid and
RNA, and then analyzed by MALDI-TOFMS. Conditions were optimized
to generate many ladder signals, between which mass differences
represent constituent units of ribonucleotides, thus the sequence of
the RNA was clearly observed. Notably, it was possible to apply these
conditions to the analysis of 2-O-methylation of siRNA. Whilst ISD,
which is a degradation reaction inside the ion source of MALDI, has
been applied to protein sequencing, this method has been rarely applied
to RNA analysis. We will report successful results of the modified
siRNA by using ISD, and introduce newly developed software for the
interpretation of results.
POS-TUE-272
PROTEIN STRUCTURE DETERMINATION FROM
PSEUDOCONTACT SHIFT
Schmitz C.1, Vernon R.2, Otting G.3, Baker D.2 and Huber T.3
1
Bijvoet Center for Biomolecular Research, Utrecht University, The
Netherlands. 2Department of Biochemistry, University of Washington,
Seattle, USA. 3Research School of Chemistry, Australian National
University, Australia.
The pseudocontact shift (PCS) effect, induced by a bound paramagnetic
lanthanide ion, is becoming widely used in protein nuclear magnetic
resonance (NMR) spectroscopy as it yields a complementary
combination of orientational and (long range) distance restraints. This
versatile effect can be accurately determined with highly sensitive
NMR experiments and has been successfully used (i) to automatically
assign NMR resonances, (ii) to determine the structure of protein-
protein complexes and protein-ligand complexes, and (iii) to refine NMR
structures. However, it has been speculated whether or not PCS data
as the only experimental restraints are sufficient for de novo structure
determination of a protein. Here we show that 3D structures of proteins
can reliably be determined using PCS data from a single metal binding
site combined with backbone chemical shifts. We present results
from a statistically meaningful number of proteins with different folds
ranging in size from 56 to 186 amino acids and show that PCS restraints
implemented in the fragment assembly step of the ROSETTA software
is highly efficient in biasing the sampling of the conformational space
towards the correct target structure. We further show that the best
structures computed have a backbone RMSD from the native structure
as low as 1.0 Ångstrom. Finally, the question whether we are performing
structure prediction assisted by experimental data, or truly de novo
structure determination is discussed.
POSTERS MONDAY & TUESDAY
POS-MON-273
INNOVATIVE STRATEGY FOR EFFICIENT PROTEIN
PRODUCTION USING A NOVEL PEPTIDE TAG DERIVED
FROM CRY TOXIN
Sakai H.1, Sato S.1, 2, Iwamoto S.2, Sudo S.2, Sakamoto Y.1, Uchida M.1,
Matsushima K.1, Kashino Y.1 and Hayakawa T.1
1
Graduate School of Natural Science and Technology, Okayama
University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan. 2Japan
Lamb CO., LTD, Department of Bioscience, Development Division,
Okayamadai-incubator 108, 1-1-1 Tsushima-Naka, Okayama 700-
0082, Japan.
We are often confronted with difficulties that some proteins cannot be
efficiently produced by ordinary recombinant DNA techniques based on
cloned genes. The difficulties may be because the recombinant protein
products are unstable or have deleterious effects on the host cell. To
overcome the difficulties in producing these difficult-to-produce proteins,
some innovative recombinant DNA techniques for heterologous protein
production are eagerly desired. We developed an innovative procedure
to produce proteins efficiently by using a novel peptide tag derived from
the insecticidal Cry toxin of a soil bacterium, Bacillus thuringiensis. Fusion
with this peptide tag, designated 4AaCter, facilitates the formation of
crystal-like inclusion bodies of glutathione S-transferase in Escherichia
coli without loosing the enzyme activity. For all the target proteins tested
to date, it was proved that the recombinant proteins were accumulated
in the crystal-like inclusion bodies and their biological activities were
also maintained. Application of 4AaCter to the production of syphilis
antigens, TpN15, TpN17 and TpN47 from Treponema pallidum, yielded
excellent results, including a dramatic increase in the production level,
simplification of the product purification and high reactivity with syphilis
antibody. The use of 4AaCter may provide an innovational strategy for
the efficient production of difficult-to-produce proteins.
POS-MON-275
CHLAMYDOMONAS REINHARDTII. - AN ALGAL
FEEDSTOCK FOR LIQUID BIOFUELS
James G., Hocart C., Hillier W. and Djordjevic M.
Plant Science Division, Research School of Biology, The Australian
National University, Canberra, Australia.
C. reinhardtii is a model algal system of intense research focus as
a feedstock for the production of biodiesel and the processing of
hydrocarbons to jet fuel (Wang et al., Eukaryot. Cell, Dec 2009; 8: 1856-
1868, Moellering et al., Eukaryot. Cell, Jan 2010; 9: 97-106, Yanto et al.,
Metabolic Engineering, doi:10.1016/j.ymben.2010.02.002,. C. reinhardtii
usually stores excess carbon as starch when nutrient deprived with
only limited production of neutral lipids (triacylglycerols). Mutations
in the starch biosynthetic pathway cause carbon to be redirected into
lipid biosynthesis and storage triacylglycerols that are suitable for liquid
biofuels. The fatty acid profiles of wild-type and starch mutants were
quantified by gas chromatography mass spectrometry. We found C.
reinhardtii starch mutants produce significantly elevated levels of 16:0,
18:1 and 18:3 fatty acids that are suited for producing biofuels. Fluorescent
spectroscopy and fluorescent activated cell sorting was used to develop
high-throughput methods for screening neutral lipid accumulation in
cells. Global transcriptional profiling by RNA-sequencing is underway
on the Illumina platform to identify a putative gene set associated with
the lipid biosynthetic pathway and to understand the molecular basis for
how cells switch to lipid synthesis. We conclude that C. reinhardtii is an
alga that can be manipulated to produce high levels of triacylglycerols.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 169
POS-TUE-274
FUNCTIONAL MICROPATTERNS TO CONTROL CELL
BEHAVIORS
Ho C., Kumar G. and Co C.
University of Cincinnati.
Microfabrication techniques are widely used in the electronic industry
to generate small features with size between 1-100 μm. This size
range is on the same order of a single cell, thus, these microsystems
are well suited for studying cell behaviors. This talk will demonstrate
some examples of using micropatterned surfaces to control local
environment around the cell. Micropatterned surfaces were used to
control the cell size and shape. Endothelial cells patterned within the
20 µm grooves formed capillary tube-like structure containing a central
lumen. Capillary networks embedded in other tissue specific cell types
can be formed on the biomaterials for creating vascularized tissue.
Patterned biomaterials can be applied to guide neurons to extend axon
and neurite for creating neuronal networks. We have also devised a
completely novel microarray-based technique to amplify the natural
directional persistence of migrating cells (MANDIP). Using MANDIP,
we can amplify this directional persistence to coerce the migration of
cells indefinitely along arbitrary paths in one preset direction without
chemoattractants, gradients in substrate adhesiveness, or external fields.
Potential applications of MANDIP include cell-sorting, drug screening,
tissue engineering, wound healing, and mechanistic studies of cell
migration, cell-cell interactions, and other cellular processes requiring
temporal and spatial regulation. We have devised and are seeking to
commercialize a simple in vitro migration assay that offers significant
improvements in reliability and ease of implementation compared to
traditional “wound healing” assays. Instead of mechanically wounding
cells, which leads to interferences caused by dead cell debris that block
cell movement and molecules released by wounded cells that alter
artificially the rate of cell migration, confluent groups of cells, initially
confined within patterns of cell-resistant polyelectrolyte, are released
by electrostatic adsorption of a second, cell adhesive polyelectrolyte.
POS-TUE-276
FLUOROMETRIC APPROACH TO SELECTIVITY
SCREENING OF MODIFIED OLIGONUCLEOTIDE
HYBRIDIZATION PROBES
Kabilov M.R. and Pyshnyi D.V.
Institute of Chemical Biology and Fundamental Medicine SB RAS.
Today various approaches to the detection of point mutations in DNA
are used. A number of them are based on principles of allele-specific
hybridization (ASH). To improve the efficiency in discrimination of
imperfect duplexes, there is a reason to look for oligonucleotide
analogues and derivatives with higher hybridization efficiency as
compared to their native precursors. In most studies, however, there
are no detail characteristics of the influence of modified probes on the
selectivity of the analysis because this problem requires the in-depth
thermodynamic analysis. Properly speaking, the efficiency of mismatch
discrimination should be characterized by comparable thermodynamic
parameters (enthalpy, entropy, and free energy) for the formation of
perfect and mismatched complexes. Just these values determine the
formation efficiency (the extent of association) of duplexes composed
by an oligonucleotide probe under given conditions. The use of the
standard real-time PCR detection system allows one to detect the
change of the fluorescence level when heating samples. The advantage
of this system is the opportunity of the parallel analysis of a large number
of probes at rather low concentrations. We have considered various
options of the fluorescent labeling of both probes and DNA templates,
which should ensure the optimal level of the signal. We attempted to
design a system, which could answer the question “is the modified
probe more selective than native one?” by the analysis of melting
curves. The parallel examination of the same oligonucleotide probes
using the UV melting technique confirmed our results. The results
demonstrate a possibility of the massive parallel analysis of selectivity
of modified oligonucleotide probes based on the thermal denaturation
of fluorescent DNA complexes.
POSTERS MONDAY & TUESDAY
POS-MON-277
ROLE OF CARBOHYDRATE BINDING DOMAIN
ON EXPRESSION, ACTIVITY AND STABILITY OF
XYLANASE C OF CLOSTRIDIUM THERMOCELLUM
Khan M.I.M., Sajjad M. and Akhtar M.W.
School of Biological Sciences, University of the Punjab, Lahore-54590,
Pakistan.
Xylanase (XynC), a major component of Clostridium thermocellum
cellulosome, was cloned and expressed with and without carbohydrate
binding domain. Four constructs with the gene encoding only the
catalytic domain (xynC CD), the catalytic domain with binding domain at
N-terminus (xynC CBD-CD), the catalytic domain with binding domain at
C-terminus (xynC CD-CBD) and the catalytic domain with binding domains
at both termini of the catalytic domain were produced (xynC CBD-CD-CBD).
The XynCCD, XynCCBD-CD, XynCCD-CBD and XynCCBD-CD-CBD expressed at
levels ~45%, ~30%, ~30% and ~33% of the total E. coli cell proteins after
induction with lactose, as analyzed by photodensitrometric scanning of
SDS-PAGE gels. The overall activities produced by XynCCD, XynCCBD-CD,
XynCCD-CBD and XynCCBD-CD-CBD were 1,488, 3,489, 3,860 and 5,732 U l-1
OD600 -1 against oat spelt xylan, respectively. All these enzymes with and
without non-catalytic domains were found to be quite stable over a broad
pH range (pH 4 - 9). XynCCBD-CD was more thermostable as it retained
70% activity on incubation at 70ºC for 2 hrs whereas XynCCD lost total
activity under these conditions. XynCCD-CBD retained 56% and XynCCBD-
CD-CBD
retained 80% of its activity under these conditions. Km values
for XynCCD, XynCCBD-CD, XynCCD-CBD, and XynCCBD-CD-CBD as determined
on soluble xylan as substrate were 3.12, 3.57, 3.12 and 1.64 mg ml-1,
respectively. Thus presence of carbohydrate binding domain on both
terminals leads to the 4 fold increase in specific activity as compared to
the native enzyme.
POS-MON-279
CHARACTERISATION OF THE GRAIN SPECIFIC
HOMEODOMAIN TRANSCRIPTION FACTORS FROM
WHEAT
Kovalchuk N., Wu W., Bazanova N., Singh R., Ismagul A., Eliby S.,
Johnson A., Hrmova M., Langridge P. and Lopato S.
Australian Centre for Plant Functional Genomics, Hartley Grove,
Urrbrae, South Australia 5064, Australia.
Two HDZip class IV genes, designated Triticum aestivum GLABRA like
7 and 9 (TaGL7 and TaGL9), were isolated using Y1H to screen a cDNA
library from developing wheat grains (0-6 DAP) with the palindromic
repeat -CATTAAATG- as bait. A predicted molecular model of TaGL9
suggested that at least five independently folded domains could be
present, although their spatial distribution is hypothetical at this stage.
The identified protein domains are currently being used as baits in the
Y2H screen of a wheat endosperm library. The 3’UTR of TaGL7 and
TaGL9 were used as probes to isolate genomic clones of orthologues/
homologues from a Triticum durum BAC library. Full length genes
containing 3 kb-long promoter regions were designated TdGL7 and
TdGL9. Spatial and temporal expression patterns of TdGL7 and TdGL9
were examined in transgenic wheat, barley and rice expressing promoter-
GUS fusion constructs. Whole-mount and histochemical GUS staining
patterns were analyzed in large numbers of independent transgenic
lines. Although very low activity of the TdGL7 promoter was detected
in most plant tissues, much stronger GUS expression was observed in
the female gametophyte before fertilization, and later in the syncytial
and starchy endosperm. The TdGL9 promoter was active only in grain.
GUS activity was initially observed on the 5th day after pollination and
persisted in the embryo until grain harvest. Small differences were
observed in the spatial patterns of GUS expression in wheat, barley and
rice. Wheat transgenic plants with constitutive overexpression of TaGL7
and also with antisense RNA expressed under the TdGL7 promoter
were generated and are currently being analysed.
Page 170 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-278
IMMUNOLOGICAL MEASUREMENT OF TROPONIN I IN
SERA FROM THE PATIENTS WITH HEART DAMAGE
DISEASES
Kim H.J.1, Kim T.K.1, Wang H.1, Oh S.W.2 and Choi E.Y.1
1
Department of Biomedical Science, Hallym University, Chuncheon,
South Korea 200-702. 2Department of Biology Education, Institute of
Fusion Science and Science Education, Chonbuk National University,
Jeonju, South Korea, 561-756.
A cardiac-specific iso-form of Troponin I (cTnI) has been known as
a marker of heart damage for more than 20 years. At present cTnI is
considered to be one of the most specific and sensitive markers of
myocardial cell death. For the preparation of antigen, we produced a
human cardiac troponin I recombinant protein. The cardiac toponin
I clones were transformed into BL21 cell for expression and the
recombinant cTn I protein was purified by affinity chromatography.
The cTnI protein was confirmed to be an expected size on an SDS-
PAGE gel. To generate monoclonal antibodies against the protein,
recombinant cTnI was injected into BALB/C mice. From the fusion
experiments, several hybridomas were selected by Western blotting and
further screened by ELISA method. With the monoclonal antibodies, we
developed a POCT type (point of care of test) immunoassay system
and conducted performance evaluation for measuring cTnI in human
serum. The linearity fell in the range 0-10ng/ml of cTnI and the analytical
detection limit was 0.5ng/ml of cTnI. The precision of intra- and inter-
assay was CVs<6% and CVs<8%. Also, we found that the amount of
cTnI protein mass in the Heart damage patients was much higher level
than that in normal population. The performance test result indicated
that newly developed immunoassay system using fluorescence bead
and lateral-flow chromatography is a simple, fast method for quantifying
the cTnI concentration in human blood. Currently we development of
an alternative method enables to measure the low amount of cTnI more
accurately.
POS-TUE-280
TELOMERASE ACTIVITY AND ITS USE IN MONITORING
BLADDER CANCER
Lalla M.1, Dezfouli S.1, Mendez R.1, Tian P.2, Elwood N.2 and Macreadie I.1
1
Sienna Cancer Diagnostics Ltd., Bio21 Institute, VIC 3010, Australia.
2
Cord Blood Stem Cell Research Institute, Murdoch Children’s
Research Institute, VIC 3052, Australia.
The shortening of chromosomes is a normal part of the aging process,
however, cancer cells are immortal and overcome lethal chromosome
shortening. Most cancer cells maintain chromosomes through the activity
of telomerase, a DNA polymerase that extends the ends of chromosomes
through the addition of (TTAGGG) repeats. The activity of telomerase
is therefore a key biomarker in monitoring cancer. Assays to monitor
telomerase activity include the in vitro extension of an oligonucleotide
substrate with (TTAGGG) repeats. Typically such assays involve the
generation of a few attomoles of telomeric extension products (TEPs)
which are amplified by the polymerase chain reaction (PCR) and then
analysed on gels. Such PCR reactions are highly unusual since they
generate products even without TEPs. A characteristic to be looked
for in the telomeric repeat amplification protocol (TRAP) is a telomeric
ladder on gels. Further refinements include measurements using real
time PCR with amplifluors, however, once again products are generated
without TEPs. Because of the high signal in telomerase negative
controls, it is considered that PCR-based assays have limitations that
may prohibit their utility for clinical assays. Work at Sienna is focused
on the development of non-PCR procedures to measure telomerase
activity in bladder cancer using exfoliated cells in urine so as to avoid
these effects. Significant challenges are the low cell numbers in urine,
the harsh environment and non-epithelial cell types. Our aim is to assay
for telomerase activity in around 100 cells, each of which may have only
20 molecules of telomerase.
POSTERS MONDAY & TUESDAY
POS-MON-281
ANALYTICAL PREREQUISITES FOR DIFFERENTIAL
AND QUANTITATIVE PROTEOMICS
Levina V.1, 2 , Cooke I.1, 2, Durand F.M.1, 2, Boyd S.E.2, 3, O’Connor L.2, 3,
Hoogenraad N.2 and Talbo G.H.1, 2
1
CRC for Biomarker Translation, La Trobe University, Melbourne, VIC,
Australia. 2La Trobe Institute for Molecular Science, La Trobe University,
Melbourne, VIC, Australia. 3AgriBio, the Centre for AgriBioscience,
Melbourne, VIC, Australia.
The molecular composition of the cell surface plasma membrane and its
dynamic changes determine how a cell interacts with its environment. As cell
surface proteins confer specific cellular functions, and are accessible, these
are the proteins of greatest interest to modern diagnostic methodology and
specifically as biomarkers. Many membrane surface proteins are glycosylated
and the chemically distinct properties of carbohydrates from those of proteins,
have led to the development of bead based protein isolation approaches,
which target cell surface glycoproteins. Plasma membrane proteins isolated
by this approach are identified by tandem mass spectrometry. Despite some
success most cell surface proteins remain undetected due to the quality of the
methodologies currently available for the isolation of cell surface glycoproteins.
This is highlighted by the relatively small number, approximately 340, of cluster
determinant (CD) cell surface protein markers1 that have been identified to date,
compared with the number of predicted human transmembrane proteins being
greater than 13,0002. Potentially, even more important than the identification
of the plasma membrane glycoproteins is determination of the relative
abundance of those proteins in diseased compared with normal states of the
cell. Therefore, we set out to develop a protocol encompassing a quantitative
workflow from “Cell to Protein Identification and Relative Protein Amount.”
In short, the glyco moiety of the outer membrane proteins was oxidised by
NaIO4, the cells were lysed and the oxidised carbohydrate covalently bound
to hydrazide coupled beads. The covalent binding of the proteins of interest
facilitates the extensive washing required to remove membrane and other
debris. The bead bound proteins were reduced, alkylated and digested by
trypsin followed by washing. The residual tryptic glycopeptide was released
by PNGase F and analysed by LC-ESI-microTofQ-MS/MS and MALDI-tof/
tof-MS. The results were reproducible; peak intensities greater than 2000
showed less than 12 % CV across 30 replicate samples. 1) Nicholson, IC,
Mavrangelos, C, Fung, K, Ayhan, M, Levichkin, I, Johnston, A, Zloa, H and
Hoogenraad, NJ. J.Immunol. Methods, 305, 84-93, 2005 2) Zola and Swart,
2003.Trends Immunol.24,353, 2003.
POS-MON-283
DISCOVERY OF NOVEL PHAGE PROTEINS FOR
BIOTECHNOLOGY BY HIGH THROUGHPUT
SEQUENCING OF PHAGE DNA FROM SOUTH AFRICAN
DEEP GOLD MINES
Mabizela N.B. and Litthauer D.
Metagenomics Platform, Department of Microbial Biochemical and
Food Biotechnology, University of the Free State. Nelson Mandela
Drive, Bloemfontein, South Africa.
Shotgun sequencing of metagenome libraries from South African mines
revealed untapped phage communities in the deep subsurface. The
majority of the clones from four mines shared no similarity to known
proteins. Pyrosequencing was used to assess the metagenomic
diversity of phage DNA from Beatrix Mine in South Africa. Annotated
data showed that approximately 75% of the proteins had no homology to
any known proteins in public databases. About 40% of the proteins that
were assigned to a specific function were of viral origin. Most of the hits
were from the Enterobacteria phages and Acanthamoeba polyphaga
mimivirus. Seven prophage regions of bigger than 4 kb were identified
using Prophage Finder. Novel viral proteins with biotechnological
functions were identified. In this study a phage DNA ligase protein was
identified among the hundreds of genes obtained. Sequence analysis
indicated that the protein is novel showing 46% similarity to its closest
relative. In addition the second, most conserved motif, SLRFPRFIRIR
was not detected; only that containing the catalytic lysine. The gene
encoding this protein was cloned and expressed in E. coli. The protein is
41 kDa in size, is ATP-dependent and ligated cohesive and blunt ended
λ-DNA fragments, the former being ligated more efficiently. Maximal
ligation of the blunt ends was achieved with added polyethelene glycol
(10%). The enzyme is active at 4 ºC, 16 ºC and 22 ºC. BamHI cut pUC19
plasmid could be ligated at 30, 40, 50, 60 and 70 ºC.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 171
POS-TUE-282
RAPID RESPONSE OF THE HUMAN NUCLEOLAR
PROTEOME TO SERUM STIMULATION
Li Z.F.1, Liang Y.M.1, Wang X.2 and So L.K.Y.3 , Lam Y.W.1
1
Dept. of Biology and Chemistry, City University of Hong Kong.
2
College of Medicine, Zhejiang University, Hangzhou, China. 3Dept. of
Biochemistry, Hong Kong University.
The nucleolus is the most prominent organelle in the cell nucleus. It is
responsible for synthesizing and assembling 60S and 40S ribosome
subunits, and plays a crucial role in the regulation of cell proliferation
and growth. The responses of the nucleolus upon external stimulations
is therefore of high interest. The high density and structural stability of
the nucleolus makes it possible to purify this nuclear organelle, and to
analyse the dynamics of the nucleolar proteomes. However, the existing
nucleolar isolation method, developed in 1963, involved relatively tedious
procedures that tend to obscure momentous biochemical changes
in this organelle. Here we developed a new and simplified nucleolar
isolation method in which cells were rapidly harvested and lysed,
thus the nucleolar proteome could be “frozen” at precisely controlled
time points. The purity of nucleoli obtained using this protocol was
comparable to those using the classical method, as judged by electron
microscopy and Western blotting. The complete proteomes of nucleoli
prepared by these two methods were also compared using SILAC-
based quantitative proteomics.To further demonstrate the applications
of this new method, we performed a time-lapse nucleolar proteomics
after serum stimulation. HeLa cells were serum starved for 24 hours and
then serum re-stimulated, and the nucleolar proteome dynamics was
followed in the first 10 minutes of serum replenishment. This approach
reveals for the first time that some nucleolar proteins responded to
serum stimulation within as short as the first 5 min. Subsets of small
and large ribosomal proteins, histones and heterogeneous nuclear
ribonucleoproteins family increased 20% to even 100% within 10 mins.
Interestingly, the eukaryotic translation initiation factor 6 (EIF6) and the
proliferation marker Ki-67 were found to be quickly accumulated in the
nucleolus within 10 mins. To the best of our knowledge, this is the first
study demonstrating these “quick triggering proteins” in the nucleolus.
The underlying mechanisms and the roles of these very early events in
cell growth will be discussed.
POS-TUE-284
SIGNIFICANT IMPROVEMENT OF THE THERMO-
STABILITY AND OPTIMUM PH RANGE OF CATALYTIC
ACTIVITY OF A LACTONE-SPECIFIC ESTERASE BY
RANDOM MUTAGENESI
Ishizuka M.1, Tsukimura W.1, Namiki K.1, Akanuma G.1 and Ushio K.2
1
Department of Applied Chemistry, Chuo University, Tokyo, Japan.
2
Department of Applied Chemistry and Biotechnology, Niihama
National College of Technology, Niihama, Japan.
Stable and high stereo-selective lipase (EC 3.1.1.3) and esterase (EC
3.1.1.1) have attracted much attention from viewpoints of industrial
usage. Pseudomonas-like bacterium isolated in our laboratory from
wastewater sample produces a thermo stable lipase by the addition of
fatty alcohols (stearyl alcohol and palmityl alcohol) as the most effective
super-inducers. The addition of fatty alcohols brought about more than
several hundred-fold enhancement of the lipase activity compared to
the case with no additive. This means several dozen-fold enhancement
of lipase activity compared with olive oil grown case. Gram per litter
lipase production has been capable. On the other hand, carboxylic
ester esterase is not induced by the addition of fatty alcohols. In this
study, we report a lactone-specific esterase expression in Escherichia
coli and significant improvement of the thermo stability and wide
optimum pH range of catalytic activity of the mesophilic esterase by
random mutagenesis and evolutionary engineering. Thermal stability
of a purified mutant esterase (R29C, Q82L, R96H, N212D, R286H)
obtained by the error-pron PCR (EP-PCR), DNA shuffling, and thermal
selection (three times) rises 12 °C in temperature (10 fold long in half-life
period) compared with the stability of the purified wild-type esterase. As
a result of site-specific mutagenesis, Contribution of R96H and N212D
for thermal stability are 5 °C and 4 °C, respectively. Additionally, wide
optimum pH range (7-9) of the mutant catalytic activity and stability were
obtained without the loss of the lactone-specificity (γ-Butyrolacton,
δ-Valerolactone, and ε-Caprolactone).
POSTERS MONDAY & TUESDAY
POS-MON-285
EFFECTIVENESS OF PEER DISCUSSION FOR
STUDENT UNDERSTANDING OF ACID-BASE
EQUILIBRIA
Watters D.J. and Love C.A.
School of Biomolecular and Biophysical Sciences, Griffith University,
Nathan Campus, Brisbane, Australia 4111.
Students frequently have difficulty in understanding the central concepts
of acid-base chemistry and buffers. The term, troublesome knowledge,
as defined by Meyer and Land, accurately describes this problem since
students are able to perform superficial tasks such as titrations, and
apply the Henderson Hasselbalch equation without understanding
the basic concepts (ritual knowledge). They also fail to transfer their
understanding of simple acids and bases to the structure and function
of proteins or other complex biological molecules. Thus pH could fit into
the category of a threshold concept which is core to the understanding
of many aspects of chemistry, cell biology, physiology and biochemistry.
We investigated the use of concept questions in class with the use of
clickers to identify student misconceptions, as well as peer discussion,
in an effort to improve student learning of these topics. Peer discussion
has previously been shown to have a positive effect on student learning.
Our results show that, in the case of threshold concepts such as acid-
base equilibria, peer discussion is not effective and merely propagates
misconceptions. These findings suggest that substantial groundwork
needs to be done to enable students to gain the necessary understanding
of the fundamental concepts of acid-base chemistry.
POS-MON-287
HOW RESEARCH INFORMS AND ENHANCES
LEARNING USING STUDENT PROJECT CASES IN THE
MONASH MBBS PROGRAMME
Samarawickrema N.A. and Macaulay J.O.
Department of Biochemistry and Molecular Biology, Monash
University, Clayton, VIC 3800.
There is a strong synergistic connection between teaching and research
because (1) research fertilises teaching with new topics and methods
(2) research provides teachers with personal engagement and (3)
academic staff research guarantees connections with developments in
the research arena. In turn, students perceive their courses to be up to
date and intellectually stimulating. Here we report the results of a study
of undergraduate student engagement in a research tutored curriculum
based on the current research of the tutor. We used Student Project
Cases (SPC) offered in second year of the Monash University medical
curriculum, which comprises a teamwork activity that emphasises
interdisciplinary learning, where the students research and present a
medical disease. The co-topics required for this SPC were specified and
the students were required to research into these and prepare a written
and an oral presentation as part of the SPC assessment process. At the
end of the SPC, students completed a questionnaire which examined
their exposure to research and research culture, their experiences on
the preparation of this SPC and participated in a Focus group. The
students stated that the knowledge that the SPC topic being a current
research interest of an academic staff member involved with the SPC
process had a positive impact on their attitude towards the topic. They
stated that being aware that the research was happening now made it
exciting to study about the topic and therefore a motivational factor.
Page 172 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-286
ACQUISITION OF SCIENTIFIC WRITING SKILLS
THROUGH ACTIVITIES EMBEDDED INTO BIOLOGICAL
LABORATORY COURSES
Tonissen K.F.1, Lee S.E.1, Woods K.J.1 and Osborne S.A.1, 2
1
School of Biomolecular and Physical Sciences, Griffith University,
Nathan, Qld, Australia 4111. 2CSIRO Livestock Industries, St Lucia,
Qld, Australia, 4067.
Scientific writing skills are important for a science career, yet specific
training can be difficult to integrate effectively into a University program.
Rather than design specific courses solely focused on writing, we
embedded writing activities into two project style third year biological
science laboratory courses where students wrote about their own data.
Students were expected to complete the writing exercises during breaks
in their experimental procedures and were given feedback during the
laboratory session. These activities were focussed on preparing figures,
tables, figure legends, and writing results and discussion paragraphs.
These exercises provided practice and a model to assist students in
writing the remainder of the report. We probed student opinions regarding
scientific writing and the use of the exercises by anonymous pre- and
post-course surveys using a combination of closed and open questions.
In the first course student confidence towards scientific writing and
performing simple writing tasks significantly improved after experiencing
the writing activities. Therefore students commenced the second
course with a higher confidence level. They related that undertaking
writing activities in more than one class helped them consolidate their
writing skills and challenged them to improve further. Students also
commented that they thought the activities helped them develop skills
relevant to future scientific careers. Independent assessors evaluated
the standard of students’ written reports that originated from the same
course held in years before and after writing activities were incorporated
into the curriculum. There was a significant improvement in scientific
writing quality that correlated with the increase in student confidence
and attitudes towards writing.
POS-TUE-288
CONSTRUCTING A GENERALIST BIOCHEMISTRY
COURSE AROUND CORE CONCEPTS: BUILDING
THE BIG PICTURE FOR A LARGE MIXED-LEARNER
COHORT
Rowland S.L., Gillam E., Hamilton S., Harrinson J., Wright T. and
Ward L.
University of Queensland.
The University of Queensland has recently revised the BSc, and
consequently we now offer a single, second-year, undergraduate
biochemistry course. This course, BIOC2000, must service over 500
students from almost 20 different programs of study. The students
have many different backgrounds, goals, and abilities. This is a radical
departure from our smaller, more “science-centric” cohorts of previous
years. In its first year of delivery, BIOC2000 uncovered serious problems
in understanding of basic chemistry concepts for many of the cohort as
evidenced by the answers to a chemical concept inventory test that we
delivered to the students, and also by the final exam results. We decided
to rebuild the course around core chemical and biochemical concepts,
rather than around content, and use a “Backwards design” approach to
the new curriculum. Developing student understanding of fundamental
chemistry concepts and their application to the molecules and structures
of life is the primary learning objective of the new curriculum. We surveyed
over 80 working scientists to determine the “fundamental concepts” of
biochemistry. We will present our data regarding (i) what these concepts
are, (ii) how we determined if the students understood them before the
course started, (iii) how we integrated these concepts into learning and
assessment activities, and (iv) how student understanding of these
concepts changed during the course. This project provides a roadmap
for generalist course design around the fundamental guiding principles
of a discipline, rather than around content. The presentation should be
useful to all course designers.
POSTERS MONDAY & TUESDAY
POS-MON-289
IMI HO’OLA POST-BACCALAUREATE PROGRAM AT
THE UNIVERSITY OF HAWAII SCHOOL OF MEDICINE
Ha C., Lee W.M. and Judd N.
University of Hawaii School of Medicine, 651 Ilalo Street, MEB306,
Honolulu, HI 96813, USA.
Imi Ho’ola (Hawaiian meaning “Those who seek to heal”) is a post
baccalaureate program established in 1972 to prepare students from
socially, educationally, or financially disadvantaged backgrounds for
the entry into the rigors of medical school education. Each year 10
students are selected to participate in the one-year program and upon
matriculation students are guaranteed acceptance into the University of
Hawaii School of Medicine as regular first year medical students. The
curriculum of the Imi Ho’ola program spans over 12 months and consists
of three phases: 1. Summer orientation and assessment phase to obtain
base-line data on students’ knowledge in the sciences, reading, and
learning skills, Phase 2. Enrichment phase to improve students’ critical
thinking skills in the content areas of biology, medical biochemistry,
scientific basis of medicine, and speech and ethics in healthcare,
Phase 3. Prematriculation phase to help students’ smooth transition into
regular medical school curriculum with introduction of clinical skills by
shadowing physicians. For the past 30 years, Imi Ho’ola program has
provided educational opportunities to disadvantaged minority students
pursuing a career in medicine. Out of 204 graduates of the program
and medical school, 96% provide health services to disadvantaged or
underserved populations in the state of Hawaii and the Pacific islands
and 72% provide primary care services such as internal medicine, family
medicine, and pediatrics. Imi Ho’ola program is a successful educational
model that contributes to the development of a diverse healthcare
workforce that provides services for the underserved communities of
Hawaii and the Pacific region.
POS-MON-291
GENE EXPRESSION PROFILING IN CIRCULATING
CELLS OF BREAST CARCINOMA PATIENTS
Zima T1, Mikulova V1, Tesarova P1, Kolostova K 2, Kubecova M2,
Rusnakova V3 and Kubista M3
1
General University Hospital and First Faculty of Medicine, Charles
University in Prague. 2 General University Hospital Královské
Vinohrady and Third Faculty of Medicine, Charles University in Prague.
3
Institute of Biotechnology, Academy of Sciences, Czech Republic.
Background: Tumor cell dissemination is an early process in breast
cancer (BC) and circulating tumor cells (CTCs) are considered potential
surrogate marker for the detection and characterization of minimal
residual disease. Here we monitored hematogenous micrometastasis
in BC patients by gene expression profiling of CTCs based on CTC-
abundance in blood and expression of 35 oncomarkers at the mRNA
level by multimarker quantitative PCR. Methods: A total of 87 patients
with diagnosed BC at stage I to III and 115 metastatic patients were
enrolled into a prospective study. Immunomagnetic enrichment of
CTCs from the 5ml of whole blood followed by cells characterization
using gene expression analysis for the presence of tumor associated
genes HER2, MUC-1 and GA 733-2 were processed using AdnaTest
BreastCancer® kit. If possible bone marrow samples were obtained for
disseminated tumor cells detection using Epimet® Kit. RNA from FFPE
tumor tissue (n=85) has been isolated. All obtained cDNA molecules
have been gene-specifically pre-amplified for multimarker qPCR analysis
measured on Biomark® microfluidic chip. Results: 286 CTC samples
have been analyzed in total. The analysis has shown that the gene
expression profiles of CTCs in primary breast cancer patients correlated
to those measured on the primary tumor, while CTCs of metastatic BC
patients had significantly different gene expression profiles. Analyzing
the gene expression data from CTC-positive patients in comparison to
CTC negative patients and FFPE samples we have revealed several
genes that were differentially expressed (p<0,05) (e.g. CK19, GA7332,
AURKA, MLF1IP, SATB1, PTEN).
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 173
POS-TUE-290
STUDENT ENGAGEMENT IN 1ST YEAR BIOSCIENCES
Nott M.W. and Poronnik P.
RMIT University, School of Medical Sciences, Melbourne.
Students typically fail to fully realise the relevance of core preliminary
subjects, and in particular the generic skills they need to develop in
1st year bioscience studies: chemistry, mathematics and statistics,
and even biology. In Pharmaceutical Sciences at RMIT University we
have devised a series of interventions that require 1st year students to
draw on and integrate their learning in each core subject. For the first
intervention, staff from each discipline agreed on the common theme:
Malaria and its Drug Treatment. The theme fulfilled criteria aimed
to enhance student engagement and their awareness of relevance of
each subject. Thus the theme is contemporary, given that many of
our students or their families come from malaria-endemic countries;
media-rich, given that malaria (and dengue) is increasingly in the
news because of global warming and the need for new strategies for its
treatment (eg, artemisinins) and eradication (eg, genetically engineered
flightless mosquitoes); and research-relevant, given that Melbourne is
a centre for immune studies approaches. The theme allowed seamless
integration of learning materials in chemistry (which stressed molecular
structures of antimalarials and their binding to protein targets); maths
and statistics (which stressed epidemiology and vector population
studies) and biology (which stressed drug-susceptible stages of the life
cycle of plasmodia). Students worked in teams to create and defend
posters, thus honing their visual and oral presentation skills as they
worked on 9 topics around the central theme. In doing so, students were
able to integrate their first year studies as well as get an early start
on developing professional capabilities according to RMIT University’s
Graduate Attributes: 1) work ready; 2) environmentally aware and
responsive; 3) global in outlook and competence; 4) culturally and
socially aware; 5) life-long learners; 6) active learners; 7) innovative.
POS-TUE-292
CHARACTERISATION OF PUTATIVE PLASMODESMATA
PROTEINS OF THE ARABIDOPSIS CALNEXIN FAMILY
Liu D.Y.T.1, Smith P.M.C.1, Day D.A.1, 2 and Overall R.L.1
1
School of Biological Sciences, The University of Sydney, Sydney
NSW 2006 Australia. 2Present address: Flinders University, Adelaide
SA 5042 Australia.
Plasmodesmata are plasma membrane-lined channels spanning the
cell wall, connecting the cytoplasm and endoplasmic reticulum (ER)
of adjacent cells. Plant survival depends upon the proper function and
regulation of plasmodesmata, since developmental signals, nutrients,
and even viruses move through these channels. However, only a few
protein constituents of plasmodesmata have been discovered. In this
study, we extend a comparative proteomics analysis of Chara (Faulkner
et al., Proteomics 5: 2866) by identifying and characterising Arabidopsis
proteins with sequence similarity to characean peptides isolated from
plasmodesmata-rich cell fractions. These proteins were screened for
plasmodesmatal localisation in Arabidopsis and Nicotiana benthamiana
using green fluorescent protein (GFP) tagging. Calnexin, one of the
proteins identified, is a type I membrane protein localised in the ER with
the main catalytic domain lying within the ER lumen where it may function
as a chaperone. The Arabidopsis calnexin protein family consists of two
members, AtCNX1 and AtCNX2, with 83% amino acid identity. Both
proteins colocalise with aniline blue-induced fluorescence of callose
at plasmodesmata, although colocalisation is reduced upon deletion
of the signal peptide. No significant morphological differences were
observed in single or double homozygous Arabidopsis T-DNA knock-
out mutants of AtCNX1 and AtCNX2. However, cell-to-cell diffusion of
GFP as well as deposition of callose at plasmodesmata were affected
in mutants, suggesting some role for calnexin in plasmodesmatal
physiology. Quantitative real-time PCR data suggested that in knock-out
mutants other chaperones may provide redundancy for CNX, including
calreticulin, an ER-lumenal homolog of calnexin. The role of calnexin at
plasmodesmata will be discussed.
POSTERS MONDAY & TUESDAY
POS-MON-293
CRITICAL ROLE OF C-ZYME ON P53IG-MEDIATED
APOPTOSIS
Kang M.Y.
Department of Bio-Materials Engineering, Graduate School and
DNA Repair Research Center, Chosun University, 375 Seosuk-dong,
Gwangju 501-759, Korea.
A number of genes involved in control of the cell cycle and apoptosis
are regulated by p53-induced genes (p53IGs). A series of p53IG have
been identified that are predicted to encode proteins that could generate
or respond to oxidative stress. C-zyme, an anti-oxidative enzyme is an
important scavenger of ROS related to p53IG activation. We found that
purified p53IG coimmunoprecipitated recombinant C-zyme suggesting
that p53IG directly binds to C-zyme. In vivo IP analysis also revealed that
endogenous p53IG bound C-zyme. A confocal microscopy examination
showed both proteins were co-localized after treatment with UV. Purified
picogram of p53IG protein significantly suppressed C-zyme activity in a
dose-dependent manner. We observed that various cell lines transfected
with p53IG exhibited elevation of ROS production. Accordingly, C-zyme
activity were significantly decreased. p53IG overexpressed cells infected
with Ad C-zyme was resistant to apoptosis. These data demonstrate
that after DNA damage, p53IG is involved in triggering apoptosis by
repressing C-zyme function. Although the exact role of p53IG in p53
pathway is not yet elucidated, p53IG may be one of the factors involved
in p53-induced apoptosis through ROS generation.
POS-MON-295
CD317/TETHERIN AND LIPID RAFT ORGANISATION
Billcliff P.1, Rollason R.1, Prior I.A.2 and Banting G.1
1
Department of Biochemistry, University of Bristol, Bristol BS8
1TD, UK. 2Division of Physiology, University of Liverpool, Crown St,
Liverpool L69 3BX, UK.
CD317 (Bst2, tetherin) has been implicated in preventing the spread of
HIV-1 virions from infected cells by tethering them to the cell surface. The
cytosolic domain of CD317 interacts indirectly with the actin cytoskeleton,
and its knockdown in polarised epithelial cells leads to a loss of the
sub-apical actin network. Whilst the N-terminus of CD317 interacts with
the actin cytoskeleton, the glycophosphatidylinositiol (GPI)-anchored
C-terminus is localised to lipid rafts; this unusual topology of CD317
could enable it to functionally unite the actin cytoskeleton and lipid rafts.
Moreover, the extracellular region of CD317 forms a parallel coiled-coil;
this rigid structure might therefore limit teh free diffusion of proteins into
and out of lipid rafts, thereby organising rafts. We present preliminary
evidence that CD317 is indeed involved in the organisation of lipid
rafts. CD317 has previously been shown to activate the NF-κB pathway
and, in line with this, increased expression of CD317 upregulated the
activity of an NF-κB-activated luciferase reporter, denoting that CD317
might aggregate signalling complexes from initally disparate rafts, and
thus augment signal transduction events. In addition, using a GPI-YFP
reporter plasmid (a lipid raft marker) in concert with fluorescence recovery
after photobleaching (FRAP) assays, the lateral diffusion mobility of
GPI-YFP was increased in cells where CD317 was knocked down - a
result consistent with CD317 playing a role in lipid raft organisation.
Further, we use electron microscopy on prepared membrane sheets to
investigate the importance of CD317 in the formation and clustering of
lipid rafts, using the raft marker GFP-tH.
Page 174 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-294
PHOSPHORYLATION OF PTP-PEST AT SER-39 IS
REGULATED THROUGH THE BINDING OF PP1 ALPHA
Palmer H.E.F., Nakamura K. and Mashima K.
Department of Life Science, Rikkyo University.
PTP-PEST is expressed in a wide variety of cell types and is an efficient
regulator of integrin-medicated signaling in adherent cells and antigen
receptor-mediated signaling in lymphocytes. PTP-PEST-associating
molecules are important in elucidating the function of PTP-PEST.
The major phosphorylation site of PTP-PEST at Ser-39, regulates
the PTP activity. Herein, we have identified protein phosphatase 1α
(PP1α) as a novel PTP-PEST binding protein, and tried to elucidate the
mechanism of Ser-39 dephosphorylation through this molecule. HEK
293 cells overexpressing exogenous PTP-PEST, were stimulated by
12-O-tetradecanoylphorbol 13-acetate (TPA) and the phosphorylation
of PTP-PEST at Ser39 was evaluated using an anti-phospho-Ser39
PTP-PEST specific antibody (anti-pS39-PEST Ab). It was demonstrated
that the phosphorylation at Ser39, detected by anti-pS39-PEST Ab,
was dependent on TPA treatment corresponding with previous reports.
Moreover, we discovered a significant inverse correlation between the
PTP activity of PTP-PEST and anti-pS39-PEST Ab-immunoreactive
band intensity quantitatively. PP1α wild-type induced dephosphorylation
of PTP-PEST at Ser39, where as, PP1α dominant-negative mutant had
no effect. TPA-induced Ser39 phosphorylation was not abrogated by
PP1α wild-type when cells were expressed with PTP-PEST without the
non-catalytic segment, indicating that the dephosphoylating action of
PP1α could possibly occur through binding at non-catalytic segment
of PTP-PEST. In conclusion, PP1α associates with the non-catalytic
segment of PTP-PEST and regulates PTP activity via dephosphorylation
of phospho-Ser39. We further seek to specify the binding site of PP1α
to PTP-PEST and clarify the cellular function of phosphorylation/
dephosphorylation of PTP-PEST at Ser39.
POS-TUE-296
A NOVEL CONTROL POINT IN CHOLESTEROL
BIOSYNTHESIS
Gill S., Stevenson J., Kristiana I. and Brown A.J.
School of Biotechnology and Biomolecular Sciences, University of
New South Wales, Sydney, NSW 2052, Australia.
Cholesterol homeostasis is maintained in part through control of its
biosynthesis. Study has focussed on 3-hydroxy-3-methyl-glutaryl-
coenzyme A reductase (HMGCR), billed as the ‘rate-limiting’ enzyme in
the pathway and target of the blockbuster statin class of drugs. In contrast,
relatively little is known about the more than twenty other enzymes
involved in the synthesis of the essential cholesterol molecule, including
squalene monooxygenase (SM). SM performs the first oxygenation
step in cholesterol synthesis, prior to cyclisation of its product into the
steroid backbone structure. We examined the regulation of SM using
a mammalian cell culture system, particularly Chinese hamster ovary
(CHO) cells. Surprisingly, cholesterol treatment caused squalene to
accumulate, indicating that SM may serve as a ‘rate-limiting’ enzyme
beyond HMGCR. This accumulation was also seen in mutant CHO cells
lacking sterol regulated transcription, implying that cholesterol regulates
SM post-transcriptionally. Indeed, we found that SM protein was degraded
within hours of cholesterol addition. Proteasomal inhibition blocked this
degradation and reversed the squalene accumulation, suggesting that
the cholesterol-induced degradation of SM is a flux control point in
cholesterol synthesis. Regulated degradation required the N-terminal
domain of SM conserved in higher organisms, but not required for
activity. This region also conferred cholesterol-regulated turnover on
heterologous fusion proteins. We have identified a significant novel
control point in the feedback regulation of cholesterol biosynthesis.
POSTERS MONDAY & TUESDAY
POS-MON-297
DIVERSITY AND BIOSYNTHESIS OF SANDALWOOD
OIL: SCENTS FROM SANTALUM
Plummer J.A.1, Jones C.G.1, 2, Moniodis J.1, Zulak K.G.1, 2, Barbour E.L.1, 3,
Bohlmann J.2 and Ghizalberti E.L.4
1
Plant Biology, The University of Western Australia, 35 Stirling Hwy,
Crawley, WA 6009 Australia. 2Michael Smith Laboratories, University
of British Columbia, 301 - 2185 East Mall, Vancouver BC V6T 1Z4,
Canada. 3Forest Products Commission of Western Australia, 117 Great
Eastern Hwy. 4Biomedical, Biomolecular and Chemical Sciences, The
University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009
Australia.
Sandalwood oil is arguably the worlds most valuable fragrance ingredient.
The heartwood of mature sandalwood trees (Santalum spp.) contains a
complex mixture of sesquiterpenes, with a composition profile reflecting
differences between and sometimes within species. Plantations relieve
harvest pressure on wild stands, however silviculture is complicated and
heartwood oil yields are often low. Our research seeks to understand
factors which control sesquiterpene biosynthesis in sandalwood. A
PCR-based cloning strategy was used to isolate cDNAs encoding
several terpene synthase genes responsible for biosynthesis of key
components of sandalwood oil from three phylogenetically divergent
species (S. album, S. spicatum and S. austocaledonicum). The encoded
proteins have been functionally characterised by over-expression in E.
coli and incubated with the universal sesquiterpene precursor FPP. The
diversity of compounds produced by these enzymes accounts for the
natural chemical diversity found across the genus. Genomic structure of
these genes is conserved, exemplifying the evolutionary importance of
sesquiterpene production in this genus. This research has applications
for oil yield improvement in this expanding industry.
POS-MON-299
SODIUM EXCLUSION GENES INCREASE SALINITY
TOLERANCE IN DURUM WHEAT
Munns R. and James R.
CSIRO Plant Industry, Canberra.
In a project aimed to increase the salt tolerance of durum wheat to
match that of bread wheat, two major genes for Na exclusion named
Nax1 and Nax2 were discovered. These lower the Na concentration in
the leaf blade by removing Na from the transpiration stream as it flows
from roots to shoots. Both genes belong to the HKT transporter family.
Nax2 (HKT1;5-A) is expressed in roots; Nax1 (HKT1;4-A2) is expressed
in both roots and shoots. Glasshouse studies showed that the Nax
genes keep leaves alive for longer, by reducing the rate at which Na
accumulates to toxic levels. This allows more photosynthate to reach
the developing grain and so increase the grain yield. The Nax genes
originated from Tricitum monocoocum and are not present in modern
wheat. When crossed into Australian durum wheat and bread wheat
cultivars, they reduced the Na+ accumulation in leaves. Field trials
validated the function of these genes in Na exclusion. Na concentrations
in the flag leaf were reduced over 100 times, e.g. from 250 to 1 umol
per g DW. These showed that the presence of Nax2 increased yield of
durum wheat in saline soil by 25%.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 175
POS-TUE-298
DELETIONS, REPEAT-INDUCED POINT MUTATIONS
AND AMINO ACID SUBSTITUTIONS DRIVE EVOLUTION
OF LINKED EFFECTORS IN A FUNGAL PLANT
PATHOGEN
van de Wouw A.P.1, Cozijnsen A.J.1, Hane J.2, Brunner P.C.3,
McDonald B.A.3, Oliver R.P.2 and Howlett B.J.1
1
School of Botany, the University of Melbourne, Vic, 3010. 2Australian
Centre for Necrotrophic Fungal Pathogens, Murdoch University, WA,
6150. 3Institute of Integrative Biology, ETH Zurich, Switzerland.
Pathogen effectors, molecules that include small secreted proteins such
as avirulence proteins, facilitate infection or induce defence responses
by plants. We report the first large scale study of evolutionary processes
affecting linked effector-like genes in a fungal plant pathogen. Mutations
affecting seven genes and four single copy non-coding regions located
in a 520 kb repetitive element-rich region of Leptosphaeria maculans,
a pathogen of Brassica napus (canola) are described. Two genes are
avirulence genes, AvrLm1 and AvrLm6, which are complementary to B.
napus resistance genes, Rlm1 and Rlm6. Analyses of 295 Australian
isolates showed that deletions, Repeat-Induced Point (RIP) mutations
and/or non-RIP derived amino acid substitutions account for rapid
evolution of four small secreted proteins. RIP was confined to three
genes and two other single copy regions and appeared to have ‘leaked’
from flanking repetitive sequences. The RIP alleles were monophyletic
and present only in isolates collected after 2004, the year when
canola cultivars with resistance conferred by Rlm1 suffered severe
yield losses. This co-incided with a large increase in the frequency
of isolates with virulence alleles of AvrLm1 and AvrLm6, even though
the canola cultivars lacked Rlm6. Evolution of these two effectors thus
appears to be influenced both by the genomic environment (flanking
repetitive elements) and by selection pressure from extensive sowing of
crop varieties with resistance genes complementary to the avirulence
effector gene.
POS-TUE-300
FLOWERING LOCUS H (FLH) AND THE
VERNALISATION PATHWAY
Seedat N. and Gendall A.
LaTrobe University Bundoora.
In wild and cultivated annual plant species flowering time is an important
trait which coordinates the plants life cycle with the local environmental
conditions. The genetic network of flowering time genes in the model
species Arabidopsis thaliana has been widely studied revealing a
complex genetic network of flowering time genes that are able to
detect the external environmental and internal signals to coordinate
reproductive development, and ensure it occurs at an optimum time
for reproductive success. FLOWERING LOCUS H (FLH) is a novel
flowering locus which enhances the vernalisation response resulting
in earlier flowering time. This study is aimed at determining FLH’s
interaction within the vernalisation pathway. Pervious work undertake
in your lab has revealed that FLH has the ability to accelerate flowering
in many of the vernalisation pathway mutants such as VERNALISATIN
5, VERNALISATION 1, VERNALISATION INSENSITIVE 3 and
FLOWERING LOCUS C. FLH has shown to have an additive affect on
these mutations, enabling these late flowering phenotypes to become
over-come returning flowering time to an early flowering phenotype
yet not as early as wild-type. FLH has also demonstrated the ability
to accelerate flowering time in mutations occurring in meristem
identity genes in the vernalisation pathway such as FLOWERING
LOCUS T (FT), CAULIFLOWER, APETALA1 and SUPPRESSOR
OF OVEREXPRESSION OF CO 1. However in the absence of
FLOWERING LOCUS D (FD), FLH was unable to over-come the late
flowering phenotype. As FT and FD interact to promote flowering, these
results suggest that that FLH is able to substitute for the loss of FT but
not the loss of FD.
POSTERS MONDAY & TUESDAY
POS-MON-301
CHARACTERIZATION OF STRAWBERRY PLANTS
OVER-EXPRESSING THE ANTHOCYANIN-REGULATING
GENE, MYB10
Lin-Wang K.1, McGhie T.K.2, Espley R.V.1, Hellens R.P.1 and Allan A.C.1
1
The New Zealand Institute for Plant & Food Research Ltd, Mt Albert
Research Centre, Auckland, New Zealand. 2Plant & Food Research,
Palmerston North, New Zealand.
Strawberry has become an ideal model for functional genomics
research in Rosaceae, because of its relatively small genome size, ease
of transformation, and genome sequence resources. Anthocyanins are
secondary metabolites found in higher plants that contribute to the
colours of flowers, fruits and seeds. The anthocyanin pigment pathway
is regulated by transcription factors that include MYB, bHLH and
WD-repeat proteins. Anthocyanin-upregulating MYBs from Fragaria
ananassa and Fragaria vesca have been successfully isolated (Lin-
Wang et al., 2010). Strawberry transgenic lines of both species over-
expressing 35S:FaMYB10 and 35S:FvMYB10 had significant increases
of anthocyanin in roots, leaves and fruits. The expression analysis of
wild-type and over-expressing lines suggests a strong correlation
between the expression of the anthocyanin biosynthetic pathway
gene CHS, DFR, F3H and UFGT and the expression of these MYB
transcription factors. HPLC analysis of fruit showed up to 2- to 4-fold
increases in anthocyanin content in 35S:FaMYB10 and 35S:FvMYB10
lines compared with the wild-type strawberry fruit.
POS-MON-303
GROWTH, PROLINE AND ION ACCUMULATION IN
SAFFLOWER CALLUS CULTURES UNDER DROUGHT-
INDUCED OSMOTIC STRESS
Karimi N., Ghasempour H.R. and Soheilikhah Z.
Department of Biology, Science Faculty, Razi University, Kermanshah,
Iran.
Safflower (Carthamus tinctorius L.) is s a highly branched, herbaceous
crop in Iran that its production affected by several biotic and abiotic stress
factors. The selection of bean cultivars adapted to salinity, drought and
other abiotic stresses is a main goal of the breeders. This will contribute
to understand the resistance mechanisms involved in different safflower
genotypes with special reference to drought. Cell cultures of different
genotypes (G1: LRV-51-51, G2: Lesaf, G3: Gila, G4: Kino-76, G5:
Isfahan) of Carthamus tinctorius were established from callus tissues
inoculated in MS liquid medium supplemented with 0.5 mg/L NAA
(naphthalene acetic acid), 0.5 mg/L BAP (6- banzeyl aminopurine) and
0.5 mg/L 2,4 D. Mannitol was added to the medium to induce water
deficit. Relative growth rate, proline, minerals and callus water content
were determined at the end of stress and relief periods. After the stress
period, calli derived from five cultivars showed a decrease in RGR, but at
lesser extent in G1 than other genotypes. Same tendency was recorded
in the callus water content under mannitol induced osmotic stress.
Moreover, the G1 genotype accumulated more K+, Na+ and N but less
Ca++ and P than the other four genotypes. The proline level showed a
positive correlation with the degree of tolerance to water stress (with
marked accumulation in G1), which suggests that proline accumulation
accompanies survival and growth in drought environment. The results
indicate that the response of the callus of G1 Cultivar is more resistance
to drought stress in comparison to that of other genotypes.
Page 176 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-302
ARTIFICIAL MICRORNA-MEDIATED
DOWNREGULATION OF STARCH BRANCHING
ENZYME IIB EXPRESSION IN RICE
Butardo V.M.1, 2, 5, Fitzgerald M.A.4, Bird T.3, Resurreccion A.P.4,
Larroque O.1, 2, Gidley M.J.5, Morell M.K.1, 2 and Rahman S.1, 2
1
CSIRO Food Futures Flagship, GPO Box 93, North Ryde, NSW 1670,
Australia. 2CSIRO Plant Industry, GPO Box 1600, ACT 2601, Australia.
3
CSIRO Food and Nutritional Sciences, PO BOX 10041, Adelaide
SA 5000, Australia. 4Grain Quality, Nutrition and Postharvest Centre,
International Rice Research Institute, Los Baños, Laguna 4031,
Philippines. 5Centre for Nutrition and Food Sciences, University of
Queensland, Brisbane, Qld 4072, Australia.
The amylose extender (ae) mutation in rice and maize is associated with
a significant reduction in the expression of starch branching enzyme IIb
(SBEIIb). In this study, we have generated transgenic Nipponbare rice
plants containing RNA silencing constructs aimed at specifically down-
regulating the expression of SBEIIb in the rice endosperm using artificial
microRNA (amiRNA) and hairpin RNA (hp-RNA). By comparing the grain
and starch properties of generated transgenic lines, we demonstrate
that the ae phenotype can be obtained by endosperm-specific silencing
of SBEIIb gene expression. Using artificial microRNA led to somewhat
greater changes in starch structure than the hairpin-RNA when
compared to wild type and ae rice starch. This study demonstrates that
down-regulation of starch enzyme expression in cereal endosperm
can be achieved using long hairpin-RNA but also by the short artificial
microRNA technique, thereby demonstrating the value of using this
RNA silencing technique in modifying starch structure, and therefore
functionality, in rice and potentially other cereals.
POS-TUE-304
STRUCTURAL AND FUNCTIONAL CHANGES INDUCED
BY TYROSINE NITRATION IN CYTOCHROME C, A BI-
FUNCTIONAL PROTEIN.
Diaz-Moreno I.1, Garcia-Heredia J.M.1, Nieto P.M.2, Orzaez M.3,
Teixeira M.4, Perez-Paya E.3, Diaz-Quintana A.1 and De La Rosa M.A.1
1
Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla
- CSIC, Spain. 2Instituto de Investigaciones Químicas. Universidad
de Sevilla - CSIC, Spain. 3Centro de Investigación Príncipe Felipe.
Valencia, Spain. 4Instituto de Tecnologia Química e Biológica.
Universidade Nova de Lisboa, Portugal.
Tyrosine nitration is one of the most common post-transcriptional
modifications of proteins, so affecting their structure and function.
Respiratory cytochrome c, with 4-6 tyrosine residues, is an excellent
case study as it is a well-known protein playing a double physiological
role in different cell compartments. On one hand, it acts as electron
carrier within the mitochondrial respiratory electron transport chain and,
on the other hand, it serves as a cytoplasmic apoptosis triggering agent.
First, we have analyzed the nitration-induced changes in secondary
structure, thermal stability, heme environment, alkaline transition
and molecular dynamics of the five monotyrosine mutants of human
cytochrome c - which have all their tyrosine residues but one replaced
by phenylalanines. The resulting data, along with the functional
analyses of the mutants, suggests that the specific nitration of Tyr46
and Tyr48 - which are both close to the heme propionate groups - and
that of the solvent-exposed Tyr74 impairs the electron transfer to (horse)
cytochrome c oxidase, enhances the peroxidase activity of cytochrome
c and blocks its ability to activate caspase-9. - See García-Heredia et al.
(2010) BBA Bioenergetics, in press.
POSTERS MONDAY & TUESDAY
POS-MON-305
APPLICATION OF LENTIL LECTIN IN THE
GLYCOPROTEOMIC PROFILING OF SERUM SAMPLES
FROM PATIENTS WITH NASOPHARYNGEAL
CARCINOMA
Seriramalu R.1, Pang W.W.2, Abdul-Rahman P.S.1, 2, Bustam A.Z.2, 3,
Khoo A.S.B.4 and Hashim O.H.1, 2
1
Department of Molecular Medicine, Faculty of Medicine, University
of Malaya, Kuala Lumpur, Malaysia. 2University of Malaya Centre for
Proteomics Research, University of Malaya, Kuala Lumpur, Malaysia.
3
Clinical Oncology Unit, University of Malaya Medical Centre, Kuala
Lumpur, Malaysia. 4Molecular Pathology Unit, Cancer Research
Centre, Institute for Medical Research, Kuala Lumpur, Malaysia.
The lentil lectin from Lens culinaris showed high specificity and
affinity for the alpha-mannose and branched fucose residues of the
N-glycans of glycoproteins. In this targeted proteomics study, we have
isolated glycoproteins from pooled serum samples from patients with
nasopharyngeal carcinoma WHO type III using immobilised Lens
culinaris lectin affinity chromatography. More than twenty clusters of
serum proteins were detected when the captured proteins were eluted
and subjected to 2-dimensional gel electrophoretic profiling. The protein
profile obtained was then compared with that similarly generated
from pooled serum samples of normal individuals and identities of the
resolved proteins were confirmed using mass spectrometry.
POS-MON-307
STRUCTURE-ACTIVITY RELATIONSHIP IN HUMAN
ANTIQUITIN
Fong W.P., Wong C.P. and Chan K.L.
Department of Biochemistry, The Chinese University of Hong Kong,
Shatin, N.T., Hong Kong, China.
Antiquitin (ALDH7) is a member of the aldehyde dehydrogenase
(ALDH) superfamily which oxidizes various aldehydes to form the
corresponding acids. Human antiquitin (ALDH7A1) is believed to play
a role in detoxification, osmoregulation and in lysine metabolism.
Clinically, mutation on antiquitin is found to be related with pyridoxine-
dependent seizures which occur as an indirect result of the failure in
oxidizing α-aminoadipic semialdehyde (α-AASA) in lysine metabolism.
In the present study, the structural basis of human antiquitin in oxidizing
α-AASA was studied. Sequence comparisons between different ALDH
families show specifically the presence of two charged amino acid
residues at the entrance of the substrate binding pocket in antiquitin.
These two amino acid residues, Glu-121 and Arg-301 in the human
enzyme, may have charge-charge interaction with the α-amino and
α-carboxylate groups of α-AASA. To confirm this hypothesis, site-
directed mutagenesis was carried out. The mutants E121A and R301A
were purified and kinetically characterized using both the specific α-AASA
and the non-specific acetaldehyde as substrate. When compared with
the wild-type enzyme, the E121A and R301A substitutions resulted in
16- and 31-fold, respectively, increase in the Km value of α-AASA. In
contrast, these substitutions caused only minor changes (< 2-fold) in
the Km value of acetaldehyde. For kcat values, the substitutions also
caused a much more significant decrease in the oxidation of α-AASA
(8.7-fold for E121A and 69-fold for R301A) than that of acetaldehyde
(1.6-fold for E121A and 7.3-fold for R301A). To conclude, Glu-121 and
Arg-301 are important in determining the substrate specificity in human
antiquitin. (This work was supported by a grant from RGC of the HKSAR,
Project No. 464407).
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 177
POS-TUE-306
STRUCTURAL DECAY OF FISH MYOGLOBIN
THROUGH THERMAL TREATMENT
Ochiai Y.
Univ. Tokyo, Japan.
The effect of thermal treatment was examined on myoglobin (Mb) purified
from the skeletal muscle of tuna by ammonium sulfate fractionation and
gel filtration chromatography. The temperature-dependent changes
of molar ellipticity or helical content obtained by circular dichroism
spectrometry suggest that Mb performed a three-step denaturation,
where small structural changes were initiated even below 20oC. On the
other hand, major structural changes took place at around 60oC and
70oC. Differential scanning calorimetry on the thermal denaturation
of this Mb demonstrated somewhat different thermal denaturation
patterns: namely, four peaks were obtained. The largest denaturation
took place at around 73oC, while slight denaturation proceeded at
around 28oC. All these results suggest that the structure of fish Mb
is subjected to perturbation even at lower temperature compared to
mammalian Mbs. Some essential differences were recognized in the
modeled tertiary structures of these Mbs, especially in the loop region
and cavity volume.
POS-TUE-308
ELECTROPHORETIC ANALYSIS OF ISOENZYMES
IN THREE CULTIVARS OF DATE PALM (PHOENIX
DACTYLIFERA L.) LEAFLETS AND ROOTS FROM AL-
AHSA AND AL-QATIF IN SAUDI ARABIA
Al-Issa A.M.1, Al-Helal A.A.2 and Al-Saad F.A.2
1
University of Dammam. 2King Saud University.
Date palm trees (Phoenix dactylifera L.) are widely distributed in the
Eastern Province of Kingdom of Saudi Arabia. There are more than
70 cultivars that have been grown there for ages, Three cultivars,
namely “khalas”, “Shaishi” and “Ruzaiz” have been selected from
each of the two localities (Al-Ahsa and Al- Qatif) and subjected to
isoenzymes electrophoretic analysis. Leaflets and roots of the three
cultivars were analyzed using the PAGE techniques for the occurrence
of the isoenzymes EST, GOT, SOD, GDH and LAP. The results show
that the three cultivars differed in their isoenzymes pattern within the
same location and between different locations. The obtained results
also signified that the isoenzymes patterns could be used as genetic
expression markers for the cultivars and their interactions with the
environmental factors in the two locations.
POSTERS MONDAY & TUESDAY
POS-MON-309
SOLUTION STRUCTURE AND DYNAMICS OF THE
RING FINGER DOMAIN FROM THE HUMAN SPLICING-
ASSOCIATED PROTEIN RBBP6 DETERMINED USING
HETERONUCLEAR NMR SPECTROSCOPY
Kappo M.A.1, 2 , Atkinson R.A.3, Ab E.4, Rees D.J.G.1 and Pugh D.J.R.1
1
Department of Biotechnology, University of the Western Cape, South
Africa. 2Department of Genetics, University of Stellenbosch, South
Africa. 3Randall Division of Cell and Molecular Biophysics, Kings
College London. 4Department of Chemistry, University of Leiden,
Netherlands.
RBBP6 is a 250-kDa multi-domain protein that interacts with both
p53 and pRb and has been implicated in mRNA splicing. The protein
contains an atypical RING finger with a C4C4 motif rather than the more
common C3HC4 motif. We report here on the solution structure and
metal exchange properties of the RING domain from human RBBP6,
using heteronuclear NMR spectroscopy. 15N- and 13C-labelled protein
samples were generated as GST-fusion by growing bacteria in minimal
media. A complete set of heteronuclear NMR data was collected at
600 MHz from which almost complete assignment of the backbone,
side-chain and aromatic resonances was achieved. Exchange of Zn2+
with 113Cd2+ confirmed the domain binds two Zn2+ ions, which were
coordinated in the expected cross-brace manner as common with
RING fingers. Structural data in the form of 2D-NOESY, 15N-separated
NOESY and 13C-separated NOESY spectra were recorded and used
to determine the structure using restrained molecular dynamics on
CYANA platform. Our results further showed the structure of the RING
domain closely resembling that of the U-boxes, particularly the U-box
from CHIP (C-terminal of Hsp70-Interacting Protein). The domain
homodimerises across the same interface as in U-boxes, and features
the same hydrophobic groove forming the binding site for E2 enzymes.
The structural similarities between the RBBP6 RING finger and the
U-box family led us to conclude that RBBP6 may, like CHIP, play a role
in protein quality control.
POS-MON-311
TARGETED PEPTIDOMIC APPROACHES TO STUDY
IMMUNE RECOGNITION
Dudek N.L.1, 2, Tan C.T.1, 2, Croft N.P.1, 2, Scull K.E.1, 2, Corbett A.J.1, 2,
Webb A.I.1, 2, Barr D.P.1, 2, Reilly C.B.1, 2, Williamson N.A.2 and Purcell
A.W.1, 2
1
Biochemistry and Molecular Biology, University of Melbourne. 2Bio21
Molecular Science and Biotechnology Institute.
The immune system has evolved to recognise foreign antigen in a
highly sensitive and specific manner. Until recently functional assays
using clonal populations of immune effector cells have been required
to aid in the discovery of new targets of immunity and to study antigen
presentation. With recent advances in mass spectrometry, and in
particular targeted approaches such as precursor scanning and multiple
reaction monitoring, the requirement for biological readouts has been
replaced by robust, relatively cheap and high-throughput assays. We will
present new strategies in the analysis of peptides involved in immune
recognition that will ultimately replace many conventional functional
cellular immunoassays.
Page 178 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-310
CRYSTAL STRUCTURE OF THE ENTIRE ECTODOMAIN
OF GP130: INSIGHTS INTO THE MOLECULAR
ASSEMBLY OF CYTOKINE RECEPTOR COMPLEXES
Xu Y., Kershaw N., Luo C.S., Soo P., Pocock M.J., Czabotar P.E.,
Hilton D.J., Nicola N.A., Garrett T.P.J. and Zhang J.-G.
The Walter + Eliza Hall Institute of Medical Research, 1G Royal
Parade, Parkville, Victoria 3052, Australia.
The cell surface receptor gp130 is a shared signalling receptor subunit
for the interleukin 6 (IL-6)-like cytokine family, which includes, IL-6,
IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary
neurotrophic factor (CNTF). This family of cytokines is involved in
inflammatory and immune responses and also plays crucial roles in
hematopoiesis, liver and neuronal regeneration, embryonic development,
and fertility. Dysregulation of signalling contributes to diseases such as
inflammatory bowel disease, osteoporosis, multiple sclerosis, multiple
myeloma, and prostate cancer. Previous X-ray structures of ligand-
receptor complexes of this family lacked the C-terminal domains
required for effective signal transduction. I will present the first crystal
structure of the entire extracellular portion of human gp130 (D1-D6) at
3.6-Å resolution. This represents the first atomic resolution structure of
the complete ectodomain of any “tall” cytokine receptor. The structure
shows that, other than a reorientation of the D1 domain, there is little
structural change in gp130 upon ligand binding. It also reveals that the
interface between the D4 and D5 domains forms an acute bend in the
gp130 structure. Key residues at this interface are highly conserved
across the entire “tall” receptor family, suggesting that this acute bend
may be a common feature of these receptors. Importantly, this geometry
positions the C-termini of the membrane-proximal FNIII domains of the
tall cytokine receptors in close proximity within the transmembrane
complex, favourable for receptor-associated Janus kinases to trans-
phosphorylate and activate each other.
POS-TUE-312
VARIABLE ROLE OF NON-CATALYTIC DOMAINS ON
ACTIVITIES OF CELLULASES AND XYLANASES
Sajjad M., Mahmood I. and Akhtar M.W.
School of Biological Sciences, University of the Punjab, Lahore,
Pakistan.
Whereas carbohydrate binding domains (CBD) are widely associated
with cellulases and xylanases, the requirement of CBD seems
unnecessary for enhancing catalytic activity of some enzymes, which
occur in nature as CBD-catalytic domain complex. Two major xylanase
components, XynC and XynZ from the anaerobic thermophilic bacterium,
Clostridium thermocellum cellulosome were cloned and expressed with
and without non-catalytic domains in E. coli. In the case of XynC the
binding domains seemed to enhance activities as well as stability of
the enzyme. These effects were more pronounced when the binding
domains were present at both the termini of the catalytic domain (pXynC-
BCB). However, for XynZ the deletion of the binding domain not only
enhanced expression level but also showed increase in specific activity
as well as thermostability significantly. The overall increase in activity
was ~9-fold higher for XynZ-C as compared to that of XynZ-BDC. The
major cellulosomal endoglucanase, CelA lacks a binding domain in the
native form. The gene encoding this enzyme was inserted into pET22b
to produce the construct pCelA-C. Binding domain of family 3a was
added at its N- and C-termini to produce the constructs pCelA-BC and
pCelA-CB, respectively. The overall endoglucanase activity expressed
in E. coli in the case of the catalytic domain only was almost twice that of
those having a binding domain. However, fusion of the binding domain
at C-terminal enhanced thermostability of the enzyme. It is possible that
the change in the physical environment from that of the native state, and
in the 3-D structure of the protein has varying effects on their activity
and stability.
POSTERS MONDAY & TUESDAY
POS-MON-313
EFFECT OF GENOTYPE, COLD PRETREATMENT
AND CULTURE MEDIUM COMPOSITION ON ANTHER
CULTURE RESPONSE OF BARELY (HORDEUM
VULGARE L.)
Ghasempour H.R.1, Kahrizi D.2 and Abdi P.1
1
Biology and Biotechnology Dept., Razi University, Baghabrisham,
67149-67346, Kermanshah, Iran. 2Biotechnology Research Institute,
Agricultural Faculty, Razi University, Kermanshah, Iran.
In this study the effects of genotype, cold pretreatment of anthers and
different 2,4-D concentrations added to the FHG barely anther culture
medium were examined. The callus induction and plant regeneration in
anther culture of two barely genotypes (G1, G2) were investigated in FHG
induction medium with different levels of 2,4-D (1,2,3 mg/l) after cold
pretreatment of spikes for 0, 7, 14, 21, 28 days at 4ْ C. Callus induction
was accomplished on FHG induction medium with different levels of 2,4-
D (1,2,3 mg/l) in combination with kinetin (0.1 mg/l). Then Calli of 2-3 mm
were transferred onto MS regeneration medium containing 1 mg/l 2,4-D
and 5 mg/l BAP. Plantlets that were regenerated from calli transferred
onto tubes with same regeneration medium but without any growth
regulator. Plants of 10 cm length were transferred to pots. Analysis
of variance showed highly significant difference between genotypes,
cold pretreatment and 2,4-D concentration on all of the androgenic
parameters (Callus, Green Plant, Albino Plant, Total Plant). Generally
three ways interacted factors showed non- significant effects. Some of
interaction effects were important for androgenesis. Results revealed
that the response of genotype to various 2,4-D concentration in media
for callus induction were significant. The green plant regeneration was
genotype dependent. Overall G1 androgenic responses were higher
than G2. FHG medium with 3 mg/l 2,4-D was most efficient in callus
induction. The optimum green plant regeneration was observed in
FHG medium containing 1mg/l 2,4-D. The optimum cold pretreatment
period for androgenic response was 14 days. Key words: anther culture,
Hordeum vulgare, Plant regeneration, Androgeneis, Cold pretreatment.
POS-MON-315
BORON TOXICITY TOLERANCE IN BARLEY THROUGH
REDUCED EXPRESSION OF THE MULTIFUNCTIONAL
AQUAPORIN HVNIP2;1
Hayes J.1, Schnurbusch T.3, Hrmova M.1, Baumann U.1, Ramesh S.A.2,
Tyerman S.D.2, Langridge P.1 and Sutton T.1
1
1Australian Centre for Plant Functional Genomics, School of
Agriculture, Food and Wine, University of Adelaide, Glen Osmond SA
5064, Australia. 2School of Agriculture, Food and Wine, University of
Adelaide, Glen Osmond SA 5064, Australia. 3Leibniz-Institute of Plant
Genetics and Crop Plant Research (IPK), Genebank Department,
Corrensstr. 3, D-06466 Gatersleben, Germany.
Boron (B) toxicity is a significant limitation to cereal crop production in
a number of regions worldwide, and although the problem has been
recognised for many years, cultivated barleys are not well adapted.
Here we describe the cloning of a gene from barley, underlying the
chromosome 6H B toxicity tolerance quantitative trait locus. It is the
second B toxicity tolerance gene identified in barley. Previously, we
identified the gene HvBot1 which functions as an efflux transporter in
B toxicity tolerant barley to move B out of the plant. The gene identified
in this work encodes HvNIP2;1, an aquaporin from the NIP subfamily
which was recently described as a silicon influx transporter (Lsi1) in
barley and rice. Here we show that a rice mutant for this gene also
shows reduced B accumulation in leaf blades compared to wild type and
that the mutant protein alters growth of yeast under high B. HvNIP2;1
facilitates significant transport of B when expressed in Xenopus oocytes
compared to controls and to another NIP (NOD26), and also in yeast
plasma membranes which appear to have relatively high B permeability.
We propose that tolerance to high soil B is mediated by reduced
expression of HvNIP2;1 to limit B uptake as well as by increased
expression of HvBot1 to remove B from roots and sensitive tissues.
Together with HvBot1, the multifunctional aquaporin HvNIP2;1 is an
important determinant of B toxicity tolerance in barley.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 179
POS-TUE-314
DIAGNOSTIC ASSESSMENT AND REMEDIATION
STRATEGIES IN INTRODUCTORY MOLECULAR LIFE
SCIENCES COURSES
Wright T.1, Hamilton S.1, Rafter M.1, Howitt S.2, Anderson T.3 and
Costa M.4
1
The Uiversity of Queensland, St Lucia. 2Australian National University,
Canberra. 3University of KwaZulu-Natal, South Africa. 44University of
Minho, Portugal.
A Molecular Life Science Concept Inventory has been completed
recently and can now be used as a diagnostic tool in introductory
biochemistry courses. The inventory contains questions drawing
on physical, chemical and biological concepts and set in molecular
life sciences contexts. The test gives the instructor information on
specific concepts that are poorly understood, as well as alternate
conceptions or misconceptions that students may have. Results from
extensive testing of the inventory, which has been carried out at three
universities in Australia and in South Africa, show that different cohorts
of students are responding very similarly, and that there are areas
where misconceptions are common. This paper will firstly examine a
selection of results from the testing of the inventory. It will then propose
some strategies to remediate student difficulties identified through the
inventory. These are presented as a module of “teaching objects” which
target one specific topic in a typical introductory biochemistry course
(protein folding), but which we wish to present as a more general model
for use in course design. In developing the module, we have considered
how to use the topic to (i) remediate student difficulties effectively around
key concepts such as bond rotation, free energy, dynamic equilibrium
and hydrophobicity; and at the same time (ii) engage students in some
of the big conceptual ideas associated with protein folding, for example
self assembly and molecular evolution, which can be developed at
increasing levels of sophistication in advanced courses.
POS-TUE-316
TIME DEPENDENT EVALUATION OF THE
CYTOTOXICITY OF PETROLEUM HYDROCARBON
BIOREMEDIATED SOIL
Ho D.H.T.1, Sanderson B.J.1, Ball A.S.2 and Schmidt L.1
1
Medical Biotechnology, Flinders University School of Medicine.
2
School of Biological Sciences, Flinders University.
Bioremediation is a process which aims to reduce the amount of
chemical contaminants in an environmental sample. One goal of
bioremediation is to reduce the hazard posed by the contaminated
sample. However, the toxicity of the sample may vary during this process
due to the breakdown of various contaminants into their metabolites.
Two in vitro cell viability assays, the MTT assay and the Crystal Violet
assay were used to evaluate the cytotoxicity of petroleum hydrocarbon
contaminated soil extracts before, during and after bioremediation.
Bioremediation was performed by a combination of bioaugmentation
and biostimulation over a period of 0-12 weeks. Chemical analysis
of the extracts was carried out using GCMS analysis. Results will be
presented comparing changes in cell viability to changes in chemical
composition. This will show if there is any relationship between toxicity
and the nature of contamination and/or breakdown products of the
contaminating material during the bioremediation process.
POSTERS MONDAY & TUESDAY
POS-MON-317
A FAMILY OF PLANT RECEPTOR-LIKE KINASES WITH
DUAL FUNCTIONING INTRACELLULAR SIGNALING
DOMAINS
Donaldson L.1, Meier S.1, Kwezi L.2, Wheeler J.2, Gehring C.1 and
Irving H.2
1
4700 King Abdullah University of Science and Technology, Thuwal
23955-6900, Saudi Arabia. 2Monash Institute of Pharmaceutical
Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052,
Australia.
The presence of the second messenger guanosine 3’5’-cyclic
monophosphate (cGMP) in plants has been unequivocally established
however the guanylyl cyclase (GC) enzymes responsible for cGMP
synthesis remain elusive. We designed a search motif using consensus
residues present in the catalytic domain of GCs from lower eukaryotes
and animals to identify putative GCs in Arabidopsis. The search returned
36 putative GCs, the majority of which are annonated as receptor-like
kinases (RLKs). We have demonstrated that a number of these RLKs
function as both GCs and kinases in vitro and in vivo. These include
WALL ASSOCIATED KINASE LIKE 10 (WAKL10), BRASSINOSTEROID
INSENSITIVE 1 (BRI1), PHYTOSULFOKINE RECEPTOR 1 (PSKR1)
and PEP1 RECEPTOR 1 (PEPR1), known for their crucial roles in
plant development and stress responses. Moreover, for BRI1 and
PSKR1, the natural ligands of these receptors were shown to stimulate
increases in cGMP in planta. Interestingly these plant receptor GCs
differ from their animal counterparts in that the GC domain overlaps
the kinase domain in plants whereas these domains remain distinct in
animals. This raises the possibility that the plant receptors might switch
between downstream cGMP-mediated or kinase-mediated signaling
to elicit desired outputs to particular stimuli. The challenge now lies in
understanding the interaction between the GC and kinase domains and
how these receptors utilize their dual functionality in planta. The large
family of RLKs with both GC and kinase domains implies that these dual
functionalities have coevolved due to the importance of both enzyme
activities in plant development and stress responses.
POS-MON-319
SCHWANN CELLS INTERACT WITH GLIAL CELLS AND
NEURONS VIA MOTILE LAMELLIPODIAL WAVES
Ekberg J.A.K., Windus L., Scott S., Lineburg K., Cornock J., Mackay-
Sim A. and St John J.A.
National Centre for Adult Stem Cell Research, Griffith University,
Nathan 4111, Brisbane, Queensland, Australia.
Implantation of olfactory ensheathing cells (OECs) or Schwann cells
(SCs) into damaged CNS tracts have led to axonal regeneration but the
results are not optimal and the favorable glial type may vary depending
on the site of injury. Therefore it is crucial to determine how these glial
cells interact with neurons so that treatments can be optimised. We have
previously shown that OECs display highly motile peripheral membrane
protrusions termed lamellipodial waves, which mediate OEC-OEC
contacts as well as OEC-axon contacts. Using time lapse imaging of
fluorescently labeled cells, we now have investigated the presence of
dynamic lamellipodial waves in SCs. The waves were similar to those
in OECs in terms of number of waves/cell and wave area but travelled
~2-fold faster. The number of waves and wave area of SCs correlated
positively with whole-cell migration rate. Both OECs and SCs interacted
directly with DRG axons via lamellipodial waves and selectively migrated
along DRG axons rather than on the surrounding substrate. Contact with
axons increased the migration rate of glial cells ~2-fold. Finally, both SCs
and OECs significantly promoted survival of DRG neurons with survival
after 3 days in a nutrient-free medium increasing from 40 % to 67 % by
SCs and to 77 % by OECs. In summary, we have demonstrated that
SCs exhibit lamellipodial waves, and that contact with axons leads to
increased wave activity and increased migration. Both OECs and SCs
promoted neuronal survival, suggesting that transplantation of both SCs
and OECs may promote regeneration of damaged sensory neurons.
Page 180 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-318
ETHANOLIC EXTRACT OF ZINGIBER OFFICINALIS
(GINGER) SUPPRESSES HEPATIC NFκB ACTIVITY
Li X.H.1, McGrath K.C.2, Heather A.K.2, 3 and Roufogalis B.D.1
1
Herbal Medicines Research and Education Centre, Faculty of Pharmacy,
The University of Sydney, NSW 2006, Australia. 2The Heart Research
Institute, Sydney, NSW 2042, Australia. 3Medical and Molecular
Biosciences, University of Technology Sydney, NSW 2007, Australia.
Objective - Lifestyle changes have resulted in an obesity epidemic
associated with increased incidence of type 2 diabetes, hypertension and
non-alcoholic fatty liver disease (NAFLD). Hepatic inflammation drives
the pathogenesis of these chronic diseases. Nuclear factor κB (NFκB) is
the master regulator of the hepatic inflammatory response. Active hepatic
IKKα/IκBα/NFκB pathway can drive the onset of insulin resistance, a
prominent feature of metabolic syndrome and type 2 diabetes. Previously,
we have demonstrated that an ethanolic extract of ginger has protects
high fat-fed rats against the development of insulin resistance. We now
explore whether this ginger extract protects against insulin resistance by
inhibiting hepatic activation of the classical NFκB signaling Bκpathway.
Methods - NFκB activity was tested using HuH7 cells ransfected an
NF luciferase reporter vector and then exposed to the ethanolic ginger
extract for 24 hrs. Cells were then activated with interleukin-1β (IL-1β for
3 hrs before luciferase activity was measured. Real-time PCR (mRNA)
and Western blot (protein) were used to measure NFκB target gene
and NFκB inhibitor protein (IκBα) expression levels. Results-Ethanolic
ginger extract significantly suppressed NFκB activity (IL-1β: 157±17 vs
IL-1β+ginger:120±10, p<0.05) and prevented the degradation of IκBα.
Ethanolic ginger extract decreased expression of NFκB target genes
including serum amyloid A1 (SAA1), interleukin 6 (IL-6) and interleukin 8
(IL-8) in a dose-dependent manner. Conclusions - Ethanolic ginger extract
containing gingerol and shogaol components suppresses NFκB activity
and downstream expression of genes that orchestrate the inflammatory
response in cultured human liver cells. This finding highlights the potential
mechanism that may underlie the protective effects of ethanolic extract of
ginger on insulin resistance in vivo. (supported by the National Institute of
Complementary Medicine (NICM) grant to BDR).
POS-TUE-320
ASSOCIATION OF SPECTRIN TO BRAIN GAMMA
TUBULIN
Shashikala S. and Sengupta S.
Rajiv Gandhi Centre for Biotechnology, Trivandrum, INDIA.
γ-tubulin, the newer member of the tubulin superfamily, is known to
mediate microtubule nucleation from the centrosome of eukaryotic cells.
The major amount of γ-tubulin is believed to be located in the centrosome.
However, a considerable amount has been found in the cytoplasm in the
form of a ring complex (γ-TuRC) along with some other proteins, whose
function is not well known. In our earlier studies by immunoprecipitation
and immunoaffinity, we found that the purified brain cytoplasmic γ-tubulin
complex contains alpha and beta non-erythroid spectrin, which were not
reported in other tissues. Spectrin is a membrane bound protein which
forms a flexible scaffold like network. Spectrin has also been reported to
interact with centractin which is a centrosomal actin like protein. In this
study, we wanted to check the role of spectrin in γ-tubulin complex. In
vitro, our tubulin polymerization studies show that the presence of spectrin
in γ-TuRC inhibits nucleation and polymerization of tubulin. But when
spectrin is complexed with anti-spectrin antibody, tubulin polymerization
is restored. With a goal to extend the study in vivo, we initially studied
the association of spectrin and γ-tubulin in brain tissue and brain derived
cell types at different conditions. Here we report the co-localization of
γ-tubulin and spectrin in tissue sections from the cortical region of the
brain by immunohistochemistry. Co-localization was also observed
by immunofluorescence experiments in both neuroblastoma and
gliablastoma cells in the extranuclear parts including the centrosomes
which showed intense labelling. Purified centrosomes from goat brains
also showed the presence of spectrin along with γ-tubulin and centrin (a
centrosomal marker). We therefore conclude that spectrin is associated
to γ-tubulin complex in the centrosomes of both neuronal and glial cells
and has important role in the function of the complex.
POSTERS MONDAY & TUESDAY
POS-MON-321
MOLECULAR MECHANISM OF RICE BRAN PHYTIC
ACID AS ANTICANCER IN HUMAN COLORECTAL
ADENOCARCINOMA CELLS
Shafie, N.H.1, Mohd-Esa, N.1, 2, Ithnin, H.1, 2, Md-Akim, A.2 and Saad, N.1
1
Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor,
Malaysia. 2Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia.
Phytic acid (inositol hexaphosphate or IP6) is one of bioactive compound
that is present in cereals, nuts and legumes. Phytic acid (IP6) is naturally
occurring polyphosphorylated carbohydrate recognized to posses
various significant health benefits including a potential as anticancer
compound. Several in vivo and in vitro studies provide convincing
evidence for the anticarcinogenic properties of IP6 in wheat bran whilst
the underlying mechanisms by which IP6 exerts anti-tumorigenic effects
are largely unknown. The purpose of this study was to investigate the
growth inhibitory effects of rice bran IP6 on human colorectal cancer cell
line, HT-29. IP6 induced marked growth inhibition in HT-29, in a dose and
time dependent manner. Flow cytometry was performed for the analysis
of cell cycle and apoptosis. Treatment of HT-29 with IP6 resulted in cell
cycle arrest. In addition, induction of early and late apoptotic cell death
in a dose- and time dependent manner was confirmed using an Annexin
V-based assay. PCR analyses indicated that IP6 upregulated proapoptotic
genes and downregulated antiapoptotic genes. IP6 is expected to exert
anticarcinogenic activity through induction of apoptosis and disruption
of cell cycle progression. Our study suggests that rice bran IP6 can be
developed as a chemopreventive agent for human colorectal cancer.
POS-MON-323
QUANTITATIVE ANALYSIS OF ANDROGENIC
INFLUENCE ON ZEB-1 GENE EXPRESSION
Anose B.M. and Benjamin D.E.
Bethel University, 3900 Bethel Drive, St. Paul, MN 55112, U.S.A.
Roughly one in six men will be diagnosed with prostate cancer during
his lifetime. Localized prostate cancer occurs when a tumor is contained
within the prostate; this prognosis is quite favorable because the tumor
can be removed surgically. The prognosis for metastatic prostate cancer
is not nearly as promising because the cancer can be life threatening
once it spreads into other tissues and disrupts normal biological function.
There is currently no diagnostic tool available to distinguish between
localized and metastatic prostate cancer. However, it has recently
been discovered that Zinc Finger E-Box Binding Homeobox-1 (ZEB-1)
could putatively be used as a biomarker for prostate cancer metastasis.
Previous research has shown that changes in the expression of ZEB-
1, a transcription factor, correlate with prostate cancer progression. To
gain further knowledge about this potential diagnostic gene, human
prostate cancer cells from the cell line 22Rv1 were treated with
dehydroandrosterone (an androgen) and flutamide (an anti-androgen)
to determine their effect on the expression of ZEB-1. After treatment,
RNA was isolated and reverse transcribed into cDNA for its use in
quantitative real time-polymerase chain reaction (qRT-PCR). qRT-PCR
revealed that dehydroandrosterone and flutamide do not significantly
influence ZEB-1 expression.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 181
POS-TUE-322
SUBCELLULAR LOCALIZATION AND ARGININE
METHYLATION OF SERBP1
Lee Y.J.1, Hsieh W.Y.2, Chen L.Y.1 and Li C.2
1
Institute of Biochemistry and Biotechnology. 2Department of Biomedical
Sciences, Chung Shan Medical University, Taichung, Taiwan.
Protein arginine methylation catalyzed by protein arginine
N-methyltransferases (PRMTs) can regulate several cellular processes.
Methylarginines are mostly found in arginine and glycine rich (GAR)
motifs or arginine rich domains in proteins. According to the GAR motif,
we identified an open reading frame (accession number AL080119) now
designated as SERBP1 (SERPINE1 mRNA binding protein 1). SERBP1
contains an arg-rich domain, an RG-rich domain, and an RGG box domain
that might be methylated by PRMTs, and a nuclear localization signal
(NLS) overlapping the RG-rich domain. In order to analyze the subcellular
localization of SERBP1, expression of GFP-fused full-length or truncated
SERBP1 in HeLa cells was analyzed by fluorescent microscopy. Full-
length SERBP1 localized mainly in the cytoplasm while proteins containing
the middle RG-rich domain with N-terminal arg-rich or C-terminal RGG
box deletions localized mainly in the nucleus with granular structures.
However, the RG-rich domain deleted protein is mostly expressed in the
cytoplasm. Treatment of a methylation inhibitor AdOx did not change the
localization of all SERBP1 proteins. Furthermore, methylarginine levels of
FLAG-fused SERBP1 proteins expressed in HeLa cells were determined
by an asymmetric dimethylarginine-specific antibody. Methylarginine
signals were detected in purified full-length or truncated FLAG-SERBP1
proteins containing RG-rich or RGG box. AdOx treatment significantly
reduced the methylation signals in these proteins. Moreover, the most
predominant type I protein arginine methyltransferase PRMT1 was co-
immunoprecipitated with SERBP1 and bound PRMT1 decreased upon
the addition of AdOx. In conclusion, the SERBP1 protein is localized in
the cytoplasm in spite of the NLS in RG-rich region. The N- and C-terminal
domains appear to be important for the cytoplasmic localization. The
protein is methylated in the RG-rich and RGG box but arginine methylation
is not related to the subcellular localization. The presence of asymmetric
dimethylarginine and interaction with PRMT1 indicate that SERBP1 is
specifically methylated by PRMT1.
POS-TUE-324
TEACHING EXPERIMENTAL, ETHICAL, AND
QUANTITATIVE (TEEQ) BIOLOGY USING CALIBRATED
PEER REVIEW™ (CPR)
Pelaez N.J.1, Dasgupta A.P.1, Gundlach E.1 and Russell A.A.2
1
Purdue University Department of Biological Sciences, West Lafayette,
IN 47907-2054, USA. 2Department of Chemistry and Biochemistry,
UCLA, Los Angeles, CA 90095-1569, USA.
CPR™ was used for Teaching Experimental, Ethical, and Quantitative
(TEEQ) Biology in a first-year course to help undergraduate students
connect what they learn in lecture to both current and historical research
endeavors. CPR is a Web-based program that enables frequent writing
assignments even in large classes. CPR was used to help students
learn to gauge their own ability and monitor their own thinking about
experimental design in primary research publications. This study
examined improvements in abilities to (1) identify the treatments in a
biological experiment; (2) present a completely randomized design to
address a research question; (3) recognize the benefit of limiting sources
of variability; and (4) describe the limitations to the scope of inference
for a biological investigation. A previously validated statistics item was
used as a measure. Students had the most difficulty with the concept of
randomization and replication while treatment identification was easier
for students. Interestingly, their textbook (Freeman, 2008; Biological
Science) illustrates each experiment without replications. Students
who had taken a statistics course in their past performed significantly
better on the pre-test than those who had no statistics training. On the
post-test, scores for students in a biology lecture course where they
used CPR were better than scores on the pre-test (including for those
who had taken statistics). In summary, CPR made it possible to teach
experimental thinking in the context of a large enrollment biology lecture
class.
POSTERS MONDAY & TUESDAY
POS-MON-325
ARABINOXYLAN ARABINOFURANOHYDROLASE
(AXAH) GENES IN CEREALS
Laidlaw H.K.C. and Jobling S.A.
CSIRO Plant Industry and Food Futures Flagship High Fibre Grains
Research Cluster, GPO Box 1600, Canberra ACT 2601 Australia.
Arabinoxylan (AX) is a key component of cereal cell walls and thought
to be involved in the remodelling of the cell wall during growth and
development. Several studies have shown changes in the structure of AX
during development and between different plant tissues. Arabinoxylan
arabinofuranosidase enzymes (AXAH; glycosyl hydrolase family 51) are
implicated in the removal of the α-L-arabinofuranosyl moieties from the
xylose backbone of AX, and are encoded by a complex and variable
gene family in cereals. The members of the AXAH gene family have
been isolated from wheat and barley and compared to their homologues
from rice, sorghum and Brachypodium. Differences in transcript levels
of the genes during grain development and between plant tissues
have been observed. Biochemical characterisation of selected AXAH
enzymes has also demonstrated activity against wheat AX.
POS-MON-327
A GENE EXPRESSION SIGNATURE FOR INSULIN
RESISTANCE MEDIATED BY EXCESS FAT
Hayward B.1, Konstantopoulos N.1, Molero-Navajas J.1, Jowett J.2 and
Walder K.1
1
Metabolic Research Unit, Deakin University, Geelong, VIC 3217.
2
Baker IDI Heart and Diabetes Institute, Melbourne, VIC 3004.
Background: A key feature of type 2 diabetes is insulin resistance.
Multiple pathways are implicated in insulin resistance, such as
hyperlipidemia, elevated levels of pro-inflammatory cytokines and/or
induction of oxidative or ER stress. The complexity of insulin resistance
hinders effective characterisation and treatment of type 2 diabetes. We
propose that small sets of genes known as gene expression signatures
(GES) that reflect the insulin resistant state can be identified. The GES
provides an unbiased transcriptional snapshot of the integrated cellular
response to an insult. Methods: Hyperlipidemia was modelled in FAO
liver cells using palmitate (PA), and global gene expression profiling
was performed using microarrays. This was followed by bioinformatics
analyses to identify the GES whose expression levels most significantly
discriminated between the insulin resistant and insulin sensitive states.
Insulin sensitivity was assessed by glucose production as the functional
endpoint. Results: In vehicle-treated cells, insulin (0.1nM, 24h)
decreased glucose production by 30±1% (P < 0.0001, n=3) indicating
an insulin sensitive state. PA treatment (75μM, 48h) impaired the ability
of insulin to decrease glucose production by ~20% (P < 0.04, n=3)
indicating a state of insulin resistance. The inhibitory effect of PA on
glucose production was reversed by the addition of metformin (0.25mM)
and sodium salicylate (2mM) in the final 24h of the PA treatment (P <
0.0001, n=3). Conclusion: This study will identify a set of genes whose
expression best defines the difference between insulin sensitivity and
PA-induced insulin resistance in FAO liver cells.
Page 182 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-326
TISSUE-SPECIFIC EXPRESSION IN TRANSGENIC TOMATO
FRUIT OF GUS GENE DRIVEN BY THE 5’ REGULATORY
SEQUENCES OF DESATURASE MESOCARP SPECIFIC OIL
PALM (ELAEIS OLEIFERA) GENE
Ismail I. 1, 2 , Saed Taha R.1, 2 and Abdullah N.3
1
School of Biosciences and Biotechnology, Faculty of Science and
Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor,
Malaysia. 2Centre for Plant Biotechnology, Universiti Kebangsaan
Malaysia, 43600, Bangi, Selangor, Malaysia. 3Department of Agriculture,
Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang,
Selangor, Malaysia.
Gene promoters are sequences of DNA that drive and regulate gene
expression. Some promoters are constitutive in that they allow for continual
transcription of associated genes whereas many others are specific that they
drive gene expression in particular space and time. In this study the oil palm
(Elaeis oleifera) mesocarp desaturase (Des) gene promoter specificity and
strength was evaluated and compared to cauliflower mosaic virus (CaMV)
35S promoter. Five deletions were purposely generated by PCR and cloned
into pCAMBIA 1301. The ability of these fragments to direct mesocarp-
specific expression was studied by fusing them to the β-glucuronidase
(GUS) reporter gene. Cotyledons and hypocotyls of tomato (Lycopersicon
esculentum) were transformed with Agrobacterium tumefaciens LBA4404
cultures carrying Des promoter and its deletions fused to GUS in pCAMBIA
1301 plasmids. The specificity of GUS expression directed by Des promoters
was performed by histochemical GUS staining of fruit and leaf tissues from
T0 plants. The results showed that all the deletions of Des promoter directed
gus gene expression in tomato mesocarp tissue, whereas no activity was
observed in any other vegetative tissues indicating the specificity of Des
promoter. The quantitative analysis of GUS expression by flourometric
assay has demonstrated much higher activity of the longest Des promoter
(~1000 bp) with an average value of 1386 pmol µg-1 min-1 compared to
CAMV 35S promoter with an average value of 347pmol µg-1 min-1. Further
analysis on the strength of the Des promoter has shown the highest GUS
activity by ~ 600 bp Des promoter, thus suggesting the possible presence of
silencing motifs upstream of the sequence.
POS-TUE-328
TBC1D13: A RAB10/RAB1 BINDING PROTEIN
INVOLVED IN INSULIN-STIMULATED GLUT4
TRANSLOCATION
Davey J.R.1, Larance M.1, Junutula J.R.2, James D.E.1 and Stoeckli J.1
1
The Garvan Institute of Medical Research, 384 Victoria St, Sydney,
NSW 2010. 2Genentech Inc., 1 DNA Way, South San Francisco, CA
94080.
In adipocytes the small GTPase Rab10 plays an important role in the
regulation of GLUT4 trafficking. Here we demonstrate that Rab10
forms a stable complex with TBC1D13, a member of the Rab GTPase
activating protein (RabGAP) family. This adds to the complexity of
Rab10 mediated trafficking because two other RabGAPs, TBC1D1
and TBC1D4, also regulate Rab10. The Rab10/TBC1D13 interaction
was GTP-dependent and specific, in that other members of the TBC
family did not bind to Rab10. Immunoprecipitation of endogenous
TBC1D13 from adipocytes resulted in co-precipitation of Rab10
and Rab1. RNAi ablation of TBC1D13 expression by siRNA reduced
insulin-stimulated glucose uptake in adipocytes and overexpression of
TBC1D13 completely inhibited insulin-stimulated GLUT4 translocation
to the plasma membrane (PM). This was specific to GLUT4 trafficking
as TBC1D13 overexpression did not affect transferrin receptor recycling
or tsVSV-G trafficking. TBC1D13 did not display any detectable in vitro
GAP activity on Rab1 or Rab10 and the catalytically inactive mutant of
TBC1D13 did not abolish the inhibitory effect on GLUT4 translocation.
These studies reveal a role for TBC1D13 in GLUT4 trafficking in
adipocytes that is independent of it’s putative GAP ability. Based upon
the Rab binding specificity of this GAP we suggest it might regulate a
trafficking step between the trans-Golgi cisternae and endosomes.
POSTERS MONDAY & TUESDAY
POS-MON-329
ANALYSIS OF THE TRANSCRIPTIONAL ACTIVITY OF
THE SOYBEAN BASIC HELIX LOOP TRANSCRIPTION
FACTOR GMSAT1 WHEN EXPRESSED IN THE YEAST
AMMONIUM TRANSPORT MUTANT 26972C
Mazurkiewicz D.1, Chiasson D.1, Loughlin P.2, Okamoto M.3 and
Kaiser B.N.1
1
School of Agriculture, Food and Wine. The University of Adelaide,
Urrbrae, SA, 5064, Australia. 2School of Biological Sciences. The
University of Sydney, Sydney, NSW, 2006, Australia. 3Australian
Centre For Plant Functional Genomics. The University of Adelaide,
Urrbrae, SA, 5064, Australia.
GmSAT1 is a soybean basic helix loop helix (bHLH) transcription
factor localised to the peribacteroid membrane and nucleus of infected
nodule cells. GmSAT1 was first identified by its ability to functionally
complement the growth of a yeast ammonium transport mutant 26972c
on low ammonium concentrations (1 mM). GmSAT1 also increases the
uptake of the toxic ammonium analogue methylammonium (MA). Like
in soybean, GmSAT1 is membrane bound in yeast and upon activation
cleaved from its C-terminal membrane anchor and enters the nucleus.
We have evaluated the transcriptional activity of GmSAT1 in 26972c
cells grown for 12 hours in media containing 1mM ammonium, using
microarray (Affymetrix Yeast Genome 2.0 Array) and quantitative RT-
PCR. We have characterised one of the genes, DMO1, upregulated
approximately 55-fold by GmSAT1. Analysis of the promoter region
of DMO1 identifies multiple bHLH E-box DNA binding motifs. DMO1,
previously uncharacterised, shows strong similarity to the predicted
drug:H+ antiporters of the DHA2 family of major facilitator proteins.
Overexpression of DMO1 in 26972c enhanced 14C-MA uptake in a SAT1
like manner, while loss of DMO1 activity in a 26972c:Δdmo1 mutant
eliminated the GmSAT1 induced enhancement of 14C-MA uptake and
related toxicity phenotype when grown at elevated MA concentrations.
We are currently exploring the binding affinities of GmSAT1 to the
promoter region of DMO1 and investigating the functional similarity of
DMO1 homologues identified in soybean.
POS-MON-331
PURIFICATION AND CHARACTERIZATION OF A NOVEL
ALKALINE SERINE PROTEASE SECRETED BY VIBRIO
METSCHNIKOVII
Park J.Y. and Lee J.S.
Department of Biotechnology and BK21 Research Team for Protein
Activity Control, Chosun University, Gwangju 501-759, Republic of
Korea
A pathogenic marine bacterium Vibrio metschnikovii (V. metschnikovii)
evokes serious symptoms, including pneumonia, leg ulcer, and diarrheal
disease. In this study, a novel extracellular alkaline serine protease
was purified and characterized. The purified Vm-AP (stands for V.
metschnikovii alkaline protease) seemed to be degraded by heating;
however, it could be inhibited by the addition of divalent cations such as
CuCl2, ZnCl2, and NiCl2. Vm-AP was composed of a single polypeptide
with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide
gel in the presence of CuCl2. The optimal temperature and the pH for
Vm-AP enzyme activity were 37°C and pH 9.5, respectively. In addition,
the maximal protease activity could be found under alkalic condition
ranging from pH 8.0 to 12.0, and the enzyme activity was inhibited by
protease inhibitors, including PMSF and aprotinin. These results suggest
that Vm-AP is an alkaline serine protease. Vm-AP could cleave various
blood coagulation-associated proteins, including plasminogen, plasmin,
prothrombin, and thrombin. In addition, the enzyme showed a powerful
fibrin(ogen)olytic activity, as it could cleave all fibrinogen chains, fibrin
polymer, and cross-linked fibrin. Taken together, the results suggest
that the pathogenic bacterium V. metschnikovii may disturb the blood
coagulation system by secreting an alkaline serine protease during the
course of its infection.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 183
POS-TUE-330
CHARACTERIZATION OF THE INTERACTION
BETWEEN THE SNARE PROTEIN SYNTAXIN AND THE
SEC/MUNC PROTEINS
Christie M.P.1, Hu S.1, Jarrott R.J.1, Latham C.F.1, 4, Lua L.H.L.2, Collins
B.M.1, James D.E.3 and Martin J.M.1
1
Institute for Molecular Biosciences, University of Queensland, St
Lucia, QLD 4072, Australia. 2Protein Expression Facility, University
of Queensland, St Lucia QLD 4072, Australia. 3Garvan Institute of
Medical Research, Darlinghurst, NSW 2010, Australia. 4Department of
Biological Science, Columbia University, New York, NY 100227, USA.
The SNARE (soluble N-ethylmaleimide-sensitive factor attachment
protein receptor) proteins are essential components of the vesicle
fusion machinery. SNAREs bring about fusion by the formation of the
SNARE ternary complex. The Sec/Munc (SM) family of proteins are
essential regulators of fusion. However, their exact role in fusion is
controversial. This is partly due to the different binding modes of SM
proteins with the SNARE protein Syntaxin. Munc18a, the SM protein
involved in neurotransmitter release acts as a negative regulator by
interacting with its cognate syntaxin, Syntaxin1 in a closed conformation
(1,2). The SM proteins Sly1p and Munc18c on the other hand interact
with their cognate syntaxins, Sed5p and Sx4 via the first ten residues
at the N-terminus (N-peptide) of Syntaxin (3,4). This is compatible with
SNARE complex assembly (5). The interaction between Munc18a and
Munc18c with the Sx N-peptide was thermodynamically characterized
using isothermal titration calorimetry. These experiments showed that
the interaction did not contribute to the specificity of SM-Sx interactions.
We were also able to show that the N-peptide interaction plays a role
in vesicle fusion regulation by SM proteins. References: 1. Misura et
al., Nature 2000 404, 355 2. Burkhardt et al., EMBO J 2008 27, 923 3.
Bracher and Weissenhorn EMBO J 2002 21, 6114 4. Hu et al., PNAS
2007 104, 8773 5. Latham et al., Traffic 2006 7, 1048.
POS-TUE-332
MULTIMODAL NANOPARTICLES FOR THE
STABILISATION OF ENZYMES AGAINST THERMAL
INACTIVATION
Clemons T.1, 3, Evans C.1, 3, Stubbs K.2 and Swaminathan I.1
1
Centre for Strategic Nano-fabrication, School of Biomedical,
Biomolecular and Chemical Sciences, University of Western Australia.
2
School of Biomedical, Biomolecular and Chemical Sciences,
University of Western Australia. 3Regenerative Neurosciences, School
of Animal Biology, University of Western Australia.
Enzyme modification and immobilisation have been extensively
studied and utilised to generate biocatalysts with improved stability
and selectivity as well as applications in sensing and enzyme-related
biotechnology. The major hindrance in developing enzyme related
therapy is its inherent thermal instability and drop in activity with
sometimes minute thermal fluctuations. This necessitates the use of
repeated injections or local infusions for a period of days to weeks
which are invasive, infection-prone, and clinically problematic. A
novel strategy for enhancing thermal stability is through non-covalent
surface interaction with the large surface area at the nanometer-scale.
Our study investigates the thermal stabilisation of a range of enzymes
through physisorption to a multimodal polymeric nanoparticle system.
The nanoparticle system contains both a fluorescent probe commonly
used in fluorescent microscopy as well as magnetite nanoparticles,
which will allow for both magnetic imaging and enzyme recovery post
treatment. The surfaces of the nanoparticles have been modified with
either polyethyleneimine (PEI) or polyethylene glycol (PEG) to ultimately
enhance not only the attachment of different enzymes to the nanoparticle
but also increase their tolerance to thermal denaturing. Preliminary
studies with a β-glucosidase and β-galactosidase, both enzymes of
significant interest to industry, have shown a strong physisorption of the
enzyme to the PEI modified nanoparticles.
POSTERS MONDAY & TUESDAY
POS-MON-333
A SERINE PROTEASE SECRETED BY
STAPHYLOCOCCUS AUREUS CAN INDUCE PRO-
INFLAMMATORY CYTOKINES AND EVOKES
VASCULAR PERMEABILITY
Park J.W. and Lee J.S.
Department of Biotechnology and BK21 Research Team for Protein
Activity Control, Chosun University, Gwangju 501-759, Republic of
Korea.
It is been well known that Staphylococcus aureus (S. aureus) is a
ubiquitous Gram-positive bacterium responsible for a majority of skin
infections as well as causing toxic shock syndrome. In this study, a
glutamate-specific serine endopeptidase (named VSPase) secreted by
a clinical isolate S. aureus sp. strain C-66 was purified and characterized
in terms of its involvement in the inductions of inflammatory response
and vascular permeability. VSPase clearly increased the transcription
levels of the genes for pro-inflammatory cytokines such as TNF-alpha
and IL-1beta, and also for an inflammatory regulator cyclooxygenase-2.
However, VSPase-derived mutant enzymes (H119L, D161A, and
S237L) that are totally deficient in proteolytic activities showed no
effects. VSPase could induce the degradation of IkappaB, resulting in
translocation of NF-kappaB proteins into nucleus, as judged by Western
blot analysis and supershift assay with anti-p65 antibody, respectively.
These results suggest that VSPase can activate NF-kappaB signaling
pathway through the degradation of IkappaB proteins, leading the
production of pro-inflammatory cytokines and an inflammatory
regulator. Interestingly, Miles assay using Guinea pig showed that
wild type VSPase could cause an increased vascular permeability in
a dose-dependent manner, whereas a mutant enzyme S237L could
not, suggesting that the permeability is also related to the proteolytic
activity of the enzyme. The data to be presented show that VSPase
plays an important role in the expression of pro-inflammatory cytokines
and inflammatory regulators through the activation of NF-kappaB, and
also can enhance a vascular permeability during the Staphylococcal
infection.
POS-MON-335
AN EXTRACELLULAR METALLOPROTEASE FROM
VIBRIO VUNIFICUS ACTIVATES TOLL-LIKE RECEPTORS
2 AND 4 TO INDUCE AN INFLAMMATORY RESPONSE
Park J.E. and Lee J.S.
Department of Biotechnology and BK21 Research Team for Protein
Activity Control,Chosun University, Gwangju 501-759, Republic of
Korea
Extracellular proteases produced by pathogenic bacteria are considered
to be important enzymes because of their implication in the pathogenesis
of these organisms. Vibrio vulnificus (V. vulnificus), a gram-negative
halophilic marine bacterium, is an opportunistic human pathogen
that causes wound infection and septicemia. We have reported that
vEP metalloprotease secreted by V. vulnificus exerts many biological
activities, such as prothrombin activation. To investigate that this enzyme
plays an additional role in pathogenesis by modulating inflammatory-
associated cytokine production, the effect of vEP on proinflammatory
cytokine induction was examined in a macrophage cell line Raw264.7.
vEP stimulated to induce typical inflammatory cytokines including TNF-
alpha and IL-1beta, and regulators such as cyclooxygenase-2 (COX-2)
and iNOS. Interestedly, the upregulation of cytokines and regulators by
vEP protease occurred only when the cells were treated with wild type
vEP having C-terminal domain (named C-ter100), while vEP lacking this
domain showed no effect. In addition, the protease could activate the
NF-kappaB signaling pathway for producing those cytokines, in which
IkappaB was phosphorylated and eventually degraded after treatment
with vEP. Toll-like receptors (TLRs)-2 and -4 seemed be involved in
the vEP-mediated NF-kappaB signaling pathway, as judged by RT-
PCR and immunoprecipitation. The C-ter100 only was also sufficient
to activate the NF-kappaB signaling for the production of those kinds of
cytokines and regulators, suggesting that the C-domain of vEP can act
as a ligand for the activation of TLRs. Moreover, the macrophage cell
lines transiently expressing RNAi molecules for TLR-2 or -4 showed
significantly decreased TNF-alpha production when they were treated
with vEP or C-ter100. These data suggest that the C-terminal domain of
vEP may play a critical role in the induction of inflammatory response.
Page 184 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-334
HSA-MIR-200B HAS TWO FUNCTIONAL PROMOTERS,
DIFFERENTIAL METHYLATION OF WHICH IS
ASSOCIATED WITH CLINICAL CHARACTERISTICS IN
BREAST CANCER
Wee E.J.H.1, Peters K.1, Hulf T.2, Stein S.3, Wagner S.3, Lee S.Y.1,
French J.1, Francis G.3, Clark S.2 and Brown M.A.1, 4
1
School of Chemistry and Molecular Biosciences, The University of
Queensland, Australia. 2Department of Pathology, Princess Alexandra
Hospital, Queensland, Australia. 3Epigenetics Group, Garvan Institute
of Medical Research, Sydney, Australia. 4UQ Diamantina Institute, The
University of Queensland, Australia.
MicroRNAs are small non-coding RNAs of ~20nt in length that are
capable of modulating gene expression post-transcriptionally. Many
miRNAs have been implicated in cancer, including breast cancer,
however, miRNA transcriptional regulation and the role of defects in this
process in cancer is not well understood. In this study, we describe an
alternate promoter of Hsa-mir-200b, P2, which is located in a CpG island
approximately 2kb upstream of its 5’ stemloop. P2 has comparable
promoter activity to the previously reported promoter, P1, and was able
to drive the expression of miR-200b in its native genomic context. DNA
methylation of both P1 and P2 is inversely associated with miR-200b
expression in 8/9 breast cancer cell lines, and in vitro methylation of
both promoters represses promoter activity in reporter assays. In
clinical samples, decreased methylation of P1 in primary breast tumours
is associated with better survival and increased DNA methylation of P2
in primary breast tumours is associated with loss of either ER, PR or
HER2. In a triple negative cohort of breast cancer cases, lymph node
metastases have lower methylation of P2 compared to primary tumours.
These data suggest the potential use of DNA methylation status of miR-
200b promoters as a prognostic marker of breast cancer progression
and survival.
POS-TUE-336
THERMO SCIENTIFIC SOLARIS QPCR GENE
EXPRESSION ASSAYS - PERFORMANCE AND
APPLICATION
Haas A.1, Jayne B.1, Haimes J.1, Covino J.1, Leake D.1, Kelley M.1 and
Gillespie J.2
1
Thermo Fisher Scientific, Dharmacon RNAi Technologies, 2650
Crescent Drive, Suite 100, Lafayette, CO 80026, USA. 2Thermo Fisher
Scientific, Abgene House, Blenheim Road, Epsom, Surrey, KT19 9AP,
UK.
Thermo Scienti‑fic SolarisTM qPCR Gene Expression Assays are gene-
speci‑fic probe and primer pairs that utilize minor groove binder (MGB)
and Superbase technologies. To test the performance of Solaris Assays,
common qPCR parameters (sensitivity, dynamic range, ampli‑fication
ef‑ficiency and r2 values) were measured. Additionally, we assessed
the stability of the Solaris reagents by comparing performance and
Cq values using reagents exposed to ambient laboratory conditions
relative to fresh reagents. We also used the Solaris Assays to measure
the consequences of RNAi-mediated knockdown where accurate
determinations of changes in target gene expression are critical for
appropriately interpreting the data. When employing the Solaris Assays
to detect gene expression and, using a Cq method for calculating relative
gene expression, we normalized to multiple reference genes to provide
further confidence in the accurate measure of target gene levels. The
results show that the Solaris qPCR Assays deliver repeatable, sensitive
and gene-specifi‑c quanti‑fication.
POSTERS MONDAY & TUESDAY
POS-MON-337
ACTIVATION OF CAMP-DEPENDENT SIGNAL
TRANSDUCTION PATHWAY IN BONE MARROW
HEMATOPOIETIC STEM CELLS OF ALPHA-
FETOPROTEIN-TREATED ANIMALS
Bogdanov A.Y.
Scientific Center for Anti-infectious Drugs, Auezov Str. 84, Almaty,
050008, Republic of Kazakhstan.
In our earlier studies it was established that the biological activity of
murine bone marrow hematopoietic stem cells (HSCs) in vitro exposed
to alpha-fetoprotein (AFP) considerably depended on activation of two
types of cAMP-dependent signal transduction pathways: cAMP/Epac1/2
and cAMP/PKA cascades. However the involvement of this pathways in
general mechanism of AFP action in bone marrow HSCs in vivo has
not been studied. In the present study we endeavored to investigate
the functioning of the discovered cAMP-dependent signal transduction
pathways in murine bone marrow HSCs in vivo under exogenous AFP
introduction. As a result it was shown that intravenous introduction of
AFP increased cAMP synthesis in every studied subpopulation of bone
marrow HSCs ex vivo as compared with control animals. Analysis of
the main components of revealed cAMP-dependent signal transduction
pathways in vitro in cytoplasm of each investigated HSCs subpopulation
demonstrated rise activity of both PKA and Rap1 in AFP-treated animals
as against the control group. The content of phosphorylated Epac1/2
in each studied HSCs subpopulation prevailed over the active Epac1/2
content in analogous HSCs subpopulations of control animals. Thereby,
the findings enable to state the activation of the revealed cAMP/Epac1/2
and cAMP/PKA pathways in the studied HSCs subpopulations in
animal bone marrow under AFP exogenous introduction. Therefore,
AFP interaction with HSCs in animal bone marrow initiates activation of
the same cAMP-dependent mechanisms of signal transduction in vitro
conditions, which, most probably, are responsible for generation of the
biological activity modifications.
POS-MON-339
THE GENERATION OF A DOXYCYCLINE-INDUCIBLE,
TISSUE-SPECIFIC AROMATASE TRANSGENIC MOUSE
Chow J.D.Y.1, 2 , Price J.3, Bills M.3, Simpson E.R.1, 3 and Boon W.C.1, 2, 4
1
Prince Henry’s Institute of Medical Research, Clayton, VIC, Australia.
2
Anatomy & Developmental Biology, Monash University, Clayton, VIC,
Australia. 3Biochemistry & Molecular Biology, Monash University, Clayton,
VIC, Australia. 4Howard Florey Institute, Melbourne, VIC, Australia.
Aromatase catalyses the biosynthesis of estrogen and is widely
expressed in the body. The aromatase knockout mouse (ArKO)
becomes estrogen-deficient and develops unexpected phenotypes
such as obesity and male-specific fatty liver and apoptosis at the
hypothalamus. As circulating levels of estrogen in males are low, we
hypothesize that local estrogen production in the brain may be important
in regulating metabolic functions (e.g. liver lipid homeostasis) by acting
in a paracrine or intracrine manner. To test this hypothesis, we are
generating a brain-specific doxycycline-inducible, aromatase transgenic
mouse. The transgene (pTetOAROM) consists of the human aromatase
cDNA (hAROM) and a luciferase marker placed under a bi-directional
tetracycline-responsive promoter (pTetO), which is regulated by
transactivators (rtTA or tTA) and doxycycline. This transgene increased
hAROM transcription (16-fold, p=0.01), aromatase activity (3.4-folds,
p=0.0008) and luciferase activity (16-fold, p=0.0006) in transfected
MBA-MB-231tet cells that stably expresses rtTA, with doxycycline
induction. Pronuclear microinjection of the transgene produced four
pTetOAROM founder mice (L1 to 4), which were bred with C57B6 WT
mice to produce transgenic F1, with the following proportions of positive
transgenics to total live births (ratios are of female to male positive
pups): founder female L1 7/12 (4:3); male L2 - 7/26 (3:4); female L3 3/8
(2:1); male L4 - 9/31 (2:1). A male pTetOAROM founder mouse was bred
with a female mammary gland specific-rtTA mouse (MTB) to produce
MTB-pTetOAROM double transgenic. Upon doxycycline treatment via
drinking water, human aromatase expression was detected (by RT-PCR)
specifically in mammary glands, salivary glands and seminal vesicles
of double transgenic mice. Luciferase expression was also detected in
these tissues by in vivo luciferase scan and in vitro luciferase assay. In
summary, we are generating a transgenic mouse model that expresses
the human aromatase in a temporal- and spatial-specific manner.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 185
POS-TUE-338
SUMO-1 MARKS LYSOSOMES IN
NEURODEGENERATIVE DISEASES AND IN ANIMAL
AND CELLULAR DISEASE MODELS
Wong M.B.1, Noe N.2, Meedeniya A.1, Richter-Landsberg C.2 and
Pountney D.L.1
1
Griffith Health Institute & ESKITIS, Griffith University, Australia.
2
Molecular Neurobiology, Carl Von Ossietzky University, Germany.
Many neurodegenerative diseases are characterised by microscopically-
visible protein aggregates, or inclusion bodies, within neural cells. The
ubiquitin homologue, SUMO-1, has been identified in sub-domains of
pathological inclusion bodies in several neurodegenerative diseases.
Inclusion bodies are believed to be formed actively in a defensive
response to soluble cytotoxic protein aggregates. We hypothesised
that SUMO-1 may become associated with lysosomes in this response.
Protein aggregation was induced in 1321N1 glioma cells by proteasome
inhibition with MG132 or by transient transfection with Q74-EGFP.
Fluorescence immunohistochemistry identified co-localisation of
SUMO-1 and the lysosomal marker, cathepsin D, in both the transfected
cells and MG132-treated cells, increasing over time at 24-96 hrs post-
transfection/treatment. SUMO-1-positive lysosomes were also detected
following MG132-treatment of 1321N1 cells expressing SUMO-1-GFP
and stained with Lysotracker dye. SUMO-1 did not mark lysosomes
in untransfected or sham treated cells. To determine if SUMO-1 also
marks lysosomes in disease, we examined 5 cases of progressive
supranuclear palsy (PSP), 5 cases of multiple system atrophy (MSA)
and a rat Parkinson’s disease model. Punctate co-localisation of
cathepsin D and SUMO-1 was consistently associated with both the
tau-positive PSP inclusions and the α-synuclein-positive MSA and rat
PD model inclusions. A similar pattern was also found with the OLN-t40
oligodendrocyte model. These findings suggest a role for SUMO-1 in
the autophagy-lysosome pathway linked to the response to protein
aggregates.
POS-TUE-340
LEARNING BIOCHEMISTRY BY USING THE PEERWISE
SYSTEM OF STUDENT CONTRIBUTED MULTIPLE
CHOICE QUESTIONS AND PEER EVALUATION
Bottomley S.1 and Denny P.2
1
School of Biomedical Sciences. Faculty of Health Sciences. Curtin
University. Western Australia. 2Computer Science Department.
University of Auckland. New Zealand.
Biochemistry students in the second year of their degree were asked
to research, design, and write their own multiple choice questions
(MCQs). In addition they were also required to answer, evaluate and
critique the MCQs written by their peers. The technology used to
support this activity was PeerWise - a freely available, innovative web-
based system that supports students in the creation of an annotated
question repository. In this study we report the implementation of, and
student responses to, the PeerWise system for a cohort of 120 second
year biomedical science students from three degree streams studying
a core biochemistry subject. The study suggests that the students are
eager participants, take an active part in their learning, develop their
higher order thinking skills, produce a large repository of MCQ’s that
they use for revision, and rate the PeerWise system highly.
POSTERS MONDAY & TUESDAY
POS-MON-341
THE CATALYTICALLY-COMPETENT COENZYME
ALLOCATION INTO THE ACTIVE SITE OF ANABAENA
FERREDOXIN NADP+-REDUCTASE
Lans I.1, Peregrina J.R.1, Gonzalez-Lafont A.2, Lluch J.M.2, Garcia-
Viloca M.2 and Medina M.1
1
Departamento de Bioquímica y Biología Molecular y Celular, Facultad
de Ciencias and Institute of Biocomputation and Physics of Complex
Systems. Universidad de Zaragoza. Spain. 2Departament de Química
and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de
Barcelona. Spain.
Ferredoxin-NADP+ reductase (FNR) catalyses the electron transfer from
Ferredoxin to NADP+ via its flavin FAD cofactor. Experimental kinetics
and QM/MM theoretical approaches are here applied to visualise the
catalytically competent interactions produced in WT Anabaena FNR
with its coenzyme, NADP+, as well as in its variant Y303S. Our data
indicate that the architecture of the WT active site precisely contributes
to the orientation of the N5 of the FAD isoalloxazine ring and the C4 of
the coenzyme nicotinamide ring in the conformation of the catalytically
competent HT complex and, therefore, to the efficiency of the process.
In the reactant complex of the Y303S variant the formation of a close
contact ionic pair FADH -:NADP+ surrounded by the polar environment of
the enzyme might be the cause of its low catalytic efficiency. However, in
WT FNR the side-chain of the C-terminal Y303 appears key to provide
the optimal geometry by reducing the stacking probability between
the isoalloxazine and nicotinamide and by providing the required co-
linearity and distance among the N5 of the flavin cofactor, the C4 of
the coenzyme nicotinamide and the hydride that has to be transferred
between them, factors highly related to the reaction efficiency,
mechanism and reversibility of the process.
POS-MON-343
THE ROLE OF L-PROLINE AND L-PROLINE
TRANSPORTERS IN THE REGULATION OF ES CELL
DIFFERENTIATION IN CULTURE
Tan B.S.N.1, Lonic A.2, Morris M.2, Rathjen P.D.1, 2 and Rathjen J.1, 2
1
Department of Zoology, University of Melbourne, Parkville, VIC, 3010.
2
School of Molecular and Biomedical, University of Adelaide, Adelaide,
S.A. 5005.
The initial step in formation of the somatic lineages from ES cells,
differentiation to primitive ectoderm, can be achieved in vitro by
formation of early primitive ectoderm-like (EPL) cells in response
the conditioned medium MEDII. L -proline, an amino acid found in
abundance in MEDII, has been identified as a bioactive component
required for EPL cell formation. Addition of L-proline to ES cells results
in changes in colony morphology, gene expression and differentiation
kinetics consistent with differentiation towards EPL cells. This activity
is restricted to L-proline as addition of other amino acids or synthetic
analogues of L-proline does not result in EPL cell formation. Here we
characterise the role of L-proline uptake and amino acid transporters
in ES cell differentiation. Characterisation of L-proline transport using
radioactive uptake assays has shown that the amino acid System A
Transporter SNAT2 (SLC38a2) is the major transporter for L -proline
transport into ES cells. Uptake is sodium and pH dependent and can be
inhibited by the addition of a molar excess of other amino acid substrates
of SNAT2. Amino acids that block L -proline uptake also block the ability
of L -proline to induce EPL cells from ES cells. This study implicates
amino acids and amino acid transporters in ES cell differentiation and
defines inexpensive culture supplements with potential applications in
improving the maintenance, and/or differentiation, of mouse and human
pluripotent cells in culture. Furthermore, this work describes a novel
and amenable model to investigate amino acid regulation of intracellular
signaling, transcriptional activity and cellular function.
Page 186 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-342
SO YOU HAVE MADE A DISCOVERY? WHAT NOW?
Tzanidis A. and YinFoo D.
Phillips Ormonde Fitzpatrick.
Life as a medical research scientist is dotted with moments of discovery.
Few will be “eureka!” moments, while others will merely provide a
satisfying moment of vindication for the efforts that were laid down
before. Whatever the response, what will you do with your discovery?
Traditionally, the pressures of building one’s track record and securing
grant funding will often lead to the publication of work in a peer-review
journal. There is certainly merit in this approach, as it aims to ensure
that only quality research reaches the public domain. However, evidence
would suggest that peer-reviewed publications alone will not attract the
commercial investment that is necessary to take your discovery from
the bench to the bedside where it is needed. Commercial investment
is likely to come only where there is valuable intellectual property (IP)
inherent in your discovery. Such value will typically take the form of
a registered patent. But how will you know whether your discovery is
necessarily patentable? What steps can you take to obtain IP protection
to attract commercial investment that will help to bring your discovery to
those that need it most? Contrary to popular belief, patentability is not
necessarily dependent on showing evidence that your discovery has
immediate application in the clinical setting. In fact, the mere discovery
of a unique signalling pathway may have practical applications that may
be patentable. Practical applications may include diagnostics, screening
assays, methods of medical treatment, pharmaceutical compositions
and so on. This paper will guide you through your next steps and
provide examples of areas of basic medical research that are inherently
patentable and common criteria for patentability.
POS-TUE-344
ROLE OF IFNγ SIGNALING IN THE PATHOGENESIS OF
HIV
Achuthan A.1, 2 , Zhou J.1, Crowe S.1, Scholz G.2 and Jaworowski A.1
1
Centre for Virology, Burnet Institute, Melbourne. 2Dept of Medicine
(RMH/WH), The University of Melbourne.
Tuberculosis (TB) is the leading cause of death among HIV-positive
individuals. Indeed, HIV and M. tuberculosis (Mtb) co-infected
individuals have a 10% annual risk of reactivating latent Mtb infection.
Macrophages are both the host cell for Mtb infection and also the cell
directly responsible for Mtb killing. Hence, evasion of macrophage
intracellular destruction mechanisms is pivotal to Mtb virulence. IFNγ
promotes macrophage immune functions (e.g. cytokine secretion and
phagocytosis) relevant to Mtb killing and immunity, in part by modulating
expression and/or function of effector proteins (e.g. SNARE, autophagy
and Rab proteins), controlling membrane vesicle fusion events. We
report here that IFNγ-induced STAT1 phosphorylation is impaired in
HIV-1-infected primary human monocyte-derived macrophages. We
found IFNγ up-regulates the expression of SNARE proteins Stx11 and
VAMP8 in macrophages. Significantly, CD14+ monocytes from HIV-1-
infected individuals displayed decreased protein levels of VAMP8 and
Stx11 as compared to controls. The effects of IFNγ-inducible Stx11 and
VAMP8 on secreting cytokines and killing Mtb in macrophages will be
presented and discussed.
POSTERS MONDAY & TUESDAY
POS-MON-345
A FLEXIBLE DEVELOPMENT ENVIRONMENT FOR THE
ANALYSIS OF LARGE SCALE BIOLOGICAL DATA
Milla L. and O’Connor L.
La Trobe University.
Biologists today are faced with the challenge of analysing increasingly
large amounts of data, sourced from a variety of instruments whose
technologies are rapidly evolving. To keep up with this constantly
changing environment, we are building a flexible development
framework that can be used to quickly create and modify data pipelines
that automate the processing and analysis of data. Using workflow
tools we can read disparate sources of information, process the data
by integrating existing algorithms or third party software, and produce
results that are further analysed and visualised with dedicated tools. This
flexible development environment allows scientists to concentrate on the
analysis and interpretation of experiments rather than the development
and maintenance of custom code to process and view the data.
POS-MON-347
MOLECULAR BASIS FOR MEMBRANE RECRUITMENT
OF THE PX DOMAIN-CONTAINING PROTEIN SNX17
AND ITS STRUCTURAL RELATIONSHIP TO SNX31 AND
SNX27
Ghai R., Mehdi M., Teasdale R., King G. and Collins B.
Institute of Molecular Bioscience, University of Queensland, ST. Lucia
Campus, Brisbane, Queensland 4067.
Transmembrane protein sorting between different membrane-bound
compartments inside the cell is critical for a number of physiological and
pathological processes. Sorting nexin 17 (SNX17) is a member of the
phox-homology (PX) domain containing family of proteins, and has been
shown to regulate the endosomal trafficking of amyloid precursor protein
(APP) and LDL receptor (LDLR) family proteins critical in Alzheimer’s
disease. In this study we show that SNX17 and other PX domain
proteins SNX27 and SNX31 define a novel sub-family of PX proteins
containing an unusual FERM-like domain. SNX17 and SNX27 have
been demonstrated to regulate the trafficking of cargo receptors from
phosphatidylinositol-3-phosphate (PtdIns(3)P)-enriched endosomal
membranes. Using isothermal titration calorimetry (ITC) and nuclear
magnetic resonance (NMR) spectroscopy we show that both SNX17
and SNX27 have absolute specificity for PtdIns(3)P interaction via their
PX domains. The structure of the SNX17 PX domain with bound SO42-
, and comparison with other PX domains reveals important insights
into the mode of PtdIns(3)P binding by the PX-FERM-like proteins
within a conserved basic cleft. This binding site was further confirmed
by NMR spectroscopy titration experiments, molecular docking and
mutagenesis. Analysis of the SNX17 PX domain structure also reveals
the presence of a novel conserved surface that may be important for
additional inter- or intra-molecular interactions. Overall we have defined
a unique sub-family of PX-FERM-like proteins, and using a combined
structural and biochemical approach have confirmed their ability to
associate with the endosomal targeting lipid PtdIns(3)P via a conserved
molecular mechanism.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 187
POS-TUE-346
EPIGALLOCATECHIN-3-GALLATE INHIBITS FIBRIL
FORMATION BY RECOMBINANT PLASMODIUM
FALCIPARUM MEROZOITE SURFACE PROTEIN 2
Adda C.G.1, Chandrashekaran I.R.2, MacRaild C.A.2, Anders R.F.1 and
Norton R.S.2
1
Department of Biochemistry, La Trobe University, Victoria 3086,
Australia. 2The Walter & Eliza Hall Institute of Medical Research, 1G
Royal Parade, Parkville, Victoria, 3052, Australia.
The merozoite surface protein 2 (MSP2) is an abundant ~28 kDa GPI-
anchored protein expressed on the surface of the maturing asexual
blood stages of the malaria parasite, Plasmodium falciparum. MSP2
is currently being assessed as a potential component of a human
malaria vaccine having shown efficacy in a Phase I/IIb clinical trial in
Papua New Guinean children. Recombinant MSP2 is an intrinsically
unstructured protein that has been shown to form fibrils that, under
physiological conditions, have many of the characteristics of amyloid.
The propensity of MSP2 to form fibrils is a potential problem for vaccine
development and small molecule inhibitors of fibril formation have
been investigated. The flavonoids, epigallocatechin-3-gallate (EGCG),
baicalein and resveratrol, were examined for their ability to inhibit
fibril formation (1). The effects on fibril formation were studied using
thioflavin T binding assays, electron microscopy, NMR spectroscopy,
circular dichroism and other biophysical methods. Unlike baicalein
and resveratrol, EGCG demonstrated the capacity to form SDS-stable
oligomeric MSP2 complexes, and inhibit fibril formation. The molecular
mechanism of fibril inhibition by EGCG involves noncovalent binding
followed by covalent modification of the protein. Although the addition of
EGCG appears to be an effective means of stabilizing MSP2 in solution,
from a regulatory perspective, EGCG-stabilized MSP2 oligomers are
unlikely to be acceptable in a vaccine formulation. However, these small
molecule inhibitors of MSP2 fibril formation may be useful as mechanistic
probes in studying oligomerization of MSP2. (1) Chandrashekaran IR,
Adda CG, MacRaild CA, Anders RF, Norton RS. (2010) Biochemistry
(in press).
POS-TUE-348
STRUCTURAL ANALYSIS OF ALPHA HAEMOGLOBIN
IN COMPLEX WITH ALPHA HAEMOGLOBIN
STABILIZING PROTEIN
Dickson C.F.1, Rich A.M.3, Krishna Kumar K.2, Lay P.A.3, Mackay J.P.2
and Gell D.A.1, 2
1
Menzies Research Institute, University of Tasmania. 2School of
Molecular Biosciences, University of Sydney. 3School of Chemistry,
University of Sydney.
Alpha haemoglobin (αHb) stabilizing protein (AHSP) is an erythroid
specific protein known to be important for the development and survival
of normal red blood cells. One role of this small protein is to sequester
and detoxify excess αHb chains that remain in the cell after haemoglobin
assembly. To fulfill this role, AHSP binds to free αHb and converts it to
an inert form in which the six available coordination sites surrounding
the haem-iron are occupied by αHb histidine sidechain or porphyrin
ligands. The structure of this final stable state is well characterized, but
the mechanism by which AHSP induces these structural changes in
the haem pocket of αHb is poorly understood. To address this, we have
performed nuclear magnetic resonance (NMR) studies of free αHb and
the αHb:AHSP complex. These data represent the first high-resolution
structure of a free Hb chain. Chemical shift analysis suggests that
upon binding AHSP, conformational change occurs in the F-G helices
of αHb. Helix G forms part of the αHb:AHSP interface, whereas the
adjacent F-helix bears the haem-coordinating H87. Thus, these data
provide a plausible mechanism by which AHSP binding effects might
be propagated to the haem pocket. Extended X-ray absorption fine
structure (EXAFS) indicate that a small but significant lengthening of
the Fe-O2 bond of oxy-αHb upon binding to AHSP, indicating that O2
dissociation is promoted. These findings fit with our hypothesis that
AHSP alters the conformation of αHb to promote autoxidation and allow
haem to simultaneously bind two axial histidine sidechain ligands.
POSTERS MONDAY & TUESDAY
POS-MON-349
THE X-RAY CRYSTAL STRUCTURE OF MURINE α2-
ANTIPLASMIN
Law R.H.P.1, Encarnacao J.1, Zhang Q.1, Horvath A.2, Buckle A.M.1,
Whisstock J.C.1 and Coughlin P.B.2
1
Department of Biochemistry and Molecular Biology, Monash
University, Clayton, Victoria 3800 Australia. 2Australian Centre for
Blood Diseases, Monash University, Alfred Medical Research Precinct,
Prahran, Victoria, 3181 Australia.
α2-Antiplasmin is a member of the serpin superfamily. It plays an
important role in the regulation of hemostasis through inhibition of
plasmin. Accordingly, deficiency in humans results in severe bleeding
episodes. Uniquely amongst human serpins, α2-antiplasmin contains
an unusual extensive N-terminal and C-terminal sequences flanking
the serpin domain. The 55-residue C-terminal extension is highly
conserved between species and contains important determinants for
exosite interactions with the target protease. Hence, α2-antiplasmin with
truncated C-terminal region reacts much more slowly with plasmin. We
study the structure and function of N-terminal truncated α2-antiplasmins.
Here we determined the 2.65Å structure of murine α2-antiplasmin in the
native and inhibitory conformation. In contrast to coagulation factors
such as antithrombin and heparin co-factor II, this structure reveals that
the reactive centre loop (RCL) is not pre-inserted into the A β-sheet.
Thus it suggests that α2-antiplasmin does not undergo co-factor
mediated conformational change. Importantly, the structure reveals that
the first 9 residues of the C-terminal sequence are tightly associated
with the serpin body and form an extra strand with the C β-sheet. Such
interaction would position the flexible plasmin-binding portion of the
C-terminus in close proximity to the RCL. In conclusion, we suggest
that the C-terminal region of α2-antiplasmin may function as a “hook”
that accelerates the interaction with the target plasmin, and disruption
of this interaction disrupt the rapid assembly of the initial plasmin / α2-
antiplasmin complex.
POS-MON-351
OXI: A NOVEL BRANCHING MUTANT IN ARABIDOPSIS
THALIANA
Meyers E.L., Brewer P.B., Mason M., Dun E.A. and Beveridge C.A.
School of Biological Sciences, University of Queensland, St Lucia,
Queensland, 4072.
Shoot branching in plants is carefully regulated by several interacting
pathways. Strigolactones are a new family of plant hormones and
are involved in controlling shoot branching. Very little is known about
strigolactone signalling pathways and only a handful of genes controlling
their biosynthesis have been characterised. Recent work has identified
several candidate genes that may be involved in shoot architecture
regulation. A knockout mutant of one of these genes, OXI, displays
a moderate branching phenotype. This phenotype is rescued by
exogenous supply of strigolactone, consistent with other strigolactone
biosynthesis mutants. Real time and GUS expression analysis has
found that OXI is co-expressed with other strigolactone pathway genes.
Grafting experiments have shown that oxi rootstocks restore branching
inhibition to other strigolactone biosynthesis branching mutant shoots.
However, unlike other branching mutants, oxi branching cannot be
rescued when grafted to a wild type rootstock. These findings suggest
that OXI is a novel player in the biosynthesis of strigolactones and
may act downstream of several previously characterised genes in the
strigolactone biosynthesis pathway. Current work to test this hypothesis
involves identifying and quantifying endogenous strigolactones in the
oxi mutant. The characterisation of OXI is important for elucidation of
the strigolactone biosynthetic pathway and to further the understanding
of shoot branching regulation.
Page 188 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-TUE-350
RECEPTOR LOCALISATION TO CAVEOLAE MODULATES
G PROTEIN COUPLING OF THE β3-ADRENOCEPTOR
Evans B.A.1, Sato M.1, Hutchinson D.S.1, Furness S.1, Bengtsson T.2
and Summers R.J.1
1
Drug Discovery Biology, Monash Institute of Pharmaceutical
Sciences, Monash University, Parkville, Victoria. 2Department of
Physiology, Wenner-Gren Institute, Stockholm University, Sweden.
Caveolae are plasma membrane invaginations, formed by recruitment
of caveolins to membrane rafts, that are known to modulate G protein-
coupled receptor signalling. For example, caveolin-3 interacts with the
β2-adrenoceptor (AR) and Gαs, Gαi, adenylate cyclase, PKA and protein
phosphatase 2A [1]. β3-AR signalling is also dependent on caveolae,
however it is not known whether caveolin-1 is associated with the β3-AR
or downstream signalling proteins [2]. We have addressed this question
by taking advantage of the distinct signalling properties of mouse β3-
AR isoforms [3]. The β3a- and β3b-AR differ only in their C-terminus,
yet the β3b-AR couples to Gs and Gi whereas the β3a-AR couples only
to Gs, suggesting β3a-AR interaction with protein(s) interfering with Gi
coupling. When CHO-β3a-AR cells are treated with filipin III to disrupt
membrane rafts, or transfected with caveolin-1 siRNA, β3a-AR signalling
becomes PTX sensitive, indicating Gi coupling. The β3a-AR C-terminus,
SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, contains a caveolin
interaction motif. Unlike the wild type receptor, β3a-ARs with F389A/
Y392A/F398A or P384S/F389A mutations display Gi coupling. A
Duolink in situ proximity ligation assay demonstrates direct interaction
between caveolin-1 and the wild type β3a-AR, but not the F389A/Y392A/
F398A or P384S/F389A mutants. In addition, endogenous β3a-ARs
acquire Gi coupling in brown adipocytes from caveolin-1 knockout mice,
or in wild type adipocytes treated with filipin III. Our data demonstrate
a functionally significant interaction between caveolin-1 and the β3a-AR
C-terminus. [1] Balijepalli et al (2006) PNAS (USA) 103, 7500-7505.
[2] Cohen et al (2005) Diabetes 54, 679-686. [3] Sato et al (2005) J
Pharmacol Exp Ther 315, 1354-1361.
POS-TUE-352
EXPLORATION OF β(1,4)-GALACTAN BIOSYNTHESIS
IN PINUS RADIATA
Weinberg C.S.1, West M.1, Phillips L.1, Flint H.1, Mast S.2 and Wagner
A.1
1
Scion, Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua, New
Zealand. 2Prozyme, 3832 Bay Center Place, Hayward, CA, 94545,
USA.
Gravitropism significantly stimulates the production of the hemicellulose
β(1,4)-galactan during secondary cell wall formation in pine. However, the
molecular and physiological function of this cell wall polymer is essentially
unknown. The goal of the project is to uncover the biological function
of β(1,4)-galactan in wood through the identification and functional
testing of genes involved in the biosynthesis of this polysaccharide
in Pinus radiata. However, genes involved in the biosynthesis of this
cell wall polymer have not been identified to date. Therefore this study
tries to explore galactan biosynthesis by using a proteomic approach
and focuses initially on the galactosyl transferase (GalT), which is
responsible for the biosynthesis of the polymer backbone. Initially, an
HPLC-based enzyme assay was established that uses fluorescently
labelled galactan oligomers as substrates. Assaying different wood
types demonstrated that GalT activity levels were virtually non-existent
in opposite wood, but high in compression wood. Our enzymatic data
indicated that production of β(1,4)-galactan in different wood types is at
least partially, if not predominantly, based on differential GalT activity
levels. Preliminary work on the purification of GalT showed that this
enzyme is likely to be associated with membrane bodies, potentially
Golgi vesicles.
POSTERS MONDAY & TUESDAY
POS-MON-353
GENETIC CONTROL OF C-GLYCOSYLATION IN
THE BIOSYNTHESIS PATHWAY OF FLAVONE-C-
DIGLYCOSIDES IN BREAD WHEAT
Wijaya G.Y.1, 2 , Asenstorfer R.1 and Mares D.J.1
1
School of Agriculture Food and Wine, University of adelaide, Glen
Osmond SA 5064, Australia. 2Biotechnology Faculty, Atma Jaya
Catholic University, Jl Jenderal Sudirman no 51 Jakarta 12930,
Indonesia.
Falvone-C-diglycosides (FCGs) have diverse and important roles
in plants, ranging from pigments and bioactive compounds in food
and beverages, to allelopathic compounds against parasitism, pest
and microbial infection. The are produced from the intermediate
naringenin, via branch of the flavonoid biosynthitic pathway, and involve
C-glycosylation of the 6th and 8th carbon of A flavone ring. To gain a
better understanding of the genetic control of FCG C-glycosylation,
two varieties of hexaploid bread wheat (Triticum aestivum vars
Tasman and Sunco, AABBDD genome), a complete set of nullisomic-
tertrasomic (NT) lines of Chinese Spring, 5 lines of the diploid species
Triticum monococcum (AmAm genome), and 5 lines of Aegilops tauschii
(DD genome) were analysed by HPLC. The results showed that
C-glycosylation in bread wheat is genetically controlled by loci on 7A,
7B, and 7D and 1B. A possible explanation for the observed variation
in FCG1/FCG2 ratio is that C-glycosyltransferase coded by genes on
these chromosome vary in their specificity for UDP-glucose or UDP-
galactose.
POS-MON-355
COMPARATIVE ANALYSIS OF FICIN STABILITY IN
DIFFERENT CHEMICAL DENATURANTS
Sidek N.A.A., Kadir H.A. and Tayyab S.
Biomolecular Research Group, Biochemistry Programme, Institute of
Biological Sciences, Faculty of Science, University of Malaya, 50603
Kuala Lumpur, Malaysia.
Ficin (E.C. 3.4.22.3) is an endopeptidase which belongs to the class,
cysteine proteases. We studied the effect of different chemical
denaturants, i.e. guanidine hydrochloride (GdnHCl), urea and ethanol
on ficin stability using far-UV CD, near-UV CD, intrinsic fluorescence,
tryptophan fluorescence, acrylamide quenching and biological activity.
Far-UV CD spectra of native ficin showed the presence of α-helical
structure which was completely lost in 6M GdnHCl but retained in 9M
urea. Loss of tertiary structure in 6M GdnHCl was also evident from
near-UV CD spectra. There was total loss of biological activity at 6M
GdnHCl. A red shift of about 10 nm was also observed in the emission
maxima in presence of 6M GdnHCl upon excitation at 280/295 nm,
suggesting major conformational change in the protein. Titration of
native ficin with increasing GdnHCl concentrations using far-UV CD
signal followed a two-state, single-step transition which yielded a value
of ∆GDH2O as 5186.6 cal/mol. Fluorescence intensity data showed lesser
changes in presence of different concentrations of urea as well as
ethanol compared to those observed with GdnHCl. Taken together, all
these results suggested a strong denaturing action of GdnHCl on ficin
among all the denaturants used.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 189
POS-TUE-354
HOW TO KILL TUMOUR CELLS THAT HAVE LOST P53
ACTIVITY: A NOVEL APOPTOTIC PATHWAY INDUCED
BY DNA DAMAGING CHEMOTHERAPEUTICS
Delbridge A.R.D.1, 2 , Scott C.1, Bouillet P.1 and Strasser A.1
1
Molecular Genetics of Cancer Division, Walter & Eliza Hall Institute.
2
Department of Medical Biology, University of Melbourne.
While a large body of knowledge has accumulated in relation to the p53-
dependent DNA damage-induced apoptotic pathway, comparatively
little is known about the p53-independent apoptotic pathways that can
be activated by DNA damage, although they are known to contribute
significantly to anti-cancer therapy induced tumour cell killing. In order
to address this we have investigated the role of the pro-apoptotic BH3-
only proteins in the response of p53-deficient thymic lymphoma derived
cell lines to DNA damage induced by γ-irradiation or chemotherapeutic
drugs. Our findings identify Bim as a critical initiator of apoptosis induced
by DNA damage inducing drugs in the absence of p53, while Puma and
Bmf may also contribute, at least in response to treatment with etoposide.
Our data also implicates several mechanisms for Bim induction in the
response of p53-deficient cells to DNA damage. This novel apoptotic
pathway also appears to play a role in the suppression of lymphoma
development. We suspect that Bim may play a crucial surveillance role
in pre-leukaemic cells, acting to eliminate cells that have sustained p53
mutations particularly when they have become genomically unstable.
Consistent with this hypothesis, our data show that the additional loss of
Bim markedly accelerates lymphoma development in p53-/- and p53+/-
mice.
POS-TUE-356
BINDING OF THE LECTIN ARTINM TO MAST CELLS
RESULTS IN CELL ACTIVATION
Lorenzi V.C.B.1, Roque-Barreira M.C.1, Pereira-Da-Silva G.2, Jamur
M.C.1 and Oliver C.1
1
Department of Cell and Molecular Biology and Pathogens Bioagents,
School of Medicine of Ribeirao Preto, University of Sao Paulo,
Ribeirao Preto, Brazil. 2School of Nursing of Ribeirao Preto, University
of Sao Paulo, Ribeirao Preto, Brazil.
Mast cells (MC) are essential cells in IgE-associated immune
responses.FcεRI is the major receptor for MC activation. FcεRI cross
linking induces MC degranulation and release of proinflammatory
mediators. We have previously shown that the lectin ArtinM induces
MC degranulation but the mechanism of ArtinM induced activation
remains unknown. The present study was undertaken to characterize
the ability of ArtinM (10μg/ml) to activate the rat MC line RBL-2H3.
By confocal microscopy, it was observed that ArtinM binds to the
MC surface and this binding is dependent on ArtinM carbohydrate
recognition domains (CRDs). Microplate assays showed that ArtinM
binds to IgE and this binding is also dependent on ArtinM CRDs. By
Western blotting of total cell lysates, ArtinM also recognizes the β
subunit of FcεRI. MC incubation with ArtinM results in the release of
β- hexosaminidase activity in the presence or absence of IgE. The
tyrosine phosphorylation of intracellular proteins was investigated.
Western blotting of IgE sensitized cells stimulated with ArtinM showed
that tyrosine phosphorylation of intracellular proteins was similar to that
of to antigen stimulated cells. These results suggest that ArtinM may
bind to glycans of FcεRI receptor and/or of the IgE (bound to FcεRI)
and that such interactions could be implicated in its ability to activate
and degranulate mast cells. In view of the well-established significance
of mast cells in allergy and inflammation, the participation of sugars as
receptors on the mast cell surface opens new possibilities to control
allergic disorders.
Page 190 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POSTERS WEDNESDAY & THURSDAY
POS-WED-01
RHOU IS REQUIRED FOR NORMAL ACTIN
DISTRIBUTION AND DIFFERENTIATION IN THE
DEFINITIVE ENDODERM
Loebel D.A.F.1, 2 , Studdert J.B.1, Jones V.1, Power M.1, Radziewic T.1
and Tam P.P.L.1, 2
1
Children’s Medical Research Institute, Westmead NSW 2145. 2Sydney
Medical School, University of Sydney, NSW 2006.
The definitive or gut endoderm forms at gastrulation and contributes to
the epithelial linings of the lungs, stomach, oesophagus and trachea, as
well as organs including the liver, pancreas, thyroid and thymus. To gain
insights into the molecular basis of development, we have performed
microarray analysis to identify endoderm-enriched transcripts in foregut
of early-somite-stage mouse embryos. We identified Rhou, encoding
an atypical Rho GTPase amongst the genes that were preferentially
expressed in the endoderm. Overexpression or knockdown of Rhou has
been shown to have dramatic effects on cell morphology and the actin
cytoskeleton in cell line models. Rhou knockdown embryonic stem (ES)
cell lines were generated and tested by in vitro differentiation as embryoid
bodies (EBs) in the presence of Activin A. Rhou-deficient cells in the
EBs display abnormal F-actin localization and impaired differentiation
of endoderm derivatives. In embryos generated from the knockdown
ES cell lines by tetraploid complementation, the foregut collapses and
the cells lining the gut lose their normal epithelial architecture. The
endodermal cells show a reduced apical concentration of F-actin but
appear to form normal adherens and tight junctions. Overexpression
of Rhou in endoderm cells by embryo electroporation also resulted in
changes in cell shape and F-actin distribution. Our data suggest that
tight regulation of levels of Rhou is required for proper F-actin distribution
in the endoderm and that disruption of the epithelial organization of the
endoderm significantly impairs endoderm differentiation.
POS-WED-03
THE ROLE OF HEDGEHOG SIGNALING IN GUT
COLONISATION BY VAGAL ENTERIC NEURAL CREST
CELLS
McConnell S., Simkin J.E., Zhang D. and Newgreen D.
Murdoch Children’s Research Institute, Flemington Rd, Parkville 3052.
The enteric nervous system is derived from a transient sub-population
of cells called the neural crest (NC), specifically from the vagal (caudal
hindbrain) level. These colonise the gut in a proximo-distal wave. Enteric
(ie. vagal) NC cells from already colonised embryonic quail gut, rapidly
colonised an abutted chick aneural gut in organ culture. But when vagal
neural tube/NC and the aneural gut were abutted in culture, there was
little NC cell migration into the gut although the NC cells were migratory
elsewhere. By altering duration of culture and duration of abutment, we
found vagal NC cells require time plus some other factor(s) to become
competent to migrate into the gut. When the entire vagal tissues (neural
tube/NC, plus somites, foregut endoderm and mesoderm) were cultured
together, subsequent colonisation of aneural gut by NC cells was
improved, relative to neural tube/NC alone. This suggested that factor(s)
in the vagal tissue, plus time to respond, enhanced the migratory
ability of the NC cells. Sonic hedgehog (Shh) expression in the foregut
endoderm at the vagal level was found exactly when and where the
vagal NC cells approach and enter the gut. Moreover, NC cells from
the entire vagal tissues failed to colonise the gut when exposed to the
hedgehog-antagonist cyclopamine. In contrast, older NC cells from gut
already colonised, could migrate into aneural gut when cyclopamine
was present. This suggests the drug is neither cytotoxic nor inhibitory
to motility, and hence is consistent with hedgehog being a competency
factor for gut colonisation, but that hedgehog activity is only critically
required during initial approach/entry of NC cells into the gut.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 191
POS-THU-02
HIGHLY SIGNIFICANT CORRELATION BETWEEN THE
PROTAMINE CONTENT, DNA INTEGRITY AND QUALITY
OF EJACULATED SPERMATOZOA FROM INFERTILE MEN
Manochantr S. 1, Chiamchanya C. 2 and Sobhon P. 3
1
Division of Cell Biology, Department of Preclinical Sciences, Faculty
of Medicine, Thammasat University, Patumthani, 12120, Thailand.
2
Department of Obstetrics and Gynecology, Faculty of Medicine,
Thammasat University, Patumthani, Bangkok 12120, Thailand.
3
Department of Anatomy, Faculty of Science, Mahidol University,
Bangkok, 10400, Thailand.
Histone-to-protamine exchange is a late spermiogenesis event,
along with acrosome formation, membrane remodeling and other
significant morphological and biochemical events that are necessary
for normal sperm function. Therefore, protamines are considered a
good marker of sperm nuclear maturity. The aims of the present study
were to investigate the condensation of chromatin and DNA integrity in
spermatozoa of infertile men and deduced the relationship with sperm
quality as measured by conventional semen parameters. Routine semen
analysis was carried out to ascertain sperm concentration, motility, and
morphology. The remaining aliquot of each sample was processed for
transmission electron microscopy, chromomysin A3 (CMA3) assay and
terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)
assay. The ultrastructural analysis of spermatozoa from infertile men
indicated nuclear maturation defect in the spermatozoa of infertile
men. Spearman’s correlation analysis indicated the positive correlation
between the percentages of CMA3- and TUNEL- positive spermatozoa.
In addition, these 2 parameters were negatively correlated with
concentration, motility and normal morphology. This study demonstrated
that the men with abnormal semen parameters carrying higher loads of
protamine deficiency and DNA-damaged spermatozoa. Therefore, the
assessment of chromatin condensation and DNA integrity appears to
be a useful tool for assessing male fertility potential.
POS-THU-04
MICRORNA SIGNALLING IN EARLY MALE GERM CELLS
McIver S.C.1, 2, 3, Roman S.D.1, 2, 3, Nixon B.1, 3 and McLaughlin E.A.1, 2, 3
1
Reproductive Science Group. 2ARC Centre of Excellence in
Biotechnology & Development. 3School of Environmental and Life
Sciences, University of Newcastle, Callaghan, Australia.
MicroRNAs are short noncoding RNA sequences which bind to the
3’ untranslated region of mRNA to control translation and aberrant
miRNA expression is linked to many disease states and developmental
abnormalities. Of particular interest to our group is the. We are focusing
on the early development of the male germ cell particularly the period
in which gonocytes differentiate to become spermatogonia. This is of
interest because in the human Carcinoma in Situ cells, the precursors
to testicular germ cell tumours have been identified as arising from
dysfunctional gonocytes which fail to differentiate into spermatogonia.
Gonocytes from post natal day 1 mice and spermatogonia from day 7-9
mice were enriched by 2-4% BSA gradient sedimentation. Total RNA
including microRNA was extracted and analysed in by Illumina miRNA
microarray. Total RNA was also reverse transcribed using specific primers
and analysed by qPCR. Three biological replicates were performed in
both the microarray and qPCR experiments. Bioinformatic analysis with
SAM (Significance analysis of Microarrays) identified seven significantly
different miRNA molecules expressed in spermatogonia and gonocytes.
qPCR analysis determined two miRNAs that were significantly
upregulated in spermatogonia (743a, 463*) and three miRNAs which
were significantly down regulated in spermatogonia (293, 290-5p,
291a-5p). Several miRNA molecules were selected for further study
(293, 290-5p, 136, and 146a) and overexpression assays in P19 cells
will help determine their function. In the future the role of these miRNA
molecules in seminoma will be analysed using overexpression within
a seminoma cell line. It is hypothesised that these miRNA molecules
control genes involved in male development and differentiation such as
stella, nanog and oct3/4 and that these molecules may also play a role
in tumour development In conclusion miRNA expression is significantly
different between gonocytes and spermatogonia and influence their
differentiation and developmental course.
POSTERS WEDNESDAY & THURSDAY
POS-WED-05
REGULATION OF FOXD3 IN THE NEURAL CREST
Mckeown S.J.1, 2 and Bronner M.2
1
University of Melbourne, VIC, Australia. 2California Institute of
Technology, Pasadena, CA, USA.
Neural crest cells are a uniquely vertebrate cell type vital for development.
They form at the border of the neural plate and epidermal ectoderm,
migrate to numerous locations and differentiate into many cell types.
Many transcription factors are involved in the generation of the neural
crest, including FoxD3. To better understand the regulatory interactions
involved in neural crest formation, we analyzed the regulatory region
of FoxD3 for the presence of enhancer elements. Elements conserved
across chick, mouse and human in the FoxD3 genomic region were
cloned into a reporter construct and electroporated into Stage 4 chick
embryos. We identified three elements upstream of FoxD3 that direct
expression in the neural crest in spatially and temporally distinct
patterns, and in combination recapitulate the entire expression of
FoxD3 in the neural crest. The first element directs expression only in
premigratory and migratory cranial neural crest. The second element
directs expression in migratory cranial neural crest and premigratory
and migratory vagal and trunk neural crest. The third element directs
expression in some premigratory vagal and trunk neural crest and a
population of spinal cord interneurons. We reduced the size of these
enhancers to identify minimal core regions and used bioinformatics
analysis to identify putative transcription factor binding sites. Site directed
mutagenesis of the premigratory cranial neural crest enhancer revealed
two binding sites critical for activity of the enhancer: a homeodomain
site and an Ets/Gata binding site. Antisense morpholinos and chromatin
immunoprecipitation are being used to identify the upstream transcription
factors that regulate these FoxD3 neural crest enhancers. The results
provide new insight into the gene regulatory mechanisms underlying
neural crest formation.
POS-WED-07
CHARACTERISATION OF ENU-INDUCED IFT140
MUTANT MICE EXHIBITING EXENCEPHALY AND
POLYDACTYLY
McMorran T.A.1, 2 and Farlie P.1
1
Murdoch Children’s Research Institute, Royal Children’s Hospital,
Parkville. 2Department of Paediatrics, University of Melbourne.
Congenital defects are abnormalities in body structure, function or
metabolism present at birth resulting from genetic or environmental
causes and are the leading cause of infant morbidity and mortality.
Approximately one third of congenital defects involve the craniofacial
structures. Affected individuals often have other abnormalities, such
as defects in limb or internal organ development. The genetic basis of
many craniofacial disorders and the developmental processes involved
are poorly understood. An ENU mutagenic screen of mice has been
undertaken to address this deficiency. A strain has been identified where
affected embryos exhibit severe neural tube and craniofacial defects
and have an abnormal number of digits on both the fore- and hindlimbs.
The features of this strain are similar to those seen a family of human
conditions including short-rib polydactyly and Jeune asphyxiating
thoracic dystrophy. The gene mutated has been identified as Ift140,
which encodes a component of the primary cilium, a cellular structure
that functions as a signalling center within the cell and coordinates the
activity of some important growth factor signalling pathways. A similar
set of features has been observed in mutant mice with defective sonic
hedgehog (Shh) signalling, one of the key signalling pathways that
functions during development. The IFT140 protein is involved in the
transport of proteins within the primary cilium and is thought to interact
with components of the Shh signalling pathway, which would explain
the similarity between the Ift140 and Shh mutants. Characterisation of
the developmental defects observed in the IFT140 mouse strain will
facilitate an investigation into the interaction between Shh and IFT140
and improve our understanding of the processes associated with
mammalian development.
Page 192 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-06
OLFACTORY GLIA ENHANCE NEONATAL AXON
REGENERATION
Chrehrehasa F., Windus L., Ekberg J., Scott D., Amaya D., Mackay-
Sim A. and St John J.
National Centre for Adult Stem Cell Research, Griffith University.
Olfactory ensheathing cells (OECs) migrate with olfactory axons that
extend from the nasal epithelium into the olfactory bulb. Unlike other glia,
OECs are thought to migrate ahead of growing axons instead of following
defined axonal paths. However it remains unknown how the presence of
axons and OECs influences the growth and migration of each other during
regeneration. We have developed a regeneration model in neonatal
mice to examine whether (i) the presence of OECs ahead of olfactory
axons affects axonal growth and (ii) the presence of olfactory axons
alters the distribution of OECs. We performed unilateral bulbectomy to
ablate olfactory axons followed by methimazole administration to further
delay neuronal growth. In this model OECs filled the cavity left by the
bulbectomy before new axons extended into the cavity. We found that
delaying axon growth increased the rate at which OECs filled the cavity.
The axons subsequently grew over a significantly larger region and
formed more distinct fascicles and glomeruli in comparison with growth
in animals that had undergone only bulbectomy. In vitro, we confirmed
(i) that olfactory axon growth was more rapid when OECs were more
widely distributed than the axons and (ii) that OECs migrated faster in
the absence of axons. These results demonstrate that the distribution of
OECs can be increased by repressing by growth of olfactory axons and
that olfactory axon growth is significantly enhanced if a permissive OEC
environment is present prior to axon growth.
POS-THU-08
TRANSGENIC APPROACHES TO ANALYSE PLANAR
CELL POLARITY/NON-CANONICAL WNT SIGNALLING
IN ZEBRAFISH EARLY ENDODERM MORPHOGENESIS
Miles L.B.1, Mizoguchi T.2, Kikuchi Y.2 and Verkade H.1
1
School of Biological Sciences, Monash University, Clayton, 3800
Victoria, Australia. 2Department of Biological Science, Graduate
School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-
Hiroshima, Hiroshima, 739-8526 Japan.
The zebrafish intestine, liver, and pancreas develop from the endodermal
cells, which start as a broad monolayer that migrates to the dorsal midline
during early development. Once at the midline the cells undergo midline
aggregation, where the monolayer of individual cells coalesces to form a
narrow endodermal rod at 18hpf. This endodermal rod will later undergo
morphogenesis and differentiation to give rise to their final organs. The
process of midline aggregation is essential for correct development
of the gut but the mechanisms used are unknown. I am investigating
the role of changes in cellular shape, behaviour, and polarity during
midline aggregation to determine the mechanisms involved. Mutants of
the non-canonical wnt / planar cell polarity (PCP) signalling pathway,
vangl2/trilobite (tri) and knypek (kny), display a distinctive phenotype
of shortened body axis and wider body plan resulting from a reduction
in convergence and extension of the mesodermal tissues that overlie
the endoderm. I will present data showing that these mutants also
have reduced convergence and extension of the endodermal cells,
producing a wider endodermal stripe during early-mid somitogenesis
stages. In addition my preliminary studies indicate that these mutants
show disrupted midline aggregation and incorrect endodermal cell
shape changes during mid-somitogenesis stages. To access the timing
specific role of PCP signalling during early endodermal migration and
midline aggregation, I have created a Tg(hsp70l-xdvlΔDEP-mCherry)
transgenic line. This line facilitates a temporal specific disruption of
the PCP signalling pathway by inducing a dominant negative form of
Dishevelled (xdvlΔDEP), the PCP signalling intracellular mediator.
POSTERS WEDNESDAY & THURSDAY
POS-WED-09
NEOGENIN 1: A NEW PLAYER IN THE SONIC
HEDGEHOG (SHH)/GLI NETWORK IN THE DEVELOPING
VERTEBRATE CENTRAL NERVOUS SYSTEM
Milla L.A.1, CortéS C.1, Wainwright B.J.2 and Palma V.1
1
Center for Genomics of the Cell (CGC), Faculty of Sciences, University
of Chile. 2Institute for Molecular Bioscience, University of Queensland,
Brisbane, Australia.
Objective Due the multiple roles played by the Shh/Gli pathway during
embryonic development and in adulthood, our work has been focused in
uncovering new direct downstream targets for this signaling network. Neo1
has been recently involved in many processes during CNS development,
has emerged as an interesting candidate. Here, we characterized the
nature of the interaction between Shh components and Neo1 in the rodent
CNS. Methods Bioinformatic analysis revealed several putative consensus
Gli binding sites (GBS) in the 5´ proximal regulatory region of neo1. To
verify in vivo and in vitro direct binding of Gli transcription factors to the
neo1 promoter, we used Chromatin Immunoprecipitation (ChIP) in mouse
embryonic neocortex (E18.5) and luciferase reporter assays. Expression
of Neo1 revealed a similar profile to Gli1 in the developing dorsal CNS. To
ascertain the relationship between Shh/Gli and Neo1 expression, we took
advantage of different experimental approaches: pharmacological gain
and loss of function experiments using mouse cerebellar primary cultures
and analysis of Shh pathway gain of function transgenic mouse models.
Results ChIP and reporter assays shows direct binding of Gli transcription
factors to 3 diffferents neo1 consensus Gli binding sites (GBS) in its
promoter, verifying our in silico analysis. Using genetic or pharmacological
Shh/Gli gain or loss of function approaches, we demonstrate a regulation of
Neo1 levels. The Shh pathway activation seems to be relevant to drive and
regulate Neo1 expression and, more importantly, to contribute thereby to
progenitor proliferation. Since Neo1 is strongly expressed in the developing
cerebellum we are currently addressing the functional consequences of
the Shh/Gli-Neo1 relationship in the Shh responsive granular precursor
cell population. Conclusion Neogenin1 is a new critical target of the Gli
Transcription Factors in vertebrate Hh signaling in the developing CNS.
POS-WED-11
THE GENETIC AND MORPHOLOGICAL
CHARACTERISATION OF A NOVEL ZEBRAFISH
DEVELOPMENT MUTANT, HL-7.2
Moore W.B.1, Sherson S.1, Ober E.2, Field H.2, Dong P.D.2, Stainier
D.Y.R.2 and Verkade H.1
1
School of Biological Sciences, Monash University, Clayton, VIC,
Australia. 2Department of Biochemistry and Biophysics, University of
California, San Francisco, USA.
The zebrafish is a useful model to examine the specification, differentiation
and proliferation of various cell types over the course of development. HL-
7.2 is a novel zebrafish mutant identified in an ENU mutagenesis screen
that aimed to identify mutants with morphological defects in endoderm
derived organs. At 36hpf, analysis using Tg(gut:GFP) transgenic, which
has GFP specifically expressed in endodermal organs revealed that
HL-7.2 mutants display dysmorphic budding of the liver and pancreas
from the endodermal rod and no intestinal looping. Other abnormalities
observed in HL-7.2 mutants include a cardiac defect, lack of pigment,
malformed brain and craniofacial defects. In situ hybridization analysis
of early stages of development up to 36hpf has demonstrated a wide
loss of expression of differentiation markers across all germ layers,
suggesting a broad developmental defect. Acridine Orange staining
revealed that HL-7.2 mutants have a significant increase in the number
of dying cells after 30hpf. I have mapped the HL-7.2 mutant locus to a
262kb genomic interval containing 6 genes. These 6 candidate genes
are currently being sequenced to identify the mutation responsible for
the HL-7.2 mutant phenotype. We hypothesize that the HL-7.2 mutant
phenotype results from a cellular arrest phenotype such as cell cycle
arrest, as HL-7.2 mutant embryos appear to developmentally arrest
at 24hpf. I am currently exploring the mechanisms responsible for
the severe abnormalities through the use of immunohistochemistry to
examine cell proliferation and to examine for mitotic abnormalities in
these mutants.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 193
POS-THU-10
TEMPORAL REGULATION OF THE SALVADOR-
WARTS-HIPPO PATHWAY IN DROSOPHILA
MELANOGASTER EYE DEVELOPMENT
Milton C.C.1, 2 , Zhang X.1, 2, Albanese N.O.1, 2 and Harvey K.F.1, 2
1
Cell Growth and Prolferation Laboratory, Peter MacCallum Cancer
Centre, East Melbourne, VIC 3002, Australia. 2Department of
Pathology, University of Melbourne, Parkville, VIC 3010, Australia.
The Salvador-Warts-Hippo (SWH) signalling pathway restricts tissue
growth and organ size during development. Loss of SWH signalling
results in tissue overgrowth as well as tumour development. The
SWH pathway is made up of multiple growth inhibitors which act
to limit activity of the Yorkie (Yki) oncoprotein. The complex fashion
by which these inhibitors act upstream of Yki is demonstrated by the
distinct phenotypes of tissue lacking different SWH pathway genes. For
example, eye tissue mutant for the core SWH complex components
salvador, warts or hippo is highly overgrown, while tissue lacking fat
or expanded is not. We analysed the relative contributions of the SWH
pathway proteins Fat, Expanded and Discs overgrown to organ size
control by investigating their temporal activity throughout Drosophila
eye development. We found that eye tissue which lacks fat, expanded
or discs overgrown displays elevated Yki activity during the larval
growth phase of development, but not in the pupal eye when apoptosis
ensues. In the pupal eye, Fat and Expanded do possess Yki-repressive
activity, but loss of fat or expanded at this stage of development can
be compensated for by Merlin. We showed that this compensation is
not due to an upregulation of Merlin protein levels in pupal eye tissue
lacking fat or expanded. It is likely that Fat represses Yki in the pupal eye
together with Expanded as pupal eye tissue lacking both of these genes
resembles single mutant tissue. This study highlights the complexity
and multiple ways by which different SWH pathway proteins can control
tissue growth and organ size during different stages of development.
POS-THU-12
STEM CELL THERAPY FOR HIRSCHSPRUNG DISEASE:
IN VIVO STUDIES USING A POST-NATAL MOUSE
MODEL
Hotta R.1, Stamp L.1, 2, Foong J.P.1, Thacker M.1, Pontell L.1, Bergner
A.J.1, Anderson R.G.1, Furness J.B.1, Newgreen D.F.2 and Young H.M.1
1
Department of Anatomy & Cell Biology, University of Melbourne,
Australia. 2Murdoch Childrens Research Institute, Royal Childrens
Hospital, Melbourne, Australia.
Gut motility is controlled by the enteric nervous system (ENS), which is
derived from the neural crest (NC). In Hirschsprung disease the ENS
is absent from the distal colon. Current treatment requires surgery to
remove the affected colon, but bowel dysmotility often persists. Cell
therapy has the potential to treat Hirschsprung disease by replacing
missing neurons. NC stem/progenitor cells can form ENS in aneural
embryonic colon in vitro. However, the clinically appropriate context is
post-natal colon in vivo; it is unknown whether such cells can colonize
and differentiate in such circumstances. To test this, ENS-derived
neurospheres were generated by FACS selection of Kikume+ or
GFP+ NC cells from gut of E14.5 transgenic mice. Neurospheres were
transplanted into the distal colon of P14-P21 wild-type and Ednrb-/-
Hirschsprung model mice. After 1-12 weeks, the neurosphere-derived
cells had migrated extensively in neural and aneural colon, formed
ganglion-like groups and differentiated as neurons (Hu and PGP9.5)
and glia (S100β). Neurons expressed nitrergic and cholinergic sub-type
markers, and projected axons into other ENS ganglia of the recipient,
with synapse-like (synaptophysin) specialisations. Electrophysiological
recording established that these neurosphere-derived neurons could
generate action potentials and display EPSPs indicative of neural
connectivity. These results show that stem/progenitor cells can migrate
and undergo neuronal and glial differentiation in the post-natal bowel in
vivo. Future studies will determine whether the transplanted cells can
restore gut motility patterns.
POSTERS WEDNESDAY & THURSDAY
POS-WED-13
THE EFFECT OF ACRYLAMIDE EXPOSURE ON EARLY
MALE GERM CELLS
Nixon B.J., Nixon B. and Roman S.D.
ARC Centre of Excellence in Biotechnology & Development, School of
Environmental & Life Sciences, Faculty of Science & IT, University of
Newcastle, Callaghan.
Acrylamide is a common industrial compound that has been found to
elicit male infertility and transgenerational toxicity in rodents through
the male germline. Acrylamide has been identified in carbohydrate-
rich foods cooked at high temperatures, thus humans may be at risk
to acrylamide exposure in the diet. The aim of this project was to
elucidate the mechanisms of acrylamide toxicity in male germ cells
of mice. Freshly isolated early male germ cells were assessed for cell
viability and aberrant morphology following exposure to acrylamide.
DNA damage in these cells was investigated using a modified version
of the Comet Assay, which allowed for adduct specificity. The regulation
of cytochrome P450 gene expression was measured using real time
PCR. Significant increases in cell death or aberrant morphology was
not found following acrylamide exposure (1μM, 18 hours). However, a
significant increase in DNA damage (125% increase in mean tail DNA
assessed by Comet) was identified; which may originate from either
the metabolic conversion of acrylamide to glycidamide, leading to
glycidamide adducts, or from oxidative stress. Additionally, early male
germ cells were found to upregulate gene expression of cytochrome
P450 enzymes in response to acrylamide treatment. The results of
this study support a genotoxic mode of action of acrylamide toxicity, in
addition to potential oxidative damage in male germ cells. However, the
mechanism by which acrylamide elicits toxicity in germ cells requires
further investigation. Future outcomes of this research may provide
insight into factors necessary for the healthy development of offspring
and aid in the risk assessment of dietary acrylamide exposure in
humans.
POS-WED-15
XENOBIOTICS; INFLUENCE ON LONG TERM OOCYTE
VIABILITY
Pye V.1, Sobinoff A.P.1, Nixon B.1, 2, Roman S.D.1, 2 and McLaughlin E.A.1, 2
1
School of Environmental and Life Sciences, The University of
Newcastle, Callaghan, NSW, Australia. 2The ARC Centre of
Excellence in Biotechnology and Development, The University of
Newcastle, Callaghan, NSW, Australia.
Mammalian females are born with a finite number of non-renewing
primordial follicles, the majority of which can remain in a quiescent
state for many years. Due to their non-renewing nature, these “resting”
oocytes are particularly vulnerable to environmental and toxic insults,
especially those which are capable of inducing oxidative stress.
Recent evidence suggests that certain synthetic chemical compounds,
known as xenobiotics, have the potential to generate oxidative stress
through the production of free oxygen radicals as a byproduct of the
cell’s detoxification process. Given the redox sensitive nature of the
mammalian oocyte, xenobiotic exposure has been hypothesized to have
long term adverse effects on oocyte viability. In this study, we attempted
to identify the effects of short term xenobiotic exposure on long term
oocyte viability. Female Swiss neonatal mice (day 4) were administered
7 daily consecutive doses of 4-Vinylcyclohexene diepoxide (40mg/kg/
daily; 80mg/kg/daily) Methoxychlor (50mg/kg/daily; 100mg/kg/daily) or
Menadione (7.5mg/kg/daily; 15mg/kg/daily). Mice were superovulated at
6wks and their oocytes collected for sperm-egg fusion assays. Sperm-
egg fusion assays revealed a significant decrease in sperm egg binding/
fusion (p<0.05) in a dose dependent manner for all three xenobiotic
treatments in vivo, signifying a decrease in oocyte membrane fluidity.
Follow-up lipid peroxidation analysis on xenobiotic cultured oocytes
also showed a dose dependent increase in membrane lipid peroxidation
in response to xenobiotic exposure. These results suggest that short
term xenobiotic exposure can cause long term oocyte dysfunction,
possibly interfering with the fluidity and/or elasticity of the oocyte plasma
membrane through lipid peroxidation.
Page 194 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-14
THE IDENTIFICATION OF A NOVEL GENE INVOLVED IN
DROSOPHILA MIDGUT MIGRATION
Pert M.C., Saint R.B. and Murray M.J.
Department of Genetics, The University of Melbourne, Australia.
Cell migration is an important process in animal development and
disease. In Drosophila embryogenesis, cell migration is necessary
for the formation of the midgut, in which epithelial patches of cells at
the anterior and posterior poles of the embryo undergo an epithelial
to mesenchymal transition and migrate towards one another using the
visceral mesoderm as a substrate. Once the two patches of cells have
met in the middle of embryo, they undergo a mesenchymal to epithelial
transition to form the midgut. Currently, there is only one group of
proteins known to be required for midgut migration; the extracellular
matrix adhesion proteins, the integrins. It is therefore of great interest
to discover new genes involved in this process. We have identified a
deficiency stock that has an embryonic phenotype in which the midgut
does not form due to a failure in cell migration. As this deficiency stock
results in the loss of a large number of genes, a deficiency screen has
been carried out to narrow down the number of possible candidates.
None of these candidates have previously been associated with midgut
migration. Currently, we are utilizing molecularly defined deletions,
genomic rescue constructs, and injection of dsRNA to determine which
of the candidate genes is responsible for the midgut phenotype. We are
also using a combination of immunostaining and live imaging to better
characterise the cellular basis for the migration defect. The findings of
these experiments will be presented.
POS-THU-16
NOVEL PARACRINE SIGNALING FROM CARDIAC
PROGENITOR CELLS TO EARLY NEURAL CREST IN
MAMMALIAN EMBRYOS
Prall O.1, Menon M.K.2, Biben C.1, Hartley L.1, Martin J.3, Imanaka-
Yoshida K.4 and Harvey R.P.2
1
Walter and Eliza Hall Institute, Melbourne, Australia. 2Victor Chang
Cardiac Research Institute, Sydney, Australia. 3Institute of Biosciences
and Technology, Texas, USA. 4Mie University, Japan.
Congenital abnormalities of the heart and face frequently occur
simultaneously due to shared aspects of their formation. In early
vertebrate embryos “neural crest” (NC) detaches from dorsal neural
tissue and migrates over substantial distances to supply cells to many
structures including facial bones and the heart. Prior to entering the
heart, NC cells exchange molecular signals with cardiac progenitors
in the pharynx. This reciprocal signaling is essential for both NC and
cardiac development. We have found evidence of two new molecular
signals that cardiac progenitors send to NC, a morphogen bone
morphogenetic protein 2 (BMP2) and an extracellular matrix protein
tenascin C (TNC). In contrast to known interactions between cardiac
progenitors and NC, this paracrine signaling occurs in cranial regions
prior to pharyngeal development. Mutation of the cardiac transcription
factor gene Nkx2-5 results in advanced migration of cranial and
cardiac NC. In Nkx2-5 mutants, Tnc and Bmp2 mRNA expression are
upregulated in cardiac progenitor cells. TNC protein is deposited in sub-
NC mesenchyme in both control and Nkx2-5 mutants, and elevated in
transgenic mice over-expressing Tnc specifically in cardiac progenitor
cells. Nkx2-5 mutants lacking the BMP signaling intermediate Smad1,
show upregulation of BMP2 in cardiac progenitors but not advanced NC
migration. This suggests that cardiac-derived BMP2 signals to neural
crest. It is possible that genetic and environmental factors perturbing
early cardiac progenitors may cause congenital abnormalities through
early indirect influences on NC behaviour.
POSTERS WEDNESDAY & THURSDAY
POS-WED-17
IDENTIFICATION AND CHARACTERISATION OF
SURFACE PROTEIN COMPLEXES IN HUMAN
SPERMATOZOA
Redgrove K.A.1, McLaughlin E.A.1, O’Bryan M.K.2, Aitken R.J.1 and
Nixon B.1
1
School of Environmental and Life Science, University of Newcastle,
Callaghan, NSW, Australia. 2School of Biological Sciences, The
Department of Anatomy and Development Biology, Monash University,
Melbourne, VIC, Australia.
Cell-cell adhesion is a highly diverse and intricate interaction that relies
on the expression, and often, coordinated action of several specialised
cell adhesion molecules. One of the most intriguing cell-cell interactions
is the binding of spermatozoa to an oocyte. Upon ejaculation, mammalian
spermatozoa are unable to recognise and bind to the female egg. They
must first undergo a maturation phase termed ‘capacitation’ during
which they are transformed into functionally competent cells. Emerging
evidence suggests that during capacitation, a multimeric protein
complex is assembled or unmasked on the sperm surface to facilitate
sperm recognition and adhesion to the egg. Through the novel use of
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have
provided evidence that human spermatozoa express a number of high
molecular weight protein complexes on their surface. In this study, we
aimed to characterise one such complex shown to comprise a number
of putative recognition molecules, including arylsulfatase a (ASA). Using
immunolocalisation techniques we demonstrated that ASA localises to
the apical region of the human sperm head, a domain that is known
to be responsible for mediating sperm-egg interactions. Furthermore,
flow cytometry analysis revealed that ASA translocates to the surface
of spermatozoa during capacitation, and that this movement may be
blocked by the addition of exogenous cholesterol to the cell. Our current
research is focused on determining the role of ASA in sperm-egg
adhesion and the cellular mechanisms that underpin it’s translocation
to the cell surface.
POS-WED-19
FUNCTIONAL ANALYSIS OF KLF2 DURING
EMBRYONIC SKELETAL DEVELOPMENT
Rodda F.A.1, 2 , Cameron T.L.1, Gordon C.T.1, Bateman J.F.1, 2 and Farlie
P.G.1, 2
1
Murdoch Childrens Research Institute, Parkville, Victoria, Australia.
2
The University of Melbourne, Parkville, Victoria, Australia.
Endochondral ossification is the process by which the majority of
the bones of the body are formed. It occurs via the differentiation of
mesenchymal cells into chondrocytes to produce a cartilage template of
the skeleton (chondrogenesis), followed by replacement of this template
with bone (osteogenesis). This complex process is incompletely
understood. Kruppel-like factor 2 (Klf2) is a zinc finger transcription
factor upregulated 30-fold during chondrogenesis. With known roles in
regulating blood vessel tone, T-cell and smooth muscle cell migration,
it as-yet has no known role in skeletal development. Here we provide
evidence for the functional significance of Klf2 in limb development.
Retroviral-driven misexpression of Klf2 in chick embryos results in
reduction of overall bone length, transformations of digit identity, and an
alteration of bone morphology coined the ‘web of bone’. We are currently
using a viral construct containing a tissue-specific promoter to assess
if the web of bone is due to a disruption of signalling from the cartilage
itself, or from the surrounding perichondrial cell layer. In addition, in
situ hybridisation analysis is being used to examine gene expression
alterations in response to Klf2 misexpression, to identify the network in
which Klf2 functions and elucidate its mode of action in chondrogenesis
and osteogenesis.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 195
POS-THU-18
INVESTIGATION OF THE ROLE OF DYNAMIN IN SPERM
SURFACE REMODELLING
Reid A.T., Roman S.D., Aitken R.J. and Nixon B.
School of Environmental and Life Sciences, University of Newcastle,
Callaghan, NSW, Australia.
Recent research from our laboratory has provided evidence that the
construction of a multimeric sperm receptor complex mediates sperm/egg
interaction. In addition, we have convincing evidence that this complex
is assembled on the sperm surface during the final phase of maturation,
a process known as capacitation. The mechanisms underpinning these
capacitation-associated surface remodelling events remain poorly
understood and are the subject of our current investigation. Specifically
we have focused on whether this process is driven by vesicle mediated,
intracellular protein trafficking. For this purpose we have investigated
the presence and physiological significance of an integral part of the
molecular machinery necessary for this form of trafficking, namely the
GTPase dynamin. Our studies revealed that mouse spermatozoa are
endowed with at least two isoforms of dynamin (1 and 2) both of which
reside within the peri-acrosomal region of the sperm head, a location
compatible with a role in sperm membrane remodelling associated with
the acquisition of the ability to engage in oocyte interaction. Consistent
with this notion, we have demonstrated that pharmacological inhibition of
dynamin activity leads to a concomitant reduction in the ability of mouse
spermatozoa to bind to the outer vestments of homologous oocytes.
Collectively these data support the novel hypothesis that dynamin does
participate indirectly in sperm membrane remodelling events by virtue
of its ability to mediate intracellular trafficking.
POS-THU-20
EXPRESSION AND FUNCTIONAL ANALYSIS OF SOX3
IN MURINE NEUROGENESIS
Rogers N.A., Banerjee K. and Thomas P.Q.
School of Molecular and Biomedical Science, The University of
Adelaide.
Sry-related HMG box transcription factor 3 (Sox3) has been associated
with human CNS defects, whereby duplication and mutations of
SOX3 have been identified in patients with X-linked hypopituitarism
(XH). Patients with XH suffer from mild intellectual disability and
hypothalamic-pituitary axis dysfunction, the developmental origin and
molecular pathology of which is poorly understood. In mice, we have
shown SOX3 is highly expressed in the progenitor/stem cells throughout
embryonic neurogenesis (12.5-18.5 dpc) and is maintained in the adult
neurogenic niches. To investigate the functional role of SOX3, we
compared the expression of cell type-specific neurogenesis markers
in Sox3 null (EGFP knock-in) mice, which phenocopy XH patients, and
wild type mice. No overt changes in dorsal telencephalic neurogenic
marker gene expression were detected indicating that the lack of SOX3
has a minimal impact on neurogenesis. Similarly, in adult mice, no
obvious defects were observed within the sub-ventricular zone or the
sub-granular zone of SOX3 null animals. Interestingly EGFP reporter
gene expression in Sox3 null mice was lost at approximately 18.5 dpc,
suggesting that SOX3 expression may be autoregulatory in neural
stem/progenitor cells. To further investigate the role of SOX3 in vitro,
we generated neurospheres from the embryonic telencephalon and
the adult neurogenic zones. No obvious difference in the proliferation
and differentiation properties of wild type and null neurospheres were
detected, although subtle differences in neurosphere growth rates were
apparent and are currently under investigation. Ultimately we hope that
these studies will provide greater understanding of the role of SOX3
within the developing CNS and the molecular pathology of XH.
POSTERS WEDNESDAY & THURSDAY
POS-WED-21
CONSISTENT NUCLEOSOME RETENTION DURING
CHROMATIN PACKAGING IN HUMAN SPERMATOZOA
Reid A.T.1, 2, McEwan K.2, Campbell D.M.1, 2, Jans D.A.1, 3 and Roman S.D.1, 2
1
ARC Centre of Excellence in Biotechnology & Development. 2Biology,
School of Environmental & Life Sciences, Faculty of Science & IT,
University of Newcastle, Callaghan NSW 2308, Australia. 3Dept of
Biochemistry & Molecular Biology, Monash University, Clayton Vic
3800, Australia.
In contrast to the histone packaging of somatic cells, spermatozoa are
predominantly packaged by the protamine proteins. However, human
spermatozoa retain ~15% histone packaging. Regions that are left
nucleosome bound could either be genes that are active shortly after
fertilisation or genes that are transcribed late during spermatogenesis.
DNA damage at histone bound loci would be of consequence to
spermatogenesis and/or to a resulting embyro post-fertilisation.
Western blot analysis confirmed the presence of acetylated H3 and H4
in sperm. Interestingly, we identified the presence of these modified
histones in isolated good quality sperm considered to have complete
packaging. This indicates that histone retention is not a consequence
of aberrant chromatin remodelling. Using a combination of ChIP
(chromatin immunoprecipitation) and tiling arrays we have obtained
evidence of regions bound by acetylated histone 3 (H3). ChIP-PCR
confirmed histone retention at several loci identified by tiling array. For
the previously identified loci in exon 1 of the TNP-2 gene we were able
to narrow the region bound to the size of one nucleosome only. Using a
modified form of ChIP known as carrier ChIP we were able to examine
histone retention in individual ejaculates. We demonstrated that humans
consistently retain acetylated H3 at the same loci. For four out of five
males the TNP-2 loci was bound in 100% of the multiple ejaculates
examined. In conclusion, retention of acetylated H3 is consistently
maintained during packaging of the genome in spermatogenesis.
POS-WED-23
THE MAMMALIAN EXPANDED HOMOLOGUE,
WILLIN, CAN NOT FUNCTIONALLY SUBSTITUTE FOR
EXPANDED IN DROSOPHILA
Sinha A.1, 2 , Gunn-Moore F.J.3 and Harvey K.F.1, 2
1
Cell Growth and Proliferation Laboratory, Peter MacCallum Cancer
Centre, St Andrews Place, East Melbourne, Victoria, Australia, 3002.
2
Department of Pathology, University of Melbourne, Parkville, Victoria,
Australia, 3010. 3Bute Building, School of Biology, University of St
Andrews, St Andrews, UK. KY16 9TS.
Studies in Drosophila melanogaster have defined a novel growth
inhibitory pathway known as the Salvador-Warts-Hippo (SWH) pathway.
This pathway is evolutionarily conserved and most of the component
members have mammalian homologues. It has been shown for a few of
these members that they can functionally substitute for their Drosophila
counterparts. Recently a sequence homologue of an upstream member
of the pathway, Expanded (Ex), was identified and is known as Willin or
FRMD6. In this study we investigated whether Willin can act in a similar
fashion to Ex in Drosophila epithelial tissues called imaginal discs.
When Willin was misexpressed using wing-specific drivers it showed
the same sub-cellular localization as Ex, but could not rescue growth
defects associated with ex deficiency. The later result was recapitulated
in a clonal assay in the eye-antennal disc as well, where pathway targets
such as ex and diap1 were high and unaffected by overexpressing Willin
in these clones. These results suggest that Willin can not functionally
substitute for the ability of ex to control SWH pathway-dependent
tissue growth in D. melanogaster. These results call into question
whether Willin executes the same function in mammals as Ex does in
D. melanogaster.
Page 196 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-22
MIGRATION OF DEFINED SUBPOPULATIONS OF
VAGAL ENTERIC NEURAL CREST CELLS
Simkin J.E., Zhang D. and Newgreen D.F.
Murdoch Childrens Research Institute, Flemington rd, Parkville, Vic
3052.
Neural crest (NC) cells arise in the embryonic brain and spinal cord.
NC from the hindbrain or vagal level (adjacent to somites s1-s7)
migrate laterally in streams from each somite level, enter the foregut,
and colonize the entire gut in a rostro-caudal wave. They then form the
enteric nervous system. Previous studies demonstrated broad regional
differences in developmental potential, depending on the NC level of
origin. This study utilizes a new focal labeling method to investigate
fine-scaled (single-somite length) differences within the vagal level.
We ask: are particular vagal subpopulations favoured in initial entry to
the gut, and is there some form of matching between subpopulation
origin and region of gut ultimately colonized? We electroporated a
genome-integrating GFP construct into single-somite lengths of E1.5
quail embryo premigratory NC. Using fluorescence time-lapse and
still imaging, we viewed the embryos at a) E2.5-E3, when the NCCs
approach/enter the foregut, and b) E6.5, when NC cells have colonized
most of the gut. Initially the subpopulations of vagal NC were unmixed,
entering a 0.5 mm long region of foregut subjacent their somite level
of origin. Later, subpopulations become intermingled, so any sub-
population could contribute stochastically to any region of the gut.
However, the s3 NC had a numerical advantage, and were more likely
to contribute large numbers of NC cells in any gut region, compared
to all other sub-populations. These results provide insight into factors
advantageous to gut colonization, which may be useful in the design of
future stem cell therapies for neurocristopathies, such as Hirschprungs
Disease (incomplete enteric nervous system).
POS-THU-24
CONSISTENT MECHANISM OF PRIMORDIAL FOLLICLE
ACTIVATION IN NEONATAL MOUSE OVOTOXICITY
Sobinoff A.P.1, Pye V.1, Nixon B.1, 2, Roman S.D.1, 2 and McLaughlin E.A.1, 2
1
School of Environmental and Life Sciences, The University of
Newcastle, Callaghan, NSW, Australia. 2The ARC Centre of Excellence
in Biotechnology and Development, The University of Newcastle,
Callaghan, NSW, Australia.
The mammalian female reproductive lifespan is largely defined by a finite
pool of primordial follicles established around the time of birth. Overall,
<1% of these follicles are destined for ovulation, with the vast majority
being lost during development in a process called atresia. While atresia
is a normal physiological process, it is now well documented that it can
be triggered through exposure to certain synthetic chemical compounds,
known as xenobiotics, causing premature ovarian senescence. In
addition to follicular atresia, new evidence suggests that aberrant
follicular activation may play a role in the xenobiotic ovotoxicity. In this
study we attempted to identify similarities between the mechanisms
of ovotoxicity for three ovotoxic agents, 4-Vinylcyclohexene Diepoxide
(VCD), Methoxychlor (MXC), and Menadione (MEN), which target
immature follicles. Microarray analysis of neonatal mouse ovaries
exposed to these xenobiotics in vitro revealed a more than two-fold
significant difference in gene expression (p<0.05) for a number of genes
associated with apoptotic cell death and primordial follicle activation.
Follow-up qPCR analysis on VCD and MXC cultured ovaries confirmed
an increase in expression for Akt1 (5.8, 6 fold), Akt2 (2.9, 1.5 fold),
and Ccnd2 (5.3, 6 fold), all three of which are involved in follicular
development. Histomorphological and immunohistological analysis
supported the microarray data, showing signs of primordial follicle
activation and pre-antral follicle atresia in vitro and in vivo. These results
indicate a consistent mechanism of primordial follicle activation in pre-
antral ovotoxicity for all three xenobiotics.
POSTERS WEDNESDAY & THURSDAY
POS-WED-25
PRIMARY MAMMARY EPITHELIAL CELLS (PMEC)
DERIVED FROM LACTATING HUMAN TISSUE HAVE
UNIQUE DIFFERENTIATION PROPERTIES IN THREE-
DIMENSIONAL CULTURE
Thomas E.C.1, 2 , Zeps N.2, Rigby P.J.1 and Hartmann P.E.1
1
School of Biomedical, Biomolecular and Chemical Sciences, The
University of Western Australia, Perth, Australia. 2School of Surgery,
QEII Medical Centre, The University of Western Australia, Perth,
Australia.
A diverse body of evidence indicates that the human mammary gland
contains a multipotent population that generates the lactating gland
during gestation. It is also believed that loss of regulation of this
population may lead to breast cancer. We have used cells expressing
markers for multipotent phenotype (CD49f, CD29, CD24, p63, CK6
and Nestin) isolated from lactating human tissue via an expressed
breastmilk sample to derive a primary cell population (PMEC) that can
be propagated through multiple passages of monolayer culture. PMEC
generate ductal-alveolus-like structures in extracellular matrix (ECM),
and display unique structural and functional features compared to non-
tumorigenic mammary epithelial cell lines MCF10A and HBL-100. Using
structurally interrupted and growth factor reduced ECM we show that
complete differentiation and organized assembly of PMEC into a basal
(CD49f-positive) and luminal (CK18-positive) ductal-alveolar structures
is dependant on intact and complete ECM. Complete differentiation is
required for luminal cell expression of milk proteins alpha-lactalbumin
and beta-casein in response to Prolactin stimulation. We also show
that treatment with developmental and lactational regulators influence
different stages of PMEC proliferation, differentiation and function. Using
quantitative mass spectrometery (iTraq) we have profiled the proteome
of PMEC-derived ductal-alveolar structures at daily intervals for seven
days to identify changes associated with proliferation and differentiation.
We suggest this will provide a model for normal differentiation of the
mammary epithelium and provides a platform to identify pathways that
may lead to aberrant growth when regulation is lost.
POS-WED-27
THE ROLES OF FOXL2 & ZDHHC17 IN SKELETAL
DEVELOPMENT
Welfare M.F. and Farlie P.G.
Murdoch Children’s Reseach Institute, Department of Paediatrics,
University of Melbourne, Royal Children’s Hospital, Parkville.
Very little is currently known about which genes control skeletal
development. In order to increase our knowledge of the genes involved,
we are performing a large scale screen of genes that are active during
skeletal development. Two of the identified genes will be examined
further to determine what role they play in skeletal development. The
first gene, Foxl2, will be overexpressed in the limbs. Initial studies
suggest that this may cause significant shortening of the limb bones and
identifying the mechanisms underpinning this phenotype (eg apoptosis
or a growth deficiency) will form a major part of the project. The Foxl2
gene will be cloned into a viral vector (RCAS) and then injected into
chicken embryos where the viral construct will cause the inserted gene
to be expressed. The second gene, Zdhhc17, will be knocked down in
the craniofacial region. This will show the effect of removing the function
of Zdhhc17 during the development of the facial and skull bones.
No previous studies have examined the role of Zdhhc17 in skeletal
development. In this case, a knockdown construct will be cloned into
RCAS. This construct encodes a RNA which is complementary to the
hosts Zdhhc17 mRNA, therefore the introduced RNA will bind to the
host mRNA and trigger its degradation. This prevents the Zdhhc17
protein from being made, effectively removing the functionality of the
Zdhhc17 gene. The understanding gained from this analysis will enable
us to further elucidate the complex mechanisms underlying skeletal
development. This in turn will then lead to an increased understanding
of many birth defects and syndromes with underlying skeletal defects.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 197
POS-THU-26
TWIST GENES REGULATE OUTGROWTH AND
PATTERNING DURING LIMB MORPHOGENESIS
Wade C., Brinas I. and Farlie P.
Murdoch Childrens Research Institute, Royal Children’s Hospital,
Parkville, Victoria, Australia 3052.
Twist1 has been demonstrated to play critical roles in the early
development of neural crest and mesodermally derived tissues.
Twist2 has been less well characterised but its relatively late onset of
expression suggests specific roles in the development of a number of
sites. We have used RCAS-mediated overexpression to investigate the
function of Twist2 in limb development. Expression of Twist2 within the
developing limbs begins prior to formation of the limb bud and persists
within the peripheral mesenchyme until digital rays condense when
Twist2 expression becomes restricted to the interdigital mesenchyme.
Viral misexpression following injection into the lateral plate mesoderm
results in a spectrum of hypoplastic limb phenotypes. These include
generalised shortening of the entire limb, fusion of the autopod skeletal
elements, loss of individual digits or distal truncation resulting in
complete loss of the autopod. These phenotypes appear to result from
a premature termination of limb outgrowth. In situ hybridisation analysis
demonstrates that many components of the Shh/Fgf/Gremlin regulatory
loop that controls early limb outgrowth are downregulated by Twist2
overexpression. This suggests that Twist2 controls limb outgrowth
through the regulation of this central growth regulatory network. Given
the downregulation of Gremlin and its complementary expression
pattern to Twist2, Gremlin is a good candidate for a transcriptional
target of Twist2, and preliminary ChIP data supports this hypothesis.
However, despite loss of AER Fgf8 and other regulatory loop factors,
Shh expression is sustained at normal levels. This raises the possibility
of Shh being a transcriptional target of Twist2. Similar data were
obtained for Twist1. These data illustrate the molecular mechanisms
through which the Twist genes control morphogenesis of the limb.
POS-THU-28
INSIGHTS INTO C-TERMINAL BINDING PROTEIN
(CTBP) VIA A MUTAGENESIS SCREEN IN C. ELEGANS
Yucel D.1, Crossley M.1, 2 and Nicholas H.1
1
University of Sydney. 2University of New South Wales.
CtBP is a transcriptional co-repressor which plays roles in development
and apoptosis. There are two highly related CtBP genes in vertebrates.
Knock-out of both CtBP1 and CtBP2 results in lethality early in
embryogenesis. Recently, we cloned a single CtBP in C. elegans.
Like its mammalian counterparts, the C. elegans CtBP, called CTBP-1,
functions as a transcriptional co-repressor and docks onto transcription
factors containing an amino acid motif of the form PXDLS. We have
analysed the expression pattern of CTBP-1 using a translational reporter
construct (CTBP-1::GFP) which has shown that CTBP-1 is expressed
in numerous cells, some of which have been identified as neurons.
We are using various reporter markers to assess whether CTBP-1
is involved in the development or specification of these neurons.One
such marker showed that ctbp-1 loss of function mutant worms have
fewer of one particular motor neuron cell type. In order to identify novel
mutations that interfere with correct CTBP-1 activity, we carried out a
mutagenesis screen.We treated the CTBP-1::GFP transgenic line with
EMS and isolated worms which showed changes in the expression
of the fusion protein.We have isolated six mutant lines, two of which
show particularly interesting phenotypes. In one of the mutants named
aus5, the expression of the CTBP-1::GFP fusion protein is dramatically
decreased. In the other mutant line, aus3, the fusion protein is ectopically
expressed.The aus5 mutant line may have a mutation in an activator
of CTBP-1 such that CTBP-1 is switched off. In the aus3 mutant line,
the mutation could be in a repressor of CTBP-1 allowing CTBP-1 to
be switched on ectopically.Whole genome sequencing is in progress
to identify the genes affected by these mutations which alter CTBP-
1 activity. Taken together, our results hold the potential to unravel the
roles of CTBP-1 in neuronal regulation and to reveal novel components
of the gene regulatory networks in which CTBP-1 is involved.
POSTERS WEDNESDAY & THURSDAY
POS-WED-29
NEURAL CREST CELL INVASION OF DEVELOPING
GUT IS PREVENTED BY EXISTING NEURAL CREST
CELLS BUT NOT BY ENTERIC NEURONS
Zhang D. and Newgreen D.F.
Murdoch Childrens Research Institute, Parkville, Victoria 3052.
The development of the enteric never system (ENS) depends on
vagal neural crest cell (NCC) invasion. We showed previously using
chick-quail gut kebab grafts that NCC invasion ability depends on
cell proliferation especially in the invasion wavefront. Using various
spatiotemporal combination gut grafts, we showed that the invasion
ability of NCCs is high when placed at the wavefront and restricted at
behind-wavefront positions. The latter position differs from the former in
that 1. the gut tissue is older, 2. NC-derived neurons are present, and
3. NCC are present. To test condition 1, we grafted quail NCC donor
gut into older gut from vagal neural tube-ablated chick embryos. The
NCCs invaded older NCC-free and neuron-free gut avidly, therefore
gut age is not primarily responsible for invasion restriction. To test
condition 2, we briefly treated older chick gut (with NCCs and neurons)
with the proliferation inhibitor mitomycin C. After 2 days the NCCs had
disappeared but abundant neurons with neurites remained. We then
grafted quail NCCs donor into the NCC-free but neuron-containing gut.
The NCCs invaded this, suggesting that enteric neurons do not restrict
NCC invasion. To test condition 3, we grafted quail NCC into chick host
gut such that chick NCC migrate caudally and quail NCC rostrally, at a
stage when neurons are almost absent. These two NCC populations
mutually impeded each other, showing that presence of NCCs in the gut
contributes to the restriction of invasion by new NCCs. In conclusion,
the restriction of invasion ability of NCCs into developing gut depends on
pre-existing NCCs but not on neurons, and this restriction is reversible
by removal of NCCs.
POS-WED-31
PHOSPHORYLATION-DEPENDENT REGULATION OF
INTRACELLULAR LOCALIZATION AND POTENTIAL
TUMOR SUPPRESSOR FUNCTION OF LKB1 BY AKT
AND 14-3-3
Liu L.1, Wang Y.2, Lam K.S.L.1 and Xu A.1, 2
1
Department of Medicine, The university of Hong Kong. 2Department of
Pharmacology, The university of Hong Kong.
Introduction: LKB1, a tumor suppressor protein kinase, is causally linked
to Peutz-Jeghers syndrome, with a predisposition to cancer. Several
studies indicated that the intracellular localization of LKB1 is very
important for its function. Scansite predicted that serine334 of LKB1
locates in both the recognition motif of 14-3-3 and the phosphorylation
site of Akt. The objectives of this study are to investigate whether LKB1
is a downstream target of Akt and a novel binding partner of 14-3-3,
and to elucidate how the intracellular localization and functions of LKB1
can be regulated. Methods: Seven GST-tagged 14-3-3 isoforms were
used for pull-down experiment to check whether 14-3-3s interact with
LKB1. In vitro phosphorylation assay, mass spectrometry (MS) analysis
and site-directed mutagenesis were employed for determination of
whether serine334 is both the phosphorylation site of Akt and 14-3-3
binding site. Results: Five of the seven 14-3-3 isoforms interact with
LKB1. The inhibition of Akt activity can cause both the decreased
interaction between 14-3-3 zeta and LKB1 and the increased cytosolic/
nuclear ratio of LKB1. In vitro phosphorylation assay result confirmed
that LKB1 is a direct target of Akt and the subsequent MS analysis
determined the precise site is serine334. Site-directed mutagenesis
revealed that mutation of LKB1 on serine334 to alanine can lead to both
the decreased interaction between LKB1 and 14-3-3zeta and increased
cytosolic localization. Conclusions: LKB1 is a downstream target of
Akt kinase and a novel interaction partner of 14-3-3. Serine334 is the
phosphorylation site of Akt and interaction site of 14-3-3. Akt regulates
the phosphorylation-dependent interaction between LKB1 and 14-3-3
and subsequently regulates the intracellular localization of LKB, which
might be very significant for its tumor suppressor functions. These
findings may provide a novel mechanism whereby the tumor suppressor
functions of LKB1 can be regulated.
Page 198 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-30
THE REGULATORY NETWORK OF TTP, MKP-1
AND MAPK IN RESPONSE TO PREADIPOCYTE
DIFFERENTIATION SIGNALS
Lin N.Y.1, Lin T.Y.2, Wang S.C.1 and Chang C.J.1, 2
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
2
Graduate Institute of Biochemical Sciences, College of Life Science,
National Taiwan University, Taipei, Taiwan.
The activation of mitogen activated protein kinases (MAPK) signaling
pathways is necessary to initiate the differentiation process of 3T3-L1
preadipocytes, and the activation needs to be turned-off to proceed to
terminal maturation. The expression of MAPK phosphatas-1 (MKP-1)
can control the timing window of MAPK activity. This study demonstrated
that the transient expression of MKP-1 was transcriptionally induced
by ERK signaling; at the same time, ERK signaling also mediated the
activation of RNA binding protein tristetraprolin (TTP) to decrease the
expression level of MKP-1 posttranscriptionally. In this study, U0126
was used to block the activation of ERK signaling and it resulted in the
down-regulation of TTP expression and enhanced stability of MKP-1
mRNAs at 1h after induction to differentiation. siRNAs against TTP
prolonged the half-life of MKP-1 mRNA. RNA-protein interaction and
functional reporter assays showed that TTP differentially bound to three
AU-rich elements (AREs) of MKP-1 mRNA and negatively regulated
the MKP-1 ARE-containing luciferase reporter activity. Interestingly, we
found that MKP-1 can associate with TTP and possibly down-regulate
TTP phosphorylation and modulate the ARE-binding activity or protein
stability of TTP. Moreover, the knockdown of MKP-1 and TTP showed
a slight but different effect on adipogenesis. The results suggested that
TTP can regulate the MKP-1 mRNA stability to subsequently control the
activation of MAPK signaling pathways in adipocyte differentiation.
POS-THU-32
INFLAMMATION IN SEPSIS: ROLE OF ENDOTHELIAL
CELLS
Low W.X.1, Muniandy S.1, Qzist R.2 and GracieOng S.Y.3
1
Department of Molecfular Medicine, Faculty of Medicine, University
of Malaya, 50603 Kuala Lumpur, Malaysia. 2Department of Medicine,
Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur,
Malaysia. 3Department of Anesthesiology, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia.
Severe sepsis and septic shock are the primary causes of the multiple
organ dysfunctions and one of the common causes of death in intensive
care units worldwide. The infecting microorganisms trigger widespread
activation of multiple pathophysiological processes resulting in the
clinical syndrome of multiple organ dysfunctions. Immune cells and
endothelial cells play crucial roles in severe sepsis. We investigated
the expression of target genes associated with triggering of endothelial
cells by inflammatory molecules present in plasma of subjects with
severe sepsis. Cultured human umbilical vein endothelial cells were
triggered with plasma from patients with gram negative bacteraemia.
Inoculation was performed at 5 different time intervals (0 hour, 4 hours,
7 hours, 12 hours and 24 hours). Results revealed that the expression
of NFκB was significantly up regulated at the 24th hours after treatment
of the endothelial cells. The expression of 84 key genes were screened
using real time reverse transcription profiler PCR array and significantly
expressed pro- and anti-inflammatory genes were identified. Prominent
pro-inflammatory cytokines namely IL8, IL6 and IFNγ were also
determined. These observations demonstrated that endothelial cells
serve as an equally important inflammatory role as other immune cells
in triggering sepsis and NFκB, inflammatory genes and inflammatory
cytokines were expressed in parallel as early as 24 hours after
treatment. Further studies into the inflammatory roles of the endothelial
cells may help in assessing the efficacy of therapeutic interventions in
near future.
POSTERS WEDNESDAY & THURSDAY
POS-WED-33
PITUITARY AND OVARIAN FUNCTION IN PRIP
KNOCKOUT MICE
Matsuda M. and Hirata M.
Laboratory of Molecular and Cellular Biochemistry, Graduate School of
Dental Science, Kyushu University.
Phospholipase C-related but catalytically inactive protein (comprising
PRIP-1 and -2) was first identified as a novel D-myo-inositol
1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein, but the biological
functions have remained elusive. We therefore generated PRIP-1
and -2 double knockout (DKO) mice to gain insight into the biological
function. DKO mice apparently grew normally and became fertile;
however, during animal maintenance, we noticed that mutant couples
exhibited decreased litter events and litter size, indicating dysfunction
of the reproductive system. Cross-mating experiments indicated that
the cause appeared to be on the female side. The observation of the
estrous cycle in mice by histological analysis of vaginal smears showed
that the estrous days were apparently increased in DKO mice. Levels of
serum LH and FSH were measured for 5-6 consecutive days, and were
significantly higher in the mutant, which was also confirmed by examining
the secretion of LH from the explant culture of anterior pituitary glands
of wild-type and DKO mice. We also examined the ability of the ovaries
in response to gonadotropins: the total number of the ovulated oocytes
were significantly decreased in mutant female mice, compared to that
seen with wild-type, which was also confirmed by histological analysis of
the ovaries after ovulation. The analysis also showed that the numbers
of follicles originally were not significantly different between WT and
DKO ovaries. These results suggest that PRIP plays an important role
in female reproductive system, especially in gonadotropin secretion and
ovarian follicle maturation.
POS-WED-35
ANALYSIS OF THE REGULATION AND FUNCTION OF
KIWIFRUIT FLOWERING LOCUS T
Moss S.M.A.1, 2 , Putterill J.1 and Varkonyi-Gasic E.2
1
School of Biological Sciences, The University of Auckland, Private
Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand. 2Plant
and Food Research, Mt Albert, Private Bag 92169, Auckland Mail
Centre, Auckland 1142, New Zealand.
Kiwifruit (Actinidia sp) requires synchronised, concentrated flowering
to obtain good fruit quality and yield. The molecular mechanisms
controlling flower initiation and development in kiwifruit are largely
unknown. The genes and pathways controlling flowering have been
elucidated in model plants, such as Arabidopsis thaliana. In Arabidopsis,
the photoperiodic, autonomous and vernalization pathways all converge
on floral integrator genes that promote the transition from vegetative to
floral development. One of the key floral integrator genes is FLOWERING
LOCUS T (FT). FT-like genes have been found to be major flowering
regulators in other model species, including rice, tomato and poplar.
In kiwifruit, a candidate FT gene (AcFT) has been identified as highly
homologous to Arabidopsis FT. Overexpression of AcFT complements
the Arabidopsis ft mutant illustrating gene functionality across species.
The aim of this research is to analyse the AcFT promoter sequence
using three techniques. The characterisation of the AcFT promoter will
provide information as to spatial and temporal AcFT expression at a
tissue-specific and cell-type specific level. Firstly, bioinformatics will be
used to identify the potential regulatory motifs of the AcFT promoter.
Secondly, the reporter genes GUS and GFP will be used in the model
plants Arabidopsis and Nicotiana to locate the sites of AcFT promoter
activation. Thirdly, a transcriptional network will be developed using dual
transient luciferase assays. The characterisation of the AcFT promoter
should help to provide insight into the initiation and maintenance of
flowering in kiwifruit, and potentially other perennial species.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 199
POS-THU-34
STRESS-INDUCED SYNTHESIS OF
PHOSPHATIDYLINOSITOL 3-PHOSPHATE IN
MYCOBACTERIA
Morita Y.S.4, Yamaryo-Botte Y.2, 3, 1, Miyanagi K.4, Crellin P.K.2, 3,
Coppel R.L.2, 3, Kinoshita T.4 and McConville M.J.1
1
Department of Biochemistry and Molecular Biology, Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne,
Parkville, Victoria 3010, Australia. 2Australian Research Council
Centre of Excellence in Structural and Functional Microbial Genomics,
Monash University, Clayton, Victoria 3800, Australia. 3Department of
Microbiology, Monash University, Clayton, Victoria 3800, Australia.
4
Research Institute for Microbial Diseases, and WPI Immunology Frontier
Research Center, Osaka University, Osaka, 565-0871, Japan.
Phosphoinositides play key roles in regulating membrane dynamics
and intracellular signaling in eukaryotic cells. However, comparable
lipid-based signaling pathways have not been identified in bacteria. In
this study, we show that Mycobacterium smegmatis can synthesize the
phosphoinositide, phosphatidylinositol 3-phosphate (PI3P), making this
organism the first example of a prokaryote that can synthesize PI3P de
novo. This lipid was transiently labeled with [3H]inositol, and its synthesis
was elevated by salt stress but not by exposure to high concentrations of
non-ionic solutes. Sensitivity of the purified lipid to alkaline phosphatase,
head group analysis by high-pressure liquid chromatography, and mass
spectrometry demonstrated that it had the structure; 1,2-[tuberculostearoyl,
octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis
of PI3P in a cell-free system was stimulated by the synthesis of CDP-
diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis,
suggesting that efficient cell-free PI3P synthesis is dependent on de
novo PI synthesis. In vitro experiments further indicated that the rapid
turnover of this lipid was mediated, at least in part, by a vanadate-sensitive
phosphatase. The transient synthesis in response to environmental stimuli
suggests that mycobacterial PI3P may be a second messenger, and we
are currently attempting to identify effector molecules that may be involved
in the signaling cascade.
POS-THU-36
A TAIL OF STAT3 SPLICEFORM DIFFERENTIAL
REGULATION
Ng I.H.W.1, 2 , Ng D.C.H.1, Jans D.A.2 and Bogoyevitch M.A.1
1
Department of Biochemistry and Molecular Biology, Bio21 Institute,
University of Melbourne, VIC, Australia. 2Department of Biochemistry
and Molecular Biology, Monash University, VIC, Australia.
STAT3 (Signal Transducer and Activator of Transcription 3) is a
transcription factor activated by a range of extracellular signals and
regulates important biological processes. Two STAT3 spliceforms,
STAT3α and STAT3β, have been identified with spliceform-specific
functions reported. Interestingly, the only structural difference noted in
the spliceforms is a unique 7 amino acid C-terminal tail in STAT3β that
replaces the 55 amino acid tail of STAT3α; this tail is a unique feature
of STAT3β when compared with other truncated STAT isoforms. We
have now demonstrated that increased expression of STAT3β leads to
a prolonged STAT3α Y705 phosphorylation in both Cos1 and HEK293
cells. This correlated with the prolonged nuclear retention of STAT3α and
suggested that STAT3β levels could influence STAT3α phosphorylation.
Since the heterodimerisation of STAT3α and STAT3β could complicate
the analysis of spliceform-specific regulations and functions, we
have developed a STAT3-inducible expression system in a STAT3 -/-
background. We have further studied the differential regulation of the
individual STAT3 spliceforms in this system and revealed the differential
phosphorylation kinetics of STAT3α and STAT3β. Thus, in the absence
of STAT3β, STAT3α Y705 phosphorylation levels appeared lower and
more transient whereas STAT3β Y705 phosphorylation levels were
strong and sustained. Furthermore, these changes in phosphorylation
correlated with the differential nuclear translocation for the individual
STAT3 spliceforms. Ultimately, this STAT3-inducible expression system
will allow the differential regulation of STAT3α and STAT3β to be
studied in greater detail with future studies such as gene regulation and
protein partner interaction studies allowing greater understanding of the
regulation and actions of these STAT3 spliceforms.
POSTERS WEDNESDAY & THURSDAY
POS-WED-37
IDENTIFICATION OF A NOVEL C-JUN N-TERMINAL
KINASE INHIBITORY PEPTIDE
Ngoei K.R.1, Catimel B.2, Lio S.1, Cheng H.C.1, Ng D.C.H.1 and
Bogoyevitch M.A.1
1
Dept. of Biochemistry and Molceular Biology, Bio21 Institute,
University of Melbourne. 2Ludwig Institute for Cancer Research,
University of Melbourne.
An improved understanding of the roles of protein kinases in cellular
signaling and disease progression has driven significant developments
in the area of protein kinase inhibitor discovery. Whilst the issues of
selectivity and specificity have become increasingly recognized as
major hurdles to overcome undesirable off-target effects of inhibitors
targeting the kinase ATP-binding site, peptide inhibitors that target the
kinase protein substrate-binding site may offer greater specificity. Here,
we report a novel c-Jun N-terminal Kinase (JNK) inhibitory peptide,
PYC71N, that inhibits JNK activity in vitro towards a range of recombinant
protein substrates including the transcription factors c-Jun, ATF2,
Elk1 and the microtubule regulatory protein DCX. Moreover, alanine
scanning replacement studies revealed the importance of two residues
within PYC71N (F9 and F11) for JNK inhibition. Intriguingly, the kinetics
data suggested that inhibition was mediated by a substrate-inhibitor
complex. Using biosensor analysis, PYC71N was shown to interact with
both non-phosphorylated JNK1 (inactive) and the substrate, cJun but
did not recognize active JNK1. In contrast, a previously characterized
JNK inhibitory peptide, TIJIP, showed stronger interaction with active
JNK1. Our results define novel properties of the PYC71N peptide,
highlight its value as a research tool to study JNK signaling, and provide
an alternative for targeting JNK for possible therapeutic benefit.
POS-WED-39
CYTOKINE RECEPTORS TURNED ON: A GENERAL
MECHANISM OF ACTIVATION
Poger D.1 and Mark A.E.1, 2
1
School of Chemistry and Molecular Biosciences, The University of
Queensland, Brisbane QLD 4067 Australia. 2Institute for Molecular
Bioscience, The University of Queensland, Brisbane QLD 4067
Australia.
Despite the structures of various cytokine receptors being known,
the mechanism of activation, in particular how the binding of ligand
mechanically transmits a signal through the plasma membrane, is
uncertain. Atomistic molecular dynamics simulations have been used to
investigate the conformational changes within the extracellular domain
(ECD) of the growth hormone receptor (GHR), the prolactin receptor
(PRLR) and the epidermal growth factor receptor (EGFR/Her1/ErbB1)
triggered by the binding of ligand (growth hormone (GH), prolactin
(PRL) and epidermal growth factor (EGF) and transforming growth
factor α (TGFα), respectively), thereby shedding light on the molecular
details on the mechanism of activation in these cases. For all three
receptors, it is shown that the removal of the cytokine from the ligand-
bound homodimeric receptor complexes results in a rotation of the two
subunits relative to each other. A relative rigid-body rotation of 45° and
25° was found in the GHR and PRLR dimers, respectively. A relative
twist by about 30° also occurred within the EGFR dimer between
the two domains I, associated with the closure of the ligand-binding
pocket between domains I and III in each ECD. In each case, control
simulations showed that the GHR, PRL and EGFR apo dimers and the
relative orientation of their monomers after the rotation were stable.
The magnitude and direction of the motion observed in the simulations
of GHR are in close agreement with experiment. The similarity of the
motions in all three cases and in multiple simulations suggests that the
relative rotation of the ECDs could represent a general mechanism of
activation of cytokine receptors.
Page 200 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-38
THE OXYGEN-SENSING ASPARAGINYL
HYDROXYLASE FACTOR INHIBITING HIF-1A (FIH) IS
AN ESSENTIAL REGULATOR OF METABOLISM
Peet D.J.1, Linke S.1, Zhang N.2, Chicher J.3, Gorman J.J.3, Poelinger L.4
and Johnson R.S.2
1
School of Molecular and Biomedical Science, University of Adelaide,
Adelaide, SA 5005, Australia. 2Molecular Biology Section, Division of
Biological Sciences, University of California, San Diego, La Jolla, CA
92093, USA. 3Protein Discovery Centre, Queensland Institute of Medical
Research, PO Royal Brisbane Hospital, QLD 4029, Australia. 4Karolinska
Institute, Stockholm S-17177, Sweden.
Oxygen sensing is essential in all metazoans to enable the control of
oxygen homeostasis. The major genomic response to hypoxia is mediated
by the hypoxic inducible transcription factors (HIFs), and is regulated
by the oxygen-dependent HIF prolyl and asparaginyl hydroxylases.
These hydroxylases are members of the 2-oxoglutarate dependent
dioxygenase family and they directly regulate activity of the HIFs by
oxygen-dependent protein hydroxylation, thus acting as cellular oxygen
sensors. The ubiquitously expressed HIF asparaginyl hydroxylase, known
as Factor Inhibiting HIF (FIH), is able to regulate the transcriptional activity
of the HIF-α proteins through oxygen-dependent hydroxylation of the
C-terminal transactivation domain. This modification directly influences
the recruitment of the CBP/p300 coactivators, thus modulating the activy
of this transactivation domain. Subsequently FIH has also been shown
to hydroxylate numerous proteins containing ankyrin repeat domains,
including IkBα and Notch. These oxygen-dependent modifications are
confined to specific, conserved asparaginyl residues, although their function
in these ankyrin repeat proteins remains unclear. The recent generation
and analysis of FIH-deficient mice has shed light on the importance of FIH,
specifically its role in regulating key aspects of metabolism. FIH deficient
mice have elevated metabolism, including respiration, cardiovascular
output, and energy metabolism; they consume more food, exercise less
and yet are leaner than wild type mice with improved glucose and lipid
homeostasis. The mutant mice are also resistant to weight gain on a high
fat diet and hepatic steatosis, and remain insulin hypersensitive, underlying
the key role and therapeutic potential of FIH in regulating metabolism.
POS-THU-40
RNAi SCREENING FOR NOVEL KINASES AND
PHOSPHATASES THAT REGULATE THE SALVADOR-
WARTS-HIPPO PATHWAY IN GROWTH CONTROL
Poon C., Cuddy C. and Harvey K.
Cell Growth & Proliferation, Peter MacCallum Cancer Centre.
The Salvador-Warts-Hippo (SWH) pathway controls tissue growth and
organ size by limiting the function of the transcriptional coactivator Yorkie
(Yki). Phosphorylation is critical to regulation of SWH activity: for example,
both the assembly and activity of the core complex, comprising protein
kinases Hippo and Warts (Wts) and their respective binding partners
Salvador and Mats, is promoted by Hpo phosphorylation. Critically, Wts-
mediated phosphorylation restricts Yki to the cytosol, thus limiting the
transcriptional output of SWH signalling. Recently, phosphoproteome
analysis of Drosophila embryos1 indicates previously uncharacterised
phosphorylation sites on Hpo pathway proteins, suggesting that
additional phosphorylation events may influence SWH signalling.
Furthermore, we have a limited understanding of which kinases and/or
phosphatases target the novel and, indeed, established phosphorylation
events described above. To identify kinases and phosphatases that
regulate the SWH pathway, we have carried out both gain- and loss- of
SWH function RNAi screens in Drosophila. In our pilot screens, we have
identified candidates that are known growth regulators, such as Akt, as
well as candidates that are yet to be described in the context of growth
control. We will validate and characterise these candidates to gain
insight into the role of phosphorylation in fine tuning SWH signalling. 1
Zhai et al, Journal of Proteome Research, 2008.
POSTERS WEDNESDAY & THURSDAY
POS-WED-41
NUCLING, A NOVEL APOPTOSIS-ASSOCIATING
PROTEIN, REGULATES NF- κB PATHWAY
Sakai T., Liu L., Tran N.H., Kim S.M. and Fukui K.
The Institute for Enzyme Research, The University of Tokushima.
Nucling is a novel protein isolated from murine embryonal carcinoma cells
with an upregulated expression during cardiac muscle differentiation.
Nucling is induced by proapoptotic stimuli and important for the following
induction of apoptosis. We further demonstrated that Nucling regulated
apoptosome complex composed of apoptotic protease activating factor
1 (Apaf-1), caspase-9 and cytochrome c following cytotoxic stimuli. We
generated Nucling-KO mice to reveal physiological functions of Nucling.
The KO mice were resistant to the damaging effects of the neurotoxin,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Upregulation of
apoptosome was attenuated in Nucling-KO mice treated with MPTP,
indicating that mitochondrial apoptosis pathway plays an important role
in the neurotoxicity of MPTP. We also reported an alternative pathway
for Nucling to regulate apoptosis, in which Nucling acts as an inhibitor
of the anti-apoptotic molecule, galectin-3. On the other hand, Nucling
is characterized as a regulator of nuclear factor-kappa B (NF-κB)
operated by its cytoplasmic retention through the physical interaction
with Nucling. We recently revealed that Nucling is important for the
regulation of NF-κB signals in liver. Defect of NF-κB signal led several
liver dysfunctions including hepatitis and cancer in Nucling-KO mice.
Our latest data concerning the regulatory machineries of Nucling in NF-
κB signal pathway will be presented.
POS-WED-43
CELL-CYCLE SENSING OF OXIDATIVE STRESS IN
SACCHAROMYCES CEREVISIAE BY OXIDATION
OF A SPECIFIC CYSTEINE RESIDUE IN THE
TRANSCRIPTION FACTOR SWI6P
Chiu J. , Tactacan C.M. , Lin R.C.Y. , Wouters M.A. and Dawes I.W.
1 1 2 3 1, 2
1
School of Biotechnology and Biomolecular Sciences, University of
New South Wales, Sydney, NSW 2052. 2The Ramaciotti Centre for
Gene Function Analysis, University of New South Wales, Sydney, NSW
2052. 3Structural and Computational Biological Program, Victor Chang
Cardiac Research Institute, Sydney, NSW 2010.
Yeast cells begin to bud and enter S phase when growth conditions
are favourable during G1 phase. When subjected to oxidative stress,
cells arrest at G1 delaying entry into the cell cycle allowing repair of
cellular damage. Hence, oxidative stress sensing is coordinated with
the regulation of cell cycle. We identified a novel function of the cell-
cycle regulator of Saccharomyces cerevisiae , Swi6p, as a redox sensor
through its intrinsic cysteine residue at position 404. Mutation of Cys404
to Ala abolished the ability of the cells to arrest at G1 upon treatment
by lipid hydroperoxide. At the protein level, Cys404 residue of Swi6p
became oxidised when cells were subjected to the oxidant. Furthermore,
microarray analysis revealed that mutation of Cys404 to Ala alleviated
the wild-type suppression of the G1 cyclins Cln1p and Pcl1p hence
promoting morphogenesis and bud emergence in S-phase when
cells were exposed to lipid hydroperoxide. However, genes involved in
DNA replication (ALG14, DUN1, CDC45, POL1) were down-regulated
in a similar manner as observed in wild-type cells indicating that the
mutation did not affect the regulation of DNA replication. In conclusion,
oxidative stress signalling for cell-cycle regulation in yeast occurs
through the oxidation of the G1/S-specific transcription factor Swi6p and
consequently leads to the suppression of G1-cyclins inhibiting entry into
the cell cycle.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 201
POS-THU-42
THE A KINASE ANCHORING PROTEIN AKAP200 CAN
SUPPRESS THE ONCOGENIC PHENOTYPES OF DV-CBL
AND RASV12 IN THE DROSOPHILA EYE
Sannang R., Robertson H. and Hime G.
Department of Anantomy and Cell Biology, The University of
Melbourne, Parkville VIC 3010, Australia.
The fruit fly Drosophila melanogaster has been an important model
organism for many years. Drosophila and humans share many genes,
and like us, they are capable of developing cancer. Two oncogenes
that act similarly in Drosophila and humans are Dv-cbl and rasV12,
and we have previously performed an enhancer/suppressor screen
in the Drosophila eye against them using the Gene Search collection
(1). In this way, a number of genes capable of suppressing Dv-cbl or
rasV12 were identified. One line was able to suppress both Dv-cbl and
rasV12. This line mapped to Akap200, an A kinase anchor protein that
was previously described as having a role in formation of ring canals
in the Drosophila ovary (2). As little is known about Akap200 function
in the eye, we went on to characterise Akap200 and its interactions
with Dv-cbl and rasV12 in eye development. Here we show detailed
analysis of Akap200 in the eye. In adults the rough eye (differentiation
defects) phenotypes, caused by over expression of Dv-cbl or rasV12, are
suppressed by Akap200 expression. We then looked in larval tissue
at the expression of proteins known to be affected by over expression
of Dv-cbl. Defects are suppressed and appear closer to the wild
type configuration when Akap200 is over expressed. This helps us
to determine which biochemical pathways or processes Akap200 is
affecting in order to suppress the Dv-cbl over expression phenotype.
We also found that Akap200 was able to suppress the cooperation of
Dv-cbl and rasV12, and plan to test its ability to suppress other oncogene
combinations. (1) Toba, G., et al., 1999. (2) Jackson, S.M. and C.A.
Berg, 2002.
POS-THU-44
CLUSTERIN INDUCES MATRIX
METALLOPROTEINASE-9 EXPRESSION VIA ERK, JNK,
AND NUCLEAR FACTOR-κB IN RAW 264.7 CELLS
Shim Y.J.1, Jeon H.S.1, Jiang L.H.1, Park I.S.2 and Min B.H.1
1
Department of Pharmacology and BK21 program for Medical Sciences,
College of Medicine, Korea University. 2Department of Anatomy and
BK21 Center for Advanced Medical Education, College of Medicine, Inha
University.
Clusterin (CLU) is a multifunctional protein that has been implicated in
tissue remodeling, metastasis, and inflammation. Since these processes
may be linked to the degradation of extracellular matrix (ECM) and cell
migration, we investigated whether CLU regulates expression of matrix
metalloproteinase-9 (MMP-9). Normally, MMP-9 expression is tightly
controlled and low or absent in most tissues. Here, we show that the
production of MMP-9 protein and mRNA is induced by CLU in murine
macrophage-like Raw 264.7 cells. CLU enhanced MMP-9 expression
in a time- and dose- dependent manner, revealed by RT-PCR, Western
blot and Gelatin zymogram. To explore the intracellular signaling routes
involved in MMP-9 gene transcription, cells were treated with different
inhibitors of major mitogen-activated protein kinase (MAPK) pathways.
CLU-stimulated MMP-9 induction was significantly attenuated through
inhibition of extracellular signal-regulated kinase (ERK1/2) and c-jun-N-
terminal kinase (JNK) by PD98059 and SP600125, respectively, but not
by SB203580 as a p38 kinase inhibitor. Indeed, CLU stimulated a time-
dependent phosphorylation of both ERK1/2 and JNK with a maximal
response within 60 min. Moreover, the CLU-induced MMP-9 gene
expression was also mediated through the translocation of nuclear factor-
κB (NF-κB) p65 into the nucleus and the degradation of IkB-alpha. The
regulation of MMP-9 induction by ERK1/2, JNK, and NF-κB was further
confirmed by luciferase reporter gene assay. MMP-9 promoter activity was
increased by CLU in cells transfected with wild-type mouse MMP-9-Luc,
which was inhibited by PD98059, SP600125. Taken together, these results
suggest that phosphorylation of ERK1/2, JNK, and NF-κB transactivation
is essential for CLU-induced MMP-9 production in Raw 264.7 cells. [This
work was supported by the Korea Science and Engineering Foundation
(KOSEF) grant funded by the Korea government (MEST) (No. 2009-009-
1418)].
POSTERS WEDNESDAY & THURSDAY

POS-WED-45 POS-THU-46
GENOME WIDE FUNCTIONAL GENOMICS
Cancelled
Thomas D., Gould K. and Simpson K.J.
Peter MacCallum Cancer Centre.

The Functional Genomics Group at the Peter MacCallum Cancer Centre

is open to the Australian medical research community for the purpose
of providing access to both small interfering (siRNA) and lentiviral-
based short hairpin microRNAi (shRNAmir) gene knockdown screens
for functional genomics studies. The Victorian Centre for Functional
Genomics (VCFG) has purchased a library of ~100,000 lentiviral based
shRNAmir constructs, representing up to 5 constructs per gene from
Open BioSystems. Researchers can access the whole human genome,
or boutique library collections that focus on apoptosis, cell cycle, growth
and proliferation, polarity and cell invasion and the kinome. We have
produced lentiviral pools of 4,608 constructs per pool that permit rapid
and successful screening at the whole genome level. The Australian
Cancer Research Foundation (ACRF) siRNA high throughput screening
facility offers a parallel screening platform using Dharmacon siRNA
technology to systematically knockdown individual genes for the entire
human (~18,500) or mouse (~17,000) genome. Such whole genome
screens are performed in high throughput (96 or 384 well format) using
our fully integrated robotics platform which includes an automated
liquid handling robot, an automated cell dispenser/plate washer, a high
throughput plate reader and a high throughput, high content image
analyser (Cellomics VTi) for cell based microscopy assays. We are
a member of the RNAi Global Initiative, a consortium of International
Institutes that have invested in the whole genome screening technology.
Efficient and cost-effective gene knockdown screens are offered along
with Standard Operating Protocols and assay development advice.

POS-WED-47
VASCULAR TONE REGULATION BY EXTRACELLULAR
UTP THROUGH P2X RECEPTOR
Sugihara M.1, Morita H.2, Matsuda M.1, Kajioka S.3, Ito Y.4, Abe K.2 and
Hirata M.1
1
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental
Science,. 2Special Patient Oral Care Unit of Kyushu University
Hospital,. 3Department of Urology, Graduate School of Medical
Sciences, Kyushu University, Fukuoka,. 4Department of Health
Science, Kumamoto Health Science University, Kumamoto Japan.
P2X receptors are non-selective cation channels gated by extracellular
ATP, and play important roles in various physiological processes.
UTP is released from sympathetic nerve terminals and various cells
surrounding smooth muscle cells in vasculature. It has been generally
considered that UTP regulates vascular tone through selective
activation of G-protein coupled P2Y receptors. However, we here
confirmed that UTP mediates contraction of vascular smooth muscle
through P2X receptor activation. We performed a tension recording with
a rat aortic ring preparation denuded of endothelium. UTP elicited a
biphasic contraction consisting of phasic and tonic components. The
phasic contraction disappeared when removal of extracellular Ca2+ or
by addition of nifedipine or TNP-ATP, whereas the tonic contraction
remained. Next, we examined native P2X receptor function using patch-
clamp method. Application of UTP (≥10μM) induced transient inward
current in arterial smooth muscle cells. The current showed inward-
rectification, which was similar to those evoked by activation of P2X
channel. The UTP-induced current was inhibited by pretreatment with
TNP-ATP, suramin and PPADS. UTP and α,β-methylene ATP (10μM),
a potent P2X receptor agonist were mutually competitive. Using RT-
PCR and Western blot analysis, we have detected high expression of
P2X1 subtype in cerebral and mesenteric arteries and aorta. P2Y2, 4, 6
receptors were also detected by RT-PCR. Taken together, our results
suggest that UTP regulate the arterial tone through dual pathways,
including P2X1-like and P2Y receptors activation.
POS-THU-48
A NOVEL NON-RAD HOMOGENEOUS ASSAY
TO MONITOR POST-TRANSLATIONAL HISTONE
MODIFICATIONS
Sutija M.1, Kreps A.2, Lipari F.2, Arias M.2, Lévesque-Sergerie J.P.2,
Rodriguez-Suarez R.2, Cosentino G.2 and Rouleau N.2
1
PerkinElmer Inc., Melbourne, Australia. 2PerkinElmer Inc., Montreal,
Quebec, Canada, H3J 1R4.
Post-translational histone modifications play a part in a wide array of
cellular processes including regulation of gene transcription, DNA
repair, and mitosis. The enzyme classes that mediate these histone
modifications include histone acetyltransferases (HATs), histone
deacetylases (HDACs), histone methyltrasferases (HMTs), and histone
demethylases. Here we report development of Alpha technology-based
assays (luminescent oxygen channelling immunoassay) to monitor
histone modifications that are mediated by the activity of HATs and
HMTs. To measure acetylation and methylation, full length histone H3
modification at lysine 9 was used as a model. The assays involved
two steps: 1) Acetylation or methylation of the histone H3K9 with P/
CAF or G9a, and 2) detection using the Alpha immunoassay. Using the
optimized buffer conditions, we were able to measure histone acetylation
at K9 using as little as 10 nM of enzyme and 300 nM of substrate. The
acetylation detection assay was also successfully used to look at the
general state of acetylated H3K9 from nuclear extract of HeLa cells
treated with a known HDAC inhibitor. G9a activity was monitored by
measuring the production of H3K9me2. A signal-to-background ratio
over 10 was obtained using as little as 10 nM enzyme. Moreover, the
evaluation of different known HMT inhibitors confirmed the specificity
of the reaction, with rank order of potency in line with those found in
the literature. Since the use of a full length H3 protein substrate may
represent certain advantages over the use an H3-derived peptide, these
novel Alpha assays will represent a powerful tool in vitro screens of
novel modulators of this emerging target class.

Page 202 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010

POSTERS WEDNESDAY & THURSDAY
POS-WED-49
GENOME-WIDE CHIP-SEQ ANALYSIS OF HISTONE
3 LYSINE 27 TRIMETHYLATION DURING SKELETAL
MUSCLE DEVELOPMENT HIGHLIGHTS CHROMOSOME
ORGANISATION AND REGULATION OF GENE
EXPRESSION
Tellam R.L.1, Byrne K.1, McWilliam S.1, Vuocolo T.1, Cockett N.2 and
Gondro C.3
1
CSIRO Livestock Industries. 2University of Utah. 3University of New
England.
Skeletal muscle undergoes a major switch in its gene expression
program and metabolism during the fetal to post-natal developmental
transition. We hypothesized that epigenetic modifications of the
genome underpin coordinated changes in gene expression which
prepare skeletal muscle for movement, support against gravity and
altered metabolism in the post-natal environment. Using Illumina GAII
sequencing we performed genome-wide mapping of Histone H3 lysine
27 trimethylation modifications (H3K27me3 CHiP-SEQ) for ovine
skeletal muscle samples taken at late fetal development (100 days; n=3)
and 12 weeks postpartum (n=3). The sequences were mapped onto
the bovine genome and processed using CisGenome. The H3K27me3
peaks fell into two major categories, local and regional peaks, with
the latter being predominant. H3K27me3 peaks were associated with
genes, transcriptional start sites and CpG islands. Gene-associated
H3K27me3 was negatively correlated with gene expression. There
were strong associations between genes with promoters enriched for
H3K27me3 and several GO terms common to both biological states.
Gene-associated H3K27me3 peaks present in the lamb samples but
absent in the fetal samples were associated with the TGF-βbeta and
WNT signaling pathways. Homeobox genes showed strong regional
enrichments for H3K27me3 in both biological states. H3K27me3 was
substantially enriched on the X chromosome of females but not males,
thereby implicating this epigenetic mark in X chromosome inactivation.
These analyses revealed remarkable modifications of the epigenome
that comment on many aspects of chromosomal organisation and gene
activity in the context of development.
POS-WED-51
VARIOUS FORMS OF THE HUMAN EXOSOME
COMPLEX CONTAIN DIFFERENTIALLY LOCALIZED
CATALYTIC SUBUNITS: HDIS3 AND HDIS3L
Tomecki R.1, Kristiansen M.S.2, Lykke-Andersen S.2, Chlebowski A.1,
Dziembowski A.1 and Jensen T.H.2
1
Institute of Biochemistry and Biophysics, Polish Academy of
Sciences; Pawinskiego 5A, 02-106 Warsaw, Poland. 2Centre for mRNP
Biogenesis and Metabolism, Department of Molecular Biology, Aarhus
University; C. F. Mollers Alle, Bldg. 1130, DK-8000 Aarhus, Denmark.
The eukaryotic RNA exosome is a multisubunit ribonucleolytic
machinery involved in virtually all aspects of RNA turnover, processing
and quality control. It is composed of a nine-subunit catalytically
inert core that serves a structural role and participates in substrate
recognition. Best defined in Saccharomyces cerevisiae, enzymatic
activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p.
The former is a nuclear and cytoplasmic protein, which possesses
both processive exo- and endonuclease activities, whereas the latter
is a distributive exonuclease restricted to the nuclear form of the yeast
complex. Although the exosome core is highly conserved, identity and
arrangements of its catalytic subunits in different vertebrates remain
elusive. Here we demonstrate the association of two different homologs
of Dis3p: hDIS3 and hDIS3L, with the human exosome core. Only hDIS3
is able to complement reduced yeast DIS3 expression. Interestingly,
the two proteins display markedly different intracellular localization
in that hDIS3 is mainly nuclear, while hDIS3L is strictly cytoplasmic.
This compartmental distribution reflects the substrate preferences of
the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases,
however, only hDIS3 has retained endonucleolytic activity. Our data
suggest that three different ribonucleases can serve as catalytic
subunits for the exosome in human cells.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 203
POS-THU-50
DEPLETION OF THE NF-κB TRANSCRIPTION FACTORS
IS REQUIRED FOR CHROMATIN REASSEMBLY AT THE
GM-CSF PROMOTER FOLLOWING GENE ACTIVATION
Poke F.S., Upcher W.C. and Holloway A.F.
Menzies Research Institute, University of Tasmania, 17 Liverpool St,
Hobart, Tasmania 7000.
Aberrant gene expression is a contributing factor to the development and
progression of disease including cancer. The chromatin environment
of a gene plays an important role in regulating gene expression,
whereby it governs access of the protein complexes, transcription
factors and transcriptional machinery which facilitate transcription, to
their binding sites. While the changes in the chromatin environment
that are associated with gene activation are well characterised, little is
known about the factors driving chromatin reassembly following gene
transcription. We have examined chromatin reassembly at the promoter
of a cytokine gene, granulocyte/macrophage colony-stimulating factor
(GM-CSF) following gene activation. GM-CSF is an important gene
involved in hematopoiesis, which becomes dysregulated in acute
myeloid leukaemias. Following activation of the GM-CSF gene in
EL-4 T cells, transcript levels decline rapidly and this occurs prior to
the progressive reassembly of the promoter nucleosome. Nucleosome
reassembly occurs independently of DNA replication and cell division,
and requires protein synthesis. Furthermore we show that nucleosome
reassembly is highly dependent on the depletion of the activating NF-κB
transcription factors. These transcription factors are often constitutively
expressed in leukaemias, and these results suggest they may incorrectly
maintain gene transcription by preventing restoration of the chromatin
environment following gene activation. This highlights the importance of
knowing how genes become “switched off” for understanding aberrant,
constitutive gene expression in cancer.
POS-THU-52
CHARACTERIZATION OF NOVEL PKD INHIBITORS
USING BIOCHEMICAL ASSAYS AND CELLULAR
STUDIES
Vantus T.1, Varga A.1, Szokol B.2, Nemeth G.2, Orfi L.2, Szantai-Kis
C.2, Horvath Z.2, Varga Z.2 and Keri G.1, 2
1
Pathobiochemistry Research Group, Hungarian Academy of
Sciences, Semmelweis University, Budapest, Hungary. 2Vichem
Chemie Research Ltd., Budapest, Hungary.
Protein kinase D (PKD) family is a novel group of serine/threonine
kinases. PKD1 was the firstly identified member of this group and also
the best characterized so far. PKD1 is critical in certain growth factor
signaling pathways and has an important role in inflammation and
angiogenesis. To further explore the signal transduction of PKD, there
is a need for a specific inhibitor. In this study, our aim was to discover
and characterize novel, small molecular PKD1 inhibitors applying
biochemical and cellular assays. Using Vichem’s Nested Chemical
Library, firstly we performed a high throughput screening (HTS) to
test molecules using in vitro kinase assay. The optimized IMAP assay
for PKD was an efficient tool to find hit molecules with different core
structures those were selected for further enzyme kinetic studies. After
enzyme kinetic measurements, we further characterized the signaling
of our, mostly ATP binding site inhibitor hit molecules in various cellular
systems including cancer cell lines and other cell types. We investigated
the effect of these inhibitors on cellular phosphorylation, cell proliferation
and on apoptosis induction, besides we determined certain early
ADME parameters as well. In summary, we optimized a cost efficient,
recombinant enzyme based HTS kinase assay platform and selected
efficient drug-like lead compounds inhibiting PKD1 with nanomolar IC50
values. We further characterized the signaling of these small inhibitory
molecules in several cellular assays. Acknowledgements: This work
has been financially supported by the NKFP-A1-0069/2006 (Inflamin)
and OM-00080/2008 (Nanodrug) grants.
POSTERS WEDNESDAY & THURSDAY
POS-WED-53
GUANINE NUCLEOTIDE EXCHANGE FACTORS FOR
RHO GTPASES IN ENDOTHELIAL CELLS: CRITICAL
TRANSDUCERS OF ANGIOGENIC SIGNALS
Carretero-Ortega J.1, Guzman-Hernandez M.L.1, Hernandez-Garcia
R.1, Vazquez-Macias A.1, Hernandez-Negrete I.1, HellerBrown J.3,
Gutkind J.S.4, Reyes-Cruz G.2 and Vazquez-Prado J.1
1
Department of Pharmacology. CINVESTAV-IPN. Mexico City.
MEXICO. 2Department of Cell Biology. CINVESTAV-IPN. Mexico City.
MEXICO. 3Department of Pharmacology, UCSD. San Diego,USA.
4
Oral & Pharyngeal Cancer Branch, NIDCR, NIH, Bethesda, USA.
G protein coupled receptors activate signaling pathways related to
endothelial cell migration and angiogenesis. We studied the role
of Gbetagamma and P-Rex1, a Rac guanine exchange factor, as
mediators of angiogenic signals. We characterized a differential
inhibitor of Gbetagamma, derived from Phosducin-like protein (PhLP-
M1-G149), able to interfere with the interaction between Gbetagamma
and PI3Kgamma, which inhibits AKT signaling, endothelial cell
migration and tubulogenesis. In addition, we tested the hypothesis that
P-Rex1 is required for the angiogenic response stimulated by SDF1 and
characterized the role of P-Rex1-interacting proteins in cell migration. We
found that P-Rex1 is an effector of the mammalian target of Rapamycin,
linking this kinase to Rac activation and cell migration, and demonstrated
a critical participation of P-Rex1 in the migration and in vitro angiogenic
response of endothelial cells stimulated with SDF-1/CXCL12. Together,
our data indicate an important role of the Gbetagamma/PI3Kgamma/P-
Rex1 signaling pathway in angiogenic responses.
POS-WED-55
NON-CANONICAL GONADOTROPIN SIGNALLING IN
OVARIAN CANCER CELL MIGRATION
Zhang H.1, 2 , Bolitho C.1, Marsh D.J.1 and Baxter R.C.1
1
Hormones and Cancer Group, Kolling Institute of Medical Research,
University of Sydney, Royal North Shore Hospital, Sydney NSW
2065 Australia. 2School of Medicine, Shanghai Jiao Tong University,
Shanghai, China.
The gonadotropin hypothesis proposes that elevated levels of follicle
stimulating hormone (FSH) and luteinising hormone (LH) seen in
postmenopausal women increase the risk of epithelial ovarian cancer
(EOC). We have reported that in EOC, gonadotropins signal through
a non-canonical, cyclic AMP-independent pathway that is Ca2+-
dependent but involves the Ca2+-independent protein kinase C isoform
PKCδ (Mertens-Walker et al. Endocr-Relat Cancer 17:335-49, 2010).
We now show that the sphingolipid sphingosine 1-phosphate (S1P)
is a key intermediate in this pathway, with a role in stimulating EOC
cell migration via activation of extracellular signal-regulated kinase
ERK1/2. Monolayer scratch-wounding and migration across transwell
membranes were employed to assess S1P involvement in gonadotropin-
induced migration of the human EOC cell lines OV207 (clear-cell) and
OVCAR-3 (serous). Cells were treated for 1 h with sphingosine kinase
(SphK) inhibitor to inhibit S1P generation (10 µM 2-(p-hydroxyanilino)-4-
(p-chlorophenyl) thiazole or 5 µM N,N-dimethylsphingosine), or 1 µg/ml
S1P blocking antibody (Dr R. Sabbadini, LPath, Inc.), then migration was
stimulated by adding 10 nM FSH or LH. Cell lysates from OV207 and
OVCAR-3 were immunoblotted for ERK1/2 activation following 10 min
treatment with either 10 nM FSH or LH, with or without inhibitors. In both
cell lines, LH and FSH stimulated ERK1/2 phosphorylation, this effect
being blocked by SphK inhibition. Gonadotropin-induced migration in
both cell lines was also inhibited by SphK inhibition or S1P blockade,
with little or no effect on basal migration. These data further delineate
the pathway of gonadotropin action in ovarian cancer, and implicate S1P
signalling in gonadotropin-dependent migration of EOC cells.
Page 204 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-54
THE ROLE OF KRüPPEL-LIKE FACTOR 3
IN B-LYMPHOCYTE DEVELOPMENT AND
TUMORIGENESIS
Vu T.1, Funnell A.1, Gatto D.2, Brink R.2, Pearson R.1, 3 and Crossley M.1, 3
1
School of Molecular Biosciences, University of Sydney,
DARLINGTON, SYDNEY NSW 2008, AUSTRALIA. 2B Cell
Immunology Group, Immunology Program, Garvan Institute of Medical
Research, DARLINGHURST, SYDNEY NSW 2010, AUSTRALIA.
3
School of Biotechnology and Biomolecular Sciences, University of
New South Wales, SYDNEY, NSW, 2052, AUSTRALIA.
Krüppel-like factor 3 (Klf3) is a member of the Krüppel-like factor family
of transcription factors. Klf3 is a transcriptional repressor that is highly
expressed in haematopoietic tissue. In addition, the Klf3 locus is a
common site of retroviral integration in murine B cell cancers [1] and
Klf3 knockout mice appears to be immuno-compromised and show
a reduced vitality. The knockout mice show a significant reduction in
percentage and absolute numbers of marginal zone and B-1a peritoneal
B cell populations. Furthermore, staining of knockout spleen sections
shows sparse distributions of follicles and thin marginal zones.
Nevertheless, the Klf3 null bone marrow has an approximate 3-fold
increase in the percentage of mature cells, accompanied by a significant
reduction in the percentage of late pre-B and immature B-cells. Given
that deregulation of Klf3 expression can lead to B-cell cancer and that
B-cell development is impaired in the knockout mice, we suggest that
Klf3 plays an important role in normal B-cell development. Microarray
analysis comparing gene expression in splenic B cells purified from wild
type and Klf3 knockout mice has revealed potential Klf3 target genes
with roles in B cell proliferation, differentiation and apoptosis. We are
currently validating the biological significance of these putative Klf3
target genes in B cell development and tumorigenesis.
POS-THU-56
GTF2IRD1, A WILLIAMS SYNDROME-RELATED
PROTEIN, IS A NOVEL SUMO SUBSTRATE
Widagdo J.1, Palmer S.J.1, Taylor K.1, Bontempo S.2 and Hardeman E.C.1
1
School of Medical Sciences, Department of Anatomy, University of New
South Wales, NSW, Australia. 2Department of Pathology, University of
Sydney, NSW, Australia.
Williams-Beuren syndrome (WBS) is a neurological disorder that
results from a hemizygous microdeletion within chromosome 7q11.23
involving 28 genes. Its features typically involve characteristic physical
abnormalities and a set of cognitive and behavioural features, known as
the Williams syndrome cognitive profile (WSCP). Studies of patients with
smaller deletions have implicated GTF2IRD1 and its evolutionary-related
homolog GTF2I in the main aspects of the WSCP. We generated a Gtf2ird1
knockout/LacZ knock-in mouse line to map its expression in the brain
and to examine the consequences of gene inactivation. These mice show
developmental abnormalities and cognitive impairment, reminiscent of the
WSCP. However, the cellular function of GTF2IRD1 still remains elusive.
Here we present the evidence that GTF2IRD1 is subjected to the post-
translational modification, sumoylation. Sumoylation involves covalent
conjugation of small ubiquitin-like modifier (SUMO) protein to a lysine
residue in the target protein, via the sequential action of SUMO-specific
E1, E2, and E3 enzymes. Overexpression of GTF2IRD1 and HA-SUMO in
COS-7 cells leads to a covalent attachment of SUMO to GTF2IRD1, shown
through western blotting and co-immunoprecipitation analyses. Through
mutagenesis experiments, we have identified the lysine residue, contained
within an evolutionary conserved motif in GTF2IRD1 that is targeted by
sumoylation. We have also confirmed the interaction of GTF2IRD1 with
the SUMO E2 conjugating enzyme UBC9 and E3 ligase PIASx through a
yeast-two-hybrid system. We are in the process of investigating the function
of GTF2IRD1 sumoylation which might affect its: subcellular localisation,
protein stability, transcription factor activity and/or its interaction with other
proteins. Whilst our investigation at the transcriptional level has already
shown that GTF2IRD1 negatively autoregulates itself, sumoylation presents
an additional mechanism. The regulations at different levels suggest that the
activity of GTF2IRD1 is finely controlled and therefore, the high likelihood of
dosage sensitivity would play a major contribution to the features of WBS.
POSTERS WEDNESDAY & THURSDAY
POS-WED-57
INVESTIGATING MECHANISMS OF POST-
TRANSCRIPTIONAL GENE REGULATION IN
ZEBRAFISH GERM CELLS
Wiszniak S.E. and Jensen K.B.
University of Adelaide, North Terrace, Adelaide, SA 5005.
Post-transcriptional gene regulation is essential in development,
especially during development of the primordial germ cells (PGCs).
In zebrafish, PGCs are specified by the inheritance of cytoplasmic
determinants, termed the germ plasm, which contains maternal
mRNAs, such as nanos and vasa. The restricted localisation of these
mRNAs to the germ plasm and subsequent PGCs is due to cis-acting
elements present in the 3’UTR. Nanos mRNA is specifically degraded
by miR-430 in the soma, while in the PGCs the RNA-binding protein
Dnd protects nanos from miR-430 mediated degradation. The Hu family
of RNA-binding proteins consists of five members in zebrafish. HuA and
HuG are ubiquitously expressed, whereas HuB, HuC and HuD show
neuron-specific expression in the adult. Interestingly, HuB also has an
independent role during early development. HuB mRNA is maternally
deposited in the embryo, and shows PGC-specific expression. A synthetic
transcript consisting of the mCherry open reading frame fused to the
HuB 3’UTR shows mCherry protein expression restricted to the PGCs
at 24 hpf. This result suggests sequence elements in the HuB 3’UTR can
restrict protein expression to the PGCs. Deletion analysis has narrowed
down the PGC-specific element to a 150nt segment of the 3’UTR, and
further mutational analysis has identified distinct regions responsible for
somatic degradation of the message, and for PGC-specific stabilisation.
There is no miR-430 site present in the HuB 3’UTR, implying a novel
mechanism is responsible for the post-transcriptional regulation of HuB
mRNA in the zebrafish embryo. Several approaches are being taken to
identify candidate RNA-binding proteins that are able to stabilise HuB
mRNA in the PGCs, including development of an RNA-tethering assay
and also RNA pull-downs combined with mass spectrometry.
POS-WED-59
CHARACTERISATION OF THE ARABIDOPSIS
DEHYDRIN, XERO2
Wright E.J. and Parish R.W.
Department of Botany, La Trobe University, Bundoora, Vic. 3086,
Australia.
Crop damage caused by frost events early in the growing season is a
major issue for Australian agriculture. One genetic approach towards
improving the frost tolerance of agronomic crops involves identifying
proteins that protect plant tissues during periods of low temperature. The
expression of these proteins can then be altered in genetically modified
varieties so that protective proteins are more active or concentrated
in plant tissues that require greater cold protection, such as sensitive
floral tissues. The Arabidopsis gene, Xero2, is a candidate gene for this
genetic approach towards improved frost tolerance. The Xero2 gene is
expressed in floral and vascular tissues and is strongly upregulated in
leaf tissues in response to cold and abscisic acid exposure. The XERO2
protein also has elements similar to other known dehydrative response
proteins. The strong expression of Xero2 in Arabidopsis florets, even
at control temperatures, suggests that XERO2 could play a role in the
protection of floral tissues from low temperature damage. Transgenic
plants have been generated to investigate the role of the XERO2
protein within the plant, however research is also being undertaken to
understand how cold responsive genes, such as Xero2, are induced and
regulated in the early stages of cold treatment. Cold treated Arabidopsis
suspension cells are being used to decipher the transcriptional changes
that take place within the plant cell after the onset of low temperature and
immediately prior to the induction of Xero2. Transcriptional information
from this study will add to our knowledge of how the strong expression
of this cold responsive gene is regulated in the early stages of cold
exposure.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 205
POS-THU-58
DIFFERENTIAL PATTERNS OF DNA METHYLATION
AND MICRORNAS IN ADPKD KIDNEY TISSUES BY
GENOME-WIDE ANALYSIS
Woo Y.M.1, Chung H.Y.2, Lee M.J.1, Bae J.B.2, Shin S.A.2, Lyu J.M.2,
Yoo K.H.1, Seo M.J.1, Kim Y.J.2 and Park J.H.1
1
Dept. of Biological Science, Sookmyung Women’s University, Seoul,
140-742, Korea. 2Dept. of Biochemistry, Yonsei University, Seoul, 120-
749, Korea.
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common
human genetic disease and characterized by the formation of multiple
fluid-filled cysts, which result in end-stage renal failure. Increased
cell proliferation is one of the key features of the disease. We already
examined the gene expression levels and methylation patterns of
genomic DNA from kidney of normal and ADPKD patients via genome-
wide analysis. As a result, overall genome of ADPKD patients was
hypermethylated compared with normal samples. While the most of
genes hypermethylated in promoter region of ADPKD patients were
correlated to proliferation, apoptosis and calcium ion channel, those
in genebody region were associated with notch signaling pathway.
Especially, genebody of PKD1 which is major gene causing ADPKD
was hypermethylated in ADPKD. To investigate how chromosome block
is prone to hypermethylated in ADPKD patients, we confirmed the gene
expression levels of the factors, which affect DNA methylation, such as
DNMTs (DNA methyltransferases), DNA demethylases, HDACs (histone
deacetylases). In this study, altered expression levels of these genes
may induce an increase of DNA methylation in ADPKD. Interestingly,
upon treatment of DNMT inhibitor, such as 5-aza-2’-dexoycytidine
and zebularine, into the ADPKD primary cells, DNA methylation and
mRNA levels were restored. Furthermore, microRNA (miRNA) DNA
methylation and its expression levels were also differential between
normal and ADPKD kidney tissues. It is suggested that epigenetic
silencing of miRNAs by CpG island hypermethylation might affect on
miRNA expression levels and result in cyst formation in ADPKD.
POS-THU-60
DISCOVERY AND CHARACTERISATION OF C-JUN
N-TERMINAL KINASE BINDING PROTEINS
Yeap Y.Y.C., Ng D.C.H. and Bogoyevitch M.A.
Department of Biochemistry and Molecular Biology, Bio21 Institute,
The University of Melbourne, VIC, Australia.
Protein-protein interactions dictate protein actions and so these
interactions have been increasingly studied to probe protein functions.
We are interested in the formation of intracellular signalling protein
complexes, and have employed the Yeast two-hybrid (Y2H) approach
to discover and characterise proteins that interact with c-Jun N-terminal
Kinase (JNK). JNK, a member of the Mitogen-activated Protein Kinase
(MAPK) proteins, can phosphorylate a variety of nuclear or non-
nuclear substrates and so mediates diverse cellular events including
cell death, differentiation or proliferation. To confirm the fidelity of our
Y2H interaction system, we included JNK1 bait with known interacting
partners (c-Jun and JIP1 prey constructs) as positive controls.
Approximately 30 million clones from a human fetal heart library were
screened with JNK1 as bait. From the positive colonies identified, we
confirmed JNK-specific interaction with 5 prey proteins: SYPL, COXIII,
NEBL, EIF2AK3 and WDR62. Retransformation and quantitative liquid
(CPRG) assay measurements confirmed interaction with JNK1 as well
as showing interaction with JNK2. Based on structural information of
the JNK1-JIP1 interaction, we also engineered and tested JNK single
mutant (R127 or E329A) and JNK double mutants (R127A/E329A) for
their interaction with the prey proteins. Quantitative liquid (CPRG) assay
measurements revealed differing effects of the R127A, E329A and
R127A/E329A changes in both JNK1 and JNK2 on their interactions
with the tested prey proteins. These results suggest different modes
of recognition of the partners by the JNKs and lay the foundation for
further structural and biochemical studies addressing detailed aspects
of these interactions.
POSTERS WEDNESDAY & THURSDAY
POS-WED-61
AN ANALYSIS OF STRESS-STIMULATED STATHMIN
(STMN) PHOSPHORYLATION BY 2D-PAGE
Yip Y.Y., Bogoyevitch M.A. and Ng D.C.H.
Department of Biochemistry and Molecular Biology, Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne, VIC
3010, Australia.
Stathmin (STMN) is a cytoplasmic phospho-protein that has critical
functions in regulating microtubule (MT) dynamics during cell
migration and mitosis. STMN triggers MT disassembly by binding and
sequestering α/β tubulin dimers. STMN activity is negatively regulated
by multi-site phosphorylation on four conserved serine residues (Ser 16,
25, 38 and 63) which renders it unable to bind or sequester free tubulin
subunits. STMN is phosphorylated by a number of kinases in response
to extracellular stimuli such as mitogenic growth factors. In addition to
growth factors, STMN is also phosphorylated in response to cellular
stress (eg. osmotic stress and heat stress). While growth factor-stimulated
STMN phosphorylation is well characterised, STMN phosphorylation
in response to cellular stress and the signaling mechanisms involved
have not been as well studied. Using a combination of 2-dimensional
gel electrophoresis (2D-PAGE) and western blotting with site-specific
phospho-STMN antibodies, we revealed differences in STMN multi-
site phosphorylation in response to hyperosmotic stress compared to
nerve growth factor (NGF) stimulation in PC12 cells. These altered
phosphorylation states allude to separate mechanisms mediating STMN
responses to environmental stressors compared to growth factors. Our
findings also highlight that STMN function in regulating microtubules in
stressed cells may differ when compared to normal growth conditions.
POS-WED-63
EVOLUTIONARY CONSERVATION AND FUNCTION OF
ESCRT-II: A KEY ENDOSOMAL SORTING COMPLEX
Ilievska J., Singh J., Dortenzio R., Annesley S., Fisher P. and Bishop N.
Department of Microbiology, La Trobe University, Bundoora VIC.
The ESCRT (endosomal complex required for transport) machinery is
essential for multivesicular body (MVB) biogenesis and degradation of
endocytosed proteins. ESCRT components also play a crucial role in
down-regulation of receptor signalling, retroviral budding and cell division.
The ESCRT machinery is comprised of four complexes 0, I, II and III in
metazoan and fungal cells. An ATPase, Vps4, plays a role in dissociation
of the ESCRT complexes from membranes, and in fission of intraluminal
vesicles within MVBs. The ESCRT-III interaction with Vps4 predates
the divergence of Archaea and Eukarya and functions in archaeal cell
division. While ESCRT complexes I-III are present in all major eukaryotic
supergroups, some clades have lost genes encoding components of
ESCRT-I and/or ESCRT-II. We present our findings on the evolutionary
history of ESCRT-II and highlight the presence of orthologs in the amoeba,
Dictyostelium discoideum. Dysfunctional ESCRTs are associated with a
number of neurodegenerative and lysosomal diseases, and functional
studies on the ESCRT-II in model organism D. discoideum are being used
to determine the contribution of ESCRT-II to these diseases.
Page 206 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-62
MARSUPIALS, MYTHS AND MASTER REGULATORS
Young L.J.1, Flenady S.1 and Belov K.2
1
Centre for Environmental Management, CQUniversity Australia.
2
Faculty of Veterinary Sciences, University of Sydney.
Members of the four-helical type I cytokine family are essential for
the regulation of adaptive immunity in mammals. Interleukin-2 (IL-2)
supports the growth, survival and differentiation of T cells. Interleukin-4
upregulates expression of antigen recognition and receptor molecules
such as Major Histocompatibility Class II and IgM and is essential for
the immunoglobulin class switch from IgG to IgE. Both ligands bind to
a common receptor, the IL-2 gamma chain (or common gamma chain
receptor), which is essential for effective downstream signalling. Despite
repeated efforts over more than a decade, the expression of neither IL-2
nor IL-4 has been identified in any marsupial species. Indeed, literature
reports have documented these difficulties and speculated about possible
implications for the complexity of immune responses in marsupials as a
consequence of the apparent lack of expression of these cytokines. Our
view, after working with cell culture systems for the tammar wallaby to
optimise in vitro T cell responses, was that the complexity of the adaptive
immune response was most likely similar to other mammals and that
reports of aberrant cell-mediated and humoral immune responses in
marsupials were attributable to other, unidentified causalities. Renewing
efforts to detect the expression of these genes, we combined information
about the conserved regions in the structure of 4-alpha helical cytokines
with nucleotide sequence data for phylogenetically distant species and
the model marsupial, Monodelphis domestica and used RNA extracted
from T cells in active blastogenesis (from our previous studies) as the
template for homologous cloning techniques. We report the identification
and expression of two master immune regulators in marsupials (IL-2 and
IL-4), along with their common receptor (IL-2Rgamma) and discuss the
implications for marsupial immunity.
POS-THU-64
MEMBRANE MICRO-DOMAIN ORGANIZATION OF
CAVEOLIN-1 MODULATES LIPID RAFT PROTEINS
Inder K.L.1, Loo D.1, Zheng Y.Z.1, 2, Foster L.J.2, Parton R.G.3 and Hill M.M.1
1
Diamantina Institute, University of Queensland and Princess
Alexandra Hospital, Brisbane, Queensland 4102, Australia. 2Centre
for High-Throughput Biology and Department of Biochemistry and
Molecular Biology, 2125 East Mall, University of British Columbia,
Vancouver, BC, Canada, V6T 1Z4. 3Institute for Molecular Bioscience,
University of Queensland, Brisbane, Queensland 4072, Australia.
Caveolin-1 is a cholesterol-binding membrane protein that forms
specialized domains on the plasma membrane termed caveolae. Until
recently, caveolins were thought to be the only proteins needed for
the formation of plasma membrane pits called caveolae. We recently
discovered that polymerase I and transcript release factor (PTRF) is
a necessary coat protein required for caveolae formation. Caveolin-1
and PTRF are proposed to mediate signal transduction through
the formation of caveolae. Several lines of evidence implicate over-
expression of caveolin-1 in progression of advanced prostate cancer,
with the mechanism still unknown. In the metastatic prostate cancer cell
line PC3, caveolin-1 is present on flat plasma membrane due to the lack
of PTRF expression. We hypothesized that non-caveolar caveolin-1
mediates metastatic ability. In agreement, ectopic expression of PTRF
in PC3 cells resulted in caveolae formation, reduced cell migration
and anchorage-independent growth. To further elucidate the caveolar
and non-caveolar functions of caveolin-1 and PTRF we performed
quantitative proteomics analysis using SILAC (stable-isotope labeling
with amino acids in culture) and liquid chromatography-tandem
mass spectrometry (LC-MS/MS) of subcellular fractionated proteins.
Quantitative proteomic analysis revealed a number of signaling proteins
differentially recruited to lipid rafts in PTRF expressing PC3 cells. Thus
the organization of caveolin-1 on the membrane modulates recruitment
of signaling proteins to membrane micro-domains. Altered caveolin
function may contribute to diseases such as prostate cancer.
POSTERS WEDNESDAY & THURSDAY
POS-WED-65
PROTEIN KINASE C CONTROLS APOLIPOPROTEIN E
SECRETION FROM HUMAN MACROPHAGES
Karunakaran D.1, Kockx M.1, Owen D.2, Gaus K.2, Jessup W.1 and
Kritharides L.1, 3
1
Macrophage Biology Group, Centre for Vascular Research, UNSW,
Sydney, Australia. 2Membrane Biology Group, Centre for Vascular
Research, UNSW, Sydney, Australia. 3Dept. of Cardiology, Concord
Repatriation General Hospital, USyd, Sydney, Australia.
Apolipoprotein E (apoE) is a multifunctional protein secreted by
macrophages. It plays an important role in reducing atherosclerosis.
However, precise signalling mechanisms regulating apoE secretion
remain unclear. Recently, we demonstrated protein kinase A (PKA),
protein phosphatase 2B (PP2B) and calcium regulate apoE secretion.
Research suggests PKA interacts with protein kinase C (PKC), and
we therefore investigated the potential role of PKC in apoE secretion
from macrophages. Human monocyte derived macrophages (HMDMs)
treated with pan-PKC inhibitors, Calphostin C (CalpC), Ro-31-8220 and
Bisindolylmaleimide-I resulted in a significant dose-dependent decrease
in apoE secretion. The PKC inhibitory peptide also significantly reduced
apoE secretion, albeit less effectively. Further, Go6976 (inhibitor of
classical PKC isoforms) resulted in ~40% decrease in apoE secretion
from HMDMs. Treatment of HMDMs with Apolipoprotein A-I (stimulator of
apoE secretion) or PMA (PKC activator) increased apoE secretion, which
was reduced with CalpC. Preliminary metabolic labelling studies, where
HMDMs were 35S-L-Met/Cys labelled and chased with or without CalpC,
suggest that CalpC has no effect on apoE synthesis or degradation but
directly inhibits the secretion of apoE. Further, apoE-vesicular movement
observed by live cell imaging of HMDMs transiently transfected with
apoE-GFP, was arrested by CalpC and Ro-31-8220. Quantification of
vesicular speed demonstrated a marked decrease in average speed of
vesicles from 0.42μm/s in non-treated HMDMs to 0.14μm/s or 0.15μm/s
in CalpC- or Ro-31-8220-treated HMDMs respectively. This is the first
report of a role for PKC in mediating basal or stimulated cellular apoE
secretion, and implies current clinical applications of PKC inhibitors in
humans may need to consider unexpected effects on atherosclerosis
and inflammatory processes.
POS-WED-67
EXPRESSION OF CALCIUM-SENSING RECEPTOR
HETERODIMERS AT THE CELL SURFACE USING THE
GABAB SORTING SYSTEM
Khan M.A., Chan R. and Conigrave A.D.
School of Molecular Bioscience, University of Sydney, NSW 2006,
Australia.
The extracellular calcium-sensing receptor (CaR) is a class C GPCR that is
expressed at the cell surface as homodimers where it selectively activates
G-proteins in response to a variety of activators including extracellular
Ca2+(Ca2+o). Mutations in the CaR may modify its trafficking and/or signalling
properties. While the effects of mutations in CaR homodimers have been
well characterised, the effects of mutations in heterodimers have not been
studied due to the presence of multiple dimeric species. The GABAB sorting
sytem has been used to deliver class C GPCR dimers to the cell surface
based on the shielding of an ER retention signal in the GABAB1 C-terminal
by the GABAB2 C-terminal. We therefore decided to investigate whether the
GABAB sorting system could be used to selectively target CaR heterodimers
to the cell surface. The following constructs were generated in pcDNA3.1:
(i) CaR-B1 in which the CaR C-tail (residues 876-1078) was replaced by the
mGABAB1 tail (residues 856-961); (ii) R185Q-B2 in which residues 901-1078
of a dysfunctional CaR mutant (R185Q) were replaced by residues 760-961
of the human GABAB2 tail. HEK 293 cells were then transfected with CaR-B1
or R185Q-B2, individually or together, to characterise receptor responses
to elevated Ca2+o using Fura 2-AM to monitor intracellular Ca2+ mobilisation.
Cells expressing CaR-B1 or R185Q-B2 individually, had reduced maximal
responses (Vmax= 9±1.4, 8±0.6, respectively) when compared with
WtCaR expressing cells (Vmax= 13±0.4) and EC50 values for Ca2+o were
markedly increased for CaR-B1 (18±5 mM) and R185Q-B2 (21±3 mM) when
compared with WtCaR (5±0.3 mM). The reduction in maximal responses
and decreased Ca2+o sensitivity for CaR-B1 indicate receptor internalisation
and for R185Q-B2 indicate impaired function. However, cells co-expressing
the CaR-B1 and R185Q-B2 constructs exhibited a significant recovery
comparable to WtCaR in the maximal response (12±0.7) as well as a recovery
in Ca2+o sensitivity (EC50=14±1.4). The results are consistent with successful
trafficking of CaR-B1 and R185Q-B2 heterodimers to the cell surface. We
conclude that the GABAB sorting system can be used to successfully traffick
CaR heterodimers to the cell surface. This system will permit the study of
specific mutations in heterodimers.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 207
POS-THU-66
DISRUPTION OF EARLY ENDOSOME INTERACTION
WITH HELICOBACTER PYLORI PHAGOSOMES
Keep S.J.1, Borlace G.N.1, 2, Butler R.N.1 and Brooks D.A.1, 2
1
Cell Biology of Diseases Research Group, Sansom Institute for Health
Research, University of South Australia. 2Department of Paediatrics
and Reproductive Medicine, University of Adelaide.
Helicobacter pylori has successfully infected over 50% of the global
human population causing gastritis, ulcers and gastric cancer. Despite
evoking a strong immune response in the host, H. pylori persists,
avoiding being killed by phagocytic cells. H. pylori may avoid degradation
and clearance by these immune effector cells by altering the process
of phagosome maturation. Primary human macrophages infected with
H. pylori for 0, 5, 15, 30 minutes, and 2 hours were investigated by
confocal immune fluorescence microscopy, using markers specific
for H. pylori and early endosomes: Rabex5, Rab5, Rabaptin5, PI(3)P
and EEA1. H. pylori phagosomes acquired Rabex5 immediately after
infection and maintained this association for 15 minutes whereupon
Rab5 became associated with the phagosomes. This recruitment of
Rabex5 and Rab5 to H. pylori phagosomes was consistent with other
models of phagosome maturation. In contrast, H. pylori phagosomes did
not acquire PI(3)P until 30 minutes after infection. Moreover, Rabaptin5
and EEA1, which are downstream effectors of Rabex5 and Rab5,
were recruited to H. pylori phagosomes immediately after infection
and were retained for at least 2 hours, which represented a significant
departure from normal phagosome maturation. These observations
indicate that H. pylori phagosomes may sequester Rabaptin5 and EEA1
independently, interfering with early endosome-phagosome interaction.
Perturbation of the molecular mechanism that results in this disrupted
phagosome maturation could reinstate the efficacy of phagocytic killing.
This has obvious significance for minimising H. pylori pathogenicity by
enhancing the killing and clearance of H. pylori following a host immune
response.
POS-THU-68
THE C-TERMINAL PROPEPTIDE OF A PLANT
DEFENSIN CONFERS CYTOPROTECTIVE AND
SUBCELLULAR TARGETING FUNCTIONS
Lay F.T.1, Heath R.L.2, Barbeta B.L.1, Poon S.2, McGinness B.2,
Connelly A.A.2 and Anderson M.A.1
1
Department of Biochemistry, La Trobe University, Melbourne VIC
3086 Australia. 2School of Botany, The University of Melbourne,
Melbourne VIC 3010 Australia.
Plant defensins are small (45-54 amino acids), basic, cysteine-rich
proteins that are found in most plant species and tissues. They can
be divided into two classes based on the presence or absence of a
C-terminal propeptide (CTPP). The antifungal floral defensin from
Nicotiana alata, NaD1, is a member of Class II. Its CTPP is removed
during maturation of the precursor and the defensin is deposited in the
vacuole. The function of the CTPP is not well understood but roles in
vacuolar targeting and/or detoxification of the mature defensin domain
have been suggested (Lay et al., 2003, Plant Physiol; Lay and Anderson,
2005, Curr Prot Pept Sci). Here we report that NaD1 accumulates in
the vacuoles of transgenic cotton plants transformed with the full-length
coding region and that removal of the CTPP prevents vacuolar deposition
of NaD1. Transgenic cotton plants produced from constructs encoding
NaD1 without the CTPP have an abnormal morphology, suggesting that
NaD1 is phytotoxic if it is not deposited in the vacuole. Furthermore, we
show that the CTPP is sufficient to direct green fluorescent protein to
the plant vacuole in transient plant expression assays.
POSTERS WEDNESDAY & THURSDAY
POS-WED-69
MITOTIC FUNCTION AND LOCALISATION OF IQGAP1
DURING GLIOBLASTOMA CELL DIVISION
Lian A.T.Y., Robinson P.J. and Chircop M.
Children’s Medical Research Institute, The University of Sydney,
Locked Bag 23, Wentworthville, Sydney, NSW 2145, Australia.
Cytokinesis is the final stage of cell division generating two independent
daughter cells. Failure of this process results in enlarged and
multinucleated cells, contributing to the initiation and/or progression of
tumourigenesis. These cells are histopathological hallmarks for many
tumours including glioblastoma multiforme (GBM). Cytokinesis in animal
cells requires membrane ingression followed by abscission. Our recent
unpublished discovery reveals a molecular pathway associated with
the completion of abscission. During cytokinesis, Ca2+ influx activates
the Ca2+- and calmodulin-dependent protein phosphatase, calcineurin
(CaN), dephosphorylating a suite of proteins such as dynamin II (dynII),
triggering abscission. This occurs at a new sub-compartment, the
Flanking Midbody Rings (FMRs), that reside on either side of the midbody
ring at the intracellular bridge between two daughter cells. IQGAP1
binds actin and calmodulin and is essential for cytokinesis in fission
yeast. We demonstrate that IQGAP1 is localised to the cleavage furrow
and the intracellular bridge of SMA-560 murine glioblastoma cells that
are undergoing cytokinesis. This is consistent with its actinomyosin ring
localisation in fission yeast cytokinesis. Western blot analysis shows
that IQGAP1 is upregulated upon mitotic entry and decreased during
cytokinesis. Overexpression of GFP-IQGAP1 leads to an increased
number of multinucleated cells. Our findings suggest that IQGAP1
participates in cytokinesis and its expression must be tightly regulated
to ensure successful completion of this process. IQGAP1 is highly
expressed in glioblastoma tumours and cytokinesis failure contributes
to genomic instability. Thus, our results provide a possible mechanism
for how IQGAP1 overexpression may increase oncogenic potential of
glioblastoma.
POS-WED-71
CHARACTERISATION OF THE ARCHITECTURE IN
MUCOPOLYSACCHARIDOSIS IIIA NEURONS
Parkinson-Lawrence E.J.1, 2 , Hemsley K.2, Khalessi-Rad M.1, Winter
M.1, Hopwood J.J.2 and Brooks D.A.1, 2
1
Sansom Institute for Health Research, University of South Australia,
Adelaide, South Australia. 2Lysosomal Diseases Research Unit, SA
pathology (at the Women’s and Children’s Hospital), North Adelaide,
South Australia.
Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder
which is caused by a deficiency in the activity of sulphamidase; an
enzyme involved in the degradation of heparan sulphate. MPS IIIA is
characterised by progressive neurological dysfunction, but the effect of
storage on neurons is largely unknown. Adult dorsal root ganglia neurons
cultured from MPS IIIA mice demonstrated significant heparan sulphate
storage together with fibrillogranular and zebra body inclusions. Neurite
outgrowth was observed for both MPS IIIA and control neurons, but there
were differences in both neurite length and network formation between
these two groups. In control cells the cytoskeleton protein F-actin was
diffusely organised with focal aggregates in the axoplasm. In MPS
IIIA cells there were was an increase in small F-actin foci indicating
cytoskeletal alterations. In control dorsal root ganglia neurons LAMP-1
staining was mainly observed in the cell body with negligible staining
in the neurites. In contrast, MPS IIIA dorsal root ganglia neurons had
a large number of punctate LAMP-1 positive vesicles in the neurites,
which correlated with the distribution of the F-actin foci. There was also
a significant increase in a high molecular weight form of LAMP-1 in MPS
IIIA compared to control mouse brain tissue. The alterations in the F-actin
cytoskeleton and differential distribution of LAMP-1-positive vesicles,
together with altered LAMP-1 processing supported the concept that
intracellular architecture and intracellular transport is altered in MPS IIIA
neurons. This could have a direct impact on neuronal function and help
explain the development of neuropathology in MPS IIIA.
Page 208 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-70
CHARACTERIZING THE ROLE OF ADAPTOR PROTEIN
1A IN THE BASOLATERAL TARGETING OF KAE1
Netsawang J.1, 2, 3, Ngaojanlar P.1, Sawasdee N.1, Malasit P. 1, 2,
Limjindaporn T.1, 4, Yenchitsomanus P.1 and Cordat E.3
1
Medical Molecular Biology Unit, Office for Research and Development,
12th Floor Adulyadejvigrom Bld., Faculty of Medicine Siriraj Hospital,
Prannok, Bangkok Noi, Bangkok, Thailand 10700. 2Department of
Immunology,11th Floor Adulyadejvigrom Bld., Faculty of Medicine Siriraj
Hospital, Prannok, Bangkok Noi, Bangkok, Thailand 10700. 3Department
of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7,
Canada. 4Department of Anatomy, Faculty of Medicine Siriraj Hospital,
Prannok, Bangkok Noi, Bangkok, Thailand 10700.
Distal renal tubular acidosis (dRTA) is characterized by defective acid
secretion by the α-intercalated cells located in distal nephron. Most of
the reported dominantly inherited forms of the disease revealed the
importance of mutations in the kidney anion exchanger 1 (kAE1) gene.
Previous studies demonstrated the crucial role of C-terminal residues in
basolateral targeting of kAE1. Our preliminary data from yeast-two hybrid
screening displayed the adaptor protein 1A (AP-1A) as one interacting
partner for the C-terminus of kAE1. AP-1A is located in the trans-golgi
network and endosomes and is involved in the cation-independent
mannose-6-phosphate receptor (CI-MPR) trafficking. We hypothesized
that AP-1A is important for kAE1 targeting to the cell surface in kidney
cells. We have generated stably transfected MDCK expressing WT kAE1
and kAE1 mutated in the 11 C-terminal residues (R901X, Y904A, and
Y904A/Y907A) in order to characterize the interaction. Endogenous AP-
1A was co-immunoprecipitated only with WT kAE1, not with the C-terminal
mutants. Far western blots confirmed the interaction, which predominantly
occurred with AP-1A, not with 1B. In addition to kAE1, erythrocyte AE1
(eAE1) also interacts with AP-1A. Immunofluorescence experiments
demonstrated that only intracellular WT kAE1 predominantly co-localized
in a perinuclear region with AP-1A. Moreover, WT kAE1 co-localized with
CI-MPR, another TGN marker that interacts with AP-1A. Pulse-chase
experiments were performed to determine when this interaction occurs.
We finally found that AP-1A was specifically immunoprecipitated with WT
kAE1 after 2 hours chase time. Therefore, we conclude that kAE1 protein
transiently interacts with AP-1A during its biosynthesis.
POS-THU-72
REGULATING SECRETION IN INNATE IMMUNITY:
DROSOPHILA 14-3-3ε’S ROLE IN ANTI-MICROBIAL
PEPTIDE SECRETION
Shandala T.1, 2, 3, Woodcock J.M.2, Ng Y.1, 2, Lopez A.F.2 and Brooks D.A.1
1
Sansom Institute for Health Research, University of South Australia,
Adelaide, SA5001, Australia. 2Division of Human Immunology, Centre
for Cancer Biology, Adelaide, SA5000, Australia. 3School of Molecular
and Biomedical Science, University of Adelaide, Adelaide, SA 5000,
Australia.
The secretion of anti-microbial peptides is recognized as an essential
step in innate immunity, but little is known about the molecular mechanism
controlling the release of these effectors from immune response cells.
Here we report that Drosophila 14-3-3ε mutants exhibited reduced
survival when infected with either Gram+ or Gram- bacteria, indicating
a functional role for 14-3-3ε in innate immunity. In Drosophila 14-3-
3ε mutants, there was an accumulation of the anti-microbial peptide
Drosomycin and Rab11-positive vesicles at the plasma membrane, of
cells from immune response tissues. This phenotype correlated with a
reduced release of Drosomycin to the hemolymph. The accumulation
of Drosomycin and Rab11-positive vesicles in immune cells from
Drosophila 14-3-3ε mutants was similar to that observed in response to
the depletion of the vesicular protein Syx1A. In contrast, RNAi silencing
of endogenous Rab11 resulted in the accumulation of Drosomycin in
the peri-nuclear region, preventing delivery to the cell surface. The
depletion of another vesicular protein Lyst caused a less pronounced
accumulation of vesicles at the plasma membrane, but enlarged
Rab11-positive vesicles within immune cells. The Lyst phenotype was
partially suppressed by the loss of 14-3-3ε, suggesting that 14-3-3ε may
act as a negative regulator of Lyst. In cells from wild-type Drosophila
immune tissue, 14-3-3ε was detected adjacent to Rab11, Hrs and
Syntaxin positive vesicles. We conclude that 14-3-3ε is required for
antimicrobial peptide secretion during an innate immune response and
for the functional interaction of Rab11 positive vesicles with the plasma
membrane.
POSTERS WEDNESDAY & THURSDAY
POS-WED-73
THE TROPOELASTIN R515 RESIDUE: ROLES IN
TROPOELASTIN ASSEMBLY AND STRUCTURE
Yeo G.C.1, Dyksterhuis L.B.2, Mithieux S.M.1 and Weiss A.S.1
1
School of Molecular Bioscience, University of Sydney, NSW 2006,
Australia. 2CSIRO Molecular and Health Technologies, Clayton, VIC
3168, Australia.
Elastic fibres provide structural integrity and confer elasticity and
resilience in tissues such as skin, lungs and blood vessels. The major
component of elastic fibres is elastin, which, in turn, is formed from its
soluble precursor, tropoelastin. The process of assembling tropoelastin
into elastic fibres consists of distinct stages of coacervation, deposition
on microfibrils, and cross-linking into mature elastin. It has been proposed
that tropoelastin may be cleaved after its arginine 515 (R515) residue at
some stage during elastin assembly. Interestingly, this R515 site resides
within a section of domains crucial for coacervation and cross-linking. It
is also proximal to a hinge region thought to contribute significantly to the
tropoelastin structure. To explore the likelihood of tropoelastin cleavage
during elastogenesis and the potential significance of the tropoelastin
R515 site, we produced a protease-resistant R515A tropoelastin mutant
and compared its assembly, cell interaction, and structural properties
with the wild-type and the cleaved species. Both the R515A and the
cleaved tropoelastin isoforms exhibited greater temperature and time
requirements for coacervation, abnormal cross-linking, and decreased
cell attachment relative to the wild-type species. Our results suggest that
tropoelastin is unlikely to be cleaved until after cross-linking has been
initiated, and that a percentage of monomers may remain uncleaved
to allow necessary elastin-cell interactions. Structural analysis of the
R515A species indicates that apart from its proposed role as a cleavage
site in tropoelastin, R515 most likely contributes to the protein structure
as part of a flexible bridge region. Mutation of this residue leads to a
significant distortion of the bridge region and consequent displacement
of the C-terminal region. Clinically, disruptions to the tropoelastin
C-terminus have been associated with elastin disease phenotypes such
as cutis laxa and supravalvular aortic stenosis. Our findings provide
direct evidence of the contributions of the R515 residue to tropoelastin
structure and function.
POS-WED-75
BINDING OF PHOSPHOLIPASE C-RELATED BUT
CATALYTICALLY INACTIVE PROTEIN TO SNARE
PROTEINS
Zhang Z., Takeuchi H., Gao J. and Hirata M.
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental
Science, Kyushu University, Fukuoka, Japan.
PRIP (Phospholipase C-related but catalytically inactive protein) was
first identified as a novel Ins(1,4,5)P3 binding protein. We have recently
found that the secretion of various peptide-hormones from multiple
organs were upregulated in PRIP knock-out mice, indicating that PRIP
negatively regulates the steps of exocytic event common to dense-core
vesicles. To explore the molecular mechanism underlying the inhibitory
role of PRIP in exocytosis, we examined the role of C2 domain of PRIP
(PRIP-C2). Although many of the functional C2 domains reported have
been shown to bind to acidic phospholipids in a Ca2+ dependent manner,
we could detect no trace of lipid binding of PRIP-C2 in a lipid overlay
assay. On the other hand, in PC12 cells, ectopically expressed EGFP-
fused PRIP-C2 was partially co-localized with endogenous SNARE
(soluble N-ethylmaleimid-sensitive factor attachment protein receptors)
complex proteins which serve as the minimum machinery of membrane
fusion. In addition, using pull-down assay and proteo-liposome
floatation assay, we found that the both isolated PRIP-C2 and full-
length PRIP interact with the components of SNARE complex including
SNAP-25 (25-kD synaptosome associated protein) and syntaxin1. The
results suggest that PRIP-C2 is involved in PRIP-mediated inhibition of
regulated exocytosis through the protein-protein interactions: SNARE
binding of PRIP might mediate recruiting PRIP which has an inhibitory
role via other domains to the site of exocytosis or competes with other
protein(s) which has a stimulatory role in exocytosis.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 209
POS-THU-74
LIGHTING UP THE INNER MEMBRANE COMPLEX OF
PLASMODIUM FALCIPARUM
Yeoman J.A.1, 2 , Dearnley M.1, Dixon M.W.A.1, Maier A.G.1, Hanssen
E.3, Baum J.4 and Tilley L.M.1, 2
1
Department of Biochemistry, La Trobe University, VIC, Australia.
2
Centre of Excellence for Coherent X-ray Science, La Trobe University,
VIC, Australia. 3Bio21 Institute, University of Melbourne, VIC, Australia.
4
Walter and Eliza Hall Institute of Medical Research, Melbourne 3050,
VIC, Australia.
The most deadly malaria parasite, Plasmodium falciparum, has a
complex life cycle consisting of asexual and sexual stages within its
host cell, the human erythrocyte. Host cell invasion is powered by an
actin/myosin motor complex (glideosome) that is linked to an inner
membrane complex (IMC) via a membrane anchor, glideosome-
associated protein-50 (GAP50). We have generated P. falciparum
transfectants expressing green fluorescent protein (GFP) chimeras
of PfGAP50 to study the formation of the IMC. Using long term live
cell imaging, we show that PfGAP50-GFP is initially located in the
endoplasmic reticulum and redistributed to a ring-like structure at the
apical end of the nascent merozoite once the parasite commences
division. Structured illumination microscopy reveals the early stage of
the IMC as a double-holed flat ellipsoid that divides to form claw-shaped
apposed structures. We have also found that PfGAP50-GFP interacts
with glideosome proteins in the previously uncharacterised gametocyte
IMC during the sexual part of the life cycle. P. falciparum gametocytes
develop a characteristic elongated banana-like shape as they mature
and our results suggest that the glideosome proteins may have a role in
this cellular modification.
POS-THU-76
PROTEIN ENGINEERING AND AMYLOID β MUTATIONS
Nisbet R., Nuttall S., Caine J., Varghese J., Sankovich S., Bartone N.
and Streltsov V.
CSIRO Molecular and Health Technologies, and Preventative Health
Flagship, 343 Royal Pde, Parkville 3052, AUSTRALIA.
Alzheimer’s disease (AD) is a highly prevalent, irreversible,
neurodegenerative disease characterised by large amyloid plaques
in the brains of affected individuals. The major constituent of amyloid
plaques in AD is the Amyloid β (Aβ) peptide which is generated by
sequential cleavage of the amyloid precursor protein (APP). Emerging
evidence suggests that it is not the insoluble amyloid plaque that is
neurotoxic in AD, but rather the soluble Aβ oligomers. We have utilised
advanced protein engineering to trap regions of Aβ within protein
scaffold domains allowing protein crystallisation. The predominant
oligomeric species is a tightly associated Aβ dimer which we believe
to be the smallest toxic species of Aβ. Several mutations within Aβ
have been shown to reduce the tendency of Aβ to aggregate in vitro.
Conversly, the Japanese familial mutation within Aβ, ΔGlu22, increases
the aggregation of Aβ. We have engineered these specific mutations
within our model system and investigated Aβ oligomerisation. Here we
show that the effect of Aβ mutation on the oligomerisation of Aβ within
the scaffolds are consistent with those previously observed providing
support for our model system.
POSTERS WEDNESDAY & THURSDAY
POS-WED-77
A TALE OF TWO SCAFFOLDS - PROTEIN
ENGINEERING OF THE AMYLOID-β PEPTIDE
Nuttall S.D., Caine J., Varghese J., Nisbet R., Sankovich S., Bartone
N. and Streltsov V.
CSIRO Molecular and Health Technologies, and Preventative Health
Flagship, 343 Royal Pde, Parkville 3052, AUSTRALIA.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder
characterized by the presence of misfolded protein depositions or
amyloid plaques. Plaques consist predominantly of amyloid β-peptide
(Aβ), which is produced by cleavage from the membrane-bound amyloid
precursor protein (APP) via the α/β/γ secretase pathway. However,
current views suggest that soluble Aβ oligomer intermediates, and not
the plaque burden, may be the major drivers of Aβ-mediated neuronal
dysfunction. Here, we utilise protein engineering using protein scaffold
domains to trap and analyse several forms of the Aβ peptide. We
describe a 2.2 Å resolution crystal structure of the P3 fragment, which
represents an unusually folded dimeric form.
POS-WED-79
TRIAZINE DERIVATIVES DECREASE LPS-INDUCED
CELL DEATH BY INHIBITING NF-κB AND COX-2 IN
NEURON-LIKE PC12 CELLS
Ramin M.R. and Khodagholi F.
Neuroscience Res Ctr., Shahid Beheshti University of Medical
Sciences, Iran
Introduction: Triazine derivatives are one group of compounds
possessing a wide array of biological activities. Some derivatives have
been introduced as anti inflammatory, radical scavenger, and β-sheet
breaker agents. Lipopolysaccharide (LPS) is a pro-inflammatory
substance present in the cell wall of Gram-negative bacteria. When
activated by LPS, macrophages produce inflammatory cytokines, which
in turn activate unprimed macrophages and other nearby cells. PC12 cell
is an established cell line that is derived from rat pheochromocytoma.
It has been used extensively as an in vitro model system to study
neuronal cell fate, including survival, proliferation, differentiation, and
apoptosis. Investigations have proved that LPS potentiates the effects
of Nerve Growth Factor (NGF) in inducing neural differentiation of PC12
cells. In this study, we investigated the effect of Triazine derivatives
on LPS induced neuron-like PC12 cell death via inhibiting caspase-3
activation. We further studied their effect on NF-κB and COX-2.
Methods: Cell viability was determined, using conventional MTT assay,
in the presence of triazine derivatives synthesized at Tehran University
of Medical Sciences. Inflammation was induced by LPS. Then western
blot analysis of caspase-3, NF-κB and COX-2 was done to determine
if these drugs inhibit cell death or not. Results: The present study
indicates that neuroinflammation resulting from LPS can be inhibited in
the presence of triazine derivatives. This protection was associated with
a marked reduction of caspase-3 activation. It shows they can inhibit
cell death. Furthermore, it was proved that NF-κB and COX-2 were also
decreased remarkably that shows the anti-inflammatory effect of triazine
derivatives. Conclusion: As inflammatory stress is a critical event in the
pathogenesis of neurodegenerative diseases, having neuroprotective
effects along with anti-inflammatory properties implies the possibility of
using triazine derivatives as a candidate for treating neurodegenerative
diseases like Alzheimer’s Disease (AD).
Page 210 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-78
CALCIUM INDUCES ALPHA-SYNUCLEIN AGGREGATES
IN SOLUTION, ON SURFACES AND IN CULTURED CELLS
Pountney D.L.1, Nath S.2, Goodwin J.1 and Engelborghs Y.2
1
School of Medical Science, Griffith University, Gold Coast, Australia.
2
Biomolecular Dynamics, Katholieke Universiteit Leuven, Leuven,
Belgium.
Parkinson’s and Parkinson’s-plus diseases are associated with
abnormal, aggregated forms of the protein, α-synuclein. We have
investigated the effects of calcium on α-synuclein aggregation in vitro
and in vivo. We treated monomeric α-synuclein with calcium in vitro
and used fluorescence imaging, fluorescence correlation and scanning
electron microscopy to investigate protein aggregation. Our in vitro
data suggests two distinct modes of aggregation: surface-dependent
aggregation and aggregation in solution, both of which are accelerated
by calcium, but at different concentrations. Incubation of monomeric
α-synuclein (24 hrs) at low concentration (10 μM) with calcium resulted in
surface aggregates (1.5±0.7 μm) saturating at a half-maximum calcium
concentration of 80 μM, whilst incubations without calcium showed few
protein aggregates. In the presence of calcium, plaques (0.5-1 μm) of
α-synuclein aggregates comprising 10-20 nm globular particles were
observed by scanning electron microscopy. Incubation of α-synuclein
at high concentration (75 μM; 6 hrs) resulted in soluble oligomeric
aggregates detected by fluorescence correlation in a calcium dependent
process, saturating at a half maximum calcium concentration of 180 μM.
In cell culture experiments, we used thapsigargin or ionophore A23187 to
induce transient increases of intracellular free calcium in human 1321N1
cells expressing an α-synuclein-GFP construct and observed calcium
flux and α-synuclein aggregation by fluorescence microscopy. The in
vivo data shows that a transient increase in intracellular free calcium
significantly increased the proportion of cells bearing cytoplasmic
α-synuclein aggregates 12 hrs post-treatment (P, 0.01). Our data
indicates that calcium accelerates α-synuclein aggregation in vitro and
in vivo and suggests that surface adsorption may play an important role
in the calcium-dependent aggregation mechanism.
POS-THU-80
AN INVESTIGATION OF THE FACTORS THAT
REGULATE MUSCARINIC RECEPTOR EXPRESSION IN
SCHIZOPHRENIA
Seo M.S.1, 2 , Scarr E.2, 3 and Dean B.1, 2
1
Department of Psychiatry, The University of Melbourne Parkville,
Victoria, Australia. 2The Rebecca L. Cooper Research Laboratories,
The Mental Health Research Institute, Parkville, Victoria, Australia.
3
Centre for Neuroscience, The University of Melbourne Parkville,
Victoria, Australia.
Muscarinic receptors (CHRM) belong to the superfamily of G-protein
coupled receptors and are crucial for normal functioning of the central
nervous system. We previously reported decreased levels of CHRM1
but not Sp1 in BA6 from subjects with schizophrenia. We have since
identified a subgroup of subjects with schizophrenia, who have a 75%
decrease in their cortical CHRM1 levels. This subgroup of subjects is
termed the muscarinic receptor deficiency schizophrenia (MRDS).
We have extended our previous study by measuring the density of
CHRMs 1 and 3 using radioligand binding with autoradiography in
BA6 in 20 MRDS subjects, 18 Non-MRDS subjects and 20 control
subjects. Levels of Sp1 and Sp3 were also measured in these subjects
using Western blotting. The density of [3H]pirenzepine binding was
decreased in both non-MRDS (p<0.05) and MRDS (p<0.001) compared
to controls. [3H]4-DAMP binding density was also decreased in MRDS
(p<0.001) compared to control subjects. However, Sp1 and Sp3 levels
were not altered in either group of subjects with schizophrenia. This
study suggests the decreased levels of CHRMs in MRDS extend
across the CHRM family, identifying further biochemical differences
between MRDS and non-MRDS subjects. However this decrease in
CHRMs does not appear to simply be regulated by local levels of Sp1
and Sp3, suggesting that other factors are involved in mediating low
cortical CHRM expression. Identifying these factors could be seminal
in the development of therapeutic agents to treat the cognitive deficits
associated with schizophrenia.
POSTERS WEDNESDAY & THURSDAY
POS-WED-81
THREE SITES PHOSPHORYLATED BY PROTEIN
KINASE A IN HUMAN TAU PROTEIN AFFECT ITS
INTERACTION WITH 14-3-3ZETA
Sluchanko N.N., Seit-Nebi A.S. and Gusev N.B.
Department of Biochemistry, School of Biology, Moscow State
University, Moscow, Russian Federation.
Neuronal specific tau protein belongs to the group of microtubule-
associated proteins and regulates formation, dynamics and functioning
of microtubules. Point mutations, limited proteolysis, oxidation of SH
groups and hyperphosphorylation promote aggregation of tau leading
to accumulation of paired-helical filaments, a hallmark of many neuronal
diseases, the so-called tauopathies and among them Alzheimer
disease. Since universal adapter protein 14-3-3zeta co-localizes with
aggregated tau, it is supposed that 14-3-3 might somehow affect
association and accumulation of insoluble aggregates. We tried to
analyze effect of tau phosphorylation on its interaction with 14-3-3.
Using native gel electrophoresis, size-exclusion chromatography and
chemical crosslinking we found that catalyzed by cAMP-dependent
protein kinase (PKA) phosphorylation of the shortest isoform of human
tau strongly increases its interaction with recombinant human 14-3-3zeta.
We supposed that phosphorylated Ser156 of the shortest tau isoform (or
homologous Ser214 of the largest isoform) located in the consensus
sequence recognized by 14-3-3 are predominantly responsible for 14-3-
3 binding. However, mutation S156A preventing phosphorylation of this
site by PKA, decreased, but did not completely prevent tight interaction
of phosphorylated tau with 14-3-3 thus indicating the presence of other
phosphorylation sites playing important role in tau-14-3-3 interaction.
By using step-by-step mutagenesis we found that phosphorylation of at
least three sites (Ser156, Ser235 and Ser267) of the shortest tau isoform
is important for 14-3-3 binding. Thus, phosphorylation-dependent sites
of 14-3-3 binding are located both in the Pro-rich region (Ser156)
and in tubulin-binding motifs (Ser235 and Ser267) of tau protein.
This work was supported by Russian Foundation for Basic Research.
POS-WED-83
CHANGING THE SOLVENT ACCESSIBILITY OF THE
PRION PROTEIN DISULFIDE BOND MARKEDLY
INFLUENCES ITS TRAFFICKING AND EFFECT ON CELL
FUNCTION
Tabrett C.A.1, Harrison C.F.2, Schmidt B.1, Bellingham S.A.2, 3, Hardy
T.1, Sanejouands Y.4, Hill A.F.2, 3 and Hogg P.J.1
1
Lowy Cancer Research Centre and Prince of Wales Clinical School,
University of New South Wales, NSW 2052 Australia. 2Department
of Biochemistry and Molecular Biology, Bio21 Molecular Science
and Biotechnology Institute, University of Melbourne, Victoria 3010
Australia. 3Mental Health Research Institute of Victoria, University of
Melbourne, Victoria 3010 Australia. 4Laboratoire de Physique, Ecole
Normale Superieure, 46 allees d’Italie, 69364 Lyon Cedex 07, France.
Prion diseases are fatal, transmissible, neurodegenerative diseases
that result from structural conversion of the prion protein into a disease-
associated isoform. The prion protein contains a single disulfide bond.
Our analysis of all NMR structures of the prion protein containing an
explicit disulfide bond reveals that the bond exists predominantly in a
stable, low-energy state, but can also adopt a high-energy configuration.
Side chains (tyrosine and phenylalanine residues) control access of
solvent to the disulfide bond by rotating away in the high-energy state.
The importance of these aromatic residues for protein function was
analysed by mutagenesis, biophysical and cell biological approaches.
While the mutant protein behaved similarly to wild-type prion protein
in recombinant systems, the mutants were retained in the endoplasmic
reticulum of mammalian cells and degraded by the proteasomal system.
The cellular behaviour of the aromatic residue mutants was similar to the
cellular behaviour of a disulfide bond mutant prion protein, a result which
is consistent with an unstable disulfide bond in the aromatic residue
mutants. These observations suggest that the conformation of the prion
protein disulfide bond may have implications for correct maturation and
function of this protein.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 211
POS-THU-82
THE BH3-ONLY PROTEIN BIM: POSSIBLE LINK
BETWEEN ER STRESS AND APOPTOSIS IN NEURAL
CELLS EXPRESSING MUTANT SOD1
Soo K.Y.1, 2, 3, Atkin J.D.1, 3, 4, Farg M.1, Walker A.K.1, 3, 4, Horne M.K.3, 4
and Nagley P.2
1
Department of Biochemistry, La Trobe University, Vic 3086, Australia.
2
Department of Biochemistry and Molecular Biology, Monash
University, Vic 3800, Australia. 3Brain Injury and Repair Group,
Howard Florey Institute, University of Melbourne, Parkville, Vic
3010, Australia. 4Centre for Neurosciences, University of Melbourne,
Parkville, Vic 3010, Australia.
In cellular models of motor neuron disease (ALS), cells bearing
mutant Cu,Zn-superoxide dismutase 1 (SOD1) inclusions undergo
mitochondrial apoptotic signaling. Dispersed SOD1 proteins, either
wildtype (WT) or mutant, were found to partially protect cells against
apoptosis; such protection is upstream of mitochondria. We previously
showed ER stress to be linked to neurotoxicity associated with mutant
SOD1 inclusions. Here, we demonstrate that the BH3-only protein,
Bim, is a direct link between ER stress and mitochondrial apoptosis.
In the murine neuroblastoma cells, Neuro2a, Bim knockdown by
siRNA significantly reduced nuclear apoptotic features in cells bearing
mutant SOD1 inclusions. After Bim knockdown, both Bax recruitment
to mitochondria and cytochrome c redistribution were also decreased
in such inclusion-bearing cells. However, CHOP translocation to
nucleus, a marker of ER stress, was not reduced by Bim knockdown.
Significantly, the neuroprotection afforded by dispersed WT SOD1 was
substantially enhanced by Bim-depletion, observed in Bim-depleted
cells exposed to various apoptotic insults. In cells not subjected to Bim
knockdown, kinetic studies indicated CHOP translocation to nucleus
to occur prior to formation of mutant SOD1 inclusions. Interestingly,
Bax recruitment to mitochondria (but not apoptotic nuclei) was also
observed before formation of mutant SOD1 inclusions. These findings
suggest that neurotoxicity is induced by a toxic structure derived from
mutant SOD1 that activates stress responses in cells much earlier than
the appearance of grossly aggregated SOD1 in inclusions.
POS-THU-84
EXPRESSION OF NF-κB REGULATED CYTOKINES TO
EVALUATE EARLY DIAGNOSIS OF NEURODEGENERATIVE
DISORDERS MAINLY HUNTINGTON’S DISEASE AND
SPINOCEREBELLAR ATAXIA
Potdar P.D. and Tatwani S.
Department of Molecular Medicine & Biology, Jaslok Hospital &
Research Centre, 15, Dr. G. Deshmukh Marg, Mumbai 400026, India.
In1999, Celera Diagnostics sequenced the first human chromosome.
Thereafter, scientists started developing molecular diagnostic tests to
define involvement of any specific gene mutation in various genetic
disorders. Huntington’s disease (HD), and Spinocerebellar ataxia
(SCA1,2,3,6,7&17) are progressive neurodegenerative disorders caused
by polyglutamine protein aggregates, which are known as “CAG triplet
repeat disorders.” However, it was observed that many times diagnosis
of these diseases, only by detecting CAG repeats, is not sufficient.
Tabrizi et al (2005) have recently shown that IL-6 is consistently
elevated in HD. Several NF-κB dependent genes are either up or down
regulated in the expanded polyglutamine expressing cells depending on
disease conditions. Recent reports have also indicated that TNF-α is up
regulated in SCAs. Though the basic works suggest the role of NF-κB
activation in the causation of these diseases, there is a need for using
these biomarkers as molecular diagnostic tests for early diagnosis
of these diseases. Our lab has already set up PCR based assays for
studying CAG repeats in HD and SCAs but it is found that addition of
these markers will help us to confirm their diagnosis more precisely. We
therefore decided to study the expression of NF-κB regulated cytokines
in HD and SCAs patients. We found that HD patients showed significant
increase in IL-6 expression whereas, SCAs patients showed significant
high expression of TNF-α with down regulation of IκB-α. Thus, this
study indicates that these biomarkers will be important for evaluating
HD & SCAs patients whose diagnosis by currently employed CAG
repeat methods is negative or uninformative.
POSTERS WEDNESDAY & THURSDAY
POS-WED-85
CHANGES IN SIGNALLING PROTEINS IN MUSCARINIC
RECEPTOR DEFICIT SCHIZOPHRENIA
Udawela M.1, 2 , Scarr E.1, 5, Hannan A.J.3, Thomas E.A.4 and Dean B.1, 5, 6
1
Rebecca L Cooper Research Laboratories, Mental Health Research
Institute of Victoria, VIC, Australia. 2Centre for Neuroscience,
University of Melbourne, VIC, Australia. 3Howard Florey Institute,
Florey Neuroscience Institutes, University of Melbourne, VIC,
Australia. 4Department of Molecular Biology, The Scripps Research
Institute, La Jolla, CA, USA. 5Department of Psychiatry, University of
Melbourne, VIC, Australia. 6Department of Psychiatry, University of
Melbourne, VIC, Australia.
Schizophrenia is a debilitating mental illness, affecting approximately 1%
of the population [1]. Cholinergic muscarinic receptors (CHRMs) have
been shown to be decreased in the CNS of subjects with schizophrenia
[2] and are thought to be implicated in cognitive deficits associated with
schizophrenia. Our laboratory recently showed in post-mortem tissue
that around 25% of subjects with schizophrenia have a 74% reduction
in binding to cortical CHRM1 in Brodmann’s Area (BA) 9 [3]. Our
recent microarray data identified decreased expression in the CHRM1
downstream signalling protein phospholipase C beta 1 (PLCβ1) mRNA
in schizophrenia in BA46, therefore to determine whether patients with
CHRM1 deficit also had disrupted downstream signalling pathways
we investigated changes in PLCβ1 in our subset of patients with low
CHRM1 binding. Using qPCR and Western blot analysis we found
PLCβ1 mRNA and protein expression is reduced in BA 9 from patients
with schizophrenia with normal levels of CHRM1 binding, but not in
those with low levels of binding, compared to age/sex matched controls.
In BA 46, mRNA was decreased in both groups of patients compared
to controls with no change in protein levels. Understanding changes in
downstream effector pathways may reveal other possible therapeutic
approaches to treating symptoms in schizophrenia. [1] Black and
Andraeson. (1994) Textbook of Psychiatry, The American Psychiatric
Press, Washington, DC [2] Raedler, T. J., et al. (2007) Mol Psychiatry
12, 232-46 [3] Scarr, E., et al. (2009) Mol Psychiatry 14, 1017-23.
POS-WED-87
IDENTIFICATION OF OLFACTORY SIGNALLING GENES
IN DROSOPHILA MELANOGASTER
Liu Y.-C., Honda T., Beale M., De Bruyne M. and Warr C.G.
School of Biological Sciences, Monash University, VIC 3800, Australia.
Drosophila melanogaster has emerged as a powerful model organism
for the study of the peripheral olfactory system due to the availability of
sophisticated molecular genetic techniques for studying gene function,
coupled with sophisticated electrophysiological tools for measuring
single olfactory receptor neuron (ORN) responses. In Drosophila odour
signals are detected by a large family of 62 seven-transmembrane
receptor proteins, the odorant receptor (Or) family. Unlike mammalian
Ors which are G protein-coupled receptors, the insect Ors appear to
encode directly ligand-gated ion channels. To identify genes involved in
peripheral olfactory function in Drosophila we have performed genetic
screens using electrophysiological recording techniques. This approach
has identified two EMS-generated mutant strains which have greatly
reduced electro-antennogram responses to all tested odours. The two
mutations show interesting differences in their electrophysiological
phenotypes, indicating one (called ll2) may affect signal transduction
in ORNs, whereas the other (called O88) may affect the function of the
accessory cells that support the ORNs. We have deficiency mapped
each mutation, the ll2 mutation maps to a region in 87A containing 12
annotated genes, and the O88 mutation to a region in 33E containing
6 annotated genes. We are using multiple approaches to identify the
gene affected in each mutant: candidate gene sequencing, expression
analysis, RNA interference experiments, and rescue experiments.
Recent in vivo RNAi experiments indicate that the gene affected in the
O88 mutation is bru-2, an essentially uncharacterised member of the
bruno family of RNA-binding proteins, and that its function is required
in the accessory cells. Rescue experiments are currently in progress to
confirm this.
Page 212 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-86
SOCS2 REGULATES NEURITE OUTGROWTH AND
NEURONAL SURVIVAL THROUGH TRK RECEPTOR
INTERACTIONS
Uren R.T., Faux C.H., Turbić A., Wong A.W., Murray S.S. and Turnley A.M.
Centre for Neuroscience, University of Melbourne, Parkville, Victoria,
3010, Australia.
Overexpression of Suppressor of Cytokine Signalling-2 (SOCS2)
promotes increases in neurite length and neurite number in PC12 cells
and cortical neurons. The mechanisms by which SOCS2 regulates the
signals that control neurite outgrowth and neuronal differentiation are
unresolved but appear to involve Trk neurotrophin receptors. Wildtype
or mutant SOCS2 proteins were expressed in PC12 cells and neurite
outgrowth examined under basal proliferative conditions and with
Nerve Growth Factor (NGF) which promotes neuronal differentiation.
Expression of full length SOCS2 promoted neurite outgrowth in both
the absence and presence of NGF and the amount of endogenously
expressed TrkA was increased. Expression of SOCS2 lacking the SOCS
box blocked NGF-induced neurite outgrowth, thus behaving as a potent
dominant negative mutation. To examine SOCS2 and Trk interactions
in primary neurons, the morphology of TrkA expressing Dorsal Root
Ganglion (DRG) neurons from SOCS2 overexpressing (SOCS2-Tg)
mice and mice lacking SOCS2 (SOCS2 -/-) was compared to wildtype
neurons. DRG neurons were obtained from 1 day post-natal mice,
dissociated neurons were cultured and neurite morphology scored. DRG
neurons from SOCS2-Tg mice demonstrated an increased proportion
of neurons with complex neurite morphology. In contrast, SOCS2 -/-
derived DRG neurons displayed reduced neurite length and number.
Furthermore, the survival profile of SOCS2-Tg and SOCS2 -/- DRG
neurons cultured with 50 ng/mL NGF was examined and the number
of viable DRG neurons (large, phase bright cell bodies) was recorded
at 24 hour intervals. At 48 and 72 hours post-plating, the number of
viable SOCS2-Tg DRG neurons was greater than controls, whereas the
number of viable SOCS2 -/- DRG neurons was reduced.
POS-THU-88
GSTP ISOLEUCINE 105 TO VALINE POLYMORPHISM
MAY PLAY A ROLE IN DETERMINING SUSCEPTIBILITY
TO PEPTIC ULCER
Kangsadalampai S., Gamnarai P., Rojpibulstit P. and Vilaichone R.K.
Faculty of Medicine, Thammasat University (Rangsit Campus), Klong-
Luang, Pathumthani, 12121. Thailand.
ABSTRACT A case-control study on a relationship between glutathione
S-transferase class pi (GSTP) Ile105Val polymorphism and a
susceptibility to gastric ulceration in Thai dyspeptic patients was carried
out. The GSTP Ile105Val genotype was determined by the Polymerase
Chain Reaction-Restriction Fragment Length Polymorphism (PCR-
RFLP). The genotype frequency in the patients with ulcer (case; n = 105)
was significantly different from that in the non-ulcer dyspeptic patients
(control; n = 194). The patients who were homozygous Val105 seemed
to have higher risk to peptic ulceration [OR = 4.290 (95% CI, 1.100-
16.722), p = 0.030]. In contrast, the Ile105Val genotype appeared to act
as a protective genotype [OR = 0.563 (95% CI, 0.333-0.952), p = 0.031].
However, other confounding factors such as sex, age, and H. pylori
infection should not be overlooked. Therefore, stratified analysis was
performed. The result of the analysis revealed that sex and age were
not the confounders but the H. pylori infection exhibited the influence on
the association. Thus, besides the genetic factors, the environmental
factors could be taken into an account when genotype susceptibility to
disease was studied.
POSTERS WEDNESDAY & THURSDAY
POS-WED-89
STUDIES ON THE ROLE OF P73 PROTEINS IN
INTESTINAL STEM CELLS AND TUMOURS
Kass L.1, Horvay K.1, Stiewe T.2, Hime G.R.3, 4 and Abud H.E.1
1
Monash University, Department of Anatomy and Developmental
Biology, Clayton VIC 3800. 2Philipps-University Marburg, Institute of
Molecular Biology and Tumor Research Marburg, Germany. 3University
of Melbourne, Department of Anatomy and Cell Biology, Parkville VIC
3010. 4ARC Centre of Excellence in Biotechnology and Development.
This research is focussed on understanding the role of a dominant-
negative form of the p73 protein (DNp73) in regulating the differentiation
of intestinal epithelial cells and the initiation of tumours. p73 is a
homologue of the p53 tumour suppressor gene and produces two major
protein isoforms via use of alternative promoters. Several studies have
suggested that the longer TAp73 protein has tumour suppressor activity
and the N-terminally truncated DNp73 protein can act as an oncogene.
Our studies show that p73 is expressed in epithelial cells at the base of
intestinal crypts which suggests that p73 may have a role in regulating
normal homeostasis within the intestinal stem cell niche. Several studies
including ours have found that the overall level of p73 is overexpressed
in human colorectal cancer (CRC) cell lines and that the DNp73/TAp73
ratio is increased. We are investigating the consequences of disrupting
the DNp73/TAp73 ratio in the murine intestine using a transgenic mouse
model that permits inducible conditional expression of DNp73 in the
intestinal epithelium. Initial analysis of the effects of overexpressing
DNp73 has revealed an interesting phenotype where there appears to
be an expansion of the progenitor pool at the expense of differentiated
cell types. We are currently analysing this phenotype in more detail.
POS-WED-91
APOPTOSOME APPARATUS AND EXPRESSION OF
ITS REGULATORS, XIAP, APIP AND NUCLING, IN NON-
SMALL CELL LUNG CANCER CELLS AND TISSUES
Moravcikova E., Krepela E., Prochazka J., Cermak J. and Benkova K.
University Hospital Bulovka, Budinova 2, 18081 Prague, Czech
Republic.
Dysfunction of apoptosome apparatus (AA) contributes to tumour growth
and progression and therapy resistance of tumours. To evaluate AA
functionality in non-small cell lung carcinoma (NSCLC) we studied AA
activation in cell-free cytosols from NSCLC cells and tissues. Although
NSCLCs expressed Apaf-1, procaspase-9, -3 and -7 proteins, AA was
activated by cytochrome-c (cyt-c) and dATP only in 2 of 6 examined
NSCLC cell lines and in 18 of 59 NSCLC tissues obtained from surgically
treated patients. Although XIAP is a direct inhibitor of some caspases,
its level in NSCLC tissue cytosols did not correlate with the endogenous
and the (cyt-c + dATP)-induced caspase-3-like activity. Moreover,
XIAP neutralizing peptides AVPIAQK or ATPFQEG derepressed only
slightly the endogenous and the (cyt-c + dATP)-induced caspase-3-
like activity in tumour cytosols. NSCLC cell lines expressed both APIP2
mRNA and protein and both nucling (UACA) mRNA and protein. NSCLC
tissues showed significantly decreased expression of both APIP and
nucling mRNAs as compared to matched lungs. In the tumours, there
was no correlation between the expression of APIP mRNA or nucling
mRNA and the endogenous or the (cyt-c + dATP)-induced caspase-
3-like activity in cytosols. In conclusion, these results indicate that AA
activation is suppressed in a high proportion of NSCLCs, that XIAP is
not the major factor responsible for apoptosome dysfunction in NSCLCs,
and that downregulation of APIP and nucling expression in NSCLCs has
no obvious impact on AA activation. Acknowledgements. Supported by
research projects NS/9715-4 and MZO00064211 from the Ministry of
Health, Czech Republic.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 213
POS-THU-90
ANTI-CANCER EFFECT BY KNOCK-DOWN OF THE
PROTEINS OVER-EXPRESSED IN LUNG CANCER
Kim J.E.1, 2, 3, Koo K.H.1, 2, 3, Kim Y.H.1, 2, 3, Sohn J.1, 2, 3 and Park Y.G.1, 2, 3
1
Dept. of Biochemistry. 2Division of Brain Korea 21 Program for
Biomedical Science. 33Cancer Research Center for Lung and Breast/
Ovarian Cancers, Korea University College of Medicine, 136-701,
Seoul.
Lung cancer is a malignant tumor with a very high incidence and
mortality. The majority of lung cancer patients have been diagnosed
at late stage and surgical resection cannot be the best choice for
successful treatment. Therefore, the therapeutic molecular targets
are necessary for improving clinical outcome of lung cancer patients.
In this study, we performed comparative proteomic studies to find the
potential therapeutic targets of lung cancer. We used the different
histopathologic types of lung cancer including squamous cell carcinoma
and adenocarcinoma. 62 proteins were up-regulated and 13 proteins
were down-regulated in lung cancer tissues. 16 of 75 identified proteins
were verified by performing western-blot analysis in the same lung
tissues used for proteomic analysis, the cell lines representing multi
stage human lung carcinogenesis and lung carcinoma cell lines. Three
proteins were selected to evaluate the roles in lung cancer cell growth,
because the three proteins were up-regulated in both lung cancer
tissues and cell lines. It has been observed down-regulation of the
proteins significantly suppressed cell proliferation and clonogenicity in
the three NSCLC cell lines, A549, H1299 and H1573. It is likely that
the three proteins contribute maintenance of lung cancer. Our results
indicate that the three proteins play a significant role in cell proliferation
and are potential therapeutic targets for lung cancer.
POS-THU-92
DNA ANALYSIS IN FEMALES WITH 46,XY KARYOTYPE-
ASSOCIATED SEX DEVELOPMENT DISORDERS
Krepelova A.1, Malikova M.1, Simandlova M.1, Plevova P.2 and
Gaillyova R.3
1
Department of Biology and Medical Genetics, University Hospital
Motol and 2nd Medical School, Prague, Czech Republic. 2Department
of Medical Genetics, University Hospital Ostrava, Czech Republic.
3
Department of Medical Genetics, University Children Hospital Brno,
Czech Republic.
Disorders of sex development (DSD) in females with karyotype 46,XY
represent a very heterogeneous group of disruptions of normal gender
ontogeny. Underlying genetic defects include disorders of genes
determining development of testes, e.g. SRY gene, disorders of
androgens synthesis or impaired function of the androgen receptor. In
many cases of 46,XY DSD the molecular basis has not been identified
yet. We studied 21 female patients with 46,XY DSD. We used PCR
amplification and direct DNA sequencing to detect mutations in several
genes including SRY, SF1, AR and 17βHSD3. We used MLPA analysis
to detect DAX2 gene duplications. We detected a novel mutation
c.146_153del8ins15 (p.Gly49fsX10) in the SRY gene in a girl with gonadal
dysgenesis. In six cases with clinical diagnosis of complete androgen
insensitivity (CAIS) we found five different mutations in the AR gene.
Three mutations are novel: c.33_37dupCCCTC (p.Arg13ProfsX23),
c.122_136del15ins5 (p.His41ProfsX130) and c.827_828dupGC
(p.Val277LeufsX17), and two were observed previously by others:
c.2194G>A (p.Asp732Asn), and c.2543dupA (p.Asn848LysfsX32). In
one CAIS patient we identified a homozygous mutation in the 17βHSD3
gene: c.[325+4A>T]+[325+4A>T]. This mutation was reported previously
to cause an abnormal mRNA splicing. In thirteen remaining patients
the molecular cause of DSD was not yet determined. In conclusion,
the heterogeneity of DSD requires to search for defects in multiple
candidate genes in order to idenfity the exact molecular basis of DSD.
This is essential for the genetic prognosis, therapeutic decision, and
psychological and social care in these patients. Supported by Grant
MZO 00064203.
POSTERS WEDNESDAY & THURSDAY
POS-WED-93
FUNCTIONAL ANALYSIS OF ZINC TRANSPORTERS OF
ZNT/SLC30A FAMILY MEMBERS AMONG A MAMMARY
GLAND DISORDER OF REDUCED ZINC SECRETION
INTO MILK
Kumar L., Michalczyk A., Freestone D. and Ackland L.
Centre for Cellular and Molecular Biology, School of life and
environmental sciences, Deakin University, VIC, 3125, Australia.
Zinc is an essential trace element required for normal growth,
development and reproduction. Zinc deficiency can cause dermatitis,
diarrhoea, alopecia, loss of appetite, hypogonadism, impaired growth
and diminished immune response. An adequate supply of zinc is required
particularly during the neonatal period. Zinc is a significant component
of breast milk, which is transported across the maternal epithelia
during lactation. Hereditary disorders of impaired zinc secretion from
the mammary gland into milk during lactation can cause acquired zinc
deficiency in infants exclusively fed on breast milk. It usually occurs in
pre term babies 27-33 week gestation due to their greater bodily demand
and lower bodily zinc stores. Mammalian zinc transporters, which
may play an important role in secretion of zinc into milk, are broadly
classified into SLC30A and SLC39A families. Transcriptional levels were
measured for identified members of mammalian SLC30 family to test the
hypothesis that defects in one or more of them may be responsible for
impaired zinc secretion into the breast milk. Significantly reduced levels
of ZnT5 and ZnT6 mRNA transcript were found in patients lymphoblast
and fibroblast compared to control cells. These finding suggested that
mutation in ZnT5 and/or ZnT6 may underline the disorder of reduced
zinc secretion into the milk. Sequence analysis of ORF was carried
out but no differences were found between the patients and control
samples. Analysis of 5/3 UTR (untranslated region), promoter region,
protein expression, localization, alkaline phosphatase activity and mi-
RNAs of both genes was carried out. In summary ZnT5 and ZnT6 may
indirectly contribute to this zinc deficiency disorder.
POS-WED-95
IDENTIFICATION AND FUNCTIONAL
CHARACTERISATION OF WDR35, A GENE MUTATED IN
SHORT RIB POLYDACTYLY SYNDROME
Amor D.J.1, Fitzpatrick E.2, Mountford H.2, Bahlo M.3, Mill P.4,
Hall E.4, Bromhead C.3, Pope K.1, Aftimos S.5 Jackson I.4, Delatycki M. 2,
Savarirayan R1 and Lockhart P.J.2
1
Victorian Clinical Genetics Service, Murdoch Childrens Research
Institute, Australia. 2The Bruce Lefroy Centre, Murdoch Childrens
Research Institute, Australia. 3Walter and Eliza Hall Institute, Australia.
4
MRC Human Genetics Unit, UK. 5Northern Regional Genetics
Service, New Zealand.
The short rib-polydactyly (SRP) disorders are a heterogenous group of
autosomal recessive lethal skeletal dysplasias characterised by short ribs
and limbs. The presence of additional clinical features such as abnormal
viscera, cranium and palate can be used to classify the disorders into
specific subtypes. We previously identified a family with an unclassifiable
SRP that displayed laterality defects suggestive of ciliary dysfunction
(1). We performed SNP analysis on five family members and identified
three regions with a LOD>1. Analysis of inferred haplotypes suggested
that a single region at chromosome 2p24.1 was homozygous by descent
in affected members. CNV analysis identified a homozygous deletion
within the linkage peak that disrupted the WDR35 gene. WDR35 encodes
a novel highly conserved protein with homology to known cilial proteins.
Immunocytochemical analysis localised WDR35 to the cilial axoneme
and basal body of control fibroblasts, however cilia were completely
absent in fibroblasts derived from our SRP patients. Targeted disruption
of wdr35 in the mouse resulted in abnormal cilia, randomised laterality,
polydactyly and neural tube defects. These features were consistent
with the human phenotype and suggested deficits in hedgehog (Hh)
signalling contributed to disease pathogenesis. Quantitative real-time
PCR analysis demonstrated that downstream effector genes, such as
Ptch1 and Gli1, were not responsive to Hh signalling in MEFs derived
from mutant mice. In conclusion, our studies have identified a novel
ciliopathy gene and extended our understanding of the pathogenesis of
SRP. (1) Kannu P et al (2007) AJMG Part A 143:26073.
Page 214 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-94
ISOMALYNGAMIDE A, A-1 AND THEIR ANALOGS
SUPPRESS CANCER CELL MIGRATION IN VITRO
Li W.-S.1, Chang T.1, More S. 1, Lu I.-H. 1, Hsu J.-C.1, Chen T.-J.2, Jen Y.1,
Jao S.-C.3 and Lu C.-K.2
1
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan. 2Division
of Herbal Drugs and Natural Products, National Research Institute
of Chinese Medicine, Taipei 112, Taiwan. 3Institute of Biological
Chemistry, Academia Sinica, Taipei 115, Taiwan.
Isomalyngamide A, A-1 and their analogs suppress cancer cell migration
in vitro Tzu Ting Chang,a Shivaji V. More,a I-Hsuan Lu,a Jui-Ching
Hsu,a Ting-Ju Chen,b Ya Ching Jen,a Shu-Chuan Jao,c Chung-Kuang
Lu,*b and Wen-Shan Li*a aInstitute of Chemistry, Academia Sinica,
Taipei 115, Taiwan, bDivision of Herbal Drugs and Natural Products,
National Research Institute of Chinese Medicine, Taipei 112, Taiwan,
and cInstitute of Biological Chemistry, Academia Sinica, Taipei 115,
Taiwan. Abstract: There are considerable pharmacological interests
associated with marine metabolites because of their demonstrated
value as antimalarial, antitubercular, anti-HIV, and antitumor agents.
However, recent advances in search of antimetastatic agents are mainly
from either natural products or small synthetic libraries, lacking the
participation of marine metabolites and their man-made analogues.
Metastasis is mutiple biological events resulting in migration of tumor
cells from their initial site to secondary organs. Inhibition of any of
these events could hamper the whole metastatic process. Therefore,
antimetastatic agents, an alternative of anticancer drugs, have attracted
much attention lately. We have identified highly potent inhibitors of tumor
progression from marine metabolites, isomalyngamide A (1) and A-1 (2),
which show therapeutic potential in breast cancer not only by bolcking
cell proliferation with low micromolar IC50 values but also by inhibiting
metastasis with low nanomolar IC50 values. Notably, since analog 9
generated in this study can present a bioactive mimic of isomalyngamide
A and A-1, it provides a channel of devotedly study integrin-mediated
antimetastatic pathway without the interference of cytotoxic effect.
POS-THU-96
IDENTIFICATION OF ACTIVATED KINASE PROTEINS IN
CHOLANGIOCARCINOMA
Loilome W.1, 2 , Dokduang H.1, 2, Juntana S.1, 2, Namwat N.1, 2 and
Yongvanit P.1, 2
1
Department of Biochemistry, Faculty of Medicine, Khon Kaen
University, Khon Kaen 40002, Thailand. 2Liver Fluke and
Cholangiocarcinoma Research Center, Faculty of Medicine, Khon
Kaen University, Khon Kaen 40002, Thailand.
Dysregulation of protein kinases activity due to overexpression and/or
mutation has been found to be implicated in various tumor types and also
in cholangiocarcinoma (CCA), the malignant tumor arising from bile duct
epithelia which is the common cancer and a major public health problem
in northeast Thailand. This study aims to identify the activated kinases
which are involved in CCA development in order to use as the drug target
in particular cancer. Using human phospho-RTKs and human phospho-
kinases array analysis demonstrated that multiple RTK and protein
kinase signaling pathways are activated in both CCA cell lines and CCA
tissues including MAPK kinase, PI-3K/Akt, Wnt/b-catenin, JAKs/STAT,
PKA and PKC signaling pathways whereas, activated RTKs which
related to tumor angiogenesis such as VEGFR, FGFR and Tie found
only in CCA tissues. We also have tested a panel of small molecule
protein kinase inhibitors including those FDA approved, clinical and pre-
clinical drugs. The multi-targeted protein kinases inhibitor, sorafenib and
sunitinib seems to be the most effective drug for inhibiting CCA cells
growth due to the lowest IC50 observed. The present work indicating
that targeting multiple kinases which are activated in CCA would be
benefit for particular tumor therapy. Although, the molecular mechanism
by which those drugs inhibiting cell growth as well as efficiency of drugs
in vivo will be further investigated.
POSTERS WEDNESDAY & THURSDAY
POS-WED-97
THE FUNCTIONAL CHARACTERISATION OF HUMAN
RMND5 PROTEINS IN PROSTATE CANCER
Louw A.1, 2 , Dawson L.1, Harvey J.2 and Bentel J.1, 2
1
Anatomical Pathology, Royal Perth Hospital. 2University of Western
Australia, Perth, Western Australia.
Human RMND5A and RMND5B are hypothetical proteins named after
their yeast orthologue, RMD5 (required for meiotic nuclear division), an
E3 ubiquitin ligase and component of the yeast Gid/Vid30c complex.
RMND5A and RMND5B contain a RING finger-like domain and LisH,
CTLH and CT-11 RanBPM (CRA) domains, the functions of which are
uncharacterised. In particular, E3 ubiquitin ligase activity of RMND5A
or RMND5B has not been demonstrated. RMND5A and RMND5B are
expressed in prostate cancer cell lines and are localised in the nucleus
and cytoplasm following transfection into these cells. In addition, each of
the human orthologues of Gid/Vid30c components, RanBPM, muskelin,
Twa1, EMLP, ARMC8α and RMND5A, are expressed in prostate cancer
cell lines, providing evidence that the human Gid/Vid30c complex
orthologue, the CTLH complex, is able to form. In LNCaP prostate
cancer cells, both RMND5A and RMND5B interact with RanBPM, whose
yeast orthologue is a proposed core component of yeast Gid/Vid30c,
further supporting formation of a human CTLH (Gid/Vid30c) complex
and potentially indicating that CTLH complexes can contain either (or
both) RMND5A, as previously reported, and the highly homologous
family member, RMND5B. RMND5A, RMND5B and RanBPM are
each localised in the nucleus and cytoplasm of prostate cancer cells
with extensive co-localisation in both cell compartments. While the
biological activity of RMND5 proteins is currently unknown, RMND5B
interacts with the prostatic tumour suppressor, NKX3.1, enhancing its
transcriptional repressor activity. Furthermore, the RMND5B gene is
located within a prostate cancer susceptibility locus and disruption of
RMND5A and RMND5B expression is detected in a variety of cancers,
indicative of important cellular functions of these highly conserved and
ubiquitously expressed proteins.
POS-WED-99
OSTEOPONTIN IS REQUIRED FOR NORMAL SKELETAL
MUSCLE DEGENERATION AND REGENERATION
Uaesoontrachoonm K., Tudor E.M., Pagel C.N. and Mackie E.J.
School of Veterinary Science, University of Melbourne, Parkville,
Victoria 3010, Australia.
Osteopontin is an extracellular matrix glycoprotein which is expressed
in muscle and regulates myoblast behaviour. To investigate the role of
osteopontin in muscle regeneration, we utilized the method of whole
muscle grafting in osteopontin-null and wildtype mice; the extensor
digitorum longus (EDL) muscle was removed from and then returned to
its bed, causing vascular disruption and muscle degeneration, followed
by muscle regeneration. Osteopontin expression as detected by
immunohistochemistry was strongly up-regulated in wildtype EDL 3 and
5 days after grafting but not at later time points. Three days after grafting,
in wildtype mice the diameter (determined as minimum Ferret’s diameter)
of the whole EDL and of individual muscle fibres was significantly
smaller in grafted than in sham-operated muscles; in osteopontin-null
mice, there was no significant difference in either parameter between
graft and sham. There were significantly fewer muscle fibres 3 days
after grafting in wildtype compared to osteopontin-null muscles; the fibre
number increased from day 3 in wildtype grafts, whereas a decrease
in fibre number was observed up to day 7 in grafted osteopontin-null
muscles. There were significantly fewer centrally nucleated muscle
fibres, a sign of regeneration, in osteopontin-null than in wildtype grafts
up to 20 days after grafting. There were fewer neutrophils at day 3 and
fewer macrophages at days 3 and 5 in osteopontin-null grafts than in
WT grafts. These results suggest that degeneration of muscle fibres is
delayed in the absence of osteopontin, which can be attributed in part to
a lack of phagocytosis resulting from reduced infiltration of osteopontin-
null grafts by macrophages and neutrophils. Furthermore, these
observations indicate that osteopontin is important in the regulation of
the early stages of muscle regeneration.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 215
POS-THU-98
CHARACTERISATION OF HUMAN ACETYL COA
CARBOXYLASE -A TARGET FOR OBESITY
THERAPEUTICS
Macaulay S.L.1, Castelli L.A.1, Jarvis K.1, Liepa A.J.1, Fernley R.T.1,
Pilling P.1, Epa V.C.1 and Kemp B.E.1, 2
1
CSIRO Preventative Health Flagship, Parkville, Vic. 2St Vincents
Institute, Fitzroy, Vic.
Acetyl CoA carboxylase (ACC) 1 and 2 are required for fatty acid
synthesis and regulation of fatty acid oxidation respectively. Both are
potential therapeutic targets for the treatment of obesity and type 2
diabetes. Difficulties in expression of full length proteins have hampered
investigation of the enzymes as targets. Two recent reports described
the expression of ACC1 and truncated ACC2 (Kim et al 2007, Cheng et
al 2007). Here we describe the expression of full length human ACC2
and ACC1 with a C-terminal FLAG tag in insect cells and inhibitors of
enzyme activity. Both proteins were active and had a Km of 12-15 μM
for acetyl CoA. The yield of protein was modest, 0.2mg/L. The proteins
were inhibited by low molecular weight compounds previously studied
as potential herbicides, with the best of the inhibitors having IC50 of
200nM. To further investigate inhibition by related compounds, FLAG
tagged carboxyltransferase (CT) domain was expressed and purified
from insect cells. This protein expressed at higher levels than the full
length enzyme (25mg/L). Activity of this domain was inhibited by the
same compounds that inhibited the full length enzyme. Crystallisation
trials of this domain are in progress together with medicinal chemistry
around the inhibitors. Structure of a similar construct of yeast CT
domain was obtained at 3.0Å with inhibitor and is being refined. The
inhibitors stimulated fatty acid oxidation in L6 muscle cells, and, in
high fat fed mice, decreased respiratory exchange ratio and improved
glucose tolerance confirming them as potential leads for therapeutic
development.
POS-THU-100
FINDING NEW SIGNALING PATHWAYS THAT
CONTRIBUTE TO BREAST CANCER PATHOGENESIS
Majewski I.J., Bosma A., Severson T., Kerkhoven R., Velds A., Linn S.C.
and Bernards R.
The Netherlands Cancer Institute (NKI-AVL).
Breast cancer is a heterogeneous disease. Our work focuses on two
very different types of breast cancer, invasive lobular carcinoma (ILC)
and triple negative breast cancer (TN), which together represent 25% of
all breast cancers. TN tumors lack expression of the estrogen receptor
(ER), the progesterone receptor (PR) and Her2 (Erbb2), greatly
restricting treatment options. Lobular breast cancers are generally ER+
and can be treated with hormonal therapies; however, these tumors
often reoccur and have a poor outcome. New therapies are desperately
required for the treatment of these cancers. Kinases are important
mediators of cell signaling and are generally considered to be good
therapeutic targets. We have developed an exon capture system for
high-throughput mutation analysis that will allow us to identify kinases
that are mutated in ILC and TN breast cancer. The kinome sequencing
technology will be validated in cell line models, but we aim to expand
this work to survey several hundred cancers from patients with ILC and
TN breast cancer.
POSTERS WEDNESDAY & THURSDAY
POS-WED-101
CLUSTERIN (APO J) AND COMMD1: CHAPERONES
THAT FACILITATE THE DEGRADATION OF THE
MAMMALIAN COPPER TRANSPORTERS, ATP7A AND
ATP7B
Materia S.1, Cater M.2, Klomp L.3, Mercer J.1 and La Fontaine S.1
1
School of Life and Environmental Sciences, Deakin University,
Burwood, Victoria, 3125, Australia. 2The Peter MacCallum Cancer
Centre, St. Andrews Place, East Melbourne, Victoria, 3002, Australia.
3
Laboratory of Metabolic and Endocrine Diseases, University Medical
Center, Utrecht, The Netherlands.
Copper is essential for human health and is important in the aetiology
and pathology of neurodegenerative diseases, such as Alzheimer’s,
Parkinson’s and prion diseases, in addition to its well-established role
in the genetically inherited disorders, Menkes and Wilson diseases.
The two mammalian copper-transporting P-type ATPases, ATP7A
and ATP7B regulate the copper status of the body. They have dual
functions that consist of copper transport into the secretory pathway for
incorporation into essential enzymes, and relocation to the cell periphery
at high copper levels to rid the cell of excess copper. Clusterin (Apo
J) and COMMD1 are two proteins that interact with the Cu-ATPases
and their misfolded variants to facilitate their degradation. Clusterin
has properties similar to the small heat shock proteins with chaperone-
like activities, binding to stressed, partially unfolded proteins. ATP7B
is predominantly degraded by the lysosomal pathway and Clusterin
overexpression facilitated its degradation. Partial degradation of ATP7B
by the proteasomal pathway involves COMMD1. Clusterin and COMMD1
overexpression and knockdown affected Cu-ATPase expression levels
and cellular copper levels as determined by a copper-sensing luciferase
reporter assay. It is emerging that both Clusterin and COMMD1 are key
components required for Cu-ATPase quality control, which in turn is
necessary for the maintenance of normal copper homeostasis.
POS-WED-103
CANINE MAMMARY TUMOURS: A MODEL FOR HUMAN
BREAST CANCER?
Milley K.M.1, Toribio R.2, Slavin J.3, Scott-Dowell K.4, Papadopoulos R.1,
Richardson S.J.1 and Danks J.A.1
1
School of Medical Sciences, RMIT University, Bundoora 3083,
Australia. 2School of Veterinary Medicine, Ohio State University,
Columbus, USA. 3St. Vincent’s Hospital, Fitzroy 3065, Australia.
4
Gribbles Pathology, Heidleberg 3084, Australia.
Canine mammary tumours (CMTs) have been suggested as a model for
human breast cancer (HMC) since the 1970s. This assertion was based
on the histopathology of CMTs compared with HMC. In addition, CMTs
mimic HMC in regards to hormone dependence, age of onset, tumour
size and cell kinetics. Unlike other animal models, dogs live in a natural
environment and are exposed to the same carcinogens as humans. To
date utilization of CMTs for research has lagged behind other animal
models such as mice and rats. Sequencing of the canine genome in
2005 has provided renewed impetus for comparative research between
humans and dogs. Our research focused on demonstrating the
application of the HMC molecular sub-typing classification system in 30
benign and malignant CMTs using immunohistochemistry. In addition,
we have localized other commonly used tumour markers such as
oestrogen receptor (ER), Ki-67, E-cadherin and β-catenin. The variation
in staining of these markers between different subtypes and histological
classifications was then compared. Both benign and malignant CMTs
demonstrated similar staining patterns to the tumour types found in
humans. In particular the reduction of staining both E-cadherin and
β-catenin in malignant tumours compared to benign tumours. The
increased proliferation of malignant tumours was demonstrated by the
higher Ki-67 indices. The results of this research further strengthen the
hypothesis that CMTs are a good model for HMC. In particular, that the
progression of both the human and dog disease follow a similar course
potentially advocating CMTs as a model for HMC.
Page 216 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-102
THE AGILENT TECHNOLOGIES’ SURESELECT TARGET
ENRICHMENT SYSTEM FOR NEXT-GENERATION
SEQUENCING DEMONSTRATES HIGH PERFORMANCE
AND ENABLES DIVERSE APPLICATIONS
Roberts D.1, Happe S.2, McInnes R.3, Pabon C.1, Giuffre A.2, Novak
B.1, Visitacion M.1, Joshi S.2, Ong J.2 and Leproust E.1
1
Agilent Technologies, Santa Clara, California. 2Agilent Technologies,
Cedar Creek, Texas. 3Agilent Technologies, Melbourne, Victoria.
The discovery of rare polymorphisms, structural variants, and novel
transcripts has been accelerated dramatically by next-generation
sequencing technologies. However, it remains cost-prohibitive to
sequence entire genomes in large cohort studies. To enable larger sample
size, Agilent Technologies has developed the SureSelect platform,
allowing focused analyses on specific genomic loci at considerable
cost savings. Agilent is continuing to expand its portfolio to increase the
number of applications available to users. First, we demonstrate high
performance across Illumina, SOLiD, and 454 platforms, as measured
by capture efficiency, uniformity, reproducibility, and SNP detection.
Custom design across all platforms and multiple model organisms is
simplified using a desktop version of the eArray software. Next, we
create multiple unique SureSelect panels such as sequences encoding
the human kinome and whole exome to highlight flexibility across a
wide range of target size and complexity. These catalog designs enable
standardized studies across multiple research sites. Third, for smaller
targeted regions, we demonstrate the ability to index samples in a single
lane, thereby further decreasing the cost of large studies. For more
comprehensive genome analysis, we reveal expanded utility and scope
of the All-Exon catalog product by increasing read depth uniformity and
target coverage to include regulatory regions, small RNAs, and content
from multiple databases including CCDS and RefSeq. SureSelect
applications will continue to expand as users harness the power of next-
generation sequencing.
POS-THU-104
INTERACTION BETWEEN THE ATAXIA
TELANGIECTASIA MUTATED (ATM) AND TUBEROUS
SCLEROSIS COMPLEX (TSC) PATHWAYS
Newman J.V.1, Crampton E.M.1, Kozlov S.2 and Watters D.1
1
ESKITIS Institute for Cell and Molecular Therapies and School of
Biomolecular and Physical Sciences, Griffith University, Nathan
Campus, Kessels Rd. Brisbane, Australia, 4111. 2Queensland Institute
of Medical Research, Herston Rd. Brisbane, Australia, 4029.
Ataxia Telangiectasia is a progressive neurodegenerative disorder
caused by mutations in the ataxia telangiectasia mutated (atm) gene.
Nuclear ATM has a well established role in response to DNA damage,
however ATM has also been localized outside the nucleus where it
has been demonstrated to participate in the insulin signalling pathway
by phosphorylating eIF-4E binding protein(4EBP1). 4EBP1 is a target
of Mammalian target of Rapamycin (mTOR) and its phosphorylation
releases it from eIF-4E enabling translation of mRNA and protein
synthesis. The Tuberous Sclerosis Complex (TSC) proteins, hamartin
(TSC1) and tuberin (TSC2) act as a heterodimer to regulate mTOR
activity. mTOR exists as two complexes, mTORC1 (rapamycin
sensitive) and mTORC2 (insensitive to short term rapamycin). These
complexes control many cellular functions including protein synthesis,
autophagy, lipid metabolism, mitochondrial biogenesis and cytoskeletal
organisation. Mutations in either of the TSC1 or TSC2 genes lead to
Tuberous Sclerosis, an autosomal dominant, multisystem disorder
of benign tumour growth and neurological abnormalities. Studies in
our laboratory have demonstrated for the first time that ATM interacts
with both tuberin and hamartin. The effects of ATM deficiency on the
mTOR pathway under different growth conditions and stresses will be
described.
POSTERS WEDNESDAY & THURSDAY
POS-WED-105
THE VITAMIN E ANALOGUE γ-TOCOTRIENOL
BLOCKS OSTEOCALSTOGENESIS AND PROMOTES
OSTEOBLAST MATURATION
Odysseos A.D.1, 2 , Liapis V.3, Hay S.3, Christou Y.A.1, 2, Maltezou E.2,
Keramidas A.2 and Evdokiou A.3
1
EPOS-IASIS, R&D, Nicosia, Cyprus. 2University of Cyprus, Nicosia,
Cyprus. 3University of Adelaide, Royal Adelaide Hospital, Adelaide,
Australia.
In this study we investigated the effects of the vitamin E analogue,
γ-Tocotrienol on (a) apoptosis induction in bone metastatic cells and
(b) osteoclast differentiation and bone resorptive activity using three
independent in vitro-model systems of osteoclastogenesis. When human
peripheral blood mononuclear cells (PBMCs) and the RAW264.7 murine
monocytic cell line were cultured with the receptor activator of nuclear
factor kappa B-ligand (RANKL), both formation of tartrate-resistant
acid phosphatase (TRAP) positive multinucleated cells and bone
resorption were increased. In combination with RANKL, γ-Tocotrienol
dose-dependently inhibited RANKL-induced osteoclastic differentiation
and bone resorption in both cell models. Similarly, it inhibited the bone
resorptive activity of mature osteoclasts that were isolated from human
Giant Cell Tumours (GCT) of bone when cultured on dentine slices.
RANKL activated osteoclastogenesis essential pathways NFKB, ERK1/2
and p38/MAPK, whereas γ-Tocotrienol profoundly inhibited RAKNL-
induced activation of NFKB and P38/MAPK but not ERK1/ERK2.
The effect of γ-tocotrienol on osteoblast function was investigated in
mineralized bone nodule-forming primary human osteoblast cultures.
γ-Tocotrienol progressively increased matrix-containing mineralized
nodules in osteoblast cultures when compared to untreated cells with
a concomitant increase in alkaline phosphatase activity. Comparable
concentrations of γ-Tocotrienol induced profound caspase-independent
apoptosis in highly metastatic osteolytic and osteoblastic breast and
prostate cancer cell lines, respectively. Taken together these results
demonstrate for the first time that γ-Tocotrienol has a combined
inhibitory effect on osteoclast activity and on survival of bone metastatic
cancer cells whereas it significantly promotes osteoblast activity serving
as promising treatment for osteolytic bone disease.
POS-WED-107
A RANDOM shRNA SCREENING STRATEGY TO
IDENTIFY NOVEL DRUG TARGETS
Ramadas R.D.1, Dawes I.W.1 and Arndt G.M.2
1
School of Biotechnology and Biomolecular Science, University of
New South Wales, Sydney 2052, Australia. 2Children’s Cancer Institute
Australia, Lowy Cancer Research Centre, University of New South
Wales, Sydney 2052, Australia.
The main goal of the project is to devise a strategy to use a random
small hairpin RNA (shRNA) library to identify novel targets to study
disease mechanisms. This strategy can be used to identify modulators
of chemotherapeutic resistance, as well as to pinpoint key factors
involved in a variety of diseases such as viral infection. A random
shRNA library was constructed using an optimized enzymatic method,
which has as its starting point an oligonucleotide incorporating 19
random nucleotides. The shRNA library was cloned into a doxycycline-
dependent retroviral vector, and transduced into HEK293 cells by
calcium phosphate transfection to establish a pooled population of cells
containing individual members of the library. The random shRNA library
was successfully constructed after several modifications to increase
the amount of shRNA-encoding DNA inserts and consequently more
clones with greater representation. The doxycycline–dependent system
was characterized using a luciferase gene target, and associated
shRNA, and the most effective transduction conditions optimized. The
selected population of cells were then treated independently with three
common chemotherapeutic agents: cisplatin, carboplatin and paclitaxel.
Cells displaying resistance to these agents (surviving cells after shRNA
induction by doxycycline addition) were enriched and studies to
characterize the resident constructs are currently underway. It is hoped
that using a random shRNA library will aid in the identification of novel
factors that may be important in overcoming cancer drug resistance.
This strategy will also be expanded to identify previously unknown
factors contributing to viral infection.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 217
POS-THU-106
MUTATIONS IN THE BHLH/PAS TRANSCRIPTION
FACTOR SIM1 MAY UNDERPIN A NOVEL MONOGENIC
OBESITY DISORDER
Raimondo A.1, Ramachandrappa S.2, Farooqi S.2 and Whitelaw M.L.1
1
School of Molecular and Biomedical Science, University of Adelaide,
Adelaide, AUSTRALIA. 2Metabolic Research Laboratories, Institute of
Metabolic Science, Addenbrooke’s Hospital, University of Cambridge,
Cambridge, UK.
Single-Minded 1 (SIM1) is a member of the basic helix-loop-helix/
Per-ARNT-SIM (bHLH/PAS) family of transcriptional regulators that
possess diverse roles in development and homeostasis. Members
of this family heterodimerise with a shared partner protein, such as
ARNT1 or ARNT2, to regulate expression of their direct target genes.
Sim1-/- mice die perinatally due to hypodevelopment of hypothalamic
neuroendocrine populations critical for appropriate feeding control.
Sim1+/- mice, however, survive to adulthood and display a severe
early onset obesity phenotype characterised by hyperphagia,
hyperinsulinemia and hyperleptinemia, without any decrease in energy
expenditure. Similar characteristics have been reported in a number
of human clinical cases involving translocations or deletions within
the SIM1 locus. We have begun functional analysis of sixteen SIM1
point mutations recently identified in the UK Genetics of Obesity Study
(GOOS). These mutations are over-represented within the GOOS
cohort relative to control individuals, and are associated with severe
early onset obesity (<10 years old) with a BMI standard deviation score
>3. Several of these mutations reproducibly result in decreased SIM1
functionality in dual luciferase reporter assay experiments, and do not
appear to do so by altering protein stability. We are therefore pursuing
other experimental lines of evidence to determine whether these mutants
are compromised in their ability to dimerise with ARNT1 and ARNT2,
and/or regulate expression of endogenous SIM1 target genes. In this
way, we hope to highlight the molecular basis of a novel, heritable form
of severe monogenic obesity, which may facilitate the development of
effective treatments in patients associated with SIM1 mutations.
POS-THU-108
CRITICAL ROLE OF THE HEPATIC GLUCOCORTICOID
RECEPTOR IN SYSTEMIC BILE SALT HOMEOSTASIS
WHICH IMPACTS UPON CHOLESTEROL-GALLSTONE
DISEASE
Rose A.J., Berriel Díaz M., Reimann A. and Herzig S.
Molecular Metabolic Control Group, German Cancer Research Center,
Heidelberg, Germany.
Normal physiological bile salt handling in the fast-feeding cycle is
essential for proper digestion of ingested fat and impacts upon systemic
energy homeostasis. On the other hand, aberrant regulation of hepatic
bile salt flux can lead to cholesterol-gallstone disease. Our preliminary
data suggested that the glucocorticoid receptor (GR) can regulate
genes involved in hepatic cholesterol and bile salt metabolism. However,
it remained unclear whether hepatic GR action exerts a regulatory
function for systemic bile salt handling in vivo. Short-term liver-specific
GR knockdown resulted in higher serum bile salt levels as well as lower
gallbladder bile volume and cholesterol solubility, especially when mice
were fed a lithogenic diet. In addition, when long-term hepatocyte-
specific GR knockdown was imposed on gallstone susceptible mice and
these mice were fed a lithogenic diet, there was a higher incidence of
cholesterol gallstones. Furthermore, the aberrant bile salt homeostasis
was recapitulated in mice with deficient GR DNA binding activity
suggesting that it is the direct genomic actions of the GR that modulate
transcriptional events of key genes in the liver that lead to this phenotype.
Overall, using several different models of liver GR deficiency, we show
that reduction of hepatic GR action results in aberrant bile salt handling
and lower gallbladder bile cholesterol solubility. Together, our studies
establish the GR signalling axis as a critical regulatory checkpoint for
bile salt homeostasis and the susceptibility for cholesterol-gallstone
disease. We are currently investigating the transcriptional mechanisms
by which the GR affects liver bile salt handling.
POSTERS WEDNESDAY & THURSDAY
POS-WED-109
INDUCTION OF METALLOTHIONEIN IN CULTURED
HUMAN KERATINOCYTES BY ZINC: DETECTION
WITH A COMMERCIALLY AVAILABLE MONOCLONAL
ANTIBODY
Ross S. and Gray P.J.
Human Protection & Performance Division, DSTO Australia.
Metallothionein is a low molecular weight protein of around 6,000 Daltons,
which has a high capacity for binding metal cations. It is expressed in
bacteria, eukaryotes and plants. It exists as several isoforms, each being
60 - 63 amino acids long; 20 of these are cysteines. It is through the thiol
groups of these cysteines that the protein can bind multiple cations (up to
seven) of various metals, including zinc, copper, cadmium and mercury.
Metallothionein is thought to have an antioxidant function. There is also
evidence for it as a factor contributing to resistance of some tumour types
to alkylating chemotherapy agents. This protective function appears to
be more complex than simply acting as a sequesterer of nucleophilic
species. Increased levels of intracellular metallothionein in a normal
cell type was proposed as a potential pre-exposure countermeasure
to the vesicant agent sulphur mustard. This chemical agent is a potent
alkylating agent, similar in action to alkylating chemotherapy drugs
- therefore, increased intracellular metallothionein in a target cell
population might improve resistance to mustard toxicity. A direct ELISA
assay for metallothionein in cell extracts was developed to monitor
intracellular expression. Protein pre-loading with free metallothionein
proved difficult - however, incubation with zinc (as chloride) induced
an increase in intracellular metallothionein in cultures of normal human
keratinocytes. Zinc chloride has a cytotoxic effect on keratinocytes at
concentrations >50μuM; however, at 20 - 40μuM, increased expression
of metallothionein of to 14-fold increase compared to control levels
over seven days of incubation was achieved. Pulse-chase experiments
showed that intracellular levels of metallothionein returned to baseline
after zinc incubation was stopped.
POS-WED-111
AN ONCOGENOMICS-BASED IN VIVO CDNA SCREEN
PINPOINTS CO-AMPLIFIED CCND1 AND FGF19 AS
THERAPEUTIC TARGETS FOR LIVER CANCER
Sawey E.T.1, Cai C.1, Bakleh A.1, Liu Q.1, French D.M.2, Finn R.S.3,
Lowe S.W.1 and Powers S.1
1
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring
Harbor, NY USA 11724. 2Department of Pathology, Genentech
Incorporated, South San Francisco, CA USA 94080. 3Department of
Medicine, Geffen School of Medicine, University of California, Los
Angeles, CA USA 90095.
Liver cancer is a leading cause of cancer death worldwide. Although
several risk factors for liver cancer are known, the exact way in which
these factors cause normal liver cells to become cancerous is only
partially understood, making this one of the most difficult-to-treat
cancers. We aimed to establish a screen to identify genes that promote
liver tumorigenesis in order to better understand the molecular basis of
liver cancer. Using array CGH analysis of hepatocellular carcinomas
(HCCs), we identified genes within focal amplicons and introduced
corresponding cDNA clones into sensitized liver progenitor cells. We
discovered 18 genes that promote tumor formation, including 10 novel
oncogenes as well as CCND1 and its neighbor FGF19. These latter two
genes, which are co-amplified in 15% of HCCs, cooperated in malignant
transformation and formed a feed-forward network whereby Fgf19
signaling increases CyclinD1 expression. RNAi-mediated inhibition
of CCND1 or FGF19 inhibited clonogenicity and tumorigenicity of
amplified HCC cells, while having no phenotypic effect in non-amplified
cells. Amplification of CCND1/FGF19 also predicted response to a
neutralizing monoclonal antibody directed against Fgf19. The result of
this work has been the identification of novel oncogenes in HCC. In
addition, these results show that gene amplification predicts response
to Fgf19 inhibition, and that oncogenomic cDNA screens are an effective
new tool to identify therapeutic strategies that are directly linked to
cancer genotypes.
Page 218 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-110
PHARMACOLOGICAL SCREENING FOR THE THERAPY
OF FRIEDREICH ATAXIA
Li L.1, Van Den Hengel S.2, Cook J.1, Sandi C.4, Pook M.4, Ioannou P.A.2, 3,
Delatycki M.B.1, 3 and Sarsero J.P.1, 2, 3
1
Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens
Research Institute, Royal Children’s Hospital, Parkville, Victoria
3052, Australia. 2Cell and Gene Therapy Research Group, Murdoch
Childrens Research Institute, Royal Children’s Hospital, Parkville,
Victoria 3052, Australia. 3Department of Paediatrics, The University
of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052,
Australia. 4Hereditary Ataxia Research Group, Centre for Cell and
Chromosome Biology, School of Health Sciences and Social Care,
Brunel University, Uxbridge UB8 3PH, United Kingdom.
Friedreich ataxia (FRDA) is characterised by neurodegeneration and
cardiomyopathy. The presence of a GAA trinucleotide repeat expansion
in the first intron of the FXN gene results in the inhibition of gene
expression and an insufficiency of the mitochondrial protein, frataxin.
There is a correlation between expansion length, the amount of residual
frataxin and the severity of disease. As the coding sequence is unaltered,
pharmacological upregulation of FXN expression may restore frataxin to
therapeutic levels in patients. To facilitate screening of compounds that
modulate FXN expression in a physiologically relevant manner, we have
established a genomic reporter assay consisting of stable HeLa cells
containing an FXN-EGFP fusion construct (in-frame fusion of the EGFP
gene with the entire normal human genomic FXN locus on a BAC clone).
The cell line was used to established a fluorometric cellular assay for
use in high throughput screening (HTS) procedures. A small chemical
library of FDA-approved compounds and natural extracts has been
screened and analysed. Compound hits identified by HTS have been
further evaluated by flow cytometry in the cellular genomic reporter
assay. The effects on FXN mRNA and frataxin protein levels have been
measured in cell lines derived from individuals with FRDA and in a GAA
expansion mouse model of FRDA. Any compound that specifically
increases frataxin levels by several-fold in FRDA patients could serve
as a potential pharmacological therapy for Friedreich ataxia.
POS-THU-112
ROLE OF EXOGENOUS ADENOSINE IN CARDIAC
REPERFUSION INJURY UNDER HIGH GLUCOSE
STRESS
Sermsuvitayawong K.1 and Wilairat P.2
1
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot
University. 2Department of Biochemistry, Faculty of Science, Mahidol
University.
The role of exogenous adenosine (0.5-1.0 mM) in regulating the
protective pathway in ischemic reperfusion injury remains unclear. Rat
cardiac myoblast H9C2 cells following 21 hr of hypoxia (1% oxygen)
and 3hr of normoxic condition in the presence of 25 mM glucose and
1 mM adenosine in DMEM demonstrated elevated apoptosis through
up-regulation of pro-apoptotic Bnip3 but not Bax and Nix compared
to normoxic condition (with high glucose but without the addition of
adenosine), a phenomenon not observed with 0.1 mM adenosine. These
results indicate that using high concentration of exogenous adenosine in
protecting against ischemic reperfusion injury may be contra-indicated.
Keywords: Reperfusion injury, high glucose, adenosine, Bnip3.
POSTERS WEDNESDAY & THURSDAY
POS-WED-113
PENTACYCLIC TRITERPENOID LANTADENE A:
ANTITUMOR ACTIVITY AND EXPRESSION OF
TRANSCRIPTION FACTORS
Sharma M.1 and Sharma N.2
1
Jaypee University of Information Technology. 2Maharaja Singh
College.
Lantadene A (LA, 22β-angeloyloxy-3-oxoolean-12-en-28-oic acid) a
pentacyclic triterpenoid isolated from leaves of obnoxious weed Lantana
camara L. was evaluated for its antitumor activity. LA showed apoptosis
in human leukemia HL-60 cell line. Typical morphological changes
including cell shrinkage, chromatin condensation and characteristic
DNA ladder formation in agarose gel electrophoresis were observed.
LA induced marked concentration and time dependant inhibition of
cancer cell proliferation with IC50 value of 19.8 ± 0.10 &956;g/ml
following 48h incubation. Flow cytometric analysis showed suppressed
cell proliferation associated with cell cycle arrest in the G0/G1 phase.
LA significantly inhibited cell proliferation of HL-60 cells and induced
cell apoptosis through down regulating Bcl-2 and up regulating Bax
expression. The peptidic caspase-3 inhibitors DEVD-CHO (NH2-Asp-
Glu-Val-Asp-CHO, 2µM), increased the viability of HL-60 cells, previously
treated with LA. The LA was further evaluated for its tumor suppression
potential in DMBA/TPA induced squamous cell carcinogenesis in
Swiss albino mice. A significant decrease in the incidence of number
of lesions in mice was observed in LA treated groups as compared to
DMBA/TPA alone. The RNA of tumors were isolated, after 20 weeks
study and evaluated for various signaling factors like p53, p65 and
c-jun. Significant increase in the protein levels of c-jun, p65, and p53 by
ELISA were observed in DMBA/TPA treated mice tumors whereas less
expression was observed in LA treated tumors. Immunohistochemical
localization of transcription factors was studied which also showed less
localization of c-jun, p65, and p53 in LA treated tumors as compared to
localization in DMBA/TPA treated tumors.
POS-WED-115
CARDIOPROTECTIVE EFFECT OF VASOENDOTHELIAL
GROWTH FACTOR AT MYOCARDIAL INFARCTION
Shurygin M.G., Shurygina I.A., Dremina N.N. and Kanya O.V.
Scientific Center of Reconstructive and Restorative Surgery SB
RAMS.
The aim is to investigate regulatory effects of vasoendothelial (VEGF)
in a reparation process by an example of experimental myocardial
infarction. Female Wistar rats (N=85, weight 220-250 g) at the age of
9 month were selected in accordance with the “Guide for the Care and
Use of Laboratory Animals” (NIH publication 85-23, revised 1985). The
experiment was carried out in accordance with the principles of humane
treatment of animals set out in the directives of the European Community.
We used myocardial infarction model with diathermocoagulation of
coronary artery. The study comprised two groups of animals: “VEGF”
(N=40), “Control” (N=45). Animals from “VEGF” group had 100 ng
VEGF164 (Sigma V3638-10UG Lot 064K1239) injection into left ventricle
at a 1.5 h period after operation. We used methods of light microscopy,
immunohistochemistry, morphometry. We found out that the high VEGF
level extended infiltrative phase of inflammation. VEGF have increased
the synthesis of collagen. The collagen volume on 30 day in “VEGF”
group was higher than in “Control” group (58.56 vs 40.51%, p<0.05).
At the same time, excessively high concentrations of VEGF led to the
development of focal cardiosclerosis in the intact myocardium, which is
a by-effect of this growth factor. VEGF provided a significant effect on
angiogenesis. We discovered that the injection of intracardiac VEGF
experimental animals had increased survivability of cardiomyocytes in
the infarction zone. VEGF has cytoprotective effect on cardiomyocytes.
Thus, VEGF is important regulators of the reparation process in a
myocardial infarction. This study was supported by a grant P1250
from the Russian Federal Purposive Program “Scientific and scientific-
pedagogical staff of innovation Russia”.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 219
POS-THU-114
CHARACTERISATION OF BCHE POLYMORPHISMS IN
SMALL AUSTRALIAN DEFENCE FORCE COHORT
Shields K.A. and Lewis J.
Defence Science and Technology Organisation, 506 Lorimer St.
Fishermans Bend, Vic 3207, Australia.
There is a wide range of chemicals and drugs that interact with the
cholinergic nervous system including: nerve agents, nerve agent
pre-treatments, insecticides and therapeutic drugs. Sensitivity to
cholinesterase inhibitors and effective nerve agent pre-treatment rely
on the activity of ACHE, BCHE and PON1, and these genes are subject
to genetic variation. Genetic variations in these genes have been shown
to alter the level and activity of their enzyme products and therefore
effective treatment may therefore be influenced by the genotype of the
individual. BCHE is a high affinity stoichiometeric scavenger of toxic
organophosphate (OP) compounds that has a role in protection against
OP exposure. Individuals carrying heterozygous or homozygous
inactivating alleles have been shown to have a lower capacity to interact
with and detoxify drugs. Our initial work has focused on characterisation
of a small cohort (n=52) of Australian Defence Force (ADF) personnel.
Using the LightCycler 480 system, a protocol was established using
High-Resolution Melting Curve Analysis to screen the BCHE gene. Any
PCR amplicons displaying altered melting profiles were subjected to
direct sequencing and variants were identified. Initial screening has
identified both known and novel polymorphisms and future work will
involve the functional characterisation of novel variants. In summary this
work provides important information in the characterisation of genetic
variation in an ADF population. It is likely that functional variants unique
to Australian populations exist and that these will need to be identified
in order to accurately predict chemical and drug response.
POS-THU-116
RECOMBINANT EXPRESSION AND
CHARACTERISATION OF PROTEASE INHIBITOR-
RESISTANT TRYPSINS FROM THE MIDGUT OF H.
PUNCTIGERA
Stevens J.A., Dunse K.M. and Anderson M.A.
Department of Biochemistry, La Trobe University, Melbourne VIC
3086.
Protease inhibitors (PIs) from the flowers of Nicotiana alata (NaPI)
show promise for the control of Helicoverpa insect pests. This has
been demonstrated using artificial diet bioassays and by expressing
NaPI in transgenic cotton plants. PIs function by blocking the digestive
proteases in the larval gut, limiting the availability of amino acids
from dietary protein. As a consequence the larvae are arrested in
development and eventually die. In addition some larvae adapt to the
presence of serine PIs in the diet by over-production of PI-insensitive
trypsins and chymotrypsins. This induction of protease expression is
a common mechanism by which herbivorous insects overcome plant
PI defenses. Even though trypsins are the major digestive proteases
of Helicoverpa species and have been a prime target for pest control,
little is known about their production, regulation and structure. We have
identified a family of PI-resistant trypsins that are produced in the gut of
H. punctigera after ingestion of serine PIs. Recombinant expression of
these trypsins is underway to provide insights into the structural features
that prevent PI inhibition. A detailed understanding of the interaction
between insect trypsin and plant PIs will assist in the rational design of
new inhibitors to protect crops from insect damage.
POSTERS WEDNESDAY & THURSDAY
POS-WED-117
CHEMOKINE BINDING TO SULFOTYROSINE RESIDUES
IN CHEMOKINE RECEPTORS
Stone M.J.1, Millard C.J.1, Clayton D.1, Zhu J.Z.2, Simpson L.S.2,
Widlanski T.S.2 and Payne R.J.3
1
Monash University. 2Indiana University. 3University of Sydney.
The trafficking of leukocytes to sites of inflammation is orchestrated by
the interactions of chemokines with chemokine receptors. Chemokine
receptors commonly contain sulfated tyrosine residues in their amino-
terminal regions, which are critical for recognition of their chemokine
ligands. Eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 are
chemokines involved in recruitment of eosinophils, basophils and Th2
cells through activation of the receptor CCR3. The amino-terminal region
of CCR3 contains two adjacent putatively sulfated tyrosine residues.
In this study, we have investigated the role of tyrosine sulfation of
N-terminal CCR3 peptides in controlling binding affinity and selectivity
for its cognate chemokines. Peptides containing sulfotyrosine were
generated by applying a novel sulfate protection strategy in Fmoc-based
solid-phase peptide synthesis. We prepared four peptides containing
sulfation on Tyr-16, Tyr-17, both Tyr residues, or neither Tyr residue. NMR
spectroscopy was then used to determine the affinity of each peptide for
each chemokine and to identify the chemokine surface interacting with
each peptide. Although all three chemokines utilise the same surface for
binding to CCR3-derived sulfopeptides, the energetic effect of sulfation
of the two adjacent tyrosine residues was either additive or cooperative
for different chemokines. The structure of a chemokine-sulfopeptide
complex obtained using isotope-filtered/edited NMR experiments will
be presented.
POS-WED-119
MARCKS EXPRESSION DURING INFLAMMATORY
PROCESS IN LIVER FLUKE-ASSOCIATED
CHOLANGIOCARCINOMA
Techasen A.1, 2 , Loilome W.1, 2, Namwat N.1, 2, Thanan R.1, 2 and
Yongvanit P.1, 2
1
Department of Biochemistry, Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand. 2Liver Fluke and Cholangiocarcinoma
Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen
40002, Thailand.
Myristoylated alanine rich C kinase substrate (MARCKS) has been implicated
in PKC-mediated membrane-cytoskeletal alterations which underlines
such LPS-induced macrophages responses. Our previous study found
that MARCKS was significantly overexpressed in CCA and inflammatory
cells. It is therefore postulated that MARCKS may involve in inflammation-
associated CCA. The aims of this study are to investigate localization
pattern of MARCKS in hamster tissue during cholangiocarcinogenesis and
examine the involvement of MARCKS in inflammation by determining the
expression and localization in Raw 264.7 macrophage cell line. The semi-
quantitative real time RT-PCR and immunofluorescence analysis were
performed to determine MARCKS mRNA level and protein localization
in hamster tissues at different time points after administrationwith either
liver fluke (Opisthorchis viverrini, Ov) or N-nitrosodimethyamine (NDMA)
or both. The expression of MARCKS induced by inflammation was also
confirmed by incubating Raw 264.7 macrophage cell line with LPS or crude
Ov antigens. Moreover, co-expresssion between MARCKS and MAC387
which is a marker for macrophage was also investigated in human CCA
tissues. Our study showed the moderate to strong expression of MARCKS
in inflammatory cells at early stage and also in bile duct cancer at the end
stage of cholangiocarcinogenesis in hamster. In addition, MARCKS mRNA
and protein levels were also induced by stimulation of LPS or Ov crude
extract as a dose- and time- dependent manner in Raw 264.7 macrophage
cell line. Moreover, the co-expression between MARCKS and MAC387
was observed in human CCA tissues. This finding can be concluded that
MARCKS was associated with inflammation-related CCA induced by Ov.
Further study on functional analysis of MARCKS is under investigation.
Page 220 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-118
ANDROGEN REGULATION OF P27 IN BREAST
CANCER CELLS
Tay J.W.1, 2 , Azzam D.G.1, 2, Harvey J.2 and Bentel J.M.1
1
Department of Anatomical Pathology, Royal Perth Hospital. 2School of
Pathology and Laboratory Medicine, University of Western Australia.
p27, an important cell cycle inhibitor that regulates the G1/S checkpoint
is frequently deregulated in breast cancers where its expression is
downregulated or the protein is mislocalised to the cytoplasm. Treatment
of MCF-7 breast cancer cells with the androgen, 5α-dihydrotestosterone
(DHT) inhibits cell proliferation in association with increased p27 levels.
Approximately 30% of breast cancers exhibit increased activity of
kinase signalling pathways and are resistant to conventional therapies,
however DHT treatment of stably transfected MCF-7 cells (Δ6B) with
elevated extracellular signal-regulated kinase (ERK1/2) activity inhibits
proliferation and increases p27 levels. The function of p27 as a cell
cycle inhibitor is dependent on its localisation in the nucleus, and the
intracellular localisation of p27 is regulated via its phosphorylation by
kinases such as AKT and ERK1/2. DHT treatment of the androgen
receptor (AR) expressing breast cancer cell lines, MCF-7, MT3, Δ6B,
T47-D, BT-474 and MDA-MB-453 results in the nuclear accumulation of
both endogenous p27 and transiently transfected GFP-p27. Increased
cytoplasmic GFP-p27 is observed in Δ6B and BT474 cells with high
ERK1/2 activity, potentially as a direct or indirect consequence of
ERK1/2 phosphorylation of p27, however DHT treatment also enhances
p27 nuclear localisation in these cells. These findings indicate that in
addition to increasing p27 protein levels, AR activation promotes the
nuclear accumulation of p27 to initiate the inhibition of cell proliferation.
Subcellular fractionation and MALDI-TOF analysis of p27 in breast
cancer cells with normal or hyperactivated ERK1/2 signalling and
following DHT treatment will identify novel phosphoresidues that direct
p27 nuclear localisation and its cell cycle inhibitory function, thereby
identifying potential therapeutic targets for human breast tumours.
POS-THU-120
CONSTRAINTS WITHIN MAJOR-HISTOCOMPATIBILITY
COMPLEX CLASS I –RESTRICTED PEPTIDES:
PRESENTATION AND CONSEQUENCES FOR T-CELL
RECOGNITION
Theodossis A.1, Guillonneau C.2, Welland A.1, Ely L.K., Clements C.S.1,
Williamson N.A., Webb A.I.3, Doherty P.C.2, McCluskey J.2, Purcell
A.W.3, Turner S.J.2 and Rossjohn J.1
1
The Protein Crystallography Unit, Department of Biochemistry and
Molecular Biology, School of Biomedical Sciences, Monash University,
Victoria 3800, Australia. 2Department of Microbiology and Immunology,
University of Melbourne, Victoria 3010, Australia. 3Department of
Biochemistry and Molecular Biology, Bio21 Molecular Science and
Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
Residues within processed protein fragments bound to major
histocompatibility complex class I (MHC-I) glycoproteins have been
considered to function as a series of “independent pegs” that either anchor
the peptide (p) to the MHC-I and/or interact with the spectrum of αβ-T-
cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining
of the extensive pMHC-I structural database established that many self-
and viral-peptides show extensive and direct inter-residue interactions, an
unexpected finding that has led us to the idea of “constrained” peptides.
Mutational analysis of two constrained peptides (the HLA B44-restricted
self-peptide (B44DPα - EEFGRAFSF) and an H2-Db-restricted influenza
peptide (DbPA, SSLENFRAYV) demonstrated that the conformation
of the prominently exposed arginine in both peptides was governed by
interactions with MHC-I-orientated flanking residues from the peptide
itself. Using reverse genetics in a murine influenza model, we revealed
that mutation of an MHC-I-orientated residue (SSLENFRAYV →
SSLENARAYV) within the constrained PA peptide resulted in a diminished
cytotoxic T-lymphocyte (CTL) response and the recruitment of a limited
pMHC-I specific TCR repertoire. Interactions between individual peptide
positions can thus impose fine control on the conformation of pMHC-I
epitopes, while the perturbation of such constraints can lead to previously
unappreciated mechanism of viral escape.
POSTERS WEDNESDAY & THURSDAY
POS-WED-121
EVALUATION OF PROSTATE CANCER ANTIGEN-1 AS A
MOLECULAR TARGET FOR PROSTATE CANCER AND
PANCREATIC CANCER
Tsujikawa K.1, Koike K.1, Hase T.1, Kitae K.1, Ueda Y.1, Shimada K.2,
Syo M.2, Konishi N.2, Nakajima Y.2 and Yamamoto H.1
1
Grad. Sch. Pharm. Sci., Osaka University, Osaka 565-0871, Japan.
2
Nara Medical University, Nara 634-8521, Japan.
We found a novel gene prostate cancer antigen (PCA)-1 that was
highly expressed in prostate carcinoma cells but not in the adjacent
normal tissues. Immunohistochemical analysis of prostate carcinoma
specimens showed that PCA-1 was strongly expressed in prostate
cancer lesions, including preneoplastic lesions, but there was little or
no expression in normal epithelium or benign prostatic hyperplasia.
Recently, high expression of PCA-1 was also detected in pancreatic
adenocarcinoma specimens. To evaluate PCA-1 as a molecular target
for these cancers, therefore, we knocked down the PCA-1 gene in both
prostate cancer cell lines (DU145 and PC3) and pancreatic cancer cell
lines (Punc-1 and MIA-Paca-2) by using the RNA interference technique.
Transfection of PCA-1 small interfering RNAs suppressed PCA-1
mRNA expression and then inhibited proliferation of these cancer cells
by apoptosis. Colony formation in soft agar was significantly inhibited
in DU145 and PC3 cells in which PCA-1 was stably knocked down.
Real-time PCR analysis indicated that PCA-1 knockdown suppressed
the expression of XIAP and urokinase-type plasminogen activator in
these cell lines. Moreover, immunoblot analysis indicated that PCA-1
knockdown induced dephosphorylation of Akt in the prostate cancer cell
lines, suggesting that PCA-1 is an important regulatory molecule in the
Akt signaling pathway. Furthermore, the administration of PCA-1 siRNA
markedly attenuated tumor formed by DU145 cells and Panc-1 cells in
nude mouse xenograft in vivo. These results indicate that PCA-1 may be
a promising molecular target for prostate cancer as well as pancreatic
cancer.
POS-WED-123
LUNG METASTATIC CHOLANGIOCARCINOMA CELL LINE
ACQUIRES HIGHER LEVEL OF CD133 EXPRESSION AND
EXHIBITS HIGHER PROLIFERATION IN VIVO
Vaeteewoottacharn K.1, 2 , Seubwai W.1, 2, Wongkham S.1, 2 and Okada S.3
1
Department of Biochemistry, Faculty of Medicine, Khon Kaen
University, Khon Kaen, Thailand. 2Liver Fluke and Cholangiocarcinoma
Research Center, Faculty of Medicine, Khon Kaen University, Khon
Kaen, Thailand. 3Division III, Center for AIDS Research, Kumamoto
University, Kumamoto, Japan.
While cancer stem cell (CSC) is rapidly progressed, the information
regarding CSC in cholangiocarcinoma (CCA), cancer of bile duct, is
limited. CCA is a rare cancer worldwide but the incidence is highest in
Thailand particularly in the Northeastern part. With a major risk factor of
liver fluke (Opisthorchis viverini) infection, CCA in Thai patients exhibit
unique genetic signatures when compared to CCA from other causes.
Even though CCA is a slow growing, early diagnosis is difficult due to
lacking of cancer associated symptoms and specific markers. In this
study, we aimed to identify the expressions of cancer stem cell markers
in CCA cell line, KKU-M214 and its lung metastatic cell line, M214L5,
and characterize cells with particular marker in vivo. Six markers (CD29,
CD34, CD44, CD90, CD117 and CD133) that have been successfully
detected in several CSC including hepatocellular carcinoma were
detected in this study. Among 6 markers, only CD44 and CD133
were differentially expressed in both cell lines. We found increased
expression of CD133 in KKU-M214L5 when compared to KKU-M214.
When CD133 expressed M214L5 cells (CD133+ M214L5) and CD133
non-expressed M214L5 (CD133- M214L5) were separated and were
subsequently injected subcutaneously into NOD/SCID Jak3 deficient
mice, tumors were detected from both CD133+ M214L5 and CD133-
M214L5 injected groups. However, tumors derived from mice injected
with CD133+ M214L5 were larger than tumor derived from CD133-
counterpart. These results indicated that CD133 in liver fluke associated
CCA might be a potential prognostic marker. Further validation is being
investigated.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 221
POS-THU-122
BIOMARKERS DISCOVERY IN THE NEOADJUVANT
BREAST CANCER TREATMENT
Tvrdik D.1, Povysil C.1, Dundr P.1, Velenska Z.1, Melcakova S.1, Skalova
H.1 and Petruzelka L.2
1
Institute of Pathology, 1st Faculty of Medicine, Charles University
and General University Hospital in Prague. 2Department of Oncology,
1st Faculty of Medicine, Charles University and General University
Hospital in Prague.
Neoadjuvant chemotherapy, also called preoperative chemotherapy, is
now widely used in the treatment of breast carcinoma. It has several
potential advantages compared to the traditional strategy of surgery
followed by adjuvant chemotherapy. Neoadjuvant chemotherapy
substantially reduces the size of the primary tumor and lymph node
metastasis in more then 80% of cases, increasing the probability that
breast-conserving surgery can be performed instead of mastectomy. The
study is aimed at investigation of the biomarkers in predicting pathologic
response to the therapy and include 10 patients with histologically
proven invasive breast cancer. Transcriptional profile of the biomarkers
were determined by DNA microarray technology with biochips including
168 human genes associated with apoptosis and multidrug resistance.
The quantification of the changes in expression of individual genes due
to the treatment was performed by qRT-PCR. In each case, analysis
of transcription profile was performed before and after the treatment,
respectively. In good prognosis group, based on pathological features
and clinical outcome, the analysis showed that the expression of FAS,
caspase 14 and DFFA were significantly up-regulated at the RNA level
after the treatment. In contrast, the expression of BCL2-related gene
MCL1 was down-regulated due to the treatment. On the other hand, in
poor prognosis group, we found over-expression of ABCC gene. As we
have shown, gene expression profiling using microarray or Real-Time
PCR assay is a valuable research tool for investigation of molecular
markers and prognostic factors which may better reflect tumor biology
and treatment response. This work was supported by Grant IGA MZ CR
No. NS10575-3/2009.
POS-THU-124
CHARACTERISATION OF PROTEIN
DISULPHIDE ISOMERASE FAMILY MEMBERS IN
NEURODEGENERATIVE DISEASES
Walker A.K.1, 2, 3, Farg M.A.1, Turner B.J.2, Soo K.Y.1, Horne M.K.2, 3, 4
and Atkin J.D.1, 2, 3
1
Department of Biochemistry, La Trobe University, Victoria. 2Howard
Florey Institute, University of Melbourne, Victoria. 3Centre for
Neuroscience, University of Melbourne, Victoria. 4Department of
Neurology, St Vincent’s Hospital, Victoria.
Protein disulphide isomerase (PDIA1) is the prototypical member of a
19-member family of human disulphide bond-modulating chaperone
proteins. Recently, differences in substrate specificity and expression
patterns of PDI family members have been identified, suggesting
different responsibilities for each protein. Amyotrophic lateral sclerosis
(ALS) is a fatal neurodegenerative disease primarily affecting motor
neurons. PDIA1 is up-regulated and shows sub-cellular redistribution
in spinal cords of ALS mouse models and human patients, and PDIA1
function is compromised in Alzheimer’s and Parkinson’s disease brains.
In neuronal cell culture, PDIA1 over-expression decreases aggregation
and toxicity associated with mutant superoxide dismutase 1 (SOD1),
which causes a subset of familial ALS, and a small molecule mimic
of the PDI active site protects motor neurons against mutant SOD1
misfolding. The largely uncharacterised family member PDIp/PDIA2
is up-regulated in Parkinson’s disease models, while ERp57/PDIA3 is
protective against misfolded prion protein and is also up-regulated in
ALS. Here we report further investigations of the protein interactions and
functions of PDI family members relevant to neurodegenerative disease.
In ALS patient spinal cord tissue, PDIA1 was found to be modified by
S-nitrosylation, which causes inactivation of the protein by inhibition of
critical active site cysteine residues. Furthermore, PDIA2 expression
was identified in spinal cord in contrast to earlier reports of pancreas-
specific expression, indicating possible involvement of this PDI family
member in ALS. These results suggest that PDIA1 and other PDI family
members are important for modulation of protein aggregation and cell
death pathways in ALS and other neurodegenerative diseases.
POSTERS WEDNESDAY & THURSDAY
POS-WED-125
EXPRESSION AND SPECIFIC ACTIVITY OF MANGANESE
SUPEROXIDE DISMUTASE (MnSOD) IN RAT BREAST AND
BLOOD DURING CHEMICAL CARCINOGESIS INDUCED
BY DIMETHYLBENZ(A)ANTHRACENE
Wanandi S.I.1, Nida K.2, Wuyung P.E.3 and Dewi S.1
1
Department of Biochemistry and Molecular Biology, Faculty of Medicine
University of Indonesia, Salemba Raya No. 6, Jakarta 10430, INDONESIA.
2
Master Program in Biomedical Sciences, Faculty of Medicine, University
of Indonesia, Salemba Raya No. 6, Jakarta 10430, INDONESIA.
3
Department of Pathology Anatomy, Faculty of Medicine University of
Indonesia, Salemba Raya No. 6, Jakarta 10430, INDONESIA.
Manganese superoxide dismutase (MnSOD) is a major antioxidant in
matrix mitochondria which plays a pivotal role on the prevention of oxidative
stress. Until now, data about the expression and activity of MnSOD in
various cancer cells are still controversial. Several studies have suggested
that MnSOD is a tumor suppressor, whereas others have reported that the
increase of MnSOD expression in cancer cells indicates a bad prognosis.
The aim of this study is to unravel the role of MnSOD during early stages of
chemical carcinogenesis. In this study, 4 mg of DMBA (7,12-dimethylbenz(a)
anthracene), a chemical carcinogen, was given twice a week for 1, 2, 3, 4,
or 5 weeks to induced breast cancer in 30 rats. Breast tissues and blood
were taken as samples and analyzed for MnSOD specific activity using
xantin oxidase inhibition assay and mRNA expression using Real Time
RT-PCR, as well as for MDA (malone dialdehyde) level using TBA assay.
Histopathology of breast tissue and determination of sialic acid level in
breast and blood resulted in gradual breast carcinogenesis. MDA level in
breast was significantly increased (5.1 – 9.3 times) during carcinogenesis
compared to that in plasma, indicating that DMBA specifically induced lipid
peroxidation in breast cancer. The mRNA expression of MnSOD in breast
was significantly enhanced (2.8 – 9.7 times) during the DMBA induction.
However, its specific activity was slightly enhanced (1.4 times) only during
the first week and then gradually decreased. Therefore, we could conclude
that the gene expression of MnSOD is up regulated during early stages
of chemical carcinogenesis in order to increase the antioxidant activity
which is suppressed by the oxidative stress. Further studies are required to
analyze the signal transduction pathway of MnSOD gene expression during
chemical carcinogenesis.
POS-WED-127
HEPCIDIN-INDEPENDENT IRON RECYCLING IN A
MOUSE MODEL OF HAEMOLYTIC ANAEMIA
Wilkins S.J., Frazer D.M., Darshan D., Whitelaw N.C., Whitelaw E.
and Anderson G.J.
The Queensland Institute of Medical Research.
The liver-derived peptide hepcidin plays a central role in body iron
homeostasis by inhibiting cellular iron export. Stimulated erythropoiesis
reduces hepcidin expression which in turn increases macrophage
iron release and intestinal iron absorption. However, in certain chronic
haemolytic anaemias, iron absorption is not elevated despite a high
erythropoietic rate. To investigate this in more detail, we studied the
MommeD7 mouse, a novel model of chronic haemolytic anaemia,
and compared it to mice with acute haemolytic anaemia induced by
phenylhydrazine (PHZ). MommeD7 carries a dominant ENU-induced
mutation, but the gene affected is unknown. Liver, spleen, duodenum and
blood were collected from six week old MommeD7 mice, from littermate
controls and from age-matched wild-type mice three days after PHZ
treatment. In both the chronic and acute anaemias, raised reticulocytes
and an enlarged spleen suggested stimulated erythropoiesis, and we
confirmed enhanced plasma iron turnover by demonstrating increased
plasma iron clearance in PHZ-treated animals. Despite increased iron
turnover, the expression of hepcidin and intestinal iron transporter
mRNAs was unaltered in MommeD7 mice, and hepatic iron levels
were normal. In contrast, PHZ treatment, a stronger erythropoietic
stimulus, led to a reduction in hepcidin expression, increased duodenal
iron transporter expression, increased iron absorption and liver iron
accumulation. These data suggest that in low-grade chronic haemolytic
anaemia, such as that in MommeD7 mice, the increased erythroid iron
requirements can be met through enhanced macrophage iron release
without the need to increase iron absorption. Since this increased iron
recycling can occur without changes in hepcidin, the data imply that
hepcidin is not the sole regulator of macrophage iron release.
Page 222 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-126
MEASUREMENT OF LIVER ALKALINE PHOSPHATASE
IN HUMAN SERUM BY IMMUNOASSAY
Wang H.T., Kim H.J., Kim T.K. and Choi E.Y.
Dept. of Biomedical Science, Hallym University, Chuncheon, Korea.
Alkaline Phosphatase(ALP) is a hydrolase enzyme responsible for
removing phosphate groups from many types of molecules, including
nucleotide, protein, and alkaloid. ALP levels in plasma will rise when
people get liver or bone disease, so ALP can be a marker for liver disease
or bone disease. Now, almost all kinds of ALP assay are used to check
total alkaline phosphatase in blood. To measure liver ALP isoenzyme
mass directly, we want to develop an immunoassay to get our goal.
Firstly, gene cloning technique was used to get positive recombinant
which can express liver ALP in E.coli; Secondly, purified liver ALP was
injected into mice to make monoclone Ab against ALP; Thirdly, ALP Ab
was labelled; Currently, we are going to check liver ALP level in serum.
Key words: liver alkaline phosphatase monoclone immunoassay.
POS-THU-128
SECRETED ADIPOCYTE FATTY ACID BINDING
PROTEIN (FABP4) FORMS THE MOLECULAR BASIS OF
THE ADIPO-INSULIN AXIS
Wu L.E.1, 2 , James D.E.1 and Cantley J.1
1
Diabetes and Obesity Program, Garvan Institute of Medical Research.
2
Bioanalytical Mass Spectrometry Facility, University of New South
Wales.
Adiposity is closely linked with fasting insulin levels and β-cell mass.
We hypothesised this to be a result of an inter-organ communication
between adipose tissue and the β cell, which may involve a secretory
component. Using in vitro models, we showed that treatment of
pancreatic islets with conditioned media from hypoxic, but not normoxic
control 3T3-L1 adipocytes led to an increase in glucose stimulated
insulin secretion of 70%. A quantitative mass spectrometry screen
was performed to identify hypoxia regulated adipokines. The adipocyte
fatty acid binding protein FABP4 was one of the few secretory proteins
that increased in conditioned media during hypoxia. Western blotting
confirmed increased secretion of FABP4 from adipocytes during
hypoxia. Serum concentrations of FABP4 were raised in high fat fed
mice, along with circulating insulin concentrations. In vitro treatment
of islets with physiologically relevant concentrations of recombinant
FABP4 enhanced glucose stimulated insulin secretion to degree similar
to hypoxic adipocyte conditioned media. Hypoxia inhibits mitochondrial
function, and the mitochondrial poisons DNP and oligomycin also
stimulated FABP4 secretion. Treatment of adipocytes with insulin during
hypoxia reduces FABP4 secretion. Removal of glucose from media
and substitution with pyruvate reverses inhibition of secretion during
hypoxia by insulin, indicating that the primary role of FABP4 secretion is
to alleviate energy crisis by increasing glucose uptake into fat through
increased insulin secretion. Taken together, these data are indicative of
an adipo-insulin axis mediated by FABP4, with the aim of coordinating
insulin secretion and glucose uptake in response to the prevailing needs
of adipose tissue.
POSTERS WEDNESDAY & THURSDAY
POS-WED-129
THE GASTRIN GENE IN HUMAN GASTRIC CANCER
CELLS IS TRANSCRIPTIONALLY ACTIVATED BY
HYPOXIA
Xiao L., Kovac S., Baldwin G. and Patel O.
The Department of Surgery, Austin Health, The University of
Melbourne, Heidelberg, VIC, Australia 3084.
Background and Aim: Hypoxia affects the transcription of approximately
1.5% of genomic genes in solid tumour cells via the activation of the
transcription factor, hypoxia-inducible factor 1 (HIF-1). Gastrin and its
precursors have been shown to promote mitogenesis and angiogenesis
in gastrointestinal cancers. However the effect of hypoxia on gastrin
gene regulation is largely unknown. Here we have investigated the effect
of hypoxia on transcription of the gastrin gene in human gastric cancer
(AGS) cells. Methods: Gastrin promoter activity under hypoxia was
examined using the dual-luciferase reporter assay, and gastrin mRNA
was measured by real-time PCR. Results: Hypoxia induced by either
low oxygen tension (0.5% oxygen) or 300µM cobalt chloride (CoCl2)
increased the gastrin promoter activity in AGS cells overexpressing the
gastrin receptor (AGS-CCK2R) by 2.4 ± 0.2 and 3.3 ± 1.3 fold (P<0.05),
respectively. The gastrin promoter activity induced by CoCl2 in AGS-
CCK2R cells was higher than in wild-type AGS cells. Hypoxia increased
Gastrin mRNA concentrations in AGS and AGS-CCK2R cells by 1.6 ±
0.2 and 6.1 ± 2.2 fold (P<0.05), respectively. Deletion of the putative
HIF-binding sites in the gastrin promoter did not affect gastrin promoter
inducibility under hypoxia, indicating that the hypoxic activation of
the gastrin gene may be HIF-1 independent. Mutational analysis of
previously identified regulatory elements in the gastrin promoter failed
to abrogate the induction of promoter activity by hypoxia. Conclusion:
Our study is the first to demonstrate that hypoxia upregulates the gastrin
gene in AGS cells. The observation that this effect is possibly enhanced
by the presence of gastrin receptors provides potential targets for GI
cancer therapy.
POS-WED-131
INDUCTION OF ETHENO-DNA ADDUCT BY
CARCINOGENIC OXYSTEROLS IN IMMORTALIZED
CHOLANGIOCYTE
Yongvanit P.1, 2 , Jusakul A.1, 2, Loilome W.1, 2, Namwat N.1, 2,
Dechakhamphu S.1, 2 and Kobayashi N.3
1
Department of Biochemistry, Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand. 2Liver Fluke and Cholangiocarcinoma
Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen
40002, Thailand. 3Department of Surgery, Okayama University Graduate
School of Medicine and Dentistry, Okayama, Japan.
Oxysterols are oxygenated derivatives of cholesterol which are produced
by non-enzymatic reaction through reactive oxygen species (ROS) and/
or reactive nitrogen species (RNS). Oxysterols, which may be produced
upon the liver fluke (Opithorchis viverrini; Ov) infection, leading to
inflammatory process and consequently overproduction of ROS, are
also suspected to play roles in genesis of cholangiocarcinoma (CCA).
Our previous data showed that the level of particular oxysterols including
cholestan-3β, 5α, 6β-triol (Triol) and cholest-4-ene-3-one (3K4), which
are categorized as carcinogenic forms, were significantly higher in liver
tissues of Ov-induced hamster CCA. We proposed to examine the effect
of oxysterols including Triol and 3K4 on the induction of DNA damage and
the alteration of cellular phenotype in human immortalized cholangiocyte
(MMNK-1). The immunocytochemical detection of etheno-DNA adduct in
oxysterols-induced MMNK-1 cell was performed to determine the level of
DNA adduct that caused by oxysterols. This study showed the increased
level of carcinogenic etheno-DNA adducts (1-N 6-ethenodeoxyadenosine
or εdA) that is formed by ROS via the reaction of lipid peroxidation. The
effect of oxysterols on cell apoptosis induction was also analyzed by using
flow cytometer. It was shown that these oxysterols induced cells death
via apoptotic pathway as a dose-dependent manner. These evidences
suggest that cell death induced by Triol and 3K4 which is generated during
carcinogenesis may have the procarcinogenic impact, since oxysterol
could also cause DNA damage via oxidative stress and if mutated clonal
expansion occurs, it may play role in the changing of cellular phenotype
from normal to malignancy. The proposed mechanistic involvement is
under investigation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 223
POS-THU-130
WNT5A ACTS AS AN ONCOGENE IN EBV-ASSOCIATED
NASOPHARYNGEAL CARCINOMA
Yap L.F.1, Munirah A.2, Zabidi M.M.1, Chai S.J.1, Chu T.L.2, Tan S.K.2,
Lim P.3, Wei W.4, Teo S.H.1, 5 and Khoo A.S.B.2
1
Cancer Research Initiatives Foundation, Sime Darby Medical Centre,
47500 Subang Jaya. Selangor. Malaysia. 2Institute for Medical Research,
Molecular Pathology Unit, 50588 Kuala Lumpur. Malaysia. 3Tung Shin
Hospital, 55100 Kuala Lumpur. Malaysia. 4Cancer Research UK Institute
for Cancer Studies, University of Birmingham, Birmingham B15 2TT.
United Kingdom. 5University of Malaya, 50603 Kuala Lumpur. Malaysia.
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr Virus (EBV)-
associated cancer which is particularly prevalent in Southern China
and Southeast Asia. In Malaysia, NPC is the fifth most common cancer
overall and third common in men. Over 70% of cases present with late
stage disease and the 5-year survival rates are less than 50%. Novel
therapeutic approaches to manage this disease are urgently required.
Using expression microarrays, we identified the Wnt5a gene as being
overexpressed in primary NPC tissue samples relative to cancer-free
controls. Further, comparison with a published microarray study using 36
normal human organs revealed that the level of Wnt5a mRNA in NPC is
significantly higher than in a wide range of normal organs. Wnt5a is one
of the most highly studied Wnts which acts primarily through the non-
canonical pathway. With respect to cancer biology, there is conflicting
evidence whether Wnt5a has a tumour-promoting or -suppressing role,
and its role in NPC has never been investigated. The upregulation of
Wnt5a was validated in 12 NPC tissue samples by quantitative PCR,
and its low expression level was confirmed in 16 normal human organs
by RT-PCR. In NPC cell lines, however, the expression of Wnt5a was
heterogenous. Nonetheless, a dramatic increase in the Wnt5a expression
was shown in the only EBV-positive line, C666.1, suggesting a potential
role of EBV in regulating the expression of Wnt5a. In addition, we
assessed the functional role of elevated Wnt5A on tumour cell behaviour
in vitro. Ectopic expression of Wnt5a in NPC cell lines significantly
promotes cell proliferation, migration and invasion. Taken together, Wnt5a
appears to function as an oncogene in NPC, and its overexpression might
be regulated by EBV. These data suggest that Wnt5a could be a useful
therapeutic molecular target for NPC.
POS-THU-132
INCIDENCE OF INOSINE TRIPHOSPHATE
PYROPHOSPHOHYDROLASE (ITPA) DEFICIENCY IN
PATIENTS WITH HAEMATOLOGICAL MALIGNANCIES
Zamzami M.1, 2 , Catley L.1, Marinaki A.3, Bowling F.1 and Duley J.1
1
The University of Queensland, Brisbane, Australia. 2King Abdulaziz
University, Jeddah, Saudi Arabia. 3St Thomas Hospital, London.
Mutations affecting genes which produce nucleotide imbalances may
represent mutational risk factors for the development of haematological
malignancies e.g., myelodysplasias (MDS), where chromosome
rearrangement or gene instability is implicated. We tested the novel
idea that low ITPase activity in ITPA carriers may be associated with
onset of haematologic malignancies common in older age-groups. We
hypothesised that accumulation of rogue nucleotides such as deoxy-
ITP may cause nuclear DNA mutations. Aim: The study assessed the
incidence of the ITPA 94C>A and IVS2+21A>C mutant polymorphisms
associated with ITPase deficiency among stable untreated patients with
e.g., chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia
(AML) and MDS. Methods: 22 MDS, 28 CLL and 18 AML patients were
screened for the ITPA 94C>A and IVS2+21A>C SNPs. The international
prognostic scoring system (IPSS) values were not available for this
patient base. Standard PCR primers were used to amplify exons 2,3
and 4 (Sumi et al, 2002). Thermocycler conditions were 95°Cx5min,
35 cycles (94°Cx20s, 56°Cx20s, 72°Cx30s), ending 72°Cx10min,
followed by sequencing. Results and discussion: For Caucasian
populations, the expected frequencies of 94C>A, and IVS2+21A>C are
6% and 13% respectively. In this study, the MDS frequency of 94C>A
was 11.4% (p=0.134) and IVS2+21A>C 20.5% (p=0.141). For CLL, these
frequencies were 7.1% (p=0.719) and 12.5% (p=0.911), and for AML
2.8% (p=0.416) and 8.3% (p=0.405) respectively. The ITPA frequencies
were not statistically different from normal for MDS, CLL and AML, but
94C>A and IVS2+21A>C frequencies were trending to significantly
higher for MDS, perhaps reflecting rogue nucleotide effects on mutation
rates. Further studies of ITPA effects on prognosis (IPSS) are needed.
POSTERS WEDNESDAY & THURSDAY
POS-WED-133
NEDD9 IS REQUIRED FOR MESENCHYMAL INVASION
OF GLIOBLASTOMA CELLS
Zhong J.1, 2 and O’Neill G.M.1, 2
1
Kids Research Institute, The Children’s Hospital at Westmead, NSW,
Australia. 2Discipline of Paediatrics and Child Health, University of
Sydney, NSW, Australia.
Glioblastoma is the most common primary brain cancer with average
patient survival of 12-15 months. Despite advances in technology and
therapies over the last two decades, there has been little improvement in
survival and the final mortality rate remains at almost 100%. Therefore,
there is an urgent need to develop new drugs to target specific molecules
in signalling pathways that have been implicated in glioblastoma. Given
that individual glioblastoma cells can diffusely infiltrate into surrounding
healthy brain tissue and thus lead to recurrence of the disease and
ultimately death, targeting migration signalling molecules could prove
promising. An exciting candidate molecule is NEDD9. The docking
molecule NEDD9/ HEF1/Cas-L is an in vivo regulator of cell migration
and has been linked to metastasis of melanoma and lung cancer and
has recently been demonstrated to promote migration in glioblastoma
cells. Thus, the goal of this study is to investigate the role of NEDD9
in glioblastoma cell migration using three-dimensional (3D) culture
systems to better recapitulate the in vivo micro-environment. We
screened 7 human glioblastoma cell lines and observed high level
NEDD9 expression in half the cell lines, correlating with mRNA levels
as determined by quantitative real-time PCR. We also established a
3D-collagen gel model which revealed distinct morphological differences
when compared with cells on 2D substrates. Subsequently, siRNA
targeting of NEDD9 was shown to cause ~80% decrease in NEDD9
expression in 3 high-expressing cell lines and siRNA-mediated down-
regulation of NEDD9 significantly inhibited glioblastoma cell migration
and invasion in 3D collagen gels. Significantly, our data is the first
demonstration that NEDD9 plays a role in 3D invasion of glioblastoma
cells.
POS-WED-135
IMPROVED PROCESSING OF SHORT HAIRPIN RNA
MOLECULES TARGETING H5N1 INFLUENZA
McLachlan K.P.1, 2 , Muralitharan M.2, Hinton T.M.1, Tyack S.G.1 and
Doran T.J.1
1
Australian Animal Health Laboratory, CSIRO, Geelong, VIC, Australia.
2
School of Life and Environmental Sciences, Deakin University,
Geelong, VIC, Australia.
RNA interference (RNAi) is a natural cellular process designed to
degrade foreign RNA. Exploitation of this pathway allows sequence
specific silencing of targeted viral genes. Vector based expression of
short hairpin RNA (shRNA) molecules is one of the strategies used to
induce RNAi. The shRNAs expressed from these vectors are processed
by the RNAi pathway into small interfering RNA duplexes (siRNAs), and
the antisense strand goes onto trigger silencing of the target gene.
Although shRNAs may be highly expressed from a vector, they are
often inefficiently processed into siRNAs- resulting in lower silencing
efficiency. As native microRNA (miRNA) hairpins are highly processed
we decided to investigate optimising the silencing and processing
efficiency of our shRNAs by modelling them on native miRNAs, starting
with the loop sequences. We selected two different miRNAs native to
the chicken based on their high expression from microarray and Solexa
sequencing data, and a third miRNA based on knowledge of it being
efficiently processed from the precursor miRNA hairpin through to the
mature miRNA duplex. We identified the loop regions of these miRNAs
and used these sequences in place of a standard nine nucleotide
sequence used in many shRNA molecules. The shRNAs selected for
optimisation are targeted to conserved regions of Influenza A viruses,
including H5N1. The silencing efficiency of the optimised molecules
(termed miRloop shRNAs) against their target genes has been assayed
using enhanced Green Fluorescent Protein fusion constructs containing
portions of the viral genes, and haemagglutination assays. The
processing of the miRloop shRNAs has been analysed using northern
blots. Results indicate that use of miRloops does have an affect on the
processing and silencing ability of shRNAs.
Page 224 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-134
CHARACTERISATION OF SH-SY5Y NEUROBLASTOMA
CELLS AS A MODEL FOR NERVE AGENT
NEUROTOXICOLOGY
Thompson G., Thirkettle-Watts D. and Mawdsley D.
Defence Science and Technology Organisation, 506 Lorimer St,
Fishermans Bend, VIC 3207, AUSTRALIA.
The molecular mechanisms that underlie long-term central nervous
system damage caused by organophosphorus (OP) nerve agents, as
well as the native mechanisms employed by neurons to combat OP
toxicity, are poorly understood. We are evaluating the SH-SY5Y human
neuroblastoma cell line as a model for nerve agent neurotoxicity that
is amenable to genetic, biochemical and cell biology analyses and
manipulation. SH-SY5Y cells have been examined to determine their
ability to model typical neuronal features and to determine quantifiable
signs of OP toxicity. Following exposure to OP chemicals, innate cellular
detoxification pathways have been examined to determine pathways
that may provide protection from toxic effects and insights into possible
novel therapeutic targets.
POS-THU-136
UTILISING A PHOTOACTIVATABLE FLUORESCENT
PROTEIN (DENDRA) TO INVESTIGATE ESSENTIAL
PROCESSES IN THE MALARIA PARASITE,
PLASMODIUM FALCIPARUM
McMillan P.J., Rozano L., Mwesigye E.E., Hanssen E. and Tilley L.
Deptartment of Biochemistry and Centre of Excellence for Coherent
X-ray Science, La Trobe University, VIC, Australia.
Photoactivatable fluorescent proteins (PAFPs) are a new class of
fluorescent proteins which are capable of undergoing an irreversible
switch in fluorescence. In this study a PAFP called DENDRA, which can
undergo a switch from a green form to a red form, is used to monitor
protein & organelle trafficking events in two vital processes in the human
malaria parasite, Plasmodium falciparum. During the symptomatic
stages of infection the parasite drastically modifies its host cell (red blood
cells; RBC) in order to survive and replicate, which it does in two ways.
Firstly, the parasite exports virulence factors to the RBC membrane that
mediate adherence of the infected cell to capillaries walls, allowing the
infected cell to avoid clearance by the spleen. As the RBC lacks any
protein trafficking machinery, the parasite generates an exomembrane
system in the host cell which is responsible for the trafficking of virulence
factors to the RBC membrane. DENDRA fusion proteins will be used to
investigate the trafficking of these virulence factors and the generation
of this exomembrane system. Secondly, the parasite ingests the host
cell cytoplasm, which primarily consists of haemoglobin, to provide
amino acids and nutrients. The haemoglobin is taken up via a process of
micropinocytosis and is degraded by a number of proteases to produce
small peptides. The haem released from this digestion is crystallised
into an inert compound called Haemozoin, which is stored in an acidic
compartment called the digestive vacuole. DENDRA will be used as a
marker for haemoglobin uptake to allow us to follow the uptake process
in vitro using confocal microscopy.
POSTERS WEDNESDAY & THURSDAY
POS-WED-137
VACUOLAR TURNOVER OF THE NUCLEUS
IN SACCHAROMYCES CEREVISIAE BY LATE
NUCLEOPHAGY
Mijaljica D., Prescott M. and Devenish R.J.
Department of Biochemistry and Molecular Biology, Monash
University, Clayton campus, Victoria 3800, Australia.
Autophagic degradation of superfluous intracellular components and
reuse of the breakdown products enables cells to survive stress from
the external environment (e.g. nutrient deprivation) as well as internal
stresses (e.g. accumulation of damaged organelles). The yeast nucleus
is subject to a form of autophagic degradation by a process called
Piecemeal Microautophagy of the Nucleus (PMN). Thus, following short
periods of nitrogen starvation (up to 3 hours), interactions between the
outer nuclear envelope membrane protein, Nvj1p, and the vacuolar
membrane protein, Vac8p, contribute to the formation of nuclear-
vacuole junctions and ultimately of membrane vesicles containing
nuclear material that are subsequently released into the vacuole.
Using a nucleoplasm-targeted fluorescent reporter, NAB35-Rosella,
we observed nucleophagy (designated LN for late nucleophagy) only
after longer periods of nitrogen starvation (20+ hours). Dual labeling of
cells with a nuclear membrane reporter, Nvj1-EYFP, and a nucleoplasm
reporter, NAB35-DsRed.T3, confirms that initiation of the delivery of the
two respective nuclear reporters to the vacuole is temporally separated.
Furthermore, our data suggest that LN occurs by a mechanistically
different process to PMN because it can occur in nvj1 or vac8 null
cells. Nevertheless, several components of the core macroautophagic
machinery are required for LN as it is efficiently inhibited in null mutants
of several genes belonging to this group. Moreover, the inhibition of LN
in these mutants is accompanied by alterations in nuclear morphology.
Collectively, these results suggest that different compartments of the
nucleus are selected for delivery to the vacuole by mechanistically
distinct forms of nucleophagy.
POS-WED-139
CYCLIC AMP DEPENDENT PROTEIN KINASE A
STABILISES BIM EL DURING APOPTOSIS
Moujalled D.1, 2, Weston R.1, Carter H.1, Goyal P.1, Ninnis R.1, Coley A.M.1, 2,
Strasser A.3 and Puthalakath H.1
1
Department of Biochemistry, La Trobe Unversity, Kingsbury Drive
Bundoora, VIC 3086, Australia. 2The Cooperative Research Centre for
Biomarker translation, Department of Biochemistry, La Trobe univesity.
3
Walter & Eliza Hall institute of medical research, IG Royal Parade,
Parkville, VIC 3050 Australia.
The pro apoptotic BH3-only protein Bim can induce apoptosis in a wide
variety of physiological settings including calcium flux, growth factor
withdrawal, endoplasmic reticulum stress, T cell receptor activation
and B cell receptor activation. Bim is considered to be the convergent
point for cyclic adenosine monophosphate (cAMP) and glucocorticoid
induced cell death pahtways (Zhang & Insel 2004). The pro-apoptotic
activity of Bim is subject to stringent control both at the transcriptional
and post-translational level. Conventionally, Bim can be phosphorylated
by MAPK/ERK and JNK pathways. To identify additional kinases
involved in its regulation, we carried out a yeast 2-hybrid screen. Here
we report that the cAMP dependant protein kinase A (PKA) is a key
regulator of BimEL(the major isoform in most, if not all cells and tissues)
function. Phosphorylation of BimEL by PKA stabilizes the protein and
thus increases the apoptotic activity of the protein. This is in contrast to
what is reported for other kinases such as ERK where the consequence
of phosphorylation is increased turnover of the protein through the
ubiquitine-proteasome pathway.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 225
POS-THU-138
NUCLEAR-LOCALIZED IMPORTIN ALPHA
REGULATES GENE EXPRESSION IN MAMMALIAN
SPERMATOGENESIS
Miyamoto Y.1, Yasuda Y.2, Yamashiro T.2, Nagai M.2, Masui A.2,
Yoneda Y.2, 3, Jans D.A.1 and Loveland K.1
1
Department of Biochemistry and Molecular Biology, School of
Biological Sciences, Monash University, Australia. 2Department of
Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka
University, Japan. 3Department of Biochemistry, Graduate School of
Medicine, Osaka University, Japan.
Importin α is known classically as a nuclear localization signal receptor.
Although it shuttles between the cytoplasm and nucleus under normal
conditions, we previously reported that importin α accumulates in the
nucleus in response to cellular stresses, such as oxidative stress,
resulting in the down-regulation of the classical nuclear import pathway
by removing importin α from the cytoplasm. In this study, we show
that nuclear-retained importin α bound a DNaseI-sensitive nuclear
component(s). Microarray analysis showed that nuclear importin α
induced the up-regulation of specific genes, including the serine/
threonine kinase 35 (STK35). Binding of importin α to the promoter
region of STK35 was demonstrated by chromatin immunoprecipitation.
Constitutive overexpression of STK35 protein was associated with cell
death under oxidative stress conditions, suggesting importin α plays a
novel role in gene expression in response to stress leading to cell death.
Interestingly, the selective and exclusive presence of importin α2 and α4
proteins in mouse within the nucleus of male germ cells in adult testes is
concordant with expression of the Stk35 transcript. In situ hybridization
analysis reveals that Stk35 gene expression is remarkably reduced in
the testis of importin α4 KO mice. Collectively, these results indicate
that nuclear importin α may play a role in quality control during sperm
production by up-regulating the Stk35 gene and thereby cell death.
POS-THU-140
IN THE PRESENCE OF HIGH LEVELS OF RIPK3,
TNF CAN CAUSE CELL DEATH INDEPENDENT OF
CASPASE 8 AND RIPK1
Moujalled D.1, 2 , Gentle I.1, Wong L.1, Evans J.1, 2, Deed S.1, Hanssen E.4,
Strasser A.3 and Vaux D.L.1, 2
1
Department of Biochemistry, La Trobe University, Kingsbury Drive,
Bundoora, VIC 3086, Australia. 2Cooperative Research centre for
Biomarker Translations (CRC), La Trobe University Bundoora, VIC,
3086 Australia. 3Walter & Eliza Hall Institute of Medical Research,
1G Royal Parade, Parkville, Victoria 3050, Australia. 4Bio21
Molecular Science and Biotechnology Institute,The University of
Melbourne,Flemington Rd, Australia.
TNF receptor super-family members bear death domains are known to
be able to recruit death domain adaptor proteins and cause caspase-8
dependent apoptosis. There are an increasing number of reports that in
some circumstances these death receptors can sometimes induce cell
death in a caspase-independent manner, for example by mechanisms
involving Receptor Interacting Protein Kinases (RIPKs) or by autophagy.
We have used a genetic approach to determine the mechanisms by
which TNF causes death of mouse embryonic fibroblasts (MEFs). In
the presence of cycloheximide, or when IAPs are depleted by an IAP
antagonist compound, TNF causes apoptosis of WT MEFs, but caspase
8 and RIPK1 deleted MEFs survive. In contrast, induction of RIPK3,
which is usually expressed at low levels, synergises strongly with TNF
to cause death of caspase 8 -/- MEFs. Interestingly, induction of RIPK3
also greatly increases the ability of TNF to kill RIPK1-/- MEFs, indicating
that the activation of caspase 8 independent death mechanisms by
TNF is enabled by RIPK3, even when RIPK1 is not present to recruit
it to the TNFR1 associated signalling complex. Furthermore, we
show that death of MEFs caused by TNF in the presence of RIPK3
was not blocked by caspase inhibitors, the RIPK1 inhibitor necrostatin,
autophagy inhibitors, or absence of pro-apoptotic Bcl-2 family proteins
Bax and Bak. Therefore, our data suggest that RIPK3 functions as the
determinant for cell death in response to TNF in a caspase 8 and RIPK1
independent manner.
POSTERS WEDNESDAY & THURSDAY
POS-WED-141
ACTIVATION OF THE CALCIUM-SENSING RECEPTOR
BY GLUTATHIONE ANALOGS: PHYSIOLOGICAL
SIGNIFICANCE AND INSIGHTS INTO THE MECHANISM
OF ACTION
Mun H.C.1, Broadhead G.K.1, Avlani V.A.1, Jourdon O.1, Christopoulos A.2,
Church W.B.3, Delbridge L.4 and Conigrave A.D.1
1
School of Molecular Bioscience, University of Sydney, NSW 2006.
2
Department of Pharmaceutical Sciences, Monash University, Victoria,
Australia. 3Faculty of Pharmacy, University of Sydney, NSW 2006,
Australia. 4University of Sydney Endocrine Surgical Unit, Royal North
Shore Hospital, St Leonard, NSW 2065, Australia.
The human calcium-sensing receptor (CaR) plays a central role in calcium
homeostasis. The CaR is activated physiologically by various L-amino
acids and pharmacologically by phenylalkylamines including the clinical
agent cinacalcet, which bind respectively in the receptor’s Venus Flytrap
and heptahelical domains. Recent molecular modeling indicates that the
amino acid side-chain binding pocket also accommodates short peptides
in which the free α-amino and α-carboxylate groups are preserved.
We have now tested the physiological significance of γ-glu-peptides
including the dipeptides γ-Glu-Cys and γ-Glu-Ala and tripeptide γ-Glu-
Cys-Gly (glutathione) and its analogs, S-methylglutathione (SMG) and
S-propylglutathione on intracellular Ca2+ (Ca2+i) mobilization and cAMP
levels in CaR-expressing HEK293 cells and Ca2+i mobilization and
parathyroid hormone (PTH) secretion in normal human parathyroid cells.
In addition, to explore the mechanism of action, we compared the effects of
SMG and L-Phenylalanine (L-Phe) on HEK293 cells that stably expressed
either the wild-type CaR or the double mutant T145A/S170T, which exhibits
selectively impaired responses to L-Phe leaving responses to elevated
the extracellular Ca2+ intact. We found that γ-glutamyl peptides are potent
positive allosteric modulators of CaR that stimulate Ca2+i mobilization and
suppress intracellular cAMP in CaR-expressing HEK293 cells and enhance
Ca2+i mobilization and inhibit PTH secretion in normal parathyroid cells.
Furthermore, T145A/S170T exhibited markedly reduced sensitivity to both
L-Phe and SMG on Ca2+i mobilization and cAMP suppression. The results
indicate that extracellular glutathione and its natural analog SMG modulate
CaR function in a physiologically relevant manner and that L-amino acids
and glutathione analogs share a common mechanism of action.
POS-WED-143
TARGETING OF THE INNATE IMMUNE REGULATORY
PROTEIN MUL1 TO MITOCHONDRIA
Ng H.1, 3, Khoo J.1, 2, 3, Jenkins K.1, 2, Mansell A.1, 2, 3, Gabriel K.1 and
Nagley P.1, 3
1
Department of Biochemistry and Molecular Biology, Monash
University, Clayton, Victoria, 3800, Australia. 2Centre for Innate
Immunity and Infectious Diseases, Monash Institute of Medical
Research, Monash University. 3ARC centre of Excellence in Structural
and Functional Microbial Genomics, Monash University.
The protein Mul1 associates with the MAVS complex on the outer
mitochondrial membrane (OMM) and is involved in regulation of the
mammalian innate immune response. Since Mul1 is a deubiquitinase
that negatively regulates innate immune anti-viral signalling via RIG-1
and MDA5, understanding the targeting of Mul1 to mitochondria and
determination of its topology will clarify the molecular basis of its role in
the MAVS complex. Mul1 (352 aa) is thought to have two transmembrane
domains, TM1 close to the N-terminus, and TM2 lying towards the
C-terminus. To test targeting roles of TM1 and TM2, we constructed a
systematic set of deletions of Mul1, each tagged N-terminally with HA
and expressed individually in HeLa cells. Immunocytochemical analysis
of HA, together with MitoTracker Red labelling of mitochondria, enabled
determination of localisation to mitochondria by confocal microscopy.
Full length Mul1 was precisely targeted to mitochondria. Mul1ΔTM1 (aa
9-28) and Mul1ΔTM1&TM2 (aa 9-28, aa 242-259) each failed to target
correctly to mitochondria. However, Mul1ΔTM2 (aa 242-259) achieved
substantial localisation to mitochondria. The data indicate that TM1 is
essential for mitochondrial targeting. Studies of the topology of Mul1
with respect to the OMM are currently in progress. Strategies include
expression of miniature derivatives of Mul1 containing TM1 alone with
appropriate N- and C- terminal tags, and incorporation of a TEV1
protease cleavage site into intact Mul1 between TM1 and TM2. A
combination of immunochemical and biochemical analyses will allow
determination of the precise orientation of the N- and C- termini of Mul1
across the OMM.
Page 226 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-142
YEAST GENOME-WIDE ANALYSIS IDENTIFIES
CELLULAR PROCESSES AFFECTING AGGREGATION
OF AMYLOID BETA 42-GFP FUSION REPORTER
Nair S., Traini M., Dawes I.W. and Perrone G.G.
School of Biotechnology and Biomolecular Sciences, UNSW, 2052
Australia.
Alzheimer’s disease is the leading cause of dementia in individuals over
65 years of age; and the risk of developing Alzheimer’s disease increases
steadily with age. A major neuropathological feature of this disease is the
presence of amyloid-beta (Aβ) plaques, which are primarily composed
of aggregates of Aβ peptides generated via amyloidogenic processing
of the amyloid precursor protein. It was recently hypothesised that
increased presence and aggregation of intracellular Aβ peptides may
play a role in the progression of Alzheimer’s disease. In our study, we
have expressed Aβ42 fused to GFP (Aβ42-GFP) in each of the mutants
of the Saccharomyces cerevisiae genome-wide deletion library. This
approach has identified on a genome-wide level the proteins and
cellular processes that affect intracellular Aβ42-GFP aggregation. In
addition, sub-cellular localisation of Aβ42-GFP was also identified in
these mutants. The data generated in this study may have important
implications for our understanding of cellular mechanisms that affect
intracellular Aβ42 aggregation and the factors affecting the disease
progression of Alzheimer’s disease.
POS-THU-144
A PLEIOTROPIC EFFECT OF SIRT1 ON HSF1-
DEPENDENT GENE ACTIVATION?
Nguyen M.T., Láng J., Bulhardt O., Csermely P. and Sőti C.
SEMMELWEIS UNIVERSITY, DEPARTMENT OF MEDICAL
CHEMISTRY, BUDAPEST, HUNGARY.
Resveratrol is polyphenolic phytoalexin that has been recognized to
promote longevity in several organisms. Its primary target is proposed
to be the Sir2 histone deacetylase. However, resveratrol has been
identified in our laboratory as a chaperone inducer and SIRT1 has been
shown to activate HSF1. Besides, in C. elegans we demonstrated that
both the genetic activation of SIR-2.1 by overexpression as well as by
a pharmacologic activation by resveratrol requires HSF1 for increased
lifespan and thermotolerance. To analyze the conservation of the genetic
interaction between Sir2 and HSF1 we have set up SIRT1 transfection in
the COS-7 mammalian cell line. Interestingly, both the wild-type and the
dominant negative mutant (deacetylase deficient) SIRT1 inhibited Hsp70
induction suggesting that it may be other, deacetylase independent
functions of SIRT1 that influence the heat shock response. To further
test the interaction we employed SIRT1 overexpressing and KO mouse
embryonic fibroblasts. We found that both SIRT1 overexpression and
KO induced Hsp70 protein expression. Moreover, the transfection of
human SIRT1 in SIRT1 KO MEFs inhibited Hsp70 promoter activation.
In summary, our findings suggest a conserved, but more complex
connection of Sir2 and the heat shock response than previously
thought.
POSTERS WEDNESDAY & THURSDAY
POS-WED-145
CHARACTERIZATION OF PUTRESCINE AND
SPERMIDINE UPTAKE BY THE HUMAN MALARIA
PARASITE, PLASMODIUM FALCIPARUM
Niemand J.1, Birkholtz L.1, Louw A.I.1 and Kirk K.2
1
Department of Biochemistry, Faculty of Natural and Agricultural
Sciences, University of Pretoria, Pretoria 0002 South Africa. 2Research
School of Biology, The Australian National University, Canberra, ACT
0200 Australia.
Polyamines and their biosynthetic enzymes are present in high levels
in proliferating cells, including cancer cells and protozoan parasites.
Inhibition of the malaria parasite’s polyamine biosynthesis pathway
causes cytostatic arrest in the trophozoite but does not cure infections
in vivo. This is possibly due to exogenous polyamine salvage from the
host which replenishes the intracellular polyamine pool. This implies that
in order to disrupt parasitic polyamine metabolism as an antimalarial
chemotherapy strategy, it may be necessary to target both polyamine
biosynthesis and transport simultaneously. Polyamine transport into
both P. falciparum infected erythrocytes and parasites isolated from the
erythrocyte were investigated using radioisotope flux techniques. While
the characteristics of transport of putrescine into infected erythrocytes
were similar to those of transport into uninfected cells; spermidine uptake
occurs via the “new permeability pathways” induced by the parasite in
the erythrocyte membrane. Once inside the erythrocyte cytoplasm,
polyamines cross the parasite membrane via a temperature-and
glucose dependent, saturable process. The uptake of both putrescine
and spermidine were competed for by the other polyamines and
biosynthesis inhibition led to increased total uptake of both putrescine
and spermidine. Polyamine uptake was pH dependent with increased
uptake upon increasing pH, but did not appear to be coupled to the
Na+, K+ or H+ gradient. Membrane potential perturbations influenced
polyamine uptake, consistent with the transport being an electrogenic
process.
POS-WED-147
CELLULAR AND EXTRACELLULAR FORMS OF
PODOCALYXIN (PODXL)
Fernandez D.2, Larrucea S.3, Nowakowski A.2, Vilar-Egea M.P.2,
Ayuso M.S.1, 2 and Parrilla R.1, 2
1
Centre of Biological Research, CSIC, Calle Ramiro de Maeztu
9, 28040 Madrid, Spain. 2CIBER de Enfermedades Raras, ISCII.
3
Hospital de Cruces, Madrid, Spain.
CHO cells were stably transfected with cDNA encoding a fusion protein
of PODXL and green fluorescence protein (PODXL-GFP). Soon after
seeding, PODXL-GFP was detected in vivo, by widefield microscopy, in
the Golgi and later on as microvesicles, randomly distributed all over the
cytoplasm and at the plasma membrane. After 10 h of incubation, the
PODXL microvesicles were preferentially located at the leading edges
of the cell, progressing along the filopodia, to finally be released into the
extracellular medium. CHO-PODXL-GFP cells showed the presence of
two distinct anti-PODXL immunoreactive bands of ~200 and ~160 KDa,
respectively. The first one reacted with anti-GFP whereas the smaller
one did not, suggesting the absence of the carboxyterminal domain of
PODXL. Those two bands were also detected in the insoluble fraction of
extracellular medium and were identified as PODXL-GFP or PODXL-Δ
by mass spectrometry analysis. An additional anti-PODXL reactive form,
of ~ 70 KDa was also detected in the soluble extracellular fraction. Either
intact or truncated forms of immunoreactive PODXL increased in the
insoluble or soluble fractions of extracellular medium upon incubation of
CHO-PODXL-GFP cells with PMA, effect that was prevented by either
PKC inhibitors, or matrix metalloproteinase inhibitors. It is concluded
that PODXL is released from the cell mainly as a cargo of microvesicles
and also as a soluble cleaved fragment of the PODXL ectodomain.
Thus, in studying the cellular actions of PODXL, the combined action
of membrane-bound and free (truncated) forms of this protein should
be considered.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 227
POS-THU-146
MID49 AND MID51, NOVEL MITOCHONDRIAL OUTER
MEMBRANE PROTEINS ACTING AS DRP1 EFFECTORS
IN MITOCHONDRIAL DIVISION
Palmer C.S.1, Osellame L.D.1, 2, Laine D.1, Koutsopoulos O.S.1, Frazier A.E.1
and Ryan M.T.1, 2
1
LaTrobe Institute for Molecular Science. 2ARC Center of Excellence
for Coherent X-ray Science, La Trobe University.
Mitochondria form appropriate cellular networks through regulated
fission, fusion and distribution events. Here, we identified two homologous
proteins termed Mitochondrial Distribution proteins of 49 kDa and 51
kDa (MiD49 and MiD51) that are anchored in the mitochondrial outer
membrane and which influence mitochondrial morphology in mammalian
cells. Overexpression of either MiD49 or MiD51 led to the mitochondria
being remodeled into perinuclear clusters with fused mitochondrial
tubules extending outwards that associated with actin. MiD49 and
MiD51 formed discrete foci and apparent rings around potential
mitochondrial constriction sites like that of the mitochondrial fission
mediator dynamin-related protein 1 (Drp1). Furthermore, expression
of MiD49 and MiD51 led to the recruitment of the cytosolic pool of
Drp1 to the mitochondrial surface. In contrast, knockdown of MiD49
and MiD51 reduced Drp1 association with mitochondria and resulted
in the formation of a mitochondrial net-like phenotype. We propose
that MiD49 and MiD51 are novel mediators of mitochondrial division
in mammals that regulate Drp1 action at the mitochondrial surface.
Further work to determine the role of Drp1 as a potential binding partner
for the MiD proteins is being pursued. Additional investigations are also
being conducted to determine the role and activity of the MiD proteins
in mitochondrial distribution and dynamics, including the generation
of truncation mutants and identification of potential protein complexes
through chemical cross-linking.
POS-THU-148
RAPID PURIFICATION OF NATIVE HISTIDINE-RICH
GLYCOPROTEIN FROM HUMAN PLASMA UTILIZING A
STEPWISE PURIFICATION METHOD
Patel K.K., Hoogenraad N.J., Poon I.H. and Hulett M.D.
La Trobe University, Department of Biochemistry, Bundoora,
Melbourne, Victoria 3086.
Histidine-rich glycoprotein (HRG), a relatively abundant (~100-150 μ/
ml) 66 kDa plasma glycoprotein, has a multidomain structure that can
interact with many ligands including heparan sulphate, phospholipids,
plasminogen, fibrinogen, IgG and complement. The ability of HRG to
interact with various ligands simultaneously has suggested that HRG
can act as an adaptor molecule and regulate numerous biological
processes such as immune complex/necrotic cell/pathogen clearance,
cell adhesion, angiogenesis, coagulation and fibrinolysis. In this
study, we have developed a rapid stepwise purification method for
HRG involving metal affinity purification, followed by Anion exchange
chromatography. The final purified protein was analysed by MALDI-MS.
The purification of HRG was assessed at each stage by measuring the
specific activity of the protein, as well as the yield and purity. We will
report on fold structure of the protein. Thus this purification procedure
provides a ready source of HRG that would enable structural studies
and functional assays to accurately assess its biological functions.
POSTERS WEDNESDAY & THURSDAY
POS-WED-149
REDOX HOMEOSTASIS AT THE CELLULAR AND
SUBCELLULAR LEVEL
Perrone G.G.1, Ayer A.1, Minard A.1, Fife C.1, Fellermeier S.1, Tan S.-X.1,
Meyer A.J.2 and Dawes I.W.1
1
School of Biotechnology and Biomolecular Sciences, UNSW,
Australia. 2Heidelberg Institute of Plant Sciences, University of
Heidelberg, Germany.
Maintenance of an optimal redox environment is critical for appropriate
functioning of cellular processes and cell survival. Despite this it is unclear
how optimal redox potential is sensed and set, and, the processes that
impact redox on a cellular/organellar level. The advent of genetically
encoded redox probes including redox-sensitive green fluorescent
protein (roGFP2) show promise in overcoming some limitations of
traditional disruptive means to analyse compartmental redox since
roGFP2 facilitates in vivo analysis of redox potential in a real time,
dynamic and relatively non-invasive manner. Compartmentally targeted
roGFP2 and the model organism Saccharomyces cerevisiae are being
exploited to investigate the biological processes and environmental
factors affecting redox homeostasis at a compartmental level. This
approach will utilise complete sets of S. cerevisiae deletion/mutation
libraries of non-essential/essential genes, and gene over-expression
libraries to identify genes and cellular processes that affect the redox
environment in each compartment of interest. Testing of a broad range
of environmental factors including nutrient source and availability, stress
conditions and/or exposure to various drugs on the compartmental
redox has also been initiated. This approach is ongoing, and to date
has revealed some surprising data indicating the cellular functions
and biochemical processes that impact on redox potential in a given
compartment. Elucidation of the underlying mechanism(s) associated
with altered compartmental redox under a given condition will contribute
to our understanding of what factors influence or help to sense/set the
redox potential in each cellular compartment and the manner in which
changes in the redox environment in one compartment influence that
in others.
POS-WED-151
REAL-TIME MONITORING AND COMPOSITION OF
THE POTASSIUM EFFLUX AT EARLY STAGES OF
APOPTOSIS IN LEUCEMIC T-CELLS
Pottosin I.1, Valencia-Cruz G.1, Bonales-Alatorre E.1, Shabala L.2,
Shabala S.2 and Dobrovinskaya O.1
1
Universidad de Colima, Colima, Mexico. 2University of Tasmania,
Hobart, Australia.
Apoptotic K+ loss contributes to the cell shrinkage and creates a low
intracellular K+ environment, favorable for the activation of apoptotic
enzymes, caspases and nucleases. The ways by which potassium ions
leave the cells are largely elusive as respective candidate channels
are assayed under artificial conditions. In this study we have adapted
unique non-invasive MIFE technique to monitor in vivo K+ fluxes from a
population of mammalian cells (human lymphoma Jurkat), undergoing
apoptosis. Apoptosis was induced either by intrinsic (staurosporine,
STS) or extrinsic (anti-CD95) stimuli. The identity of K+ channels involved
was verified by the use of channels´ blockers and cross-checked by
patch-clamp measurements in the whole cell mode. Caspase 3 activity
and apoptotic volume decrease were monitored in parallel. Anti-
CD95 caused a relatively slow (peaked at 2 hrs) K+ efflux, immediately
preceding the caspase-3 activation. On the contrary, 1 μM STS caused
a rapid transient (peaked at 15 min) and massive K+ influx, followed by
a slower phase. Combination of patch-clamp and MIFE, and use of K+
channels blockers suggested that the early component of K+ efflux is
mediated by TRESK-like double pore K+ channels, recently identified
by our group in Jurkat cells. These channels transiently activated upon
STS-stimulation and completely inactivated after 0.5 hr, leading to the
membrane depolarization. From that moment, the apoptotic K+ efflux
was dominated by margatoxin-sensitive Kv1.3 channels. Channel-
mediated K+ efflux provoked a substantial cellular shrinkage and affected
the caspases activation. Supported by CONACyT (Mexico) and small
grants from Universities of Colima and Tasmania.
Page 228 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-150
LOCALIZATION OF SERINE
HYDROXYMETHYLTRANSFERASE ISOFORMS IN
PLASMODIUM FALCIPARUM
Pornthanakasem W., Leartsakulpanich U., Uthaipibull C.,
Kongkasuriyachai D. and Yuthavong Y.
National Center for Genetic Engineering and Biotechnology, 113
Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120
THAILAND.
Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-
dependent enzyme in the dTMP cycle, converts serine and
tetrahydrofolate to glycine and methylene tetrahydrofolate. Since
Plasmodium malaria parasite relies solely on de novo pyrimidine
biosynthesis pathway which differs from human, SHMT has been
proposed to be a potential antimalarial drug target. Based on the DNA
sequence search from PlasmoDB, there are two genes encoding
SHMT in P. falciparum; one of which has a putative transit peptide at
N-terminus. The presence of two isozymes in P. falciparum that may
implicate the drug development is yet to be established. Along with
other studies on gene disruption and enzyme characterization to
validate these proteins as the drug targets, the localization study of
these isoforms is investigated. The putative cytosolic and mitochondria
SHMT genes were cloned in frame with DsRed and green fluorescence
protein respectively. These plasmids were successfully transfected
into P. falciparum and SHMT-fusion proteins were expressed. Results
from laser-scanning confocal microscope showed different distribution
patterns of SHMT-fusion proteins in transfected parasites with specific
organelle localization. Additionally, IFA is being used to confirm the
results and expression levels of proteins in each blood stage will be
measured by RT-PCR. This study provides evidence that these proteins
are function at different compartments.
POS-THU-152
MALATE TRANSPORTERS IN THE SOYBEAN (GLYCINE
MAX)- RHIZOBIA SYMBIOSIS
Qu Y.1, Clarke V.C.1, Loughlin P.C.1, Day D.A.2 and Smith P.M.C.1
1
School of Biological Sciences, University of Sydney, Sydney, 2006.
2
Flinders University, Adelaide, SA, 5001.
The legume-rhizobia symbiosis describes the phenomenon of bacteria
infecting legume root cortex cells and forming a novel organ known
as the nodule after being engulfed by the host plant. The host plant
can grow better by the grace of atmospheric nitrogen being fixed into a
biologically available form by the differentiated rhizobia (bacteroid) within
the nodule. In return for nitrogen, the plant provides bacteroids with
nutrients to support its growth. A specialised, plant derived membrane,
termed the symbiosome membrane (SM), surrounds the bacteroids
and plays an integral role in regulating the exchange process, therefore
making the study of transporters on the SM an important topic. Amongst
all these nutrients, malate is thought to be the primary carbon energy
source provided by the plant to the bacteroid, though a transporter for
it on the SM has yet to be identified. Using a bioinformatic analysis
based on previously characterised malate transporters in Arabidopsis
and Medicago we have identified several potential soybean malate
transporters. The expression pattern of candidate transporters has
been investigated using qRT-PCR and candidate genes with enhanced
nodule expression will be studied using GFP-fusion constructs to
determine their localisation. Functional analysis will be done to confirm
that our candidates can transport malate.
POSTERS WEDNESDAY & THURSDAY
POS-WED-153
CELLULAR ROLES OF THE PUF1/PUF2 RNA BINDING
PROTEINS IN YEAST
Quenault T.L. and Traven A.
Monash University.
Puf proteins are a family of RNA-binding proteins that regulate mRNA
translation and stability. Highly conserved in eukaryotes, Puf proteins are
characterised by the presence of repeated Pumilio-homology domains,
which form an arc-like structure that binds to the 3’-untranslated regions
of target mRNAs. We are investigating the roles of the Puf proteins in
two distantly related yeast species, the model yeast Saccharomyces
cerevisiae and the fungal pathogen Candida albicans. We are focusing
on two highly homologous S. cerevisiae Pufs; Puf1 and Puf2. Puf1 and
Puf2 have been shown to bind mRNA targets that primarily encode
membrane-associated proteins and could therefore be involved in the
biogenesis of fungal membranes and cell walls. Biosynthesis of fungal
membrane and wall components is targeted by the two main classes
of antifungal drugs, the azoles and the echinocandins. Our data
indicates that Puf1 modulates the sensitivity of S. cerevisiae cells to the
echinocandin caspofungin. Our current research aims at identifying the
relevant mRNA target of Puf1 for its role in caspofungin tolerance. We are
also studying the conservation of Puf1 functions between S. cerevisiae
and C. albicans at the genomic level through to the transcriptome and
proteome, to understand how posttranscriptional regulation evolved
between these two distantly related yeasts.
POS-WED-155
IDENTIFICATION OF LIGAND(S) FOR HUMAN
LEUKOCYTE IMMUNOGLOBULIN LIKE RECEPTOR 4
(LILRA3)
Rajasekariah P., An H., Bryant K. and Tedla N.
Infalmmation and Infection Research Centre, School of Medical
Sciences, University of New South Wales, Kensington, NSW 2052.
Leukocyte Immunoglobulin like Receptors (LILRs) are a superfamily
of cell-surface immunoregulatory receptors. They are expressed in a
variety of leukocytes, macrophages, dendritic cells, B lymphocytes, and
mast cell progenitors. Based on their structure, these are classified into
activating, inhibitory and soluble forms. LILRA3 is expressed as a soluble
form which lacks transmembrane domain (1). To date the functional role
and ligands for LILRA3 are not clearly known although the deletion of the
functional gene (1-7 exons) is implicated in Multiple Sclerosis (MS) and
Sjögren’s syndrome (2, 3). Using Alkaline Phospatase tagged LILRA3
(APLILRA3) recombinant protein, Alkaline Phosphate (AP) binding
assays and cytochemistry assays, we have demonstrated that LILRA3
binds to Peripheral Blood Mononuclear Cells (PBMCs) and U937 cells.
Two cDNA libraries were constructed using these cell types in pMT21
mammalian expression vector and DNA from several pools of these
libraries was transfected into COS1 cells. The transient transfectants
were screened with APLILRA3 recombinant protein and the positively
stained pools were rescreened. After a few rounds of screening, several
cDNA clones were selected. Currently these cDNA clones are being
sequenced in order to establish their identity. References: 1.Arm, J.P.,
et.al., (1997) J. Immunol. 159, 2342-2349. 2.Koch, S., et.al., (2005)
Genes Immun. 6, 445-447. 3.Kabalak, G., et.al., (2009) Arthritis and
Rheumatism 60, 2923-2925.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 229
POS-THU-154
A NOVEL INHIBITOR OF RIBOSOMAL RNA GENE
TRANSCRIPTION IDENTIFIES COUPLING OF POLI
TRANSCRIPTION TO MULTIPLE CELL CYCLE
CHECKPOINTS AND SENESCENCE
Quin J.1, 2 , Sanij E.1, Bywater M.1, 2, Drygin D.3, Anderes K.3,
McArthur G.1, 4, Pearson R.1, 2, 5 and Hannan R.1, 2, 5
1
Research, Peter MacCallum Cancer Centre, Melbourne, VIC,
Australia. 2Department of Biochemistry and Molecular Biology,
University of Melbourne, Melbourne, VIC, Australia. 3Cylene
Pharmaceuticals, San Diego, CA, USA. 4Department of Medicine,
St Vincents Hospital, Melbourne, VIC, Australia. 5Department of
Biochemistry and Molecular Biology, Monash University, Melbourne,
VIC, Australia.
The transcription of the 45S ribosomal RNA (rRNA) genes by RNA
Polymerase I (Pol I) is a major rate limiting step for ribosomal biogenesis,
and consequently for cell growth and proliferation. Dysregulation of Pol
I transcription of the rRNA genes is an almost universal feature of cell
transformation and cancer, and work from our laboratory has shown
that targeting this process is a promising new approach for cancer
therapy (M.J. Bywater, Unpublished Results). A small molecule inhibitor
of Pol I transcription (CX-5461) has recently been developed by Cylene
Pharmaceuticals, which provides an opportunity to examine the acute
response of both non-malignant and transformed cells to perturbation of
rDNA transcription. We have utilized CX-5461 to examine the response
of BJ (hTert) immortalized primary human fibroblasts and isogenically
matched BJ cell lines, at defined stages of transformation, to inhibition
of Pol I transcription. Following inhibition of Pol I transcription, BJ (hTert)
cells display a p53-independent proliferation defect, which is associated
with both G1 and G2 cell cycle arrest, and senescence. Pre-malignant
BJ (Large-T, hTert) cells display increased sensitivity to the inhibitor,
and undergo apoptosis. However, transformed BJ (Large-T, Small-T,
hTert, Ras) cells are resistant to CX-5461 treatment. We will present
results of our ongoing studies, which include experiments to address
the molecular mechanisms underlying these differing responses.
POS-THU-156
CHARACTERIZATION OF THE α-SYN EXPRESSION
PATTERN BY IMMUNOFLUORESCENCE ASSAY
WITH COMMERCIALLY AVAILABLE ANTI-α-SYN
ANTIBODIES THAT RECOGNIZE DISTINCT REGIONS
OF α-SYN
Nam M.K., Han J.H., Park D.W., Kim S.S., Kim G.Y., Shin H.A., Moon J.M.
and Rhim H.
Department of Biomedical Sciences/ Research Institute of Molecular
Genetics The Catholic University of Korea.
Alpha-synuclein (α-Syn) is one of the major components of Lewy bodies
observed in the brains with Parkinson’s disease. To date, various
antibodies (Abs) against α-Syn have been commercially available
for western blotting and immunofluoresence assays (IFA) to detect
α-Syn. Nevertheless, there are no reports for epitope mapping and
immunostaining patterns of α-Syn using those Abs. We used various
purified recombinant α-Syn fragments to identify the region specifically
recognized by three different Abs and compared staining patterns of
α-Syn in various cell lines by IFA. We also confirmed the specificity
of three Abs by an Ab depletion assay incubating the specific Ab with
the recombinant α-Syn protein. Broad expression patterns of α-Syn
in the cytosol were observed by the monoclonal Ab (A mAb) which
recognizes the region containing the NAC domain. Although both
polyclonal Ab (B pAb) and another C mAb recognize the following
same fragments containing the C-terminal region of α-Syn: aa 1-140,
61-140, 50-140, and 96-140, tubular-like structured- and punctuated-
expression patterns of α-Syn were observed by staining with the B pAb
and C mAb, respectively. Additionally, a nuclear pattern of α-Syn was
able to detect by staining with the B pAb and C mAb, but not with the
A mAb. In this study, we have demonstrated that several commercially
available anti-α-Syn Abs that recognize different region of α-Syn show
different subcellular localization of α-Syn. These results suggest that
conformational changes of α-Syn may occur in the different cellular
compartments and influence the differential functions of α-Syn in the
cell.
POSTERS WEDNESDAY & THURSDAY
POS-WED-157
BIOCHEMICAL ANALYSIS OF FLAVOPROTEIN
SUBUNIT ISOFORMS IN HUMAN MITOCHONDRIAL
COMPLEX II (SUCCINATE-UBIQUINONE REDUCTASE)
Sakai C.1, Tomitsuka E.1, Miyagishi M.2 and Kita K.1
1
Department of Biomedical Chemistry, Graduate School of Medicine,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan. 221st Century COE Program, Graduate School of Medicine,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan.
We previously reported the presence of two isoforms of flavoprotein
(Fp) subunit of human mitochondrial complex II (succinate-ubiquinone
reductase, SQR). Type I Fp and type II Fp differs in two amino acid
residues and the difference of the biochemical features of two isoforms
are almost unknown. In this study, we constructed cell strains that
express predominantly type I Fp or type II Fp by RNA interference to
investigate the biochemical properties of two isoforms. Although SQR
activities of the complex II with each isoform are almost the same, the
complex II with type I Fp (type I complex II) showed a pH optimum with
the physiological pH of mitochondrial matrix (pH 8) while that for the
complex II with type II Fp (type II complex II) was pH 7.5. Moreover,
the type I complex II has about 3.5 times higher affinity for succinate
than the type II complex II at pH 8.0. On the other hand, the type II
complex II showed about 3.7 times higher affinity for succinate than
the type I complex II at pH 7.5. These results indicate that the type I
complex II mainly functions as SQR in mitochondria of the cells under
normal condition and the type II complex II may function in the cells
with relatively low pH condition of mitochondrial matrix in such cases as
hypoxia or nutrition deprivation. The investigation of the physiological
role of type II Fp is now in progress.
POS-WED-159
INFLUENCE OF P38 MAPK INHIBITOR ON
INFLAMMATION AFTER SURGICAL WOUND
Shurygina I.A., Shurygin M.G., Zelenin N.V., Granina G.B. and
Lepekhova S.A.
Scientific Center of Reconstructive and Restorative Surgery SB
RAMS.
To study the role of p38 MAPK mechanisms in the regulation of
inflammatory response and on this basis to develop ways to control
inflammation. 60 Male Wistar rats were selected in accordance with
the “Guide for the Care and Use of Laboratory Animals”. The study
was approved by the Local Ethics Committee. We studied the effect
of an inhibitor of p38 MAP-kinase SB203580 in the development of
inflammatory response in models of skin-muscle wounds. We used
methods of light microscopy, immunohistochemistry, tensiometry.
We revealed that the inhibition of p38 cascade significantly affects
the duration and severity of the phases of inflammation, modifies the
formation of connective tissue scar in the wound process. Application of
p38 blockers reduced the duration and severity of neutrophil infiltration
of the damaged area. The application of SB203580 to the end of
observation (30 days) in the area of surgical wound formed tender scar,
the degree of development of connective tissue in the scar area was
significantly lower than in the controls group. Blocker p38 substantially
alter the mechanical characteristics of the forming scar in the skin-
muscle wounds. Thus, the use of blocker p38 allowed to reach a 30
day wound process indicators modulus, breaking load and the transition
point of elastic deformation of the plastic, similar to that of the intact
skin. The results show the importance of studying regulators of cell
differentiation as potential drugs significantly affecting the outcome of
the pathological processes. This study was supported by a grant P803
from the Russian Federal Purposive Program “Scientific and scientific-
pedagogical cadres innovation Russia”.
Page 230 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-158
DECODING THE MECHANISM OF CELL-ELASTIN
ADHESION BY MODIFYING TROPOELASTIN
Seneviratne M.G., Mithieux S.M., Bax D.V. and Weiss A.S.
School of Molecular Bioscience, The University of Sydney.
Elastin is an extracellular matrix protein composed of a mesh of cross-
linked tropoelastin molecules, which provides elasticity to a variety of
tissues and organs including the skin, lungs and arteries (Vrhovski &
Weiss, 1998). Although a range of cell types have been shown to adhere
to tropoelastin (Broekelmann et al., 2005), the adhesion mechanism is
not fully understood. Recent evidence points towards an interaction
between αvβ3 integrins on the cell surface and a highly-conserved RKRK
motif at the C-terminus of the tropoelastin molecule (Bax et al., 2009). We
aim to better understand the role of this RKRK motif. Using a synthetic
human tropoelastin gene developed by our lab, we generate a panel of
tropoelastin constructs with deletion and insertion modifications to the
RKRK sequence. Experiments are underway to evaluate these synthetic
proteins on the basis of cell adhesion, spreading and integrin binding, in
order to elucidate the biochemistry of tropoelastin adhesion signaling.
Developing a better understanding of this adhesion mechanism, and
our potential ability to regulate it, has important implications for elastin-
based biomaterials used as scaffolds for tissue regeneration.
POS-THU-160
IMMUNE-ENHANCING ANTITUMOUR RNA
INTERFERENCE THERAPY FOR CERVICAL CANCER
Singhania R., Gu W. and McMillan N.
UQ Diamantina Institute for Cancer, Immunology and Metabolic
Medicine.
Cervical cancer, caused by the over-expression of human papillomavirus E6
and E7 oncogenes, contributes to almost 12% of the global cancer burden
in women. Even though prophylactic vaccines have been developed, they
are of limited use to women who are already infected. Also, owing to the
prolonged course of cervical carcinogenesis, cancer burden will not be
reduced significantly for several years even after the availability of these
preventive vaccines. So, an effective therapeutic vaccination strategy is
urgently required. In recent past, RNA Interference (RNAi) technology has
emerged as an effective way to silence cancer-causing genes. The recent
findings of our lab show that when short hairpin RNA is targeted downstream
of the cytotoxic T cell epitope in E7 gene, 5’ mRNA intermediates are
generated, which get translated into incomplete proteins and result in
increased presentation of the target protein and generation of a tumour-
protective immune response in mice. It is known that certain viral proteins
are rapidly presented to the immune system than normal proteins due to the
production of defective ribosomal products (DRiPs) by the virus. Our work
aims to bring the concept of RNAi and DRiP together and we hypothesize
that RNAi-enhanced adaptive immune response against tumour antigens
occur via increased presentation of defective proteins. Accordingly, we
have investigated the fate of RNAi-cleaved mRNA and found that shRNA
treatment reduced the half-life of full-length and 3’ mRNA fragment of the
E6/7 and TRP2 mRNA, but the 5’ mRNA fragment appeared to be more
stable. This would favour its translation into truncated protein, which we will
show from polysome analysis and identify the sub-cellular locations of 5’
mRNA fragment and its truncated protein using RNA-immunoprecipitation
and confocal microscopy, respectively. Also, we will investigate the levels
of peptides presented on the cell surface after RNAi treatment by mass
spectrometry to address the hypothesis that immune-enhancement occurs
via increased presentation of defective proteins to the immune system.
In summary, the results till now indicate that this therapy need not kill or
be delivered to every cancer cell to be effective, thus demonstrating its
potential to overcome the current efficacy and delivery constraints.
POSTERS WEDNESDAY & THURSDAY
POS-WED-161
INDUCED POLYPLOIDISATION AND MUTAGENESIS
FOR GENERATION OF NOVEL PASTURE GRASS
FUNGAL ENDOPHYTES
Ludlow E.1, 2, Guthridge K.M.1, 2, Forster J.W.1, 2, 3 and Spangenberg G.C.1, 2, 3
1
Department of Primary Industries, Victorian AgriBiosciences Centre, La
Trobe University Research and Development Park, Bundoora, Victoria
3083, Australia. 2Molecular Plant Breeding Cooperative Research Centre,
Australia. 3Department of Botany, La Trobe University, Bundoora, Victoria
3086, Australia.
Fungal species of the genus Neotyphodium form endophytic symbioses with
agronomically important pasture grass species such as perennial ryegrass
(Lolium perenne L.) and tall fescue (Lolium arundinaceum [Schreb.] Darbysh.).
The fungal grass endophyte association results in both beneficial and
deleterious agronomic effects for pasture grass production. Lolitrem B and
ergovaline alkaloids produced by the fungal endophyte are responsible for
herbivore toxicity syndromes (ryegrass staggers and fescue toxicosis), while
deterrence of invertebrate herbivore feeding is attributable to alkaloids such
as loline and peramine. Novel variants of the perennial ryegrass endophyte
Neotyphodium lolii are being generated through induced polyploidisation
and mutagenesis. N. lolii mycelium has been exposed to the mitotic spindle
inhibitor, colchicine (0 - 0.2% [w/v]), which has been shown to be capable
of successfully inducing autopolyploidisation in higher plants and a limited
selection of fungal species. Protoplast preparations enabled recovery of single
cells and analysis of ploidy changes. Flow cytometry studies have identified
N. lolii populations with altered ploidy status and permitted assessment of
the stability of ploidy changes over a number of generations. Phenotypic
variation attributable to ploidy changes will be evaluated by assessment of
toxin biosynthesis and other traits following isogenic inoculation into specific
host plant genotypes. Ionising radiation, historically the first mutagenic agent
to be described, is capable of inducing a broad range of mutagenic lesions
and has been found to be very effective for a wide range of species. N. lolii
mycelia were exposed to a range of ionising radiation doses and assessed
for recovery and regeneration from single cells. Screening for mutations,
in particular deletions within the lolitrem biosynthesis gene cluster, was
optimised using a modified non-radioactive colony screening method. Novel
variation within the lolitrem biosynthesis gene cluster will be further assessed
following isogenic inoculation into specific host plant genotypes.
POS-WED-163
ROLE OF APP COPPER BINDING DOMAIN IN APP
METABOLISM AND STRUCTURE
Spoerri L.1, 2 , Vella L.J.1, 2, Pham C.L.L.1, 2, Barnham K.J.1, 2, 3 and
Cappai R.1, 2
1
The Department of Pathology, The University of Melbourne, Parkville,
VIC, Australia. 2The Bio21 Molecular Science and Biotechnology
Institute, The University of Melbourne, Parkville, VIC, Australia. 3The
Mental Health Research Institute of Victoria, Parkville, VIC, Australia.
Two major pathological characteristics of Alzheimer’s disease (AD)
include accumulation of β-amyloid (Aβ), a toxic peptide derived from
the Amyloid Precursor Protein (APP), and abnormal copper distribution
in the brain. A relationship exists between copper and APP, whereby
copper can modulate APP processing and APP can regulate copper
homeostasis. A better understanding of the molecular mechanisms
involved in these events could lead to novel therapeutics to treat the
disease. The aim of this work was to investigate the potential role of the
putative copper binding residues of the APP N-terminal copper binding
domain (CuBD) on APP metabolism. HEK293 cell lines overexpressing
APP WT and APP CuBD mutants were employed to study this process.
This investigation was complemented by structural studies using a
yeast expressed recombinant protein encompassing the APP CuBD.
We demonstrate that His149 and His151, located on the α-helix of
the APP CuBD, are important for APP metabolism and structural
stability. Replacement of these residues results in modulation of APP
localization, impairment of APP maturation and decreased sAPPα
and sAPPβ generation in HEK293 cell line. The decreased sAPPβ
formation may imply decreased Aβ generation. No contribution to this
effect is observed by replacement of other residues on the same α-helix
(Leu148, Thr152 and Glu160), indicating a specific role for His149 and
His151. Replacement of His149 and His151 on the recombinant protein
results in changes in secondary structure. Further studies will be aimed
to elucidate the mechanisms underlying these modulations.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 231
POS-THU-162
REGULATION OF NA+ IN THE INTRAERYTHROCYTIC
MALARIA PARASITE
Spillman N.J., Allen R.J.W. and Kirk K.
Research School of Biology, The Australian National University,
Canberra, ACT, 0200.
All cells maintain tight control over their ionic composition and the malaria
parasite, Plasmodium falciparum, is no exception. When the parasite
first invades the erythrocyte host it encounters a low extracellular [Na+].
However, approximately 12-16 hours after invasion, there appear in the
red blood cell membrane ‘New Permeability Pathways’ which increase
the permeability to a large variety of unrelated solutes, including
Na+. The increased host permeability to Na+ leads to an increase in
the intraerythrocytic [Na+] and the mature stages of the parasite are
therefore exposed to a high Na+ environment. The trophozoite form
of the parasite maintains a low intracellular [Na+] of ~10 mM despite a
high extracellular [Na+] (~130 mM) in the erythrocyte host cytosol. The
resulting electrochemical gradient is important for the survival of the
parasite, energising the uptake of essential nutrients, such as inorganic
phosphate, via Na+-coupled symport processes. The mechanisms
by which the malaria parasite regulates its intracellular [Na+], were
investigated using the Na+-sensitive, fluorescent dye sodium-binding
benzofuran isophthalate (SBFI). The functional characterisation of
Na+ efflux pathways involved investigation of: pH sensitivity, inhibitor
sensitivity, ATP requirements and kinetics. The data are consistent
with two Na+ extrusion mechanisms operating in parallel in the mature,
trophozoite stage of Plasmodium falciparum, postulated to be a Na+/H+
exchanger and a P-type Na+-ATPase.
POS-THU-164
INHIBITION OF BAK ACTIVATION BY VDAC2 IS
DEPENDENT ON THE BAK TRANSMEMBRANE
ANCHOR
Stojanovski D.1, Lazarou M.1, Frazier A.E.1, Kotevski A.1, Dewson G.2,
Kluck R.M.2, Vaux D.L.1 and Ryan M.T.1
1
La Trobe Institute for Molecular Science, La Trobe University,
Melbourne 3086, Australia. 2The Walter and Eliza Hall Institute of
Medical Research, Melbourne 3052, Australia.
Bax and Bak are pro-apoptotic factors that are required for cell death
by the mitochondrial or intrinsic pathway. Bax is found in an inactive
state in the cytosol, and upon activation is targeted to the mitochondrial
outer membrane, where it releases cytochrome c and other factors that
cause caspase activation. While Bak functions in the same way as Bax,
it is constitutively localized to the mitochondrial outer membrane. Here,
its activation is believed to be inhibited by the voltage dependent anion
channel isoform 2 (VDAC2), but it is not known how it does so. Using
blue native gel electrophoresis, we show that endogenous, inactive
Bak exists in a novel 400 kDa complex that requires the presence
of VDAC2. Activation of Bak correlated with its release from the 400
kDa complex and the formation of lower molecular weight species.
Furthermore, substitution of the Bak transmembrane anchor with that
of the mitochondrial outer membrane tail-anchored protein hFis1,
prevented association of Bak with the VDAC2 complex, and increased
the sensitivity of cells to an apoptotic stimulus. Our results suggest that
VDAC2 sequesters Bak in an inactive state in the mitochondrial outer
membrane, thereby raising the threshold necessary for permeabilization
of the mitochondrial outer membrane and cell death.
POSTERS WEDNESDAY & THURSDAY
POS-WED-165
RECLINOMONAS AMERICANA: A SNAPSHOT OF
MITOCHONDRIAL EVOLUTION
Tong J., Gabriel K. and Lithgow T.
Department of Biochemistry and Molecular Biology, Monash
University, Clayton VIC, Australia.
Mitochondria has evolved from an engulfed bacterial symbiont and
throughout evolution, the mitochondrial genome has undergone radical
changes. In mammals, the mitochondrial genome encodes only 13
proteins. Curiously, a unicellular eukaryote known as Reclinomonas
americana harbours a mitochondrial genome with the largest gene set,
encoding a total of 67 proteins. Using a combination of yeast genetics,
biochemical and genomic analyses, we have examined some of the
genes in the R. americana mitochondrial genome. We have identified
the existence of both bacterial and classic mitochondrial translocation
machineries that are encoded in the mitochondrial genome of this
unusual organism. This might reflect an evolutionary snapshot in
mitochondrial evolution. Furthermore, one of these genes encodes a
protein with homology to Cox11, which is encoded for by the nuclear
genome in “higher” eukaryotes. In mammals and fungi, Cox11 has an
amino-terminal targeting signal that directs its translocation into the
mitochondria from the cytosol. Although the R. americana Cox11 protein
does not possess such a clearly defined targeting signal, it is still able to
enter yeast mitochondria when expressed in the cytosol. This indicates
that at the time of gene transfer to the nucleus, some proteins may have
had sufficient signals for targeting back to mitochondria. Over time
these targeting signals may have become more elaborate and efficient.
POS-WED-167
REGULATORY ROLE OF PRIP IN THE BONE
FORMATION
Tsutsumi K.1, Matsuda M.1, Jimi E.2 and Hirata M.1
1
Lab of Mol & Cell Biochem, Fac of Dent Sci, Kyushu Univ. 2Dept of
Biosci, Cent for Oral Bio Research, Kyushu Dental College.
PRIP (phospholipase C-related, but catalytically inactive protein) is
a novel signaling protein isolated in this laboratory. PRIP-deficient
mice (PRIP-KO) mice showed an increased gonadotropin, but a
decreased sex steroid hormone. The difference was similar to that
for the cause of bone disease, such as osteoporosis. In the present
study, therefore, we analyzed bone condition of PRIP-KO mice. We
first performed three dimensional analysis of the femur of female mice.
The bone mineral density and trabecular bone volume was higher in
the mutant mice. We further performed histomorphometrical assay of
bone formation parameters: bone formation rate, mineral apposition
rate, osteoid thickness and osteoblast number were accelerated in
the mutant, indicating that the increased bone mass is caused by the
enhancement of bone formation ability. We then performed the primary
culture of calvaria prepared from both genotypes. In the mutant mice,
osteoblast differentiation as assessed by alkaline phosphatase activity
was accelerated. These results indicate that PRIP is implicated in the
regulation of the bone formation.
Page 232 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-166
HAMLET BINDING TO α-ACTININ DISRUPTS FOCAL
ADHESIONS AND FACILITATES TUMOR CELL
DETACHMENT
Trulsson M.1, Hao Y.1, Gisselsson L.2, Chao Y.3, Urbano A.3, Aits S.1,
Mossberg A.K.1, Mok K.H.4 and Svanborg C.1, 3
1
Institute of Laboratory Medicine, MIG, Lund Univerity, Lund, Sweden.
2
Phase Holographic Imaging, Lund, Sweden. 3Singapore Immunology
Network (SIgN), A*STAR, Singapore. 4Trinity College Dublin, University
of Dublin, Dublin, Ireland.
HAMLET is a protein-lipid complex that triggers tumor cell death, in
parallel with a change from a distended to a rounded morphology and
from an adherent to a detached state. In this study, HAMLET was shown
to recognize α-actinin-4, a major F-actin cross-linking protein and
focal adhesion constituent. Synthetic peptide mapping revealed that
HAMLET recognizes the N-terminal actin-binding domain of α-actinin-4,
suggesting that HAMLET might disrupt the α-actinin-4-dependent
scaffold between the actin cytoskeleton and β integrins. HAMLET
was shown by co-immunoprecipitation to interact with α-actinins in
tumor cell extracts and to colocalize with α-actinin-4 at the membrane
of carcinoma cells. Focal adhesions containing paxillin, vinculin and
α-actinin-4 were readily observed by confocal microscopy in adherent
tumor cells but were rapidly lost after HAMLET treatment. In addition,
FAK and ERK1/2 phosphorylation decreased and β1integrin staining was
lost in detached cells, consistent with a disruption of focal adhesions.
Cell detachment in response to HAMLET was previously observed
in bladder cancer patients following intra-vesical HAMLET instillation
and cell detachment in response to HAMLET was confirmed in vitro,
using adherent carcinoma cells. α-Actinin siRNA increased detachment
of tumor cells while α-actinin-4 over-expression significantly delayed
morphological changes in response to HAMLET. The results suggest
that the interaction between HAMLET and α-actinins may disrupt focal
adhesion complexes, leading to tumor cell detachment, in parallel with
their cell death response.
POS-THU-168
THE ROLE OF PRIP IN THE REGULATION OF
AUTOPHAGY
Umebayashi H.1, Matsuda M.1, Kanematsu T.2 and Hirata M.1
1
Lab. of Mol.and Cell. Biochem. Fact. of Dent. Sci. Kyushu Univ. 2Dept.
of Dent.Pharmacol. Grad. Sch. of Biomed. Sci. Hiroshima Univ.
Autophagy is an intracellular bulk degradation system, through which
a portion of cytoplasm is delivered to lysosomes to be degraded under
starvation and stress. Of 33 autophagy-related (ATG) proteins identified
so far, ATG8 (MAP1LC3 in mammal) is used as a specific marker to monitor
autophagy. We isolated a novel protein as an inositol 1,4,5-triphosphate
binding protein molecule similar to phospholipase C, but catalytically
inactive, then we termed it PRIP (PLC-related catalytically inactive
protein). Later, we found that PRIP bound GABARAP (GABA receptor
associated protein), a homologue of MAP1LC3. Therefore, we studied
the possible involvement of PRIP in autophagy. We first confirmed the
interaction between PRIP and MAP1LC3. PRIP had an interaction with
not only GABARAP but also MAP1LC3. Then, autophagy activities were
monitored by Western blotting analysis using lysates obtained from wild-
type (WT) and PRIP- deficient (PRIP-KO) mouse embryonic fibroblast
(MEF) starved by amino acid-deprivation. The level of LC3-II, a lipidation
form of MAP1LC3, was more increased in PRIP-KO MEF. Furthermore,
we generated WT and PRIP-KO mice, expressing transgene of GFP-
LC3 by mating and prepared the MEF. Enhanced autophagic activity,
as assessed by more GFP-LC3 puncta observed under a confocal
microscopy, was observed in the PRIP-KO GFP-LC3 MEF. These
results suggest that PRIP plays a nagative role in autophagy.
POSTERS WEDNESDAY & THURSDAY
POS-WED-169
INHIBITION OF ATP-BINDING CASSETTE
TRANSPORTER G2 INCREASED MITOCHONDRIAL
LOCALIZATION OF PPIX IN ALA TREATED CELLS AND
ENHANCED THEIR APOPTOTIC CELL DEATH INDUCED
BY PHOTODYNAMIC TREATMENT
Utsumi K.1, Fujita H.1, Uchida M.1, Yamamoto M.1, Inoue K.2, Utsumi T.3
and Sasaki J.1
1
Okayama University Graduate School, Medicine, Dentistry and
Pharmaceutical Sciences. 2Kochi Medical School. 3Graduate School of
Medicine, Yamaguchi University.
Since protoporphyrin IX (PpIX) preferentially accumulates in cancer cells
treated with 5-aminolevulinic acid (ALA), photodynamic therapy (PDT)
has been applied for patients with cancer. In this context, apoptotic
but not necrotic cell death of the targeted cancer cells is required to
increase the clinical availability. It has been postulated that intracellular
localization of photoactive ligands, such as PpIX, plays critical role in the
induction of apoptosis of cells subjected to PDT. Recent study revealed
that a family of ATP binding cassette transporters (ABC) plays important
roles in intracellular localization and accumulation of ALA-derived PpIX
and that ABCG2 distributed in mitochondrial cristae. The present work
shows that silencing of ABCG2 using siRNA and inhibition of ABCG2
by reserpin, a pan-ABC inhibiter, increased the accumulation of ALA-
mediated PpIX in ABCG2 expressed cells, such as T24, HSC-4 and
Cos-1 cells. Furthermore, treatment of cells with reserpin increased
PpIX levels preferentially in mitochondria (but not in cytosol and plasma
membranes) and enhanced PDT-induced apoptosis of cells.
POS-WED-171
THE ROLE OF MITOCHONDRIAL PROTEIN, CISD2, IN
ADIPOGENESIS
Wang C.H.1, Chen Y.F.2, Wu C.Y.2, Tsai T.F.2 and Wei Y.H.1, 3
1
Institute of Biochemistry and Molecular Biology, National Yang-Ming
University, Taipei 112, Taiwan. 2Department of Life Sciences and
Institute of Genome Sciences, National Yang-Ming University, Taipei
112, Taiwan. 3Department of Medicine, Mackay Medical College,
Sanjhih, Taipei County 252, Taiwan.
Cisd2, the causative gene for Wolfram syndrome 2 (WFS2), is located
on human chromosome 4q, where a genetic component for longevity
has been mapped. In a previous study, we demonstrated that Cisd2
is related to the life-span of the animals. Cisd2 is a mitochondrial
protein and the Cisd2 deficiency causes mitochondrial breakdown and
dysfunction accompanied by autophagic cell death, and leads to a wide
spectrum of premature aging phenotypes in mice. However, the functions
of Cisd2 in a specific cell type remain unclear. Here, we report that the
conditional Cisd2 knockout mice displayed lower weight of epididymal
white adipose tissues and interscapular brown adipocytes as compared
with age-matched control mice. These findings suggest that Cisd2
deficiency leads to defect in the formation or maintenance of adipose
tissues. We then investigated whether the loss of Cisd2 causes defect
in adipogenesis. We observed that the mRNA and protein expression
levels of Cisd2 were up-regulated during adipogenesis. In addition, we
examined the adipogenesis of mouse embryonic fibroblasts (MEFs)
from the mouse embryos of different Cisd2 genotypes including wild-
type, heterozygous and double-knockout. The results showed that the
content of triglycerides and the expression levels of some adipogenic
genes were decreased in the adipocytes differentiated from the MEFs
of mice with heterozygous mutation or double-knockout of Cisd2 gene,
respectively. In summary, in animal and cell models we observed that
Cisd2 is required for adipogenesis.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 233
POS-THU-170
EVIDENCE FOR A NOVEL, DEVELOPMENTALLY
REGULATED, AMYLOID PRECURSOR PROTEIN
PROCESSING PATHWAY
Vella L.J.1, Munter L.M.3, Fodero-Tavoletti M.T.1, Roberts R.2, Masters C.L.2,
Barnham K.J.1, 2 and Cappai R.1, 2
1
Department of Pathology, The Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Australia. 2The
Mental Health Research Institute of Victoria, Parkville, Victoria. 3Institut
fur Chemie und Biochemie, Freie Universitat Berlin, Germany.
A major pathological characteristic of Alzheimer’s disease are the
extracellular plaques composed of β-amyloid (Aβ) peptides, which
are derived from the larger Amyloid Precursor Protein (APP). While
much is known about the production of β-amyloid (Aβ), many questions
remain concerning APP function and APP metabolism. In this study,
we investigated the occurrence of novel N-terminal APP (NTFs)
fragments in human and mouse tissue and aimed to establish cell
models to investigate the cellular pathways implicated in the generation
of these fragments. Our results suggest a novel, developmentally
regulated, processing pathway produce the 17-28kDa APP NTFs. As
APP is a known neurite outgrowth promoting molecule, we suspect
that the secreted NTFs may perform this function and promote
neuronal proliferation and differentiation. Our data suggests that APP
transmembrane dimerisation may be important for APP N-terminal
processing and consequently the function of APP. Further studies will
be aimed at determining the relevance of these isoforms to the normal
cellular function of APP and Alzheimer’s disease pathophysiology.
POS-THU-172
THE CHARACTERIZATION OF C10ORF65 A PUTATIVE
COMPLEX I ASSEMBLY FACTOR
Wang X., Wang W., McKenzie M. and Ryan M.T.
Department of Biochemistry, La Trobe University.
Mitochondrial complex I (NADH: ubiquinone oxidoreductase) is the
largest enzyme complex in the mitochondrial respiratory chain. It is the
entrance point for electrons into the respiratory complexes, which couple
electron transport with proton pumping to create an electrochemical
gradient to drive ATP generation. In order to achieve its enzyme activity,
a total number of 45 subunits need to be assembled correctly to form
the mature complex. To aid this assembly, a group of proteins, termed
as “complex I assembly factors”, exist. C10orf65 is a putative assembly
factor recently identified by phylogenetic profiling of different species
containing or lacking complex I, however, its function is poorly understood.
After translation in the cytosol, C10orf65 is imported into mitochondria
via the protein import machinery of mitochondria. In vitro import assays
revealed that C10orf65 has a cleaved pre-sequence which is removed
to form the mature protein following import. Mitochondrial localization
of C10orf65 was further confirmed by generating a C-terminal GFP-
tagged construct and epi-fluorescence microscopy, while, sonication
and sodium carbonate treatment of isolated mitochondria showed that
C10orf65 is partially associated with the inner membrane. Blue Native
(BN)-PAGE analysis revealed that GFP-tagged C10orf65 was present in
a ~400 and ~500 kDa complexes which may be assembly intermediates
of complex I. The function of C10orf65 in complex I biogenesis requires
further investigation, however, its study may provide insights into
complex I biogenesis and how disruption of this process can lead to
complex I deficiency and mitochondrial disease.
POSTERS WEDNESDAY & THURSDAY
POS-WED-173
DECIPHERING THE MOLECULAR DETAILS OF TYPE
V SECRETION SYSTEMS IN ENTEROPATHOGENIC
ESCHERICHIA COLI (EPEC)
Webb C.T., Selkrig J.P., Celik N. and Lithgow T.J.
Host-Pathogen Molecular Biology Unit, Department of Biochemistry
and Molecular Biology, Monash University, Clayton, 3800, Australia.
In order to colonise human hosts and initiate productive infections,
pathogenic bacteria rely on protein transport systems. The proteins
secreted by these systems, ”effectors”, function as adhesins, toxins, and
in other activities that contribute to virulence. Understanding how these
protein transport systems work is essential to understanding bacterial
pathogenesis. Consequently some of these systems constitute attractive
drug targets to disarm Gram-negative bacteria. Type V secretion systems
(T5SS) are elegant devices that secrete effector proteins resulting in a
myriad of disease states such as meningitis, sepsis, pneumonia and
diarrhoea. Using a bioinformatic approach, we are exploring the T5SS
proteins from the recently sequenced enteropathogenic Escherichia coli
strain, E2348/69. Several novel proteins have been identified and we are
using these to dissect the inter-related processes of protein insertion in
the outer membrane and effector domain assembly. Here we present our
current findings for one such candidate which we hypothesise to belong
to the subfamily of functionally and structurally related autotransporters,
SAATs (self-associating autotransporters).
POS-WED-175
THE EFFECTS OF WEAKENED MITOTIC
CHECKPOINTS: MOLECULAR EVENTS THAT FOLLOW
AND THE RELEVANCE TO CANCER THERAPY
Wong H.1, Murray M.2, Gregory S.1 and Saint R.1
1
University of Melbourne. 2University of Adelaide.
The spindle assembly checkpoint (SAC) is the major cell cycle control
mechanism that ensures correct chromosome segregation in mitosis.
Its disruption causes genomic instability and is linked to neoplastic
transformation in cancer. Drugs targeting mitosis are commonly used
in cancer therapy and their mode of action is dependent on a functional
SAC. The lost of checkpoint functions are linked to drug resistance
in clinical settings, but the cellular fates following a weakened SAC
remains unclear. Using Drosophila melanogaster, this project follows
the fate of cells with weakened Mitotic Arrest Deficient 2 (MAD2), a key
SAC component, using flies expressing a tissue specific and inducible
RNAi construct targeting MAD2. The project screens a set of 450 kinase
and phosphatase RNAi lines for those that enhance the phenotype of
MAD2 knockdown to lethality. In this screen we have identified MAD2
interactors for genes involved in the regulation of centromeres, histones
and telomeres, JNK signalling, DNA damage responses and growth,
metabolic and stress signaling. With almost no knowledge of the link
between a defective SAC and apoptosis, and because apoptosis is the
preferred clinical outcome of cancer treatment, the components of the
JNK apoptotic pathways are key candidates for further characterization.
Using fixed tissue analysis and fluorescent activated cell sorting, we are
now analyzing the cellular role of these MAD2 interactors in cells with
a defective SAC.
Page 234 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-174
ANTIPROLIFERATIVE AND PROAPOPTOTIC EFFECTS
OF CURCIN FROM JATROPHA CURCAS ON HUMAN
SMALL CELL LUNG CANCER CELL LINE
Wetprasit N.1, Bhorncharoennop C.1, Uyen U.1, Abhornsuwan T.2,
Promboon A.3, Roytrakul S.4 and Ratanapo S.3
1
Department of Biotechnology, Faculty of science, Ramkhamhaeng
University, Bangkok 10240, Thailand. 2Department of Medical
Technology, Faculty of Allied Health Sciences, Thammasat University,
Pathumthani 12120, Thailand. 3Department of Biochemistry, Faculty
of Science, Kasetsart University, Bangkok 10900, Thailand. 4Genome
Institute, National Center for Genetic Engineering and Biotechnology,
Pathumthani 12120, Thailand.
Curcin is one of type I ribosome-inactivating proteins which can inhibit
growth of several tumor cells but the molecular mechanism of this
effect remains elusive. The cDNA encoding curcin gene from leaves of
Jatropha curcas KUBP 78, was cloned and overexpressed in Escherichia
coli. The partially purified protein was obtained by filtration through
membrane with molecular weight cut off 50 kDa. In order to understand a
possible involvement of genes regulating apoptotic signaling pathways,
transcriptional expression levels of Caspase-3, -7, -8 and -9 genes were
analyzed after incubating partial purified curcin with NCI-H187 cell line
(human small cell lung carcinoma) at various times using Real-time
PCR. Partially purified curcin was able to inhibit the growth of NCI-H187
cell line even at a very low concentration. Incubation of target cells with
curcin could enhance the caspase-7 and -9 expressions which could be
observed since day 1 and reached the peak at day 3 and 5, respectively.
Delayed up-regulation of Caspase-3 at day 3 was also found but the
level of Caspase-8 was not changed. These results indicated that curcin
may induce apoptosis in NCI-H187 via caspase dependent pathway.
POS-THU-176
OPTIMIZATION OF HETEROGENEOUS ION CHANNEL
EXPRESSION IN HEK-293T CELLS
Yaakob N.S., Exintaris B. and Irving H.R.
Monash Institute of Pharmaceutical Sciences, Monash University, 381
Royal Parade, Parkville VIC 3052, Australia.
The 5-hydroxytryptamine receptor subtype 3 (5-HT3) is the only
member of the serotonin receptor family that functions as a ligand-gated
ion channel (LGIC). The 5-HT3 receptor has the potential for complex
heterogeneity as five HTR3-like genes have been discovered that code
for 5-HT3A, 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E subunits. Functional
receptors consist of pentamers of 5-HT3A subunits only or combinations
of the 5-HT3A and other subunits. The goal of our research is to compare
the electrical activities between different receptor subunit combinations
expressed in HEK-293T cells using patch-clamping. However, to
perform efficient patch-clamping, high expression levels of the receptors
are required. We have manipulated a range of parameters to optimize
expression of the 5-HT3 receptor subunits in HEK-293T cells using
green fluorescent protein as a marker for transfection efficiency. We
are currently expressing tagged heterogenous receptor combinations to
determine relative expression levels using a multi-plasmid co-transfection
approach. We are also preparing multi-cistronic vectors to express two
or three subunit genes in a single vector. We will report on comparisons
between the two methods by assessing transfection efficiency, protein
expression levels via western blotting and immunohistochemistry and
finally patch clamp experiments.
POSTERS WEDNESDAY & THURSDAY
POS-WED-177
A SINGLE AMINO ACID POINT MUTATION IN
FIBROBLAST ACTIVATION PROTEIN (FAP) IN HUMANS
IS ASSOCIATED WITH LOSS OF FUNCTION AND CELL
SURFACE LOCALISATION
Yao T.W.1, Osborne B.1, Wang X.M.1, Yu D.M.T.1, Topaloglu K.A.2 and
Gorrell M.D.1
1
Centenary Institute DO6, University of Sydney, NSW 2006, Australia.
2
Cukurova University, Faculty of Medicine, Department of Pediatric
Endocrinology and Metabolism, Adana, Turkey.
Fibroblast activation protein (FAP) is a type II cell surface glycoprotein
of the prolyl oligopeptidase family. FAP possesses a rare catalytic
activity, hydrolysis of the post-proline bond two or more residues from
the N-terminus of target substrates. FAP is undetectable in normal
adult tissues, but its expression is significantly induced in tumours,
inflammation and at sites of the interface of tissue fibrosis1. We have
previously shown in vitro that FAP overexpression in LX2 and HEK293T
cell lines has an important role in cell adhesion, apoptosis, proliferation
and cell migration2. The FAP gene of 26 exons spans 72.8 kb at human
2q24.3. A homozygous loss-of-function single nucleotide mutation in
this FAP gene encoding a residue in the 6th blade of the beta-propeller
domain of the protein was identified. We demonstrated that this mutation
caused ablation of FAP enzyme activity and an accumulation of FAP in
the ER rather than at the cell surface. This mutation was associated with
ER stress and unfolded protein response, as indicated by upregulation
of the ER chaperone protein, BiP/GRP78, in both a heterologous
expression system [transfected HEK293 cells] and cultured patient
fibroblasts. A monoclonal antibody that binds to a conformational
epitope of FAP bound poorly to the mutant FAP protein, suggesting
a conformational change in the FAP structure. This is the first loss
of function single amino acid point mutation outside the catalytic site
characterised in the prolyl oligopeptidase gene family. 1. Wang XM, Yao
T-W, Nadvi NA, Osborne B, McCaughan GW, Gorrell MD 2008 Front
Biosci 13:3168-80. 2. Wang, XM, DMT Yu, GW McCaughan, MD Gorrell
2005 Hepatology 42: 935-45.
POS-WED-179
THE ROLE OF WW AND C-TERMINAL DOMAINS IN
YORKIE/YES-ASSOCIATED PROTEIN
Zhang X., Milton C.C., Humbert P.O. and Harvey K.F.
Peter MacCallum Cancer Centre.
The Salvador/Warts/Hippo (SWH) pathway regulates tissue growth
through inhibiting the activity of Yorkie (Yki) in D. melanogaster and Yes-
associated protein (YAP) in mammals, which act as transcriptional co-
activators. Yki and YAP have a conserved N-terminal domain, through
which they associate and cooperate with the transcription factors,
Scalloped (Sd)/TEAD, to promote tissue growth. In addition, Yki and YAP
possess two conserved WW domains that mediate interaction between
several proteins that either promote or inhibit YAP’s ability to drive
transcription. Furthermore, the C-terminus of YAP includes three further
motifs: a SH3 domain-binding motif, transcription activation domain and a
PDZ domain-binding motif, none of which are conserved in Yki. To study
the role of the Yki/YAP WW domains and the YAP C-terminus, several
Yki/YAP mutants were constructed and their function was analyzed
with assays for tissue growth in Drosophila, or with assays for colony
growth, cell transformation, migration and invasion in MCF10A or NIH-
3T3 cells. Results showed that: 1. WW domain played an inhibitory role
in migration and invasion in MCF10A cells, but promoted NIH-3T3 cell
growth and transformation and drived tissue growth in D. melanogaster.
2. Deletion of YAP’s transcription activation domain abolished its ability
to activate TEAD2, as well as the ability to stimulate invasion of MCF10A
cells. However, this mutant promoted growth and transformation of both
MCF10A and NIH-3T3 cells. 3. Mutation of YAP’s PDZ domain-binding
motif lowered its ability to promote invasive behavior of MCF10A cells, but
promoted 3D growth and transformation. Taken together, these results
suggest that YAP is able to mediate tissue growth, transformation and
invasion through different domains and the total effect of YAP on growth
and invasion is dependent on the coordination of protein interactions
via its WW and transcriptional activation domains, as well as its PDZ
domain-binding motif.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 235
POS-THU-178
THROMBIN-TREATED CYCLOOXYGENASE-2
EXPRESSION AND PROSTAGLANDIN E2 PRODUCTION
BY PRIMARY MYOBLASTS IS MEDIATED BY
PROTEASE-ACTIVATED RECEPTOR-1
Yoo H.J.1, Pagel C.N.1, Pike R.N.2 and Mackie E.J.1
1
School of Veterinary Science, The University of Melbourne, Cnr of
Flemington Rd and Park Dr, Parkville, Victoria, Australia. 2Department
of Biochemistry and Molecular Biology, Monash University, Wellington
Rd, Clayton, Victoria, Australia.
Thrombin treatment of primary skeletal muscle myoblasts stimulates
proliferation and intracellular calcium mobilisation and inhibits
differentiation and apoptosis induced by serum deprivation. Previous
studies have demonstrated that the effect of thrombin on myoblast
apoptosis and differentiation are not mediated by the main thrombin
receptor, protease-activated receptor-1 (PAR-1). The aim of this project
was to identify the role of thrombin and PAR-1 in myoblast function.
Thrombin treatment of serum-deprived primary myoblasts increased
cyclooxygenase-2 (COX-2) gene expression, and prostaglandin E2
(PGE2) and interleukin-6 (IL-6) production. In addition, PGE2 inhibited
apoptosis and differentiation induced by serum deprivation in primary
myoblast cultures. We hypothesised that PGE2 production may be
related to the mechanism of thrombin’s inhibitory effect on myoblast
apoptosis and differentiation. However stimulation of COX-2 gene
expression and both PGE2 and IL-6 production by thrombin occurred in
PAR-1 dependent manners. This result suggests that COX and PGE2
may be important mediators of PAR-1 dependent effects of thrombin
on myoblasts.
POS-THU-180
LARGE-CONDUCTANCE CA 2+-ACTIVATED
POTASSIUM AND ETHER-à-GO-GO POTASSIUM
CHANNELS REGULATE PROLIFERATION OF HUMAN
MESENCHYMAL STEM CELLS
Zhang Y.Y. and Li G.R.
Department of Medicine, The University of Hong Kong, Hong Kong
SAR China.
Bone marrow-derived mesenchymal stem cells (MSCs) are a promising
cell source for regenerative medicine; however, cellular physiology
is not fully understood in human MSCs. The present study was to
determine the potential role of the dominant functional ion channels,
large-conductance Ca2+-activated potassium (BKCa) channel, ether-à-
go-go potassium (hEAG1) channel, and sodium channel, in regulating
proliferation of human MSCs using electrophysiological, cell proliferation
assay, and biochemical approaches. We found that paxilline (1 μM),
astemizole (1 μM), and tetrodotoxin (TTX, 30 nM) respectively reduced
BKCa, hEAG1, and sodium current in human MSCs. The cell proliferation
assay with MTT and 3H-thymidine incorporation methods revealed that
the inhibition of BKCa with paxilline and hEAG1 with astemizole, but not
sodium current with TTX, decreased cell proliferation and reduced DNA
synthesis rate in a concentration-dependent manner. Flowcytometry
analysis displayed that paxilline and astemizole accumulated human
MSCs at G0/G1 phase, and decreased cell population of S phase.
Moreover, lentivirus-based shRNAs targeted to BKCa or hEAG1 channel
remarkably reduced both mRNA and protein expression of BKCa or
hEAG1 channel; and proliferation of human MSCs was also reduced
by BKCa-shRNAs or hEAG1-shRNAs. These results demonstrate the
novel information that BKCa and hEAG1 channels, but not sodium
channel, participate in the regulation of cell proliferation by promoting
G0/G1 cells into cell cycling progression in human MSCs.
POSTERS WEDNESDAY & THURSDAY
POS-WED-181
CHARACTERISING THE INHIBITION OF LEGUME
NODULATION CAUSED BY LOW pH CONDITIONS
Lin M.H., Ferguson B.J., Indrasumunar A. and Gresshoff P.M.
ARC Centre of Excellence for Integrative Legume Research, The
University of Queensland, St. Lucia, Brisbane, QLD, Australia.
Lateral organ differentiation and its internal and external control are
critical frontiers of developmental plant genetics. Legume nodulation
offers an elegant system to study the genetic and biochemical basis
of these processes where mutational analysis has coupled structure
to function. Legumes form nitrogen-fixing root organs known as
nodules after establishment of a symbiotic relationship with rhizobia.
Nodule formation is tightly regulated by the plant and can be inhibited
by a number of external factors, such as soil acidity. This is of global
importance as many of the world’s legume crops, such as soybean, are
grown in acidic soils. Despite this, the precise plant mechanism(s) by
which acidic conditions inhibit soybean nodule development remains
poorly characterised. Here, we present evidence that the inhibition
of soybean nodulation by acidic conditions may be controlled by the
plant and that this control may have a systemic component. In addition,
a separate inoculation-independent inhibition pathway may also
exist. Furthermore, we show that the inhibition of nodulation in acidic
conditions likely acts during the early stages of signal perception during
nodule meristem establishment. Next generation deep sequencing
will be used to determine RNA expression changes and novel genes
functioning in acid-stressed soybean roots inoculated with or without a
compatible Bradyrhizobium strain. Transgenic soybean chimeric plants
expressing a visual reporter system containing the red fluorescent
protein, DsRed2, were generated, and the nodulation-deficient mutant
nod49 was successfully complemented by over-expression of the
soybean Nod Factor Receptor, GmNFR1α. This transformation system
will be used to investigate the role of the soybean GmNFR1α, in wild
type and GmNFR1α-complemented nod49 plants grown under acid
stress.
POS-WED-183
INVESTIGATING THE ROLE OF SHORT RNAS IN
TOMATO FRUIT DEVELOPMENT
Lopez-Gomollon S. and Dalmay T.
University of East Anglia, Norwich, NR4 7TJ, UK.
The availability and consumption of fresh fruit depends upon the
quality attributes and physiological integrity of these products. For
all fruit products it is essential that the quality characteristics of color,
flavour and especially texture meet consumer expectations.Tomato
is the model system for studying development and ripening in fleshy
fruits due to excellent genetic and genomic resources. Regulatory short
RNAs have been recently discovered and become one of the most
intensively studied fields in plant molecular biology. However, to date
this field has been restricted mainly to Arabidopsis and it is now time to
investigate the role of short regulatory RNAs in crop plants, like tomato.
Understanding the molecular mechanisms of specific plant traits such
as fleshy fruit development and ripening is key in future breeding
programs. One of the most recently discovered gene regulation
mechanisms is post-transcriptional and involves 21-24 nucleotide small
RNA molecules (sRNA). To get a complete picture of sRNA profiles
in fruit ripening and development, 10 separate libraries from different
stages of fruit development were created. We found that the pattern
of 10 known miRNA change across the timeserie, suggesting a role
in fruit development. Moreover, 6 new miRNA identified for first time
in tomato, show a expression profile that change during development.
These results were confirmed by Northern blot. To study the role of
the new miRNA in vivo, tomato plants overexpressing the precursor of
each miRNA will be developed, as well as plant overexpressing target
mimicries, to inhibit the activity of each miRNA. Our aim is to develop
a platform to study regulatory networks involved in development and
maturation of commercially important fruits.
Page 236 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-182
CHARACTERIZING A NOVEL LEA PROTEIN FROM
RESURRECTION GRASS
Ling H.Y., Blomstedt C.K., Hamill J.D., Walmsley A.M. and Neale A.D.
School of Biological Sciences, Monash University, Melbourne,
Australia.
The grass Sporobolus stapfianus Gandoger exhibits homochlorophyllous
desiccation tolerance, with plants able to resist to cellular degradation
due to severe water deficit. This resurrection plant can revive to a fully
hydrated state within 24 hours after loss of more than 95% of its total
water content. This rare trait is found only about 0.2% of all vascular plant
species but is widespread in primitive clades for example algae, lichens
and bryophytes. A novel LEA (late embryogenesis abundant) protein
was identified from dehydration-stressed leaf tissue of S. stapfianus.
LEA proteins were first described in orthodox seed development and
have a crucial role as a protectant in vegetative plants, invertebrates
and bacteria during water deficit. The expression of LEA proteins from
S. stapfianus during dehydration and rehydration has been compared
with the LEA mRNA expression profile in leaf tissue. In addition,
subcellular localisation of the LEA protein using transient expression
in Nicotiana benthamiana revealed high accumulation of this protein
in the chloroplast. The LEA gene was introduced into Arabidopsis to
elucidate its role in improving dehydration tolerance and in response to
other abiotic stresses. The ability of the LEA protein to protect enzyme
activity in vitro using a lactate dehydrogenase enzymatic assay was
investigated. This study is important to provide an understanding of the
LEA protein in desiccation tolerance.
POS-THU-184
CHARACTERISATION OF THE PHYTOPHTHORA
ZOOSPORE SECRETOME
Ludowici V.A., Blackman L.M. and Hardham A.R.
Plant Sciences Division, Research School of Biology, The Australian
National University, Canberra, ACT 2601, Australia.
Phytophthora species are fungus-like plant pathogens in the Class
Oomycetes. They cause millions of dollars of losses to agricultural
industries and threaten natural ecosystems across the globe. Because
of their phylogenetic distance from true fungi, chemical controls that
target fungi are not effective against Phytophthora pathogens. A greater
understanding of the molecular and cellular mechanisms of host infection
could lead to the development of novel control measures. During initial
plant infection, motile Phytophthora zoospores encyst and the contents
of three types of peripheral vesicles undergo regulated secretion. One of
the proteins secreted during this stage is a complement control protein
(Ccp) which is found in so-called large peripheral vesicles. The objectives
of this project are to investigate the role of the large peripheral vesicles
during plant infection and also to identify other proteins secreted from
zoospores during encystment. Expression of PnCcp in P. nicotianae
during development has been analysed by quantitative real-time PCR
and showed that this gene is more highly expressed during sporulation
and in zoospores than in vegetative hyphae or 3-hour germinated cysts.
Immunofluorescence microscopy showed that biogenesis of large
peripheral vesicles begins prior to the development of ventral surface
vesicles. Immunofluorescence microscopy was also used to study the
timing of secretion from these vesicles and indicated that PnCcp is
secreted within 2 minutes after induction of encystment. The secretion
of two other peripheral vesicle proteins, Vsv1 found in the ventral surface
vesicles and Cpa found in the dorsal peripheral vesicles, also occurred
within this time period. To identify other secretome components, proteins
from young cysts will be solubilised and proteomic techniques employed
for their analysis.
POSTERS WEDNESDAY & THURSDAY
POS-WED-185
TOWARDS THE DOMESTICATION OF A WILD RICE
RELATIVE, MICROLAENA STIPOIDES USING LARGE
SCALE GENE SEQUENCING
Malory S.1, Shapter F.M.1, Elphinstone M.S.1, Chivers I.H.2 and Henry
R.J.1
1
Centre for Plant Conservation Genetics, Southern Cross University,
Lismore, NSW. 2Native Seeds Pty Ltd, Cheltenham, VIC.
Microlaena stipoides, commonly known as weeping grass, is a distant
relative of rice. It is a drought, frost and shade tolerant perennial
evergreen plant and produces seeds similar to rice. M. stipoides can be
used for grain production and additionally it can be grazed as a pasture.
This species responds well to nitrogen application and also regular
irrigation, making commercial production possible and making it a target
for domestication. Extensive sequencing and comparative mapping
has established a high degree of conservation between rice and other
grasses, allowing the isolation of the corresponding homologues of
important rice genes in other grasses. In this study the rice genome
sequence is being used for comparison with corresponding genes in M.
stipoides. A high range of variability occurs within the natural populations
for domestication traits and this can be harnessed for selective breeding
programs. An induced mutation population has also been established
to capture desired traits for crop improvement. High-throughput next
generation sequencing using the Illumina Genome Analyser IIx is
being used to discover single nucleotide polymorphisms (SNPs) in M.
stipoides which can be used in establishing domesticated lines of M.
stipoides. Once domesticated, M. stipoides will become a new cereal
crop for commercial food production. This technique can also be utilised
for other wild grasses to screen for desirable domestication traits and
possibly to create new crops for food consumption.
POS-WED-187
EXAMINATION OF RESIDUES THAT INFLUENCE
INTERACTION BETWEEN RUBISCO AND RUBISCO
ACTIVASE
Milward S.E. and Whitney S.M.
Division of Plant Sciences, Research School of Biology. RN Robertson
Building 46, Australian National University, Canberra, ACT.
Ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) is the key
CO2 fixing-enzyme in the biosphere and frequently limits photosynthetic
carbon assimilation in higher plants. In addition to a unique activation
process (requiring non-substrate CO2 and Mg2+ binding) the activity of
Rubisco in higher plants is regulated by a nuclear-encoded “helper”
protein, Rubisco activase (RA). Using energy generated from ATP
hydrolysis, RA facilitates the removal of inhibitory sugar phosphate
ligands from the Rubisco active sites via a poorly understood interactive
mechanism (hindered by the inability to obtain a crystal structure of RA).
Prior mutagenic studies suggest Rubisco-RA interactions are defined by
surface residues 89 to 94 in the catalytic Rubisco large (L-) subunit and
those in the C-terminal domain between codons 311 and 314 of RA. To
examine Rubisco-RA interactions in tobacco, three transplastomic lines
producing Rubisco with L-subunit mutations at codons 89 and 94 have
been generated. Light-transient CO2-assimilation assays suggest that
while both single L-subunit mutations have little effect on RA activation
capacity (at 25°C),in the double mutant activation is impaired by ~25%.
Presented here is an update of gas exchange and biochemical analysis
of the effects of these mutations on the interaction between tobacco
Rubisco and RA.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 237
POS-THU-186
A QUIESCENT MIR159C:TARGET GENE REGULATORY
MODULE IMPLIES THE ANCIENT MIR159 FAMILY HAS
VERY FEW TARGETS OF BIOLOGICAL RELEVANCE IN
ARABIDOPSIS
Allen R.S.1, 2, Li J.1, 2, Alsonso-Peral M.M.1, White R.G.2, Gubler F.2 and
Millar A.A.1
1
Research School of Biology, Australian National University, Canberra,
ACT. 2CSIRO, Division of Plant Industry, Canberra, ACT.
A current challenge regarding microRNA (miRNA) biology is the
identification of biologically relevant target genes. Bioinformatics
and molecular approaches have predicted that the Arabidopsis
miR159 family may regulate more than twenty such genes. However,
genetic analysis has revealed the functional specificity of the major
family members, miR159a and miR159b appears limited to only two
genes, MYB33 and MYB65. Here we show that the expression of the
remaining family member, MIR159c, overlaps with a subset of these
predicted target genes in anthers. Despite this, a mir159c mutant
does not display any anther defects and a mir159abc triple mutant is
indistinguishable from mir159ab. Consistent with this, transcription of
MIR159c was low and processing of pre-MIR159c poor, resulting in a
low abundance of miR159c and no deregulation of potential miR159
target genes was evident in mir159c. Conversely, examination of
potential miR159c targets, MYB101 and MYB120, found that despite the
detection of miR159-guided cleavage products, neither their spatial nor
temporal expression domain appeared miR159-regulated. Furthermore
expression of miR159-resistant versions of MYB101 or MYB120 does
not lead to anther defects. Thus the potential miR159c-MYB101/MYB120
regulatory relationship appears to be of little or no biological importance,
but may represent the remains of a former miRNA:target module. Such
genetic remnants from the death of conserved miRNA modules may
be leading to the overestimation of the complexity of miRNA regulation
and illustrates the dynamic nature of regulatory relationships that exists
even within ancient miRNA families.
POS-THU-188
OVER EXPRESSION OF SNAKIN-1 AND SNAKIN-2
GENES UNDER A POTATO LIGHT INDUCIBLE LHCA3
PROMOTER IN TRANSGENIC POTATOES FOR BROAD
SPECTRUM DISEASE RESISTANCE
Mohan S.1, 2 , Meiyalaghan S.1, Jones E.E.2, Jacobs J.M.E.1, Pringle J.M.1
and Conner A.J.1, 2
1
New Zealand Institute for Plant & Food Research. 2Agriculture and
Life Science Division, Lincoln University.
Abstract Snakin-1 (SN1) and Snakin-2 (SN2) are low-molecular
weight antimicrobial peptides produced in potato tubers. Such proteins
are thought to play important roles in the innate defence against
invading microorganisms. Over-expression of StSN genes is known
to provide broad spectrum activity against a wide range of bacterial
and fungal pathogens in potato. Based on homology to other genes,
these proteins have also been hypothesised to be involved in diverse
biological processes, including: cell division, cell elongation, cell
growth, transition to flowering, and signalling pathways. StSN1 and
StSN2 genes were isolated from potato cv. Iwa. The coding regions
of these genes were individually cloned into the intragenic expression
cassette harbouring the Lhca3 (cab) promoter and the Lhca3 (cab)
terminator. The resulting intragenic cab5’-StSN-cab3’ chimeric genes
were cloned into transgenic binary vector pMOA33. The binary vectors
pMOA33cabStSN1 and pMOA33cabStSN2 were individually used for
Agrobacterium-mediated transformation of potato. Thirty-three and
thirty-two independently derived SN1- and SN2-transgenic plants were
regenerated respectively. Multiplex-PCR confirmed the presence of the
nptII selectable marker gene and the specific gene in all regenerated
lines. Based on quantitative RT-PCR of each SN gene, seven lines were
selected for each of the SN1- and SN2-transgenic plant populations
for disease resistance assessment. Pathogenicity bioassays showed
that the over-expression of either StSN1 gene or StSN2 gene from the
intragenic cassettes conferred resistance against the enterobacterial
phytopathogen Pectobacterium atrosepticum (formally Erwinia
carotovora ssp. atroseptica).
POSTERS WEDNESDAY & THURSDAY
POS-WED-189
THE ROLE OF ATMYB5 AND ATMYB23 IN SEED COAT
AND TRICHOME DEVELOPMENT
Napoli R., Parish R. and Li S.
The Department of Botany, LaTrobe University, Melbourne, Victoria,
Australia.
The cell wall and mucilage (Pectin) are important in protecting seeds
from damage, maintaining dormancy and facilitating germination under
favourable conditions. Knowledge gained on trichome development may
lead to modifictaions which improve crop pest ressistance. MYB genes
encode transcription factors which regulate several developmental
and metabolic pathways in plants. MYB transcription factors function
by activating or repressing other genes involved in secondary
metabolism, cell development, signaling pathways, disease resistance
and responses to environmental stresses. Arabidopsis is well suited to
study the function, structure and regulation of MYB genes. This project
aims to characterise the AtMYB5 and AtMYB23 regulatory pathways
in seed coat and trichome development. The AtMYB5 and AtMYB23
MYB transcription factors are found in the model plant Arabidopsis.
AtMYB5 and AtMYB23 are both expressed in the developing seed and
leaf trichomes. An atmyb5/atmyb23 double mutant revealed a possible
redundancy between the two proteins in trichome development. Both
MYB transcription factors interact with bHLH and HD-ZIP proteins
to form an activation complex capable of activating target genes.
Characterisation of these pathways involves expression analysis of
AtMYB5/AtMYB23 target genes in trichome development. Such putative
target genes were identified by microarray data from immature seeds
of the atmyb5 mutant. These genes include GLYCOSYL HYDROLASE
810 (GH10810) which encodes a putative xylanase enzyme and two
hydrolase genes ABE1 and ABE4. EMSA analysis is being utilised to
study protein-DNA interaction between AtMYB5 and the promoters of
selected target genes. The AtMYB5 and AtMYB23 pathways provide a
useful system for the study of cell wall synthesis.
POS-WED-191
ENVIRONMENTAL REGULATION OF THE CYANOGENIC
GLYCOSIDE, DHURRIN, IN SORGHUM
O’Donnell N.1, Blomstedt C.1, Stuart P.2, Neale A.1, Hamill J.1 and
Gleadow R.1
1
School of Biological Sciences, Monash University, VIC Australia.
2
Pacific Seeds, QLD Australia.
Sorghum is a C4 plant with high water use efficiency, making it drought
and heat tolerant. Forage sorghum is used as fodder for cattle in the
dry tropics, but is cyanogenic, releasing hydrogen cyanide (HCN) when
plant tissue is disrupted. The HCN potential (HCNp) of sorghum differs
greatly between varieties and within a variety sorghum is subject to
spatial and temporal regulation. The HCNp of sorghum is also affected
by environmental factors and varieties can differ from one season to the
next, HCNp is known to be influenced by numerous factors, including
maturity, drought, frost and soil fertility. Experienced farmers know
that young sorghum plants, or older plants that have been subjected to
drought, are often toxic and can kill cattle. However there are no known
scientific articles that have shown that HCNp in sorghum is increased
under drought conditions. We have used various concentrations of
polyethelene glycol (PEG), an osmoticant, added to the nutrient solution
of sorghum plants grown in hydroponics, and have found a positive
correlation between PEG and HCNp. The increase in HCNp under
higher PEG concentrations could be due to either the up-regulation of
the cyanogenesis genes, or the constitutive expression of cyanogenesis
genes and subsequent accumulation of dhurrin due to reduced growth,
or both. Although we have found a definite correlation between HCNp
and PEG concentration, we found that the HCNp only increased when
the plants were grown in the PEG for a number of weeks. These findings
are relevant to the efficient production and use of sorghum for farmers
globally.
Page 238 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-190
CHARACTERISATION OF THE SERK GENE FAMILY
IN LEGUMES INVOLVED IN DEVELOPMENT AND
DEFENSE
Nolan K.E. and Rose R.J.
ARC CILR, School of Environmental and Life Sciences, University of
Newcastle. Australia.
SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK)
genes are part of the regulation of diverse signalling events in plants.
Current evidence shows SERK proteins to function both in developmental
and defense signalling pathways, which occur in response to both peptide
and steroid ligands. SERKs are generally present as small gene families
in plants, with five SERK genes in Arabidopsis. Knowledge gained
primarily through work on Arabidopsis SERKs indicates that these
proteins probably interact with a wide range of other receptor kinases
and form a fundamental part of many essential signalling pathways. This
seemingly broad requirement for this small family of genes indicates
their underlying importance for plant growth and defense. The SERK1
gene of the model legume, Medicago truncatula functions in somatic and
zygotic embryogenesis, and during many phases of plant development,
including nodule and lateral root formation [1, 2]. However, other SERK
genes in M. truncatula and other legumes are largely unidentified and
their functions are unknown. To aid the understanding of signalling
pathways in M. truncatula, we have attempted to identify and annotate
all SERK genes in this species. Using degenerate PCR and database
mining, eight more SERK-like genes have been identified and these
have been shown to be expressed. One of them was found to contain a
number of splice variants, which has not previously been reported in this
gene family. The sequence information obtained from M. truncatula was
used to identify potential SERK family genes in the recently sequenced
soybean genome. The phylogenetic relationship of members of the
SERK gene family to development and defense function is presented. 1.
Nolan et al (2003) Plant Physiology, 133 218-230. 2. Nolan et al (2009)
Journal of Experimental Botany, 60 1759-1771.
POS-THU-192
ONTOGENETIC VARIATION IN EUCALYPTUS DEFENSE
PROFILES
O’Gorman K.L.1, Miller R.E.1, Cody N.1, Burns A.E.1, Foley W.J.2 and
Gleadow R.M.1
1
School of Biological Sciences, Monash University, Clayton, VIC,
Australia. 22. Evolution, Ecology and Genetics, Research School of
Biology, The Australian National University, Canberra, ACT, Australia.
Plants have evolved a range of physical and chemical mechanisms
which have been shown to play a role as herbivore deterrents. Chemical
defences are broadly categorised as carbon-based or nitrogen-based.
According to the Carbon Nutrient Balance Theory, carbon-based
compounds tend to be more prevalent in plants which have evolved
in non-light limiting, nutrient poor, environments. Variations in defence
strategies also occur within an individual during its development.
Eucalyptus, a dominant Australian genus, is typically well defended
with carbon-based compounds, and a few species also invest in the
nitrogen-based defence cyanogenesis. Polymorphism exists in the
expression of the cyanogenic glycoside, prunasin, even within closely
related populations. Recently, variation in the expression of cyanogenic
glycosides has been found to change with ontogeny. The objective of
this study was to characterise ontogenetic variation in chemical and
physical defenses in seven closely related Eucalyptus species over a
two year period. As the plants developed they were found to increase
the amount of foliar carbon relative to nitrogen. This was associated
with an increase in the concentration of carbon-based defences, and
leaf thickness. Two species were found to be cyanogenic at 6 months
of age, and three were cyanogenic by 15 months of age, suggesting
ontogenetic variation in the ability to ‘switch on’ cyanogenesis. These
species appear to have “defence syndromes” which differ in the extent
of ontogenetic control on defence traits.
POSTERS WEDNESDAY & THURSDAY
POS-WED-193
THE CONSEQUENCES OF ALTERING N-TERMINAL
POST-TRANSLATIONAL PROCESSING OF RUBISCO IN
TOBACCO CHLOROPLASTS
Orr D.J.1, Houtz R.L.2 and Whitney S.M.1
1
Plant Sciences, Research School of Biology, Australian National
University, GPO Box 475, Canberra, ACT, 0200. 2Department of
Horticulture, University of Kentucky, 401D Plant Science Building
Lexington KY 40546-0312 USA.
The co- and post-translational modification of proteins during maturation
is an almost ubiquitous process which has long been observed to
be essential for protein regulation, activation, and degradation in a
large range of organisms. In higher plant chloroplasts assembly of
the photosynthetic CO2-fixing enzyme, Rubisco, is a highly complex
process that necessitates the co-ordinated expression and assembly
of its 8 large (L) and 8 small (S) subunits into a hexadecamer (L8S8).
The L subunits house the catalytic sites and are subject to the post-
translational aminopeptidase removal of Met-1 and Ser-2, and the
acetylation of the new N-terminal Pro-3. The function of these
modifications are undefined. The L-subunit gene (rbcL) is located in
the chloroplast genome (plastome) and can be genetically manipulated
in tobacco with surgical precision via the homologous recombinatorial
method of plastome transformation. We have generated transplastomic
tobacco lines containing mutations in rbcL at codons Ser-2 and Pro-3.
Changes in the N-terminal processing of the mutant L-subunits have
been identified by mass-spectrometry. Presented will be an update on
how these alterations in N-terminal processing influence leaf L-subunit
translation, Rubisco content and the growth of these transgenic tobacco
lines.
POS-WED-195
DICHOTOMY IN THE NRT GENE FAMILIES OF
ARABIDOPSIS AND CEREAL SPECIES
Plett D.1, Toubia J.1, Baumann U.1, Garnett T.1, Tester M.1 and Kaiser B.N.2
1
Australian Centre for Plant Functional Genomics, The University of
Adelaide, Waite Campus, Urrbrae, South Australia, 5064, Australia.
2
School of Agriculture Food and Wine, The University of Adelaide,
Waite Campus, Urrbrae, South Australia, 5064, Australia.
A large proportion of the nitrate (NO3-) acquired by plants from
soil is actively transported via members of the NRT families of NO3-
transporters. In Arabidopsis, the NRT1 family has 7 functionally
characterized members and is predominantly comprised of low-affinity
transporters; the NRT2 family contains 7 members which appear to
be high-affinity transporters; and there are two NRT3 (NAR2) family
members which are known to participate in high-affinity transport. Our
bioinformatic analyses of four fully sequenced cereal genomes (maize,
rice, sorghum, Brachypodium) indicates fundamental differences
between Arabidopsis and the cereal species in the gene number and
family structure of all three families of NRT transporters. Quantitative-
PCR analysis of the expression of the NRT genes in maize adds to the
apparent dichotomy between Arabidopsis and the cereals and begs the
question: Is Arabidopsis a suitable model for NO3- transport in cereal
crop species?.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 239
POS-THU-194
PROTEIN AND DNA ARE TAKEN UP BY PLANT ROOTS
Paungfoo-Lonhienne C.1, Lonhienne T.G.A.2, Rentsch D.3, Mudge S.R.1,
Webb R.4 and Schmidt S.1
1
1School of Biological Sciences, The University of Queensland,
St. Lucia QLD, 4072, Australia. 2ARC Centre of Excellence
for Integrative Legume Research, School of Biochemistry and
Molecular Biosciences, The University of Queensland, QLD, 4072,
Australia. 3Institute of Plant Sciences, University of Bern, 3013 Bern,
Switzerland. 4Centre for Microscopy and Microanalysis, The University
of Queensland, QLD, 4072.
We examined the long-held view that plants rely on inorganic forms
of nutrients and depend on microbial conversion of soil organic
nitrogen (N) and phosphorus (P) prior to uptake into roots. Using green
fluorescence protein (GFP) and fluorescently labelled phosphorothioate
oligonucleotides (S-DNA) as sources of organic N and P, we show that
protein and DNA are taken up by plant roots. Comparable results were
obtained using pollen tubes as analogues of root hairs as both have
similar elongated and polarized growth. We further show that addition
of DNA to growth medium containing inorganic P enhanced the growth
of lateral roots and root hairs even though plants were P replete and had
similar biomass as plants supplied with inorganic P only. These findings
change our view of the spectrum of the N and P sources accessible
to plants and demonstrate that DNA triggers morphological responses
which may enhance root proliferation in organic matter-rich soil. Our
research challenges the current paradigm that plants rely on microbes
and soil fauna for break-down of organic matter and provide further
evidence for heterotrophy in plants.
POS-THU-196
GENETIC ARCHITECTURE OF ROOT SYSTEMS IN
COTTON
Prodhan M.A., McGee P.A. and Saleeba J.A.
School of Biological Sciences, The University of Sydney, NSW 2006,
AUSTRALIA.
Genetic architecture refers to the underlying genetic basis of a phenotypic
trait. The spatial spread of roots or root system architecture (RSA) is an
agronomically important trait that has been investigated in a few plants.
Here we summarize the progress in determining genetic architecture of
RSA of cotton. We chose several genes influencing RSA in Arabidopsis
thaliana and assayed the RSA of their knock out (KO) lines. Data
analysis revealed that KO and WT lines vary in RSA indicating the
effect of the corresponding gene on RSA. The gene sequences were
compared with the nucleotide and protein databases of Gossypium
(cotton) at The National Center for Biotechnology Information. Results
suggested that at least two of the genes have homologues in cotton,
which prompted us to investigate them further as candidate genes for
cotton RSA. Progress will be reported on the role of the genes in root
system development in cotton.
POSTERS WEDNESDAY & THURSDAY
POS-WED-197
MODELLING SCENARIOS FOR BIOMASS
OPPORTUNITIES IN SUGARCANE
Rae A.L.1, Lam D.1 and O’Shea M.G.2
1
CSIRO Plant Industry, 306 Carmody Rd, St. Lucia, Qld. 4067,
Australia. 2BSES Limited, 50 Meiers Rd, Indooroopilly, Qld. 4068,
Australia.
Sugarcane has potential as a biomass feedstock to provide materials
and energy for the future. New technologies for producing ethanol from
cellulose may enable more profitable use of residual fibre. In this study,
an economic model was used to assess the profitability of a theoretical
biorefinery producing sugar, electricity and ethanol from cellulose. The
model takes into account all costs and profits relating to sugarcane
production, and has the capacity to predict returns if these values
change. The aim of the study was to highlight areas where research
into plant variety development should be targeted. Therefore the model
was used to test a number of scenarios based on changes to feedstock
characteristics. The outputs defined the levels of change in each
parameter which would be required for profitability. The feasibility of
achieving these changes was tested by comparison to data on current
and experimental plant varieties. The results showed that the scale
of biomass input was a critical factor. For an average sized mill area,
increasing the cane yield (t/ha), the bagasse yield (% dry matter) or
the ethanol yield (L/t) individually were shown to achieve a profitable
system. However, the increases required for some of these factors
were outside the range of feasibility. Varying two factors in conjunction
was a more feasible way to achieve profitability. Plant varieties with
characteristics that can address these targets could be selected from
existing germplasm or developed by breeding and selection.
POS-WED-199
CHARACTERISATION OF THE WHEAT MULTI-
PATHOGEN RESISTANCE GENE Lr34/Yr18/Pm8
Risk J.1, Solomon P.2, Richardson T.1, Viccars L.1, Chandramohan S.1,
Tabe L.1, Spielmeyer W.1 and Lagudah E.1
1
CSIRO Plant Industry, Black Mountain Site, GPO Box 1600, Canberra
ACT 2601, Australia. 2Research School of Biology, RN Robertson
Building, The Australian National University, Canberra 0200 ACT,
Australia.
According to the Food and Agricultural Organization of the United
Nations over one billion people are reliant on wheat for food and their
livelihoods. Wheat biotrophic pathogens, such as rust and powdery
mildew, can have a devastating effect on crop yields resulting in up to
60% yield loss. Recently a rust resistance gene, Lr34, that also confers
multi-pathogen resistance (Yr18/Pm38), was identified and shown to be
integral to durable wheat resistance to fungal pathogens for over 100
years (Krattinger et al., 2009, Science. 323, 1360-1363). Analysis of this
gene revealed that Lr34 encodes an adenosine triphosphate-binding
cassette transporter (ABC transporter) and that it shows homology to
the Arabidopsis plasma membrane-localized protein PENETRATION3
(PEN3) (Stein et al., 2006, The Plant Cell. 18, 731-746.). While both
genes are involved in pathogen resistance, LR34 confers resistance
against adapted pathogens whereas PEN3 is associated with non-host
resistance. Several approaches are being taken to gain insight into LR34
conferred resistance. Analysis of LR34 expression is being investigated
using GFP-tagged LR34 and anti-LR34 antibodies. Brachypodium and
barley transformant lines are also being developed to ascertain whether
resistance can be gained from addition of Lr34 in other cereals, and
to provide alternate model systems for Lr34 characterisation. Finally,
metabolite profiling using GC-MS is being used to analyse whole leaf
extracts, ultimately leading to the identification of the metabolite being
exported by LR34.
Page 240 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-198
CLE PEPTIDE INDUCTION OF SOYBEAN NODULE
REGULATION
Reid D.E., Li D., Hayashi S., Ferguson B. and Gresshoff P.
ARC Centre of Excellence for Integrative Legume Research, University
of Queensland.
The ability of legumes to enter into a symbiotic relationship with nitrogen
fixing bacteria is one of their defining characteristics. The host plant
maintains control of this symbiosis in response to external and internal
stimuli, including environmental nitrate availability, and via a systemic
autoregulation of nodulation (AON) mechanism. The current model
for these regulatory mechanisms involves a root-derived cue (Q) that
is perceived by an LRR receptor kinase called GmNARK in soybean.
Nitrate-induced Q appears to be perceived locally, whereas rhizobia-
induced Q is transported in the xylem to the shoot where it is perceived
by GmNARK. This activity triggers the production of a shoot-derived
inhibitor that is transported back to the roots where it prevents further
nodule development. Due to the high degree of molecular similarity
between GmNARK and the LRR receptor-like kinase CLAVATA1 in
Arabidopsis, it has been proposed that GmNARK-dependent regulation
may be induced by a similar ligand/s to that interacting with CLAVATA1,
a small peptide called CLAVATA3. We have identified candidate CLEs
that respond to inoculation and/or nitrate treatment and which inhibit
nodulation in a GmNARK-dependent manner when expressed in the
root. In order to confirm if these or other candidates are required for the
initiation of AON, we have developed a bioassay to detect nodulation-
dependent signals in xylem sap. This bioassay is reliant on differential
gene expression responses in the leaves of soybean plants fed with
xylem sap presumed to either contain Q, or be devoid of it. To identify the
differential responses, complete transcriptome sequencing of soybean
leaves using the Illumina GAIIx has been employed.
POS-THU-200
AN INVESTIGATION ON THE INFLUENCE OF DGAT1
AND POLYOLEOSIN ON TRIACYLGLYCERIDE
PRODUCTION
Roldan M.B., Winichayakul S., Scott R. and Roberts N.J.
AgResearch Grasslands, Forage Biotechnology, Palmerston North
4442, New Zealand.
Diacylglycerol acyltransferase (DGAT) is an enzyme that catalyses
the last and only committed step in the synthesis of triacylglycerides
(TAG). We are currently using a quadruple mutant of Saccharomyces
cerevisiae, which is incapable of producing storage lipids, to investigate
potential differences between monocot and dicot DGATs. Differences
observed in the TAG profiles will be presented in this poster. We have
also co-expressed a mutated form of the dicot DGAT1 with several
forms of polyoleosin (a novel protein invented at AgResearch) in the
leaves of Arabidopsis. TAG accumulation in the leaves was analysed
by Gas Chromatography-Mass Spectrometry. The preliminary results
indicate that one form results in the accumulation of substantial amounts
of TAG, compared to control plants and plants transformed with other
forms of polyoleosin. These results are also presented and the potential
to apply this technology in forages is discussed.
POSTERS WEDNESDAY & THURSDAY
POS-WED-201
AN UNUSUAL GA AND ABA SYNERGISM
PROMOTES SOMATIC EMBRYOGENESIS IN
MEDICAGO TRUNCATULA 2HA: IMPLICATIONS FOR
TRANSCRIPTIONAL REGULATION
Rose R.J., Kurdyukov S., Zhang X.Y. and Nolan K.E.
ARC CILR School of Environmental and Life Sciences, The University
of Newcastle, NSW 2308, Australia.
In higher plants somatic embryogenesis (SE) induction in the appropriate
genotype and tissue commonly requires auxin (in Arabidopsis) or
auxin plus cytokinin (in Medicago) and has been studied since the
1950s. A focus with SE studies in Arabidopsis has been the LEAFY
COTYLEDON transcription factors LEC1 and LEC2 which when
overexpressed are able to induce SE. It has been suggested that LEC
transcription factors (TFs) may make cells competent to respond to
auxin and undergo SE by catabolising gibberellic acid (GA). Additional
studies involving the MADS box TF AGL15 has lead to the hypothesis
in Arabidopsis involving GA/ABA ratios; with GA suppressing SE and
abscisic acid (ABA) acting antagonistically to enhance SE. How GA
and ABA influence embryonic competence is not known. However, SE
induced in the pickle (pkl) mutant of Arabidopsis can be suppressed
by GA. The PKL gene encodes a chromatin remodelling protein. In M.
truncatula what have been surprising results are the SE dependence
on ethylene and new data that GA and ABA act synergistically to
enhance SE induced by auxin plus cytokinin severalfold. LEC1 and
LEC1-LIKE are not expressed during the induction of SE and are only
expressed in the developing embryo. LEC2 has not been demonstrated
to be present in M. truncatula. We have argued that the TF MtSERF1
whose transcription is auxin, cytokinin and ethylene dependent acts as
a nexus between stress-induced ethylene and auxin and cytokinin to
promote SE. However, we also have evidence that the 2HA line is an
epigenetic mutant that is weakly ethylene insensitive with the MtEIL1 TF
down-regulated. We suggest that in Medicago ABA and GA modulate
a different set of TFs compared to Arabidopsis to enable the induction
and then development of embryos in vitro, with modulation of ethylene
signalling being critical.
POS-WED-203
P-BODY PARTICIPATION IN CELLULAR
REPROGRAMMING OF CULTURED MESOPHYLL
PROTOPLASTS
Bhullar D.S., Sheahan M.B. and Rose R.J.
ARC CILR, School of Environmental and Life Sciences, The University
of Newcastle, Callaghan, NSW, Australia, 2308.
Regeneration initiated from somatic cells of plants represents an
important mechanism of morphogenic adaptation to stress and entails
the acquisition of a totipotent state. The acquisition of totipotency
requires dedifferentiation, a process that reprograms somatic cells to a
stem cell-like state. Inherent to this reprogramming are massive changes
in gene expression. Here we investigated processes that influence
transcriptome reprogramming during the regeneration of tobacco
mesophyll protoplasts. Processing bodies (P-bodies) are cytoplasmic
RNA-protein complexes that both store and degrade mRNA and play
an important role in developmental transitions. Moreover, P-bodies are
dynamic structures that can change in response to environmental stress.
We therefore investigated P-body dynamics during dedifferentiation
and cell division initiation of tobacco mesophyll protoplasts. To visualise
P-bodies, we created stable tobacco transformants expressing
a YFP fusion to the C-terminal domain of VARICOSE (VCSc), a
P-body localised protein that facilitates mRNA decapping. YFP-VCSc
labelled spherical structures that ranged in size from 7-150 μm in leaf
epidermal cells. In protoplasts, P-bodies exhibited variable size but
also abundance per cell. While average P-bodies size in protoplasts
remained relatively constant during culture, the number of P-bodies per
cell increased significantly during early (24 h) culture and then again
preceding cell division. P-body size and abundance was significantly
reduced by cycloheximide and actinomycin D treatments suggesting
P-bodies require continual recruitment of translationally-repressed
mRNA. Transiently expressing a fusion of DCP2 (a decapping enzyme)
to CFP, revealed an apparent heterogeneity in the P-body population
with co-expression of VCSc promoting localisation of DCP2 to P-bodies.
Our results suggest P-bodies are dynamic structures that potentially
participate in reprogramming the transcriptome of mesophyll cells.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 241
POS-THU-202
IN PLANTA PRODUCTION OF RECOMBINANT
ENZYMES FOR THE PROCESSING OF BIOFUEL
FEEDSTOCKS
Scott R.W., Bartho J.D. and Roberts N.J.
Forage Biotechnology, AgResearch Grasslands, Palmerston North,
New Zealand.
Up to 50  % of the cost of producing ethanol-based biofuels from
cellulosic feedstocks (maize, wood, etc) can be attributed to the cost of
the cellulase enzymes.  Cellulases break down the complex cellulose
molecules into simple sugars, which can be subsequently fermented
into alcohols.  Expression of recombinant cellulases in plants offers
a means of producing the enzymes in a low maintenance organism,
with little or no processing required prior to being added to the
primary feedstock.  Previous studies have shown that it is possible to
express recombinant cellulases in plants.  In order to develop a cost-
effective system we are investigating methods for sequestering high
concentrations of the enzymes in planta.   Four subcellular regions
were chosen for targeting: (1) endoplasmic reticulum,  (2) chloroplast, 
(3) protein storage vacuole,  and  (4) cytoplasm via fusion to oleosins
(oil body proteins).  Supporting this research we are also investigating
the potential of utilising recombinant cellulases originally isolated from
the rumen bacterium Butyrivibrio proteoclasticus.  The cellulases
chosen for study were:   (A)  GA1 (predicted endocellulase)  and 
(B)  GA25 (predicted endocellulase).  Cellulases from the industrial
standard cellulolytic fungus, Trichoderma reesei, were used as positive
controls:  (C) Cel7a (exocellulase)  and  (D) Cel7b (endocellulase).  The
presentation discusses the preliminary results and our plans to enhance
production and enzyme activities.
POS-THU-204
ROOT GROWTH RESPONSES IN BARLEY (HORDEUM
VULGARE L.) TO SALT STRESS
Shelden M.C.1, Roessner U.1, Tester M.2 and Bacic A.1
1
Australian Centre for Plant Functional Genomics, School of Botany,
University of Melbourne, Parkville, 3010 VIC. 2Australian Centre for
Plant Functional Genomics, University of Adelaide, Glen Osmond,
5064 SA.
Soil salinity is a major agriculture problem resulting in decreased
growth and yield of crops. Cereal crops, such as barley and wheat, are
of major economic importance to the Australian agricultural industry.
The root system is essential for plant growth and plays a critical role in
determining the crop yield. In response to salt stress (osmotic stress)
there is an immediate reduction in the root elongation rate (RER). Often
this is followed by a partial recovery and a new steady rate of elongation
is obtained. Root growth occurs within a few mm of tissue from the
root apices, in the root elongation zone (REZ). How root elongation is
maintained in saline soils is largely unknown. The aim of this study is to
identify candidate genes involved in the maintenance of root elongation
in response to salt stress. To identify the mechanisms that regulate root
elongation we are utilizing both physiological and molecular techniques.
Currently we are screening a variety of barley genotypes to determine
the effect of salt (NaCl) on RER. Barley seedlings are grown at different
NaCl concentrations on agar plates and seminal root growth measured.
Cultivars able to maintain root elongation in response to salt stress
will be selected for molecular profiling of the root zones (root cap,
elongation zone, maturation zone). The identification of genes involved
in salt tolerance is necessary for improving crop productivity.
POSTERS WEDNESDAY & THURSDAY
POS-WED-205
A COMPARATIVE GENOMICS APPROACH FOR
IDENTIFYING EFFECTORS IN VENTURIA INAEQUALIS
Shiller J.B.1, Mesarich C.2, Jones D.1, Bowen J.2, Templeton M.2 and
Plummer K.1
1
La Trobe University. 2Plant and Food Research, New Zealand. 3The
University of Auckland.
Venturia inaequalis is a hemi-biotrophic fungus that causes apple
scab disease. The disease is widespread, and is found in almost every
country where apples are grown. Currently, control of this disease is
through fungicide use and resistant cultivars. However, there are races
of V. inaequalis that can infect these previously resistant cultivars and
there are also emerging cases of fungicide resistance in V. inaequalis.
Whether a race of V. inaequalis is able to cause disease on a given
cultivar of apple is governed by the ability of the host to detect the
invading pathogen and mount a successful resistance response. At a
genetic level this is dependent on a gene-for-gene relationship involving
effector genes in the pathogen and resistance genes in the host.
Identifying the effector genes (or gene mutations) in V. inaequalis that
enable the fungus to evade the host immune system and cause disease
will allow for rational development of resistant cultivars in the future.
Whole genome sequences, obtained by pyrosequencing will soon be
available for V. inaequalis and the closely related pear pathogen V.
pirina. Using bioinformatics and functional genomics we will attempt
to identify, characterise and compare effectors from these fungi. This
poster will outline our progress in the project so far.
POS-WED-207
BROWNING OF CINNAMON MYRTLE (BACKHOUSIA
MYRTIFOLIA) LEAVES
Sommano S.1, Joyce D.1, D’Arcy B.1 and Joyce P.2
1
The University of Queensland, Centre for Native Floriculture, School
of Land, Crop and Food Sciences, Gatton, Qld. 4343, Australia. 2BSES
Limited, 50 Meiers Road, Indooroopilly, Qld. 4068, Australia.
Physiology and biochemistry potentially associated with unsightly
browning of harvested Cinnamon Myrtle (Backhousia myrtifolia)
leaves were investigated. Chlorophyll content (Chl a & b), chlorophyll
fluorescence (CF), electrolyte leakage (EL), malondialdehyde
(MDA) content, polyphenol oxidase (PPO), peroxidase (POD) and
phenylalanine ammonialyase (PAL) enzyme activities, total phenolic
content and antioxidant capacity (DPPH) were measured for heat-
induced browning. Green versus yellowish leaves were contrasted
because of observed differential susceptibility to browning. After
heating at 60°C for 60 min, yellow leaf tissue browned more than did
green leaf tissue. Green leaves contained more Chl. a & b than did
yellow leaves. Low CF ratios (Fv/Fm) in both leaf types suggested that
this sub-tropical species suffered from low temperature stress (Gemel
et al. 1997) as the potted plants were grown in the open during winter.
Heat treatment of harvested leaves had generally similar effects on
physiological and biochemical parameters in both leaf types. However,
increased browning susceptibility was associated with greater post-
treatment EL, relatively high pre-treatment (initial) PPO activity, and high
pre- and post-treatment PAL activity in yellow tissue. High PPO activity
has been associated (Steffens et al. 1994; Thipyapong et al. 2004) with
elevated levels of reactive oxygen species (ROS) potentially leading to
chlorophyll bleaching and browning susceptibility. References Gemel J
et al. (1997) Plants for Environmental Studies p. 209. Steffens JC et al.
(1994) Recent Advances in Phytochemistry p. 275. Thipyapong P et al.
(2004) Plant Science 167, 693-703.
Page 242 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-206
MANIPULATION OF THE NON-PHOSPHORYLATING
PATHWAY ALTERS IONIC HOMEOSTASIS
Smith C.A.1, Barthet M.M.2, Whelan J.3, Smith P.M.C.2, Day D.A.1 and
Soole K.L.1
1
School of Biological Sciences, Flinders University, SA, 5042. 2School
of Biological Sciences, University of Sydney NSW 2006. 3ARC Centre
of Excellence in Plant Energy Biology, University of Western Australia,
WA, 6009.
The alternative respiratory pathway of the mitochondria is an essential
bypass to adenylate control of redox poise in the cell. In most animals,
reduction of mitochondrial NADH occurs through the cytochrome
c pathway of cellular respiration by the rotenone-sensitive NADH
dehydrogenase. In the model plant Arabidopsis thaliana, type II NDs are
encoded by a small gene family with a total of seven members, NDB1-
4, NDA1-2, and NDC1, as well as Alternative Oxidase (AOX). Although
the nature of these enzymes as a bypass to adenylate control of cellular
redox regulation implies a critical role for these enzymes in plant function,
little is currently known regarding the impact of these enzymes on whole
plant and cellular physiology. Using A. thaliana plants with knockdown
of Atndb4 and up regulation of Atndb2 and Ataox1a, we have observed
an alteration in the ionic state of these plants with increased levels of
zinc. Microarray analyses on these lines at both 6 days and 5 weeks,
showed an increase in nicotianamine synthase at 6 days which is the
enzyme responsible for synthesizing nicotianamine. Nicotianamine has
been linked to improved uptake and mobilisation of metal ions around
the plant, and may explain why higher levels of zinc have been found in
the Atndb4 knockdown lines as compared to wild type plants. A further
exacerbation of this altered ionic homeostasis was observed in these
plant lines during salinity stress, where in the presence of NaCl, leaves
has higher calcium and lower boron and sodium levels.
POS-THU-208
FROM ZYGOTE TO OIL AND PROTEIN BODIES IN
THE SEED OF THE MODEL LEGUME MEDICAGO
TRUNCATULA
Song Y.1, Wang X.-D.1, Garg M.2 and Rose R.1
1
ARC CILR School of Environmental and Life Sciences, The University
of Newcastle, NSW 2308, Australia. 2School of Biomedical Sciences
and Pharmacy, The University of Newcastle, NSW 2308, Australia.
Medicago truncatula is a genetic and genomic model for legumes and is
used to study seed development as well as plant-microbe interactions.
However, the developmental and cell biology of embryo development to
produce the oil and protein rich cotyledons has received little attention
in M. truncatula. We have carried out a baseline study of the developing
embryo and related this to oil and protein biosynthesis. Embryo sac
and zygote development is similar to the classical models. The embryo
however develops an extensive multicellular suspensor and there
is extensive early development of the procambium connecting the
shoot and root apical meristems. What was of particular interest in the
mature cotyledon was the organization of the oil and protein bodies.
Electron microscopy showed that the oil bodies 0.5-2.0 um in diameter
are aligned around the periphery of the protein bodies and along the
plasma membrane in an apparent specific cellular arrangement. The
fatty acid composition is C16:0, C18:1n-9, C18:2n-6, and C18:3n-3. The
major component is C18:3n-3, accounting for 35% of the total. Fatty acid
accumulation is evident as the cotyledons start to develop. An OLEOSIN
gene, which encodes an oleosin protein inserted in the phospholipid
monolayer enclosing the triacylglycerols of the oil bodies, prevents
coalescence of the oil bodies and is expressed at a similar stage.
Genes expressing the storage protein vicillin commence expression at
a similar time to OLEOSIN but LEGUMIN A initiates expression earlier
in development. These data will assist in linking the biochemistry and
cell biology to the genetic regulation of the biogenesis of oil and protein
bodies.
POSTERS WEDNESDAY & THURSDAY
POS-WED-209
AN AUTOACTIVE FLAX R PROTEIN IS PURIFIED WITH
ATP BOUND IN ITS NUCLEOTIDE BINDING POCKET
Williams S.J.1, Sornaraj P.1, Decourcy-Ireland E.1, Menz R.I.1, Kobe B.2,
Ellis J.G.3, Dodds P.N.3 and Anderson P.A.1
1
The School of Biological Sciences, Flinders University, Adelaide,
Australia. 2School of Chemistry & Molecular Bioscience; Institute for
Molecular Bioscience, University of Queensland, Brisbane, Australia.
3
CSIRO, Plant Industry, Canberra.
The flax rust resistance protein, M, confers immunity in flax to strains
of the flax rust fungus that encode the effector molecule, AvrM. The M
protein is a member of the most common class of resistance proteins,
containing a central NB-ARC domain and a c-terminal leucine-rich
repeat (LRR). When strains of the rust pathogen carrying the AvrM
effector encounter a flax plant carrying the M rust resistance gene, a
recognition event takes place that culminates in localised cell death at
the infection site. This recognition event involves the direct interaction
between the M and AvrM and presumably results in the activation of the
M protein to instigate cell death signalling. We have previously reported
the purification of flax M and L6 resistance proteins using the Pichia
pastoris expression system (Schmidt et al 2007). A simplified method of
protein production has been developed and with this we have found that
purified recombinant M protein is associated with ADP. A mutation in the
P-loop motif of M, shown to inactivate the protein in planta, abolishes this
ADP association. Furthermore, purified M protein containing a mutation
in the conserved MHD motif of the ARC2 subdomain, that gives rise
to an autoactive M protein in planta, is associated with higher levels of
ATP than ADP, and this association is also dependent on the integrity
of the P-loop. Our data validates aspects of the current preferred model
of NB-ARC-LRR protein activation, suggesting that ADP/ATP binding
controls the inactive/active, state of the protein (Tameling et al 2006).
Schmidt et al (2007) Plant J. 50:1107-1117 Tameling et al (2006) Plant
Physiol. 140:1233-1245.
POS-WED-211
A TRANSCRIPTIONAL AND ENZYMATIC EVALUATION
OF MALIC ACID METABOLISM IN DEVELOPING
WINEGRAPES
Sweetman C.1, Ford C.M.2 and Soole K.L.1
1
Flinders University of South Australia, Sturt Road, Bedford Park, SA
5042. 2University of Adelaide, Hartley Grove, Glen Osmond, SA 5064.
The acidity of Vitis vinifera berries is largely determined by tartaric acid
and malic acid. Unlike tartaric acid, malic acid is intricately involved in
cellular metabolism, and is degraded as the fruit ripens. As such, the
concentration of malic acid in winegrapes is often an influential factor for
harvesting and winemaking decisions, and can ultimately affect sensory
qualities of the wine. The importance of malic acid in berry ripening can
be demonstrated by the significant change in metabolism at veraison,
whereby net malic acid accumulation gives way to net degradation.
This metabolic “switch” coincides with the initiation of berry softening,
sugar accumulation and pigment development. The aim of this research
is to improve understanding of the control of malic acid metabolism
during berry development, particularly at the metabolic switch. As
malic acid has numerous metabolic roles (including photosynthesis and
respiration), specific activities of several enzymes have been measured
to determine which pathways of metabolism may be favoured during
malate accumulation and degradation phases, and the potential functions
of these pathways. Enzyme assays were tested from mitochondrial and
cellular isolates of grape berries at various stages of development and
expression of genes of key enzymes were determined using qRT-PCR.
Data are used to evaluate known metabolic functions of malic acid in
plants, and to determine which enzymes may be important in regulating
the content of malic acid in developing grape berries.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 243
POS-THU-210
JASMONIC ACID AND ETHYLENE SIGNALING
PATHWAYS ARE INVOLVED IN CITRUS DEFENSES
AGAINST A. ALTERNATA “TANGERINE PATHOTYPE”
Stuart R.M.1, 2, Kubo K.S.1, 2, Boava L.P.2, Bastianel M.2 and Machado M.A.2
1
State University of Campinas UNICAMP, Campinas, SP, Brazil. 2Centro
de Citricultura Sylvio Moreira, Cordeiropolis, SP, Brazil.
Alternaria alternata “tangerine pathotype” causes the disease known
as Alternaria brown spot (ABS), which affects mandarins and several
of their hybrids. The symptoms include brown to black necrotic spots
surrounded by a yellow halo, and affect twigs, young leaves and fruits.
The fungus produces the host-selective toxin (HST) ACT, which is
responsible for the pathogenesis. The molecular mechanisms involved
in plant susceptibility or resistance are obscure. In order to better
understand the plant defense responses to ABS, key genes involved
in salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways
were analyzed by RT-qPCR in response to A. alternata “tangerine
pathotype” inoculation. Leaves of susceptible Murcott tangor (Citrus
sinensis x C. reticulata), resistant Cleopatra mandarin (C. reshni), and
resistant Clementine (C. clementine) were inoculated with fungal spores
suspension. cDNA was synthesized from the total RNA extracted from
leaves after 6 and 12 hours post inoculation. Resistant Clementine
and Cleopatra mandarin showed up regulated genes involved in JA/
ET pathways, like RlemLOX2 (lipoxigenase), RlemMPL2 (miraculin
like protein 2), PR-3 (Chitinase II), and ACC oxidase after inoculation.
In contrast, genes involved in JA/ET pathways, like RlemLOX2 and
PR-3, and also genes involved in SA/flavonoids pathways, like MPK1
(MAP kinase phosphatase 1), PAL (Phenylalanine ammonia lyase),
CHS (chalcone synthase), were down regulated in susceptible Murcott
tangor. This suggests that JA/ET play important roles in plant resistance
against A. alternata. Financial support: FAPESP (07/00931-3) and
CNPq (INCT).
POS-THU-212
INVESTIGATING THE ROLES OF BARLEY CESA
GENES IN CELL WALL BIOSYNTHESIS
Tan A.H.T., Burton R.A. and Fincher G.B.
Australian Centre for Plant Functional Genomics, School of
Agriculture, Food and Wine, The University of Adelaide, Urrbrae, SA.
Barley contains at least eight cellulose synthase (CesA) genes of which
have been transcript profiled in a range of tissues (Burton et al., 2004).
CesA1, CesA2 and CesA6 have higher transcript levels in tissues more
likely to be undergoing the synthesis of primary cell walls, while CesA4,
CesA5/7 and CesA8 are more likely to be involved in the synthesis of
secondary cell walls (Burton et al., 2004). Growing and expanding cells
usually consist of primary walls and secondary cell walls are usually
deposited upon cessation of cell growth (McNeil et al., 1984). In this
study, a gain-of-function approach was used to study the roles of the
CesA genes in barley cell wall biosynthesis. Five sets of transformed
barley plants transgenic for constructs carrying the CesA1, CesA2,
CesA4, CesA6 or CesA8 genes driven by the constitutive 35S promoter
were analysed for transcript levels, phenotype and tissue morphology.
Preliminary results showed that transcript levels of the CesA transgenes
were lower than expected even though they were driven by a strong
constitutive promoter. The transgenic lines transformed with the
secondary cell wall CesA4 exhibited a phenotype where xylem cells
in the vascular tissue appear to be irregular in shape and are partially
collapsed. This resembles the irx mutants described in Arabidopsis
(Taylor et al., 1999). A “brittle node” phenotype was also observed in
barley transgenic for the 35S:HvCesA8 which also may be affecting
secondary cell wall synthesis. Further studies will include stem strength
measurements and cellulose content assays of the transgenic barley
stems.
POSTERS WEDNESDAY & THURSDAY
POS-WED-213
HIGH ENERGY FORAGE WITH ELEVATED AND
PROTECTED TRIACYLGLYCERIDES
Winichayakul S., Scott R., Roldan M., Cookson R., Richardson K.,
Bryan G. and Roberts N.
AgResearch Grasslands, Forage Biotechnology, Palmerston North,
New Zealand.
To ensure benefits to farming both financially and environmentally while
maintaining low input agricultural systems, our main goal is to elevate
the metabolisable energy available in forages to the grazing ruminants.
Raising dietary intake of lipids mostly in the form of triacylglycerides
(TAGs) is standard practice in the feed lot industry and results in a
significant positive influence on the feed conversion efficiency of sheep
and beef as well as an increase in milk production and reproductive
performance. We proposed to emulate this practice via accumulation of
TAG in the leaves of forage species by over-expressing diacylglycerol
acyltransferase1 (DGAT1), an enzyme that catalyses the last and only
committed step in the synthesis of TAG. Our first modification in ryegrass
showed approximately 30-40% of total lipids increased in the leaves
over-expressing Arabidopsis thaliana DGAT1, under the control of the
CaMV35S promoter. Further improvements included the optimisation
of the DGAT1 nucleotide sequence for transcription stability, mutation
of the peptide sequence to prevent posttranslational regulation, placing
of the gene under control of a leaf specific promoter, and co-expression
of DGAT1 with modified oleosin - a unique protein that encapsulates
TAG forming discrete oil bodies (OBs) in the seed. Modified oleosins
are engineered in our laboratory to stabilise OB integrity and protect
TAG from (1) remobilisation within the leaf, (2) biohydrogenation within
the rumen. The efficacy of these modifications was determined in
Arabidopsis and showed that one of our modifications leads to 70%
increase in total leaf lipids and a substantial portion of this was in the
form of TAG. Results to date and our projection for future work are
presented here.
POS-WED-215
GENOTYPE AND ENVIRONMENT EFFECTS ON
PROTEIN COMPOSITION OF WHEAT GRAINS
Md Zin N.H., Copeland L. and Wilkes M.
Faculty of Agriculture, Food and Natural Resources, University of
Sydney, NSW 2006.
The amount and type of proteins are important in determining the
quality of wheat grains for specific uses in foods. Protein composition is
determined by the variety of grains and environment in which it is grown.
To increase our knowledge of genotype by environment interactions
on quality proteins, three wheat varieties were grown in four different
locations in New South Wales, Australia. Proteins were extracted from
the grains and separated by SDS-PAGE and bands were identified by
peptide mass fingerprinting. Quantitative changes were observed in
electrophoretic patterns of the polypeptides of molecular mass in the 32
kDa to 60 kDa range from the different wheat varieties grown in different
locations. Proteins in this size range are known to be associated with
quality characteristics of wheat grains. In comparison, polypeptides with
molecular mass smaller than 32 kDa or greater than 60 kDa showed
essentially no variability of expression in gels, indicating the influences
of genotype by environment interactions on protein composition were
selective.
Page 244 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-214
CHARACTERIZATION OF REGULATED PROTEIN
SECRETION IN PHYTOPHTHORA ZOOSPORES
Zhang W., Hardham A.R. and Blackman L.M.
Plant Sciences Division, Research School of Biology, The Australian
National University, Canberra, ACT 2601, Australia.
Motile zoospores are key players in establishing infection in many
species of Phytophthora and other pathogenic oomycetes. Three types
of vesicles in the peripheral cytoplasm of zoospores may play important
roles during infection. Having reached a potential host, Phytophthora
zoospores encyst and become immobilised on the plant surface. During
zoospore encystment, ventral and dorsal vesicles are exocytosed rapidly,
delivering adhesives and a putative protective coating onto the surface
of the cysts. By contrast, during encystment large peripheral vesicles
move away from the plasma membrane, become randomly distributed
within the cyst cytoplasm and after several hours are degraded. In
recent research, we have found that a 12 kDa protein, designated
PnCcp, within the large peripheral vesicles is selectively secreted
during encystment. This discovery provides evidence of a highly novel
mechanism for differential protein secretion in eukaryotes. Studies have
shown that in sporangia and zoospores PnCcp colocalises with Lpv, a
high molecular weight glycoprotein also resident in the large peripheral
vesicles. However, in hyphae, in some cases the large peripheral
vesicles contain only Lpv and in some cases, PnCcp appears to form a
layer surrounding Lpv within the vesicles. These observations suggest
that the two proteins may be transported to the large peripheral vesicles
at different times during their development. Constructs encoding GFP-
tagged versions of PnCcp are being made and will be transformed
into Phytophthora nicotianae to facilitate studies of the synthesis and
dynamic movement of the large peripheral vesicles and the timing of
PnCcp secretion.
POS-THU-216
TERMINATION OF DNA REPLICATION IN E. COLI:
NOT EVERY TER SITE MEDIATES TERMINATION OF
REPLICATION
Moreau M.J. and Schaeffer P.M.
James Cook University, School of Pharmacy and Molecular Sciences,
Angus Smith Drive, Townsville, QLD, 4811.
The replication of the circular chromosome of E. coli starts at the
unique origin of replication oriC where two replisomes are assembled
and progress in opposite directions to replicate the chromosome.
Termination of replication occurs roughly opposite to oriC and is
mediated by the Tus protein bound to 21 bp sequences called Ter.
Fourteen Ter sites have been identified but only 10 (TerA-J) were shown
to mediate DNA replication fork arrest. The 10 primary Ter sites are
arranged in two clusters of five, each cluster being on either side of the
region directly opposite to oriC. These clusters act in a polar manner to
constrain forks meeting in the terminus region of the chromosome and
prevent fork progression in the terminus to origin direction by forming a
Tus-Ter-Lock structure. We studied the affinity and kinetics of complex
formation of Tus with the 10 primary Ter sites and their Tus-Ter-Lock
variants using surface plasmon resonance and our new GFP-based
protein stability assay (GFP-Basta). GFP-Basta measures the increased
stability of a protein induced by its binding to a specific ligand. It is a
powerful technique capable of detecting very small changes in binding
affinity (Moreau et al. (2010) submitted to Molecular BioSystems). Both
methods showed for the first time that Tus binds differently to the 10 Ter
sites and the Tus-Ter-Lock variants. The results also revealed that not
all Ter sites are able to form the Tus-Ter-Lock structure. The significance
of these new results, along with the development of GFP-Basta will be
discussed in detail.
POSTERS WEDNESDAY & THURSDAY
POS-WED-217
STRATEGY FOR COMPREHENSIVE IDENTIFICATION
OF FUNCTIONAL HUMAN N-MYRISTOYLATED
PROTEINS USING AN INSECT CELL-FREE PROTEIN
SYNTHESIS SYSTEM
Moriya K.1, Suzuki T.2, Ando E.2 and Utsumi T.1
1
Graduate School of Medicine, Yamaguchi University. 2Shimadzu
Corporation.
To establish a strategy for the comprehensive identification of functional
human N-myristoylated proteins, the susceptibility of human cDNA
clones to protein N-myristoylation was evaluated by metabolic labeling
and mass spectrometric analyses of proteins expressed in an insect
cell-free protein synthesis system. One-hundred-and-forty-one cDNA
clones with N-terminal Met-Gly motifs were selected as potential
candidates from ~2,000 KOP (Kazusa ORFeome project) human
cDNA clones, and their susceptibility to protein N-myristoylation was
evaluated using fusion proteins, in which the N-terminal 10 amino
acid residues were fused to an epitope-tagged model protein. As a
result, the products of 29 out of 141 cDNA clones were found to be
effectively N-myristoylated. The metabolic labeling experiments both in
an insect cell-free protein synthesis system and in transfected COS-1
cells using full-length cDNA revealed that 27 out of 29 proteins were in
fact N-myristoylated. Database searches with these 27 cDNA clones
revealed that 18 out of 27 proteins are novel N-myristoylated proteins
that have not been reported previously to be N-myristoylated. Thirteen
proteins out of these 18 novel N-myristoylated proteins were functionally
unknown proteins. The rest of the proteins were key players in various
cellular signal transduction pathways, such as phosphatase, ubiquitin
E3-ligase, cytoskeletal regulatory protein, apoptosis-related protein, and
amino acid transporter. Analysis of intracellular localization of EGFP-
fusion proteins of wild type and G2A-mutant of these proteins revealed
that protein N-myristoylation strongly affected intracellular localization of
many of these proteins. These results indicate that the strategy proposed
in this study is useful for the comprehensive identification of functional
human N-myristoylated proteins from human cDNA resources.
POS-WED-219
RATIONAL DRUG DESIGN OF INHIBITORS FOR
THE SCHIZOPHRENIA THERAPEUTIC TARGET
KYNURENINE AMINOTRANSFERASE-I
Nadvi N.A.1, 2 , Akladios F.1, Salam N.K.1, Park J.H.2, Hibbs D.E.1,
Hanrahan J.R.1, Kapoor V.3, Gorrell M.D.2 and Church W.B.1
1
Faculty of Pharmacy, University of Sydney NSW. 2Centenary Institute,
University of Sydney NSW. 3School of Medicine and Pharmacology,
University of Western Australia, Perth, WA.
Kynurenic acid (KA) is an intermediate derived from the Kynurenine
pathway of tryptophan metabolism. It is an endogenous antagonist at
the glutamate-binding site of the NMDA receptor and high levels of KA
often accompanies the occurrence of several neurological disorders
including Schizophrenia. Overactivity in the enzyme human Kynurenine
Aminotransferase-I (hKAT-I) which produces this metabolite, is believed
to be an important link in the pathophysiology and makes the kynurenine
pathway a valuable target for the treatment of Schizophrenia. We
require to express large amounts of purified hKAT-I to obtain co-crystal
structures of enzyme-inhibitor complexes, test the lead compounds
and continue subsequent optimisation to design potent inhibitors.
Recombinant hKAT-I was expressed using the baculovirus-insect cell
system. Computational biochemistry and bioinformatics have been used
to generate potential inhibitors for hKAT-I. In particular in silico docking
calculations involving both ligand and structure-based descriptors have
been used to predict lead compounds, and a number synthesized. Two
sets of recombinant hKAT-I have been prepared: a semi-purified sample
for high-throughput screening of lead inhibitors and a very pure, crystal-
grade sample for crystallisation trials and comprehensive investigation
into the inhibition study with the most promising lead inhibitors. In
preliminary assays eight out of 11 candidate compounds were found to
inhibit KAT-I, with the strongest possessing an apparent Ki of 2.8 μmol/l.
These assays were performed with semi-purified recombinant hKAT-I
and further compounds are available for testing. An in-depth analysis of
inhibition kinetics with crystal-grade hKAT-I samples and corresponding
co-crystal structures will aid in elucidating and designing novel KAT-I
specific inhibitors for therapeutic treatment of Schizophrenia.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 245
POS-THU-218
β7-STRAND IS ESSENTIAL FOR MONOMER
INTERACTION IN HOMO- AND HETEROOLIGOMERS
OF HUMAN SMALL HEAT SHOCK PROTEINS
Mymrikov E.V., Bukach O.V., Seit-Nebi A.S. and Gusev N.B.
Department of Biochemistry, School of Biology, Moscow State
University.
Ten small heat shock proteins (sHsp) form a family of chaperones
widely expressed in different human tissues. sHsp prevent aggregation
of denatured proteins and regulation of many intracellular processes. All
sHsp contain conservative α-crystallin domain and tend to form homo-
and heterooligomers. Irregular oligomer composition and high mobility
prevent crystallization of full-size sHsp and make desirable development
of alternative approaches for investigation of protein-protein interaction
within sHsp oligomers. In order to solve this problem we obtained point
mutants of human Hsp20, Hsp22 and αB-crystallin with replacement of
all endogenous Cys residues by Ser and a single Cys residue inserted
in position homologous to Cys137 of human Hsp27. This unique Cys
residue is located in the β7-strand of α-crystallin domain, i.e. in position
of putative monomer contacts within Hsp27 homooligomers. Mutations
introduced do not affect secondary, tertiary or quaternary structure and
chaperone-like activity of human sHsp and therefore are suitable for
investigation of monomer interaction. Mild oxidation of thus obtained
Cys-mutant of sHsp leads to formation of disulfide cross-linked
dimers in the case of homooligomers of Hsp20, Hsp22, αB-crystallin
and wild type Hsp27. Effective disulfide crosslinking was observed
in heterooligomers containing Hsp20, Hsp27 and αB-crystallin. In
heterooligomers containing Hsp22 and any other sHsp formation of
crosslinked heterodimers was significantly reduced. Thus, the β7-strand
of α-crystallin domain is essential for monomer contacts in homo- and
heterooligomers of sHsp and Hsp22 is less effective in formation of
heterooligomers than any other sHsp analyzed. Acknowledgement. This
investigation was supported by Russian Foundation for Basic Science.
.
POS-THU-220
DESIGNER SINGLE-STRANDED RNA-BINDING
PROTEINS
Nguyen C.D., Weisbrodt S., Mansfield R. and Mackay J.P.
School of Molecular Bioscience, University of Sydney, NSW, Australia.
It has become clear that a large number of RNA species are widely
involved in gene regulation and diseases in complex organisms. Their
functions are regulated by RNA-binding proteins (RBPs). Therefore,
engineered RNA-binding proteins with the capacity to target any chosen
RNA sequence would be powerful tools with which to manipulate gene
expression for both therapeutic and research applications. Given
spectacular achievements of designer DNA-binding proteins assembled
from zinc fingers (ZnFs) and preliminary data from our laboratory,
we propose that classical ZnFs and RanBP2 ZnFs can be excellent
scaffolds from which to create sequence-specific RBPs. Mutagenesis
has been extensively used to make variants of ZRANB2 ZnFs which
is a member of RanBP2 ZnFs family, then their binding affinities to
relevant single-stranded RNA (ssRNA) sequences are measured by
using fluorescence anisotropy. So far, a couple of mutants have been
found to have specificity for different ssRNA triplets. Nuclear magnetic
resonance spectroscopy and/or X-ray crystallography will be recruited
to determine the structural basis for these protein-RNA recognitions.
Moreover, the composition and length of inter-domain linker will be
optimized in order to construct modular single-stranded RNA-binding
proteins that can recognize desired six/nine/twelve-base ssRNA
sequences.
POSTERS WEDNESDAY & THURSDAY
POS-WED-221
NATURAL SUBSTRATES OF THE N-END RULE
PATHWAY IN ESCHERICHIA COLI
Ninnis R., Spall S., Talbo G., Truscott K. and Dougan D.
La Trobe Institute for Molecular Science, La Trobe University,
Bundoora, VIC, Australia.
The N-end rule pathway is conserved from bacteria to man and
determines the half-life of a protein based on its N-terminal amino acid.
In Escherichia coli, model substrates bearing a destabilising N-terminal
amino acid (N-degron) are degraded by ClpAP in an ATP-dependent
manner. The degradation of these substrates is controlled by the
adaptor protein ClpS, which is directly responsible for recognition of the
N-degron and its delivery to the ClpAP protease(1). We have isolated
23 ClpS interacting proteins from E. coli. Our data demonstrate that
two of these proteins, DNA protection during starvation protein (Dps)
and putrescine aminotransferase (PATase), are post-translationally
modified to generate an N-degron. In the first example, the N-degron of
Dps appears to result from an endoproteolytic cleavage by an unknown
protease. In the second case, the N-degron of PATase is generated
by the addition of primary destabilising residues Leu and Phe to the
N-terminus of the protein. In this case generation of the N-degron is
dependent on leucyl/phenylalanyl-tRNA protein transferase (LFTR)
and determines the stability of the protein in vivo. Independent of the
type of post translational modification, both N-end rule substrates are
degraded by ClpAP in a ClpS dependent manner. We conclude that the
modification of PATase, by LFTR, is directly responsible for its ClpAPS-
mediated turnover in E. coli and hence the N-end rule pathway may
play an important role in putrescine homeostasis. (1)Erbse A, Schmidt
R, Bornemann T, Schneider-Mergener J, Mogk A, Zahn R, Dougan
DA, & Bukau B 2006 ClpS is an essential component of the N-end rule
pathway in Escherichia coli. Nature 439, 753-756.
POS-WED-223
STRUCTURE-FUNCTION STUDIES OF ANNEXINS IN
INFECTIOUS DISEASES
Osman A. and Hofmann A.
Structural Chemistry Program, Eskitis Institute for Cell & Molecular
Therapies, Griffith University, Don Young Road, Nathan, Brisbane,
4111 Qld, Australia.
Many biochemical and histological properties are shared within the
annexin family of proteins, because of their highly conserved three-
dimensional fold. The landmark feature of annexins is the calcium-
dependent binding to acidic phospholipid membranes. Recently, parasite
annexins have attracted attention due to their immunogenic properties
in the case of cysticercosis and giardiasis. It is hypothesized that their
localization at the cyst wall (Taenia solium) and adhesive ventral disk
(Giardia lamblia) provides a potential link to the parasite attachment to
or invasion of the host. As such, parasite annexins may be novel targets
to combat infectious diseases. In this study, the crystallization and
biochemical properties (membrane and glycosaminoglycan binding) of
parasite annexins are investigated. Crystals of alpha-1 giardin from G.
lamblia were obtained and diffracted to 1.9 Å. The crystals belong to
the orthorhombic space group P212121. The determination of a parasite
annexin structure is essential to provide better insight into their biological
roles and to support drug discovery efforts against infectious diseases.
Only few biochemical characteristics of parasite annexins are known to
date. Recent studies of parasite annexins in our lab demonstrate lectin
properties for alpha-1 giardin and annexin B1 from T. solium, but not
for annexin (Sm)1 from Schistosoma mansoni and alpha-3 giardin from
G. lamblia. Similarly, membrane binding varies for the different parasite
annexins, depending on the presence of the canonical calcium binding
motif, the endonexin sequence. The difference in the binding ability of
parasite annexins towards glycosaminoglycan and lipid membranes
may suggest diverse biological functions.
Page 246 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-222
ANALYZING LIM-DOMAIN MEDIATED INTERACTIONS
Nisevic I., O’Connell M., Mansfield R. and Matthews J.M.
School of Molecular Bioscience, The University of Sydney, NSW 2006.
LIM-homeodomain (LIM-HD) proteins are transcription factors involved
in cell specification in many different tissue types. The twelve LIM-HD
proteins in mammals are expressed in different combinations in different
cells at various stages of development, thereby forming a combinatorial
“LIM-code”. Each LIM-HD protein has 2 N-terminal tandem LIM-domains
(LIM1 and LIM2) and a central homeodomain. The essential cofactor
protein LIM-domain binding protein 1 (Ldb1) contains a ~30-residue
region, known as the LIM-interaction domain (LID) which allows it to
bind the tandem LIM domains of all members of the LIM-HD family. We
want to determine the binding affinities for all of these interactions to
establish which LIM-HD proteins are most likely to bind to the cofactor
Ldb1. It is not straight-forward to achieve this aim because the isolated
LIM domains from these proteins tend to be insoluble and/or aggregate
in solution. Fortunately, they are stabilised by the presence of Ldb1-
LID or other binding partners. Thus, we have been assessing the use
of several different types of competition assays, including ELISAs
and Fluoresence-based assays. The implementation of these assays
and different strategies for fluorescently labelling the proteins will be
discussed.
POS-THU-224
AN INVESTIGATION IN PARTNERSHIPS:
PARASPECKLE PROTEIN- RNA INTERACTIONS
Passon D.M.1, 2, Lee M.1, Bythell-Douglas R.R.1, Fox A.H.2 and
Bond C.S.1
1
School of Biomedical, Biomolecular and Chemical Sciences,
University of Western Australia, Crawley, 6009 WA. 2Western
Australian Institute of Medical Research, University of Western
Australia, Perth 6000 WA.
Paraspeckles are dynamic sub-nuclear bodies, located next to splicing
speckles, within eukaryotic cells. The formation of paraspeckles depends
both on the transcription of a long non-coding RNA, NEAT1 and on the
presence of core paraspeckle proteins. The best evidence thus far for
paraspeckle function points to a role in the control of gene expression
via nuclear retention of certain mRNA species. This is mediated by
paraspeckle proteins binding to adenosine-to-inosine edited inverted
repeat motifs and targeting of the RNA transcript to paraspeckles. The
molecular mechanisms behind these processes are still unclear. Our
hypothesis is that the characterisation of paraspeckle proteins and their
RNA interaction partners will give functional and mechanistic insights
into this method for controlling gene expression. The core paraspeckle
proteins PSP1 and P54nrb are located in paraspeckles in an RNA-
dependent manner. Both proteins have highly similar sequences,
contain RNA-recognition motifs and form a heterodimer. I will present
preliminary data of potential RNA binding partners of PSP1 and p54nrb
and I will describe experiments aimed at establishing in vitro and in vivo
RNA-protein interaction assays in order to identify potential RNA binding
partners for subsequent crystallization of protein-RNA complexes.
POSTERS WEDNESDAY & THURSDAY
POS-WED-225
THE ANTIFUNGAL ACTIVITY OF A PLANT DEFENSIN
REQUIRES THE FUNGAL CELL WALL
Payne J.A., Hulett M., Anderson M.A. and Van der Weerden N.
Department of Biochemistry, La Trobe University, Plenty Road,
Bundoora 3086.
Plant defensins are small, cysteine-rich proteins involved in the innate
immune system of plants. NaD1, a plant defensin from the flowers of the
ornamental tobacco, Nicotiana alata, has potent antifungal activity and
is able to protect transgenic cotton from fungal infection in the field. The
antifungal activity of NaD1 involves a multi-step mode of action whereby
NaD1 interacts specifically with the cell wall, permeabilises the plasma
membrane and ultimately enters the cytoplasm. Unlike some other
antifungal molecules, NaD1 requires an intact cell wall for its antifungal
activity. The fungal cell wall is composed of three layers; a thin layer of
chitin adjacent to the plasma membrane, a layer of (1-3)-β-glucan and
an outer layer of glycoproteins. We are currently investigating how NaD1
interacts with each of these components in an attempt to understand
why cell wall binding is essential before NaD1 can permeabilise the
plasma membrane and enter the cell.
POS-WED-227
OLIGOMERIC STRUCTURE OF DAP DECARBOXYLASE
FROM DIVERSE BACTERIAL SPECIES
Peverelli M.G., Dogovski C. and Perugini M.A.
Bio21 Institute, 30 Flemington Road, Parkville VIC 3010 Australia,
Department of Biochemistry and Molecular Biology, University of
Melbourne.
Diaminopimelate decarboxylase (DAP-DC) plays a critical role in
the lysine biosynthesis pathway of bacteria and plants. The enzyme
catalyses the final step in the pathway, namely, the decarboxylation of
meso-diaminopimelate to form L-lysine. The structure of the enzyme has
been characterised from several bacterial species, including, E. coli, M.
jannaschii, and M. tuberculosis. The enzyme forms different oligomeric
structures in the crystal form of these species. Moreover, DAP-DC from
the Gram-negative eubacterium E. coli is monomeric; the enzyme from
the Gram-negative archaea M. jannaschii is dimeric; whilst DAP-DC
from the eubacterium M. tuberculosis forms a tetramer in the crystal
form. We hypothesise that the diversity of the enzyme’s quaternary
structure is linked to species diversity and/or environment. Accordingly,
the aim of this study is to characterise the quaternary structure of DAP-
DC in solution and crystal forms from diverse eubacterial and archaeal
species. We have thus cloned, expressed, and purified DAP-DC from
Bacillus anthracis and are currently conducting structural studies
comparing B. anthracis DAP-DC with the enzyme from E. coli, M.
jannaschii, and M. tuberculosis.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 247
POS-THU-226
DYNOLE 34-2 IS A POTENT INHIBITOR OF DYNAMIN
THAT INDUCES CELL DEATH FOLLOWING
CYTOKINESIS FAILURE AND SUPPRESSES
GLIOBLASTOMA TUMOUR GROWTH IN VIVO
Chircop M.1, Perera S.1, Ma M.P.C.1, Lau H.2, Gilbert J.3, Jones N.C.2,
Dabacco G.2, Gordon C.P.4, Young K.A.4, Morokoff A.2, Sakoff J. 3, O’Brien
T. 2, McCluskey A. 4 and Robinson P.J. 1
1
Children’s Medical Research Institute, The University of Sydney, 214
Hawkesbury Road, Westmead, NSW 2145, Australia. 2Department of
Medicine, Royal Melbourne Hospital, University of Melbourne, Parkville,
3050, Victoria, Australia. 3Senior Hospital Scientist, Department of
Medical Oncology, Newcastle Mater Misericordiae Hospital, Edith Street,
Waratah, NSW 2298, Australia. 4Chemistry, School of Environmental & Life
Sciences, The University of Newcastle, Callaghan NSW 2308, Australia.
Glioblastoma brain tumours are one of the most lethal and difficult forms of
cancer to treat. With optimal therapy the median survival period is 40-60
weeks. Several inhibitors of mitotic proteins (e.g. Aurora kinase, Plk) have
emerged in preclinical or early clinical development for the treatment of
cancer. Cytokinesis is the final stage of mitosis resulting in the generation
of two independent daughter cells. Our recent discoveries highlight the
endocytic protein dynamin II (dynII) as a novel cancer therapeutic target.
DynII is required for the abscission phase of cytokinesis – a stage in mitosis
not yet targeted for cancer treatment. One of the difficulties in treating brain
cancers is the ability of small molecules to cross the blood brain barrier.
The dynole class of dynamin inhibitors represent such molecules. Here, we
reveal that analogous to MiTMABs, the five most potent dynoles, 25, 26, 33,
34-2 and 35, induce cytokinesis failure at the point of abscission, consistent
with inhibition of dynamin. Dynoles inhibited cell proliferation (MTT and
colony formation assays) with dynole 34-2 being the most potent and the
SMA-560 glioblastoma cells the most sensitive, compared to ten other
cancer cell lines screened. Dynole 34-2 also induced apoptosis as evident
by PARP cleavage. Cell death was specifically induced in cells following
cytokinesis failure, suggesting that dynole 34-2 selectively targets dividing
cells. Moreover, this compound inhibited glioblastoma tumour volume by
>80% in an in vivo othotopic glioblastoma mouse model with no adverse
toxicity affects at clinically relevant doses. We conclude that dynamin
inhibitors are a new class of anti-mitotic agents with a novel mechanism of
action – targeting cytokinesis. Thus, dynamin inhibitors are a new approach
for the treatment of cancers, particularly brain tumours that require the rare
ability of chemotherapeutic agents to cross the blood brain barrier.
POS-THU-228
DISTINCT PARTITIONING OF POLYQ AND POLYA-
EXPANDED PROTEINS IN CELLS
Polling S., Ramdzan Y.M., Hill A.F. and Hatters D.M.
Biochemistry and Molecular Biology, University of Melbourne, VIC
3010.
Nine human congenital disorders result from polyalanine (polyA)
sequence expansions in nine different host proteins. In addition, nine
other neurodegenerative diseases, including Huntington’s, are caused
by polyglutamine (polyQ) expansions in different host proteins. While
polyQ-expansions are well-understood to lead to the aggregation of
the host proteins into amyloid like fibrils, which is predicted as a major
source of toxicity to cells, the corresponding influence of polyA on
proteins remains largely unknown. Here, we examined the biophysical
properties of model proteins containing polyA expansions and compared
them to proteins with polyQ expansions. In cells co-expressing polyA
and polyQ-containing proteins, we found that polyA-expanded proteins
formed punctate structures, predicted to contain aggregated protein,
that accumulated into mutually exclusive cellular locations relative to
polyQ-containing protein aggregates. In contrast, different proteins that
contained polyA expansions, and likewise with the polyQ expansions,
always accumulated together within the same punctate structures in
cells. The differential partitioning of polyA proteins to polyQ proteins
in cells, suggest the presence of multiple cellular processes for the
trafficking and handling of protein aggregates formed by different
proteins.
POSTERS WEDNESDAY & THURSDAY
POS-WED-229
SYNTHESIS OF THE RECOMBINANT CHIMERIC
TRANSTHYRETIN FOR ITS FUNCTIONAL STUDY
Poodproh R., Leelawatwattana L. and Prapunpoj P.
Department of Biochemistry, Faculty of Science, Prince of Songkla
University, Hat Yai, Songkhla 90112, Thailand.
Transthyretin (TTR) is a homo-tetrameric protein that plays major
role in distribution of thyroid hormones (THs), and transport of retinol
via binding to retinol binding protein (RBP). Besides, its proteolytic
catalysis was recently demonstrated and has been proved to associate
to human pathology. However, the amino acid residues participate in the
catalysis could not be identified. To obtain more insight the underlying
mechanism, the comparative effect of changes in structure on the
catalysis of TTR could be a useful tool. In this research, cDNAs coding
for TTRs having altered amino acid sequence particular in the N- and
C-terminal regions were constructed and produced in Pichia pastoris.
These include Crocodylus porosus TTR with N-terminal sequence of
Xenopus TTR and C-terminal sequence of pig TTR, human TTR with N-
and C-terminal sequences of Xenopus and pig TTR, respectively, and
human TTR with the C-terminal segment of pig TTR. The recombinant
TTRs were successfully synthesized and extracellularly secreted into
culture medium, and all of them had general physicochemical properties
such as subunit mass, tetrameric formation and the electrophoretic
mobility under native condition similar to TTRs found in nature. The
cleavage ability of the chimeric TTRs was examined on either universal
substrate such as casein or natural substrate such as apolipoprotein A-I.
By comparing the catalysis of the chimeric TTRs to that of the unaltered
TTRs, the results revealed the effect and confirmed the relationship
between these structural segments and function of TTR.
POS-WED-231
MITOGENIC AND METABOLIC SIGNALLING VIA THE
INSULIN RECPTOR ISOFORM-A
Rajapaksha K.H., Alvino C.L., Wallace J.C. and Forbes B.E.
School of Molecular and Biomedical Sciences, University of Adelaide.
There are two insulin receptor isoforms (IR-A and IR-B) which arise
from alternative splicing. The IR-B is responsible for metabolic control
more commonly associated with IR action. The IR-A isoform lacks 12
amino acids normally encoded by exon 11 and it binds insulin and IGF-
II with high affinity. Interestingly, IGF-II is able to stimulate mitogenic
responses via the IR-A. The IR is a heterodimer of 2α and 2β subunits.
The ligand binding site is found in the extracellular α subunits whereas
the kinase domain and phosphorylation sites involved in signalling are
found on the intracellular portions of the β subunits. Phosphorylation of
the IR β chain in response to insulin has previously been detected on 7
of the possible 14 Tyr residues, 12 of the 32 Ser residues and a single
Thr. The total pattern of phosphorylation of the IR-A in response to IGF-
II has never been reported. We are aiming to define receptor mitogenic
and metabolic phosphorylation barcodes using an innovative mass
spectrometry approach. Cells expressing IR-A were treated with insulin
and IGF-II. Phosphorylation barcodes were defined in response to these
two proteins by purifying stimulated receptors from cell lysates and
labelling using Isotope-Coded Protein Labelling (ICPL) methodology.
The intracellular domain was trypsin digested and phosphorylated and
nonphosphorylated peptides were quantitated by mass spectrometry.
Page 248 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-230
DEVELOPMENT OF A PEPTIDE INHIBITOR OF
MATRIPTASE FOR IN VIVO TESTING
Quimbar Dominguez P.M.1, Daly N.L.2, Dunse K.M.1, Hulett M.D.1,
Craik D.J.2 and Anderson M.A.1
1
Department of Biochemistry, La Trobe University, Melbourne, 3086,
Australia. 2Institute for Molecular Bioscience, Division of Chemistry
and Structural Biology, The University of Queensland, Brisbane, 4072,
Australia.
Matriptase, a member of the type II transmembrane serine protease
family, has been implicated in the progression of some epithelial
cancers. Matriptase is thought to aid tumour progression through
activation of hepatocyte growth factor which regulates the proliferation
and migration of epithelial cells. Therefore discovery of potent inhibitors
of tumour associated matriptase activity presents the potential to
develop anti-cancer drugs. We are investigating the ability of a group
of cyclic peptides to inhibit matriptase in order to develop inhibitors
that block metastasis. Cyclic peptides are ideal candidates for cancer
therapeutics due to their proteolytic stability and small size. Preliminary
tests have shown that squash trypsin inhibitor MCoTI-II has a higher
binding affinity for matriptase than the cyclic sunflower trypsin inhibitor 1
(SFTI-1). Comparison of these two inhibitors may aid in the development
of more potent inhibitors of matriptase. We are currently investigating
the potential of these inhibitors using in vitro cell migration assays.
POS-THU-232
ULTRACELLULAR ALTERATIONS OF THE
HEPATOCELLULAR CARCINOMA CELL LINE INDUCED
BY Hep88 mAb: A NOVEL HARMFUL mAb
Rojpibulstit P.1, Manochan S.1, Puthong S.3, Gamnarai P.1,
Kittisenachai S.2, Kangsadalampai S.1 and Roitrakul S.2
1
Faculty of Medicine, Thammasat University (Rangsit Campus),
Klong-Luang, Pathumthani, 12121 Thailand. 2Thailand National Center
for Genetic Engineering and Biotechnology (BIOTEC), Thailand
Science Park, Phahonyothin Road, Klong Luang, Pathumthani
12120 THAILAND. 3Antibody Production Research Unit, Institute of
Biotechnology and Genetic Engineering, Chulalongkorn University,
Patumwan, Bangkok, 10330.
Abstract Hepatocellular carcinoma (HCC) is the most leading cause of
cancer- death in the world. Nowadays many targeted therapy particularly
an immunotherapy via monoclonal antibodies (mAbs) specific to tumor-
associated antigen is continually explored. In this regard, Hep88
mAb was established and subsequently revealed its cytotoxicity on
hepatocellular carcinoma cell line (Hep G2 cell line) and normal liver
cell line (Chang liver). We are then further focused on the localization
of Hep88 mAb’s specific protein from hepatocellular carcinoma Hep G2
cell line by western blot analysis. Moreover, 3 days incubation of Hep88
mAb on Hep G2 cell line was also investigated by using a transmission
electron microscope. The result demonstrated that the recognizing
proteins of molecular weights about 53-60 kDa and 70-90 kDa have been
found not only on cell membrane but also on cytoplasmic compartment.
The cytotoxicity’s result at its IC50 concentration against HepG2 cell
line showed its tumoricidal effect on hepatocellular carcinoma cell line
(Hep G2 cell line) with harmful-less to the normal liver cell line (Chang
liver). Furthermore, the transmission electron micrograph illustrated an
intracellular vacuolization with mitochondria and endoplasmic reticulum
dilatation. These fascinating results from this study imply that Hep88
mAb might be a promising tool to a development of an effective treatment
of HCC in the next decade.
POSTERS WEDNESDAY & THURSDAY
POS-WED-233
THE EXTRACURRICULAR ACTIVITIES OF UPSTREAM
OPEN READING FRAMES
Friend L.1, Hillman K.1, Skarshewski A.1, Wang X.Q.1, Gorman J.1, Smith R.1
and Rothnagel J.A.1
1
School of Chemistry and Molecular Biosciences, University of
Queensland, Qld, 4072, Australia.
Many eukaryote transcripts contain at least one AUG codon upstream
(uAUG) of the main start codon. In mammals, approximately 30% of these
are followed by open reading frames (uORFs) of 20 or more codons. We
developed a web-based program to rapidly identify conserved uORFs
and their putative bioactive peptides. We identified over 200 uORFs that
are strongly conserved. An examination of the consensus sequences
surrounding their uAUGs found that ~20% were in an optimal context for
ribosomal initiation and therefore predicted to be efficiently translated.
We hypothesize that these uORFs encode functional peptides which
we have termed upstream peptides (uPEPs). Several uPEP sequences
were prioritized for further analysis based on their predicted likelihood of
being expressed. Ten uPEP sequences were amplified by RT-PCR and
cloned into a GFP plasmid expression vector. After appropriate quality
assurance, these plasmids were transfected into HeLa cells and GFP
expression visualised by confocal microscopy. Surprisingly, we found
that eight exhibited distinct cellular localisation patterns that can provide
insight into their cellular roles. Four uPEPs showed nuclear localisation,
four showed cytoplasmic localisation and two were indistinguishable from
the GFP alone control. In only one case was the localisation predicted
by an examination of the sequence; this uPEP contained 5 positively
charged residues reminiscent of a nuclear localisation sequence and
was specifically localized to the nucleolus of transfected cells. The use
of bioinformatic tools and in vivo cell analysis has provided insights
into an over-looked component of the proteome. While not all uORFs
encode bioactive peptides, a subset will have important cellular roles
and aberrations in their expression will result in disease.
POS-WED-235
THE STRUCTURE AND VACCINE POTENTIAL OF AN
IMMUNOMODULATORY SALIVARY CYSTATIN FROM A
SOFT TICK
Salat J.1, Paesen G.C.2, Rezacova P.3, 4, Kotsyfakis M.1, 5, Kovarova Z.3, 6,
Sanda M.3, Andersen J.F.5, Kopacek P.1, Kopecky J.1 and Mares M.3
1
Biology Centre ASCR and Faculty of Sciences, University of South
Bohemia, Czech Republic. 2Centre for Ecology and Hydrology,
Mansfield Road, Oxford, UK. 3Institute of Organic Chemistry and
Biochemistry, ASCR, Czech Republic. 4Institute of Molecular Genetics,
ASCR, Czech Republic. 5Laboratory of Malaria and Vector Research,
NIH, Rockville, USA. 6Department of Biochemistry, Faculty of Science,
Charles University, Czech Republic.
The saliva of blood-feeding parasites is a rich source of peptidase
inhibitors that help overcome the host’s defense during host-parasite
interactions. Using proteomic analysis, the cystatin OmC2 was
demonstrated in the saliva of the soft tick Ornithodoros moubata,
an important disease-vector transmitting African swine fever virus
and the spirochaete Borrelia duttoni. A structural, biochemical and
biological characterization of this peptidase inhibitor was undertaken.
Recombinant OmC2 was screened against a panel of physiologically
relevant peptidases and found to be an effective broad-specificity
inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L
and S) and exopeptidases (cathepsins B, C and H). The crystal structure
of OmC2 was determined at a resolution of 2.45 Å and used to describe
the structure-inhibitory activity relationship. The biological impact
of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected
the function of antigen-presenting mouse dendritic cells by reducing
the production of the proinflammatory cytokines, and proliferation of
antigen-specific T-cells. This suggests that OmC2 may suppress the
host’s adaptive immune response. Immunization of mice with OmC2
significantly suppressed the survival of ticks in infestation experiments.
We conclude that OmC2 is a promising target for the development of a
novel anti-tick vaccine to control O. moubata populations and combat
the spread of associated diseases.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 249
POS-THU-234
INHIBITION OF MUSHROOM TYROSINASE BY
NOVEL MONOFUNCTIONAL AND BIFUNCTIONAL
DITHIOCARBAMATES SODIUM SALTS
Saboury A.A. and Amin E.
Institute of Biochemistry and Biophysics, University of Tehran, Tehran,
Iran.
Two novel monofunctional dithiocarbamates, C6H5NHCSSNa (I)
and C6H5CH2NHCSSNa (II), and two bifunctional dithiocarbamates,
NaSSCNHC6H4NHCSSNa (III) and NaSSCNHCH2C6H4CH2NHCSSNa
(IV), were synthesized and characterized by spectroscopic
techniques. These water-soluble compounds were examined for
their inhibition of both activities of mushroom tyrosinase (MT). L-3,
4-dihydroxyphenylalanine and L-tyrosine were used as substrates for
the catecholase and cresolase enzyme reactions, respectively. Kinetic
studies showed noncompetitive inhibition of I and II and mixed type of III
and IV on both activities of MT. The inhibition constant (KI) value of II is
smaller that of I. Also, the inhibition constant value of IV is smaller that of
III. Moreover, the inhibition constant value of III is smaller that of I, as the
same as IV is smaller than of II. It seems bifunctional dithiocarbamates
due to two charged head groups chelate active site and are potent MT
inhibitors. Raising temperature caused decreasing KI values of I and
II and increasing those of III and IV. The binding process for inhibition
of monofunctional dithiocarbamates (I and II) is entropy driven,
which means that the predominant interaction in the active site of the
enzyme is hydrophobic, meanwhile the electrostatic interaction can be
important for inhibition of bifunctional dithiocarbamates (III and IV) due
to the enthalpy driven binding process. In order to find more information
about mechanism of inhibition of the enzyme, we studied structural
investigations on the effect of our four synthesized compounds on MT.
Fluorescence studies showed probably conformation of inhibitor-bound
MT is stable and inflexible than of uninhibited MT. Circular Dichroism
studies did not show any considerable secondary structure changes
of MT.
POS-THU-236
CHARACTERISATION OF OXIDIZED RECOMBINANT
HUMAN GALECTIN-1 STRUCTURE AND FUNCTION
Scott S.A. and Blanchard H.
Institute for Glycomics, Griffith University, Gold Coast Campus,
Australia.
Galectin-1 protein is over-expressed by many aggressive human
cancers. The lectin mediated apoptotic affects galectin-1 has upon T cells
suggests that secretion of galectin-1 into the tumour stroma indirectly
contributes to tumour survival and growth by essentially creating a
shield from immune surveillance. Futhermore, human galectin-1 lectin
activity promotes cell migration and adhesion, two critical processes in
angiogenesis and the invasion of metastatic cancer cells. Galectin-1
lectin activity is exhibited by the reduced homodimeric (~ 30 kDa)
form of the protein and to maintain this lectin activity the cysteine
residues within each 14.5 kDa subunit must be kept from oxidizing to
form disulphide bonds. Cysteine oxidation however does not render
galectin-1 inactive with respect to all biological functions. An oxidized
form of human galectin-1 can heal injured and diseased axons. Given
the well-established link between oxidative stress and pathological
conditions such as injury and disease, greater understanding of the
oxidized human galectin-1 structure and function is required. We have
detected two forms of oxidized human galectin-1, an oligomer of 68 kDa
and a smaller protein species of apparent molecular weight 17 kDa.
The 68 kDa form contains inter- and intra-molecular disulphide bonds,
whereas the 17 kDa form contains only intra-molecular disulphide
bonds. The oligomer is formed under higher protein concentration and
lower salt concentration, whereas the smaller species is formed under
opposite conditions. Both forms exhibit lectin-independent activity upon
a cancerous immune cell line and reveal affinity for the fatty acid portion
of an intracellular binding partner.
POSTERS WEDNESDAY & THURSDAY
POS-WED-237
IDENTIFICATION AND CHARACTERIZATION OF
PROTEIN KINASES THAT BIND TO N-TERMINAL
DOMAIN OF DNA METHYLTRANSFERASE 1
Sekiguchi M., Sugiyama Y., Sueyoshi N. and Kameshita I.
Department of Life Sciences, Kagawa University, Kagawa 761-0795,
Japan.
DNA methylation plays an important role in transcriptional repression
of imprinted genes in mammals. DNA methyltransferase 1 (Dnmt1)
is an enzyme that recognizes and methylates hemimethylated DNA
during DNA replication to maintain methylation patterns. The N-terminal
region of Dnmt1 is known to form an independent domain structure
that interacts with various regulatory proteins and DNA. In the present
study, we explored protein kinases in the mouse brain that could bind
and phosphorylate the N-terminal regulatory domain of Dnmt1. Dnmt1-
binding protein fraction was prepared from mouse brain extract using
Dnmt1(1-290)-affinity column. This fraction was further subjected to
DNA-cellulose column chromatography and analyzed by Western
blotting with kinase-specific antibodies. The protein kinase activity
toward Dnmt1(1-290) was also examined using separated fractions by
gel filtration column chromatography. These analyses revealed that
Dnmt1-binding protein fraction contained two Ser/Thr protein kinases
with molecular masses of approximately 110 kDa and 45 kDa. When
these proteins were subjected to LC-MS/MS analysis, 110-kDa kinase
and 45-kDa kinase were identified as cyclin-dependent kinase-like 5
(CDKL5) and casein kinase 1 (CK1), respectively. Both CDKL5 and CK1
could be coprecipitated with Dnmt1 when they were analyzed by GST
pull down experiments. CDKL5 and Dnmt1 were found to colocalize in
the nucleus and appeared to interact with each other. Catalytically active
form of CDKL5, CDKL5(1-352) phosphorylated the N-terminal domain
of Dnmt1 in the presence of DNA. CK1 also phosphorylated this domain
much more significantly than CDKL5, and phosphorylation of Dnmt1 by
CK1 decreased the affinity for DNA. These results suggest that CDKL5
and CK1 bind to and phosphorylate the N-terminal domain of Dnmt1 and
may regulate the functions of Dnmt1.
POS-WED-239
FLUORESCENCE-ACTIVATED CELL SORTING
ANALYSIS OF THIOREDOXIN IN CANCER CELLS
UNDER DIFFERENT OXYGEN CONDITIONS
Shah F.L.1, 2 , Di Trapani G.1, Karlenius T.C.1, 2, Clarke F.M.1, 2 and
Tonissen K.F.1, 2
1
School of Biomolecular and Physical Sciences, Griffith University,
Nathan QLD 4111, Australia. 2Eskitis Institute for Cell and Molecular
Therapies, Griffith University.
Thioredoxin is a redox capable protein that is integral to the functioning
of cells. It performs several roles within the intracellular environment,
including the scavenging of reactive oxygen species or ROS. Oxidative
stress is caused when the cell contains excessive levels of ROS, which
can lead to inflammation, ischemic tissue damage, carcinogenesis
and cancer progression, or even cell death. Thioredoxin expression
increases under conditions of oxidative stress, which can also cause
thioredoxin to be secreted into the extracellular environment. Another
condition that causes an increase in thioredoxin levels is hypoxia, which
is defined as a loss of oxygen in cells and tissues and is widespread in
solid tumours. While it is not known how hypoxia leads to an increase
in thioredoxin expression, there is evidence that ROS has roles to
play within the hypoxic cellular environment, which may be related to
the increased thioredoxin levels. There is not much known about the
increase of thioredoxin in hypoxic conditions, or whether intracellular
thioredoxin increase corresponds to increased thioredoxin on the
surface of cells. Therefore, the aim of this project is to quantitatively look
at the levels of thioredoxin both within and on the surface of cancer cells
under different oxygen conditions (oxidative stress, normoxia, hypoxia)
via FACS analysis. Immunoflourescence was performed as a visual
complement to the FACS analysis.
Page 250 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-238
KNOCKDOWN OF CALMODULIN-DEPENDENT
PROTEIN KINASE I-DELTA CAUSES DEVELOPMENTAL
ABNORMALITIES IN ZEBRAFISH
Senga Y., Nagamine T., Nimura T., Sueyoshi N. and Kameshita I.
Department of Life Sciences, Kagawa University, Kagawa 761-0795,
Japan.
Ca2+/calmodulin-dependent protein kinase I-delta (CaMKIδ), a fourth
isoform of CaMKI, is known to be expressed ubiquitously in various
tissues. However, the functional roles of this enzyme in vivo have not been
fully clarified yet. To investigate the biological significance of CaMKIδ
during zebrafish embryogenesis, we cloned and characterized two splice
variants of zebrafish CaMKIδ, zCaMKIδ-L (392 AA) and zCaMKIδ-S
(368 AA). Although recombinant proteins expressed in Escherichia
coli showed weak protein kinase activities, they were activated when
phosphorylated by mouse Ca2+/calmodulin-dependent protein kinase
kinase alpha. Activated CaMKIδ significantly phosphorylated various
proteins including CREB, histones, and myelin basic protein in a
Ca2+/CaM-dependent manner. In COS7 cells, transiently expressed
zCaMKIδ-L and zCaMKIδ-S were localized in the cytoplasm as in case
of mouse CaMKIδ. During zebrafish embryogenesis, zCaMKIδ-L and
zCaMKIδ-S were detected in the embryos after 36 hpf and 60 hpf,
respectively, by Western blotting using specific antibodies. Western
blotting analysis in adult zebrafish demonstrated wide expression of
zCaMKIδ-L in the brain, eyes, ovary, and skeletal muscle. In contrast,
zCaMKIδ-S specifically localized in the brain. The knockdown of the
zCaMKIδ genes with morpholino-based antisense oligonucleotide
resulted in an increase of abnormal embryos with small heads. These
results clearly show the significance of zCaMKIδ-L and zCaMKIδ-S
during zebrafish embryogenesis.
POS-THU-240
QUASI-IRREVERSIBLE INSULIN-RECEPTOR
COMPLEXES: EVIDENCE FOR THE FORMATION OF
DISULPHIDE BONDS BETWEEN INSULIN AND THE
INSULIN RECEPTOR?
Shelton B.P.1, 2 and Helmerhorst E.1
1
Western Australian Biomedical Research Institute & Curtin Health
Innovation Research Institute, School of Biomedical Sciences, Curtin
University of Technology, Bentley Western Australia 6012. 2Lung
Institute of Western Australia, QEII Medical Centre Nedlands Western
Australia 6009.
The binding of insulin to its cell surface receptor initiates a signalling
cascade that mediates insulin’s cellular effects. While it is well established
that insulin binding induces a conformational change in the insulin
receptor, the exact mechanism involved in insulin binding and receptor
activation is still poorly understood. Complex binding kinetics, which are
not fully described by current models of biomolecular interactions, typify
the interaction between insulin its receptor. A negative cooperativity
model has been widely reported to describe the ligand-induced
enhancement of insulin dissociating from its receptor. Further, evidence
from our laboratory and others demonstrates a reduced dissociation
of insulin from its receptor as a function of increased association
time, suggesting a time-dependent transition from a rapid to a slow
dissociating complex. Careful analysis of data in our laboratory shows
that this reduced dissociation is only partially explained by changes in
the dissociation rates per se. Instead, these effects appear dependent
on the amount of complex available for dissociation, with a population
of complexes displaying binding that does not dissociate even after
24hrs. This quasi-irreversible population of ligand- receptor complexes
is sensitive to reducing, oxidising and alkylating agents, suggesting that
disulphide bonds may be involved. This quasi-irreversible population is
also sensitive to changes in ionic strength, independent of the salt used,
suggesting that key ionic interactions may be involved in the formation
of the quasi-irreversible state. The existence of a non-dissociating
population of receptor complexes may have significant physiological
implications and must be considered when modelling the insulin receptor
interaction.
POSTERS WEDNESDAY & THURSDAY
POS-WED-241
THE BIOMOLECULAR CHANNELLING OF A REACTIVE
INTERMEDIATE
Smith N.E., Vrielink A., Corry B. and Attwood P.
University of Western Australia, M310, 35 Stirling Highway, Crawley
WA 6009.
The channelling of intermediates through buried molecular tunnels in
multi-enzyme subunits is a topic of considerable interest as it is thought
to occur in many different enzymes. This process shields reactive or
poisonous intermediates from the rest of the organism and ensures
direct, rapid transport from one active site to the other. Many of these
molecular channels have been identified using techniques such as
X-ray crystallography, but little is known about the internal mechanisms
they use to transport their intermediates. In this case, computational
and kinetic methods have been applied to study the bifunctional enzyme
4-hydroxy-2-ketovalerate aldolase-aldehyde dehydrogenase (acylating)
(DmpFG) and its proposed channelling activity. Previous crystallographic
studies have found that DmpFG has a 29 Å channel linking its two
active sites. This raises questions about whether it functions as a
conduit for the poisonous intermediate acetaldehyde. In the case of this
project computational techniques, including both standard molecular
dynamics and metadynamics, have been used to investigate whether
it is energetically feasible for the intermediate to be transported in
this way between the two active sites and to verify the role of proposed
checkpoints at the entry and exit of the channel. The kinetic assays on
wild type DmpFG are designed to determine the kinetic parameters of
both the active site 1 and 2 reactions and the channelling event. Both
computational and kinetic data will be presented.
POS-WED-243
PLASMODIUM SERINE HYDROXYMETHYLTRANSFERASE
IS DIFFERENT IN SUBSTRATE SPECIFICITY FROM ITS
HOMOLOGUES - A POTENTIAL ANTIMALARIAL TARGET
Sopitthummakhun K.1, Leartsakulpanich U.2, Yuthavong Y.2 and
Chaiyen P.1
1
Center for Excellence in Protein Structure & Function, Department of
Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road,
Bangkok, 10400, Thailand. 2National Center for Genetic Engineering
and Biotechnology, National Science and Technology Development
Agency, 113 Paholyothin Road, Pathumthani 12120, Thailand.
Abstract World malaria report in 2008 estimated the widespread of
malaria as 247 million cases among 3.3 billion people with nearly a million
deaths and most of cases were in children less than 5 years. Plasmodium
falciparum (Pf) was among the leading causes of death worldwide from
a single infectious agent, whereas P. vivax (Pv) was the cause of chronic
malaria. The effective control remains chemotherapy. However, increasing
numbers of reports of drug resistance in parasites press the need to find
new effective anti-malarial compounds. Deoxythymidylate synthesis
pathway is vital for Plasmodium which consists of the bi-functional
enzyme, dihydrofolate reductase-thymidylate synthase, and serine
hydroxymethyltransferase (SHMT). The latter is less well-characterized.
We previously showed that both recombinant Pf- and PvSHMT are PLP
enzyme that catalyze the conversion of serine and tetrahydrofolate to
glycine and methylene tetrahydrofolate. Here, the ability of PvSHMT
to use other substrates in THF-independent reaction was studied. The
results showed that PvSHMT can catalyze the retro-aldol cleavage toward
3-hydroxy amino acids, L-threonine, allo-threonine and β-phenylserine.
Moreover, the enzyme catalyzed the aminotransferase reaction of
D-alanine and pyridoxal phosphate to pyruvate and pyridoxamine.
Interestingly, PvSHMT can use D-serine as substrate in addition to
L-serine which is unlike that of the eukaryotic enzymes. Based on the
distinct property, selective inhibitors against Plasmodium enzyme such
as D-amino acid analogs can be designed. We currently are developing
an assay to increase the throughput for inhibitor screening. Inhibition
study with various derivatives of folate and D-amino acid analogs will be
reported.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 251
POS-THU-242
CROSS-REACTIVITY BETWEEN LUPIN AND PEANUT
Soo L.1, Smith P.M.C.1, Mir G.A.1, Smith W.B.2, Prescott S.L.3, Soutter V.4
and Loblay R.H.4
1
School of Biological Sciences, University of Sydney. 2School of
Paediatrics & Child Health Research, UWA, Princess Margaret
Hospital Perth, WA. 3Royal Adelaide Hospital, South Australia. 4Allergy
Unit, RPA Hospital Sydney.
The number of immunoglobulin E (IgE) mediated allergic reactions to
lupin has increased incrementally with the increasing inclusion of lupin
products in human foods. Although some individuals are allergic to
only lupin, there is evidence that some individuals who are allergic to
peanuts are more likely to be allergic to lupin. This suggests that lupin
and peanut allergens may share common IgE epitopes ie. they cross-
react. We have conducted lupin food challenges on individuals who are
allergic to peanut. Approximately 6.5% of peanut allergic individuals
also react to lupin. In an inhibition assay we used peanut proteins to
inhibit binding of IgE to lupin proteins suggesting that they share cross-
reactive epitopes. Further characterization identified that the peanut
protein extract inhibited binding to proteins of 25 and 34 kDa. We are
currently expressing the major seed storage proteins of lupin, that have
been identified as allergenic, in a cell-free protein synthesis system.
The recombinant proteins will be used in inhibition assays to identify the
cross-reactive proteins. A site-directed mutagenesis will then be used
to characterize the shared IgE epitopes. Findings from this work will
contribute to the understanding of lupin allergy and the cross-reactivity
between lupin and peanut allergens, and possibly other legumes. These
results will also assist in designing better diagnostic and therapeutic
approaches for food hypersensitivity reactions. This work was funded
by Grain Food CRC.
POS-THU-244
A PROPHAGE BASED GENETIC SELECTION FOR USE
IN PROTEIN STRUCTURE FUNCTION STUDIES
Murchland I., Schubert R.A., Dodd I.B. and Shearwin K.E.
School of Molecular and Biomedical Science, The University of
Adelaide.
Using the principles of synthetic biology, we have developed a modular
genetic selection system designed to allow the isolation of mutant
proteins defective in particular types of interactions. The system is
based on the observation that an E. coli strain carrying an integrase
deficient (int-) lysogen of bacteriophage 186 shows a very high level of
cell death upon induction (derepression) of the lysogen. By customised
construction of genetic circuits which interface with those of the
resident int- lysogen, we have adapted the system to isolate collections
of mutants for a number of different proteins, in each case targeting
a specific function of that protein. For example, we have isolated
repressor protein mutants defective in self association at particular
interfaces, transcriptional activator proteins defective in their interaction
with RNA polymerase, and anti-repressor mutants unable to interact
with their target repressor. In each case, the genetic selection also
contains a modification to ensure that only full length mutant proteins
are isolated. The various sets of mutants have provided valuable data
for deciphering the molecular mechanisms by which these proteins
carry out their biological actions.
POSTERS WEDNESDAY & THURSDAY
POS-WED-245
ENAMIDE ANALOGUES OF PANTOTHENATE (VITAMIN
B5) INHIBIT PROLIFERATION OF THE HUMAN
MALARIA PARASITE PLASMODIUM FALCIPARUM
Spry C.1, Villa M.V.J.3, Marquez R.3 and Saliba K.J.1, 2
1
Research School of Biology, The Australian National University,
Canberra, Australia. 2Medical School, The Australian National
University, Canberra, Australia. 3Department of Chemistry, University
of Glasgow, Glasgow, Scotland.
Survival of the virulent human malaria parasite Plasmodium falciparum
during intraerythrocytic development is dependent on an extracellular
supply of pantothenate (vitamin B5), a precursor of the fundamental
enzyme cofactor coenzyme A (CoA). Previously, a naturally occurring
pantothenate analogue called CJ-15,801 was shown to inhibit proliferation
of P. falciparum in vitro (IC50 of 39 μM). This analogue differs in structure
from pantothenate only by containing a carbon-carbon double bond in
place of a carbon-carbon single bond, which, by virtue of its position
relative to an amide group, forms part of an enamide moiety. CJ-15,801
was shown to exert its antiplasmodial effect specifically by interfering
with parasite pantothenate utilisation and to be less toxic to mammalian
cells. Inspired by the activity of CJ-15,801, we synthesised and tested
43 novel fully-synthetic enamide-bearing pantothenate analogues for
antiplasmodial activity. Twenty-eight antiplasmodial analogues were
identified, including two with improved potency relative to CJ-15,801 (IC50
values of 4 and 14 μM). The effect of pantothenate on the antiplasmodial
activity of the enamides was investigated to identify those exerting an
antiplasmodial effect by competitively inhibiting pantothenate utilisation.
Inhibition of pantothenate utilisation by the enamides was explored
further by testing their activity against P. falciparum pantothenate
kinase(s)-catalysed pantothenate phosphorylation (the first step in
the metabolism of pantothenate to CoA). The differential activity of the
analogues in this series has shed light on the key structural features
required for inhibition of pantothenate utilisation and parasite growth,
which should facilitate the future development of pantothenate utilisation
inhibitors with improved antiplasmodial activity.
POS-WED-247
CRYSTAL STRUCTURE OF IMPORTIN α COMPLEXED
WITH THE DNA REPAIR PROTEIN KU70 NLS PEPTIDE
Takeda A.A.S.1, 2 , Kobe B.2 and Fontes M.R.M.1
1
Departamento de Física e Biofísica, Instituto de Biociências, UNESP -
Univ Estadual Paulista, Botucatu, SP, Brazil. 2School of Chemistry and
Molecular Biosciences, Institute for Molecular Bioscience and Centre
for Infectious Disease Research, University of Queensland, Brisbane,
Queensland 4072, Australia.
Importin α (Impα) plays a role in the classical nuclear import pathway,
binding to cargo proteins with activities in the nucleus. Those proteins
contain nuclear localization sequences (NLS), which are characterized
by one or two clusters of basic amino acids (monopartite and bipartite
NLSs, respectively). In this work we present the expression, purification
and the crystal structure of Impα from M. musculus (residues 70-
529, lacking the autoinhibitory domain) and the NLS peptide from the
DNA repair protein Ku70 to provide more information about the NLS
recognition by Impα. Impα (70-529) sample was obtained in E. coli
expression system and purified by affinity chromatography followed by
an ion exchange chromatography. A single crystal of Impα-Ku70NLS
complex was grown by hanging drop method. The data were collected
at 100K using a Synchrotron Radiation Source (Laboratório Nacional
de Luz Synchrotron, LNLS, Brazil) and processed to 2.6 Å. Molecular
replacement techniques determined the crystal structure. Results of
the Impα-Ku70NLS complex indicated an electron density available
only in the major binding site corresponding to the Ku70 NLS peptide. It
bounded to the ImpA similarly to the phosphorilated version of the simian
virus 40 (SV40) large tumour (T)-antigen NLS showing the possibility of
this peptide being a monopartite type in spite of being a bipartite as
suggested in previous studies. Keywords: Importin α, nuclear import,
NLS, X-ray crystallography, Ku70. Supported by: FAPESP and CNPq.
Page 252 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-246
ACCUMULATION OF ENDOGENOUS LOW MOLECULAR
WEIGHT CRYSTALLIN PEPTIDES IN THE HUMAN LENS:
A KEY EVENT IN THE AGING PROCESS
Su S.-P.1, 2 , Truscott R.J.W.2, McArthur J.D.1 and Aquilina J.A.1
1
School of Biological Sciences, University of Wollongong, NSW 2522.
2
Save Sight Institute, University of Sydney, NSW 2001.
Age-dependent modification of crystallin proteins is a major
contributor to cataract in human lenses. Recent studies have shown
that endogenous low molecular weight (LMW) peptides, derived
from the breakdown of crystallins, accumulate in old and cataractous
lenses, with some possessing natural anti-chaperone abilities against
α-crystallin - the molecular chaperone in the lens. Furthermore, during
aging and/or the onset of cataract, α-crystallin has been shown to
associate increasingly with the lens fibre cell membrane. To investigate
structural and functional changes of crystallins in the aging lens, we
characterized and quantified crystallin-derived LMW peptides present
in water-soluble and water-insoluble extracts of human lenses aged 20
to 86 years using MALDI-MS and nanoLC-ESI-MS/MS. The prominent
LMW peptides (αB1(2)-18, αA66(67)-80, γS2-22 and βA3189-215) present in the
lens were mapped using MALDI-imaging MS. Our results indicate that
LMW peptides emerged during early middle-age and are confined to
nuclear fibre cells. Interestingly, in older lenses these peptides were
present in both cortical and nuclear regions. Using fluorescently-tagged
(Alexa350) α-crystallin subunits in direct membrane-binding assays, we
showed that the presence of the LMW crystallin peptides (αB1(2)-18 and
αA66(67)-80) can influence the binding of α-crystallin subunits with lens
fibre cell membranes. Taken together, these results demonstrate that
LMW crystallin peptides accumulate in the human lens during the aging
process and can affect the molecular chaperone function of α-crystallin
and its interaction with fibre cell membranes. This process can directly
contribute to the aggregation of water-insoluble lens protein complexes,
leading to the opacification commonly observed in old lenses.
POS-THU-248
PURIFIED MOUSE CYP27B1 CAN HYDROXYLATE
20,23-DIHYDROXYVITAMIN D3 PRODUCING 1α,20,23-
TRIHYDROXYVITAMIN WHICH HAS ALTERED
BIOLOGICAL ACTIVITY
Tang E.K.Y.1, Janjetovic Z.2, Li W.3, Nguyen M.N.1, Slominski A.2 and
Tuckey R.C.1
1
The University of UWA (School of Biomedical, Biomolecular and
Chemical Sciences). 2University of Tennessee Health Science Center
(Department of Pathology and Laboratory Medicine). 3University of
Tennesse Health Science Center (Department of Pharmaceutical
Sciences, College of Pharmacy).
20,23-Dihydroxyvitamin D3 is a biologically active metabolite produced
via the action of cytochrome P450scc (CYP11A1) on vitamin D3. It inhibits
keratinocyte proliferation, stimulates differentiation and inhibits NF-κB
activity working as a vitamin D receptor agonist. We have tested the ability
of purified mouse 25-hydroxyvitamin D3 1α -hydroxylase (CYP27B1) to
add a 1α-hydroxyl group to this vitamin D analogue and whether this
altered its biological activity. 20,23-Dihydroxyvitamin D3 incorporated
into phospholipid vesicles was converted to a single product by CYP27B1
which was confirmed to be 1α,20,23-trihydroxyvitamin D3 by NMR. The
20,23-dihydroxyvitamin D3 was a relatively poor substrate for CYP27B1
compared to the normal substrate, 25-hydroxyvitamin D3, displaying a
5-fold higher Km and 8-fold lower kcat value. Like 20,23-dihydroxyvitamin
D3, 1α,20,23-trihydroxyvitamin D3 decreased keratinocyte proliferation.
It caused little stimulation of the expression of the vitamin D receptor
at the mRNA level compared to the 30-fold induction observed with
the same concentration (100 nM) of 1α,25-dihydroxyvitamin D3 at 24
h. Addition of the 1α-hydroxyl group to 20,23-dihydroxyvitamin D3
greatly enhanced its ability to stimulate the expression of the CYP24
gene but not to the extent seen with 1α,25-dihydroxyvitamin D3. This
study shows that purified CYP27B1 can be used to enzymatically add
a 1α-hydroxyl group to 20,23-dihydroxyvitamin D3 with the product
showing altered biological activity, especially for the stimulation of
CYP24 gene expression. Supported by NIH grant R01AR052190.
POSTERS WEDNESDAY & THURSDAY
POS-WED-249
ANS FLUORESCENCE STUDIES ON FLEXIBILITY AND
SUBSTRATE-INDUCED CONFORMATIONAL CHANGES
OF THE PSYCHROTROPHIC AND MESOPHILIC
ACETATE KINASES
Tang A.K.1, 2, 3, Motoshima H.1 and Watanabe K.1, 2
1
Department of Applied Biochemistry and Food Science, Faculty of
Agriculture, Saga University, 1-Honjo-machi, Saga 840-8502, Japan.
2
The United Graduate School of Agricultural Sciences, Kagoshima
University, Kagoshima 890-0065, Japan. 3Department of Applied
Nutritin and Food Technology, Islamic University, Kushtia, Bangladesh.
The acetate kinase from the Antarctic psychrotroph Shewanella sp.
AS-11 (SAK) has a significantly higher activity at low temperatures
when compared with the mesophilic homologue from Escherichia coli
K-12 (EAK). The SAK and EAK were investigated by fluorescence
spectroscopy of the hydrophobic probe 8-anilino-1-naphthalene
sulfonate (ANS) to compare their flexibility and substrate-induced
conformational changes. The fluorescence maximum wavelength of
ANS with SAK and EAK were observed at 458 and 480 nm, respectively,
when excited at 360 nm. The maximum ANS fluorescence intensity with
SAK was 5.8-fold higher than that with EAK. The binding of ANS had no
effect on the kinase activity of both enzymes. The fluorescence intensity
with EAK increased with decreasing temperatures, but that with SAK
decreased. When ATP bound to SAK, almost all ANS fluorescence was
quenched, whereas bound to EAK, only 60% was quenched. These
data suggest that SAK has a more hydrophobic ANS binding site with a
more flexible structure when compared with EAK. The bindings of ATP
to free enzymes resulted in substantial conformational changes, which
can be interpreted as the transition from an open to closed form. This
substrate-induced conformational change in SAK appears to be larger
than that in EAK. It is likely that these structural features of SAK may
contribute to its high activity at low temperatures.
POS-WED-251
EXPRESSION, PURIFICATION AND
CHARACTERIZATION OF CLONING-GRADE ZINC
FINGER NUCLEASE
Tovkach A.1 and Tzfira T.2
1
CSIRO Plant Industry, Canberra, ACT, Australia. 2University of
Michigan, Ann Arbor, MI, USA.
The limited number of naturally occurring rare-cutting restriction enzymes
and the slow and tedious engineering of existing restriction enzymes
for novel specificities have prompted the design of new strategies for
the development of restriction enzymes with specificities for long DNA
sequences. One possibility is using zinc finger nucleases (ZFNs) -
synthetic restriction enzymes that are custom-designed to target and
cleave long DNA sequences and which have been recently shown useful
for DNA cloning. Here we report on the purification and biochemical
analysis of ZFN-10, a custom-made ZFN. We show that Ni-affinity and
gel-filtration purification methods are sufficient to produce a cloning-
grade enzyme. We show that ZFN-10 can function as an accurate and
reliable ZFN using the same reagents and protocols used for naturally
occurring and commercially available recombinant restriction enzymes.
We also show that ZFN-10 tolerates a set of target-site substitutions
which can be predicted from the specificities of recognition helices
incorporated into the structure of its DNA-binding domain. The relative
simplicity of ZFN-10 design, expression, purification and analysis
suggests that novel ZFNs can potentially be designed and applied for
various recombinant DNA applications.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 253
POS-THU-250
MOLECULAR BASIS OF DEATH DOMAIN
INTERACTIONS THAT REGULATE ASSEMBLY OF
PROINFLAMMATORY CASPASE-1 ACTIVATING
COMPLEXES
Vajjhala P.R., Kaiser S., Mirams R.E. and Hill J.H.
School of Chemistry and Molecular Biosciences, The University of
Queensland, St. Lucia, Qld 4072, Australia.
The assembly of proinflammatory caspase-1 activating complexes,
known as inflammasomes, is initiated in response to intracellular
pathogens and various cellular stress conditions. These complexes
process proinflammatory cytokines such as pro-interleukin-1β into
active forms that trigger an inflammatory response. Inflammasome
assembly is mediated by interactions between death domains present
in components of these complexes. Death domains consist of six
antiparallel α-helices and typically mediate homotypic interactions
between members of the same subtype. Two subtypes of death domains
are present in inflammasomes; the pyrin domain (PYD) and the caspase
activation and recruitment domain (CARD). Despite the importance
of PYD domains in both inflammasome assembly and regulation,
nothing is known about the molecular details of their interactions. We
have identified molecular determinants important for interaction of
the PYD domain of ASC, a critical adaptor protein for inflammasome
assembly. In addition to interacting with inflammasome components,
ASC interacts with several regulatory proteins via its PYD. Moreover,
ASC is a dimer and dimerisation is proposed to be mediated via the
PYD. To identify residues that are important for ASC PYD interactions,
we have individually mutated 17 surface exposed residues on the ASC
PYD and tested the effect of these mutations on ASC self-association
and interaction with inflammasome-related proteins. We have done this
using a range of biochemical, biophysical and cell-based techniques.
We have also identified the complementary binding surface on the
ASC-interacting protein, pyrin. Here we will present our data and the
implications for inflammasome assembly and regulation.
POS-THU-252
INHIBITORS OF VIRAL PROTEIN NUCLEAR IMPORT
AS ANTI-VIRAL AGENTS
Wagstaff K.M., Rawlinson S.M. and Jans D.A.
Dept. Biochemistry and Molecular Biology, Monash University,
Clayton, Vic 3800 Australia.
Infectious diseases, such as those caused by human immunodeficiency
virus (HIV) and Dengue Virus (DENV) remain highly significant health
burdens world-wide. Due to a lack of effective treatments and the ability
of many viruses to develop resistance to anti-viral agents, there is an
urgent need for the identification/development of new anti-viral drugs.
Significantly, a number of viral proteins, even from cytoplasmically
replicating viruses such as HIV or DENV, localise in the host cell
nucleus as an essential part of the infectious cycle, making viral protein
nuclear import an attractive target for therapeutic intervention. We
have developed a novel high-throughput ALPHAScreen-based assay
to identify inhibitors of important biological protein-protein interactions
from compound library screens. Since nuclear import of HIV Integrase
protein (IN) is critical to HIV function we conducted a pilot screen to
identify inhibitors of IN binding to its nuclear import receptor protein
Importin (Imp) α/β. Through a series of counter/cross-screen we were
able to identify several potent and specific IN:Imp α/β inhibitors, which
significantly inhibit IN nuclear accumulation in transfected cells, but
have no effect on the nuclear accumulation of other nuclear proteins.
In addition we have identified several general inhibitors, which potently
inhibit all nuclear import of proteins mediated by Imp α/β, but do not
effect nuclear import mediated by alternative pathways. Significantly,
several of these compounds have potent anti-viral action. Identifying
inhibitors of nuclear transport through our approaches is clearly a viable
approach to tackle many of the world’s significant viral pathogens.
POSTERS WEDNESDAY & THURSDAY
POS-WED-253
CHARGED REPETITIVE PROTEINS UNITE DIVERSE
EUKARYOTES
Waller R.F., Gould S.B. and McFadden G.I.
School of Botany, University of Melbourne.
Human parasites of the phylum Apicomplexa (e.g. the malarial parasite
Plasmodium) belong to the supergroup Alveolata that also includes
ciliates (e.g. Tetrahymena thermophila)) and dinoflagellate algae. This
diverse group of important organisms share a common cell pellicular
organization of flattened membrane sacs (alveoli) beneath the plasma
membrane and associated with a membrane-anchored cytoskeleton.
Despite diverse functional requirements of the pellicles of the different
Alveolata members, a common family of proteins (called “Alveolins”)
that associate with these alveoli have recently been identified and
are unique to Alveolata. To further our knowledge of the organization
and makeup of this Alveolata-specific cell feature we have performed
a proteomic analysis of purified cell pellicles from the model ciliate
Tetrahymena thermophila. Purified pellicle fractions were analysed by
electrospray ionisation on a QSTAR Elite hybrid quadrupole time-of-
flight mass spectrometer, peptides searched against the T. thermophila
genome and 1178 proteins identified by two or more peptides. 272 of
these proteins were of unknown function, but contain similar proteins
when searched against Apicomplexan genomic data, implying a broad
suite of common pellicular proteins. A conspicuous feature of almost
two thirds of these common proteins is the presence of highly charged
repetitive motifs. Through localization of such proteins we have identified
novel components of pellicular structures involved in host invasion
in apicomplexan parasites and the oral apparatus of ciliates. Thus it
appears that despite the different roles of the cell pellicular structures
of Alveolates, a similar suite of repetitive proteins contribute to their
diverse functionality.
POS-WED-255
SWITCHING THE CATALYTIC PROPERTIES OF C3 AND
C4 PLANT RUBISCOS
Whitney S.M.
Plants Sciences, Research School of Biology, The Australian National
University.
The catalytic properties of the photosynthetic CO2-fixing enzyme,
Rubisco, have a profound impact on photosynthetic carbon assimilation.
In C4 plants the protein machinery and leaf physiology have evolved
to append to C3 photosynthesis systems that concentrate CO2 around
Rubisco to enhance its activity as well as plant productivity, water and
nitrogen use efficiency. Despite sharing the same complex catalytic
chemistry, improvements in the carboxylation rate (vcmax) of Rubisco
from C4 plants have generally come at the expense of CO2 affinity.
The homologous-recombination mechanism of chloroplast genomes
has been used to identify the structural switches that lead to the higher
vcmax of Rubisco from C 4-plants. The catalytic Rubisco large (L) subunit
gene (rbcL) in tobacco has been replaced with the rbcL from either C3
or C4 Flaveria species. Sequence and catalytic analysis of the resulting
hybrid L8S8 Rubiscos (comprising eight Flaveria L-subunits and eight
native tobacco small (S) subunits) have identified the L-subunit residues
responsible for the catalytic disparity between these C3 and C4 Rubiscos.
Introducing these changes into the tobacco L-subunit also endow
this C3-Rubisco with C 4 catalytic features, that is, an improvement in
vcmax and a higher Km for CO2. Identifying structural switches that can
increase the catalytic turnover of C3-Rubiscos may assist ongoing CO2
transgenic approaches (i.e. engineering strategies to concentrate CO2
around Rubisco in C3-plants to minimize wasteful photorespiration)
aimed at improving yield potential and resource use efficiency.
Page 254 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-254
CHARACTERISATION OF A RECOMBINANT PLANT-
MADE SOLUBLE RECEPTOR FOR ADVANCED
GLYCATION ENDPRODUCTS (SRAGE)
Webster D.E.1, Mulcair M.1, Madhavan S.2, Bialkowski K.3, Talbo G.3,
Premaratne R.1, Cooper M.E.3, Forbes J.M.3 and Thomas M.C.3
1
School of Biological Sciences, Monash University, Vic 3800, Australia.
2
Burnet Institute, Vic 3004, Australia. 3Baker IDI Heart and Diabetes
Institute, Vic 3004, Australia.
Advanced Glycation End-products (AGEs) play a key role in the
development and progression of human disease. The pathogenic activity
of AGEs is partly mediated by activation of AGE receptors, the best known
of which is the Receptor for AGEs (RAGE), a multi-ligand glycoprotein
receptor from the immunoglobulin superfamily. Activation of RAGE can
be prevented by a truncated ‘dummy receptor’ (soluble RAGE, sRAGE)
and this strategy has been successfully used to prevent disease in vivo.
The development and optimisation of an efficient method of sRAGE
production and its detailed characterisation represents an important
step in the practical application of sRAGE. Plants represent a viable
production system, which is safe, scalable, affordable and produces
high-quality recombinant protein. Plant-made proteins are functionally
and structurally comparable to native protein, with similar patterns of
glycosylation. Our research with plant-based production systems has
shown that sRAGE can be expressed by stable transgenic plants and
hairy root cultures. Plant-made sRAGE is stable, appropriately folded
and glycosylated and retains its ability to form polymeric complexes
required for ligand binding. In addition, we have shown that plant-made
sRAGE can bind a range of putative RAGE-ligands in a specific manner
and inhibit activation of RAGE in cell-based assays. Further studies have
optimised a purification protocol and undertaken secondary structure
determination by CD spectroscopy. The results will be discussed in the
context of in planta protein engineering with the aim of improving the
bioactivity and half-life of sRAGE.
POS-THU-256
BIOPHYSICAL CHARACTERISATION OF PROTEIN-RNA
INTERACTIONS
Wilce J.A.1, Kim H.1, Yoga Y.1, Cowieson N.2, Scanlon M.J.3, Headey S.J.3,
Gorospe M.4, Williams B.5 and Wilce M.C.J.1
1
Biochemistry and Molecular Biology, Monash University, Clayton,
Australia. 2Australian Synchrotron, Clayton, Australia. 3Monash
Institute of Pharmaceutical Sciences, Parkville, Australia. 4NIH,
Baltimore USA. 5Monash Institute of Medical Research, Clayton,
Australia.
The control of mRNA stability is a key factor in the regulation of gene
expression. The competing events of translation and mRNA decay
ultimately dictate the level of protein expressed and are readily affected
in response to changes in cellular conditions. mRNA transcripts bearing
AU-rich sequences in their 3’UTR, in particular, appear to be under the
control of many factors that determine whether the transcript is destined
for translation, degradation or held in a translationally repressed
state. These factors include RNA-binding proteins such Hu proteins
which are protective against degradation and TIA proteins which are
involved in sequestering mRNA into stress granules under conditions
of cellular stress. Exactly how these proteins recognise and interact
with AU-rich sequences is only partially understood, including whether
they actually compete for the same target mRNAs. We have focused
on two of these proteins, HuR and TIAR which are both triple RNA
recognition motif (RRM) proteins that bind AU-rich RNA with nM affinity.
We report our studies using SPR that reveal similar mRNA target
sequence preferences, yet differences in their stringency of interaction.
We also explore the importance of the separate RRMs, as well as the
protein regions beyond the RRMs, for the mRNA interaction using
NMR spectroscopy and differences in molecular shape of the protein/
RNA complex using SAXS. Together these data bring us closer to an
understanding of the dynamic interplay that takes place in the cell in the
regulation of gene expression at the level of mRNA.
POSTERS WEDNESDAY & THURSDAY
POS-WED-257
CHARACTERISATION OF SUBSTRATE SPECIFICITY
OF THE OXYGEN-DEPENDENT ASPARAGINYL
HYDROXYLASE FIH-1
Wilkins S.E.1, Hyvarinen J.2, Koivunen P.2 and Peet D.J.1
1
School of Molecular and Biomedical Science, University of Adelaide,
South Australia. 2Department of Medical Biochemistry and Molecular
Biology, University of Oulu, Finland.
FIH-1 (Factor Inhibiting HIF-1) is an oxygen-dependent asparaginyl
hydroxylase that plays a role in cellular oxygen homeostasis. It functions
as an oxygen sensor and regulates the activity of essential mediators of
the chronic hypoxic response, the hypoxia-inducible transcription factors
(HIFs), which have been implicated in a number of human diseases
including cancer, vascular disease, and cerebral and myocardial
ischemia. In normoxia, FIH-1 hydroxylates the transactivation domain
of HIF-α, resulting in transcriptional repression. FIH-1 has also been
found to hydroxylate numerous ankyrin domain-containing proteins, and
work by our laboratory has identified members of the Notch receptor
family as substrates of FIH-1. We present data that identify Notch1-3,
but not Notch4, as substrates of FIH-1. Binding studies indicate that
FIH-1 has a much higher affinity for Notch substrates than for HIF-1α,
although HIF-1α demonstrates a higher efficiency of hydroxylation.
Interestingly, FIH-1 is able to bind to Notch4, even though it is not a
substrate, suggesting a possible role for FIH-1 that is independent of
hydroxylation. Analyses of chimeric substrate proteins indicate that
secondary structure is an important determinant of both binding affinity
and hydroxylation efficiency, providing an explanation for observed
differences in hydroxylation of the Notch ankyrin domain compared
with HIF. Site-directed mutagenesis shows that substrate recognition
is substantially influenced by the amino acids directly adjacent to the
target asparagine, a surprising result considering the observed variation
in these residues between different substrates. Characterisation of
substrate specificity will aid in identification of the full array of substrates
and is essential to predict and interpret the functional consequences of
therapeutic targeting of FIH-1.
POS-WED-259
INTERACTION OF DENGUE VIRUS ENVELOPE
PROTEIN WITH ENDOPLASMIC RETICULUM-RESIDENT
CHAPERONES FACILITATES DENGUE VIRUS
PRODUCTION
Wongwiwat W.1, 2 , Limjindaporn T.1, 2, 3, Noisakran S.2, 4,
Yenchitsomanus P.2, 4 and Malasit P.2, 4
1
Department of Immunology, Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, Thailand. 2Office for Research and
Development (Medical Molecular Biology Unit), Faculty of Medicine
Siriraj Hospital, Mahidol University, Bangkok, Thailand. 3Department
of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok, Thailand. 4Medical Biotechnology Unit, National Center
for Genetic Engineering and Biotechnology, National Science and
Technology Development Agency, Bangkok, Thailand.
Dengue virus infection is an important mosquito-borne disease and a
public health problem worldwide. A better understanding of interactions
between human cellular host and dengue virus proteins will provide
insight into dengue virus replication and cellular pathogenesis. The
glycosylated envelope protein of dengue virus, DENV E, is processed
in the endoplasmic reticulum of host cells and therefore reliant on host
processing functions. The complement of host ER functions involved
and nature of the interactions with DENV E has not been thoroughly
investigated. By employing a yeast two-hybrid assay, we found that
domain III of DENV E interacts with human immunoglobulin heavy chain
binding protein (BiP). The relevance of this interaction was demonstrated
by co-immunoprecipitation and co-localization of BiP and DENV E in
dengue virus-infected cells. Using the same approach, association of
DENV E with two other chaperones, calnexin and calreticulin was also
observed. Knocking-down expression of BiP, calnexin, or calreticulin
by siRNA significantly decreased the production of infectious dengue
virions. These results indicate that the interaction of these three
chaperones with DENV E plays an important role in virion production,
likely facilitating proper folding and assembly of dengue proteins.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 255
POS-THU-258
NEW TRICKS TO MEASURE HOW GENETIC
MODIFIERS OF MUTANT HUNTINGTIN TOXICITY
AFFECT HUNTINGTIN AGGREGATION IN DROSOPHILA
Wong Y.Q.1, 2, 3, Quinn L.M.2, Bottomley S.P.3 and Hatters D.M.1
1
1 Bio21 Molecular Science and Biotechnology Institute, Department
of Biochemistry and Molecular Biology, University of Melbourne.
2
Department of Anatomy and Cell Biology, University of Melbourne.
3
Department of Biochemistry, Monash University.
Huntington’s Disease (HD) is caused by mutations that expand a
polyglutamine (polyQ) sequence within the first exon of the huntingtin
protein. Mutated, polyQ-expanded Huntingtin forms visible aggregates,
known as inclusions, within neurons as a key marker of pathology. While
in vitro studies have provided insight into the kinetics of aggregation, how
aggregation occurs in cells and relates to cellular dysfunction remains
poorly understood. This problem is compounded by studies indicating
inclusions are actively formed by cells as a protective response to
sequester smaller diffuse forms of huntingtin such as monomers
and submicroscopic oligomers. We have developed new methods
to quantitatively measure the size, localization and heterogeneity of
all huntingtin molecules in cells and are using them to examine how
previously validated genetic modifiers of mutant huntingtin toxicity affect
in-cell aggregation directly in vivo in Drosophila models of HD. In a work
in progress, we describe our new methodology and their implementation
in Drosophila. We describe preliminary studies on candidate genes
in the Wnt pathway known to modulate huntingtin toxicity and bind to
huntingtin on their role in inclusion formation.
POS-THU-260
STRUCTURE OF A DIMERIC ENZYME FROM A
BACTERIAL PSYCHROPHILE PROVIDES INSIGHT
INTO MOLECULAR EVOLUTION AT THE QUATERNARY
LEVEL
Wubben J.M.1, 2 , Dogovski C.1, 2, Dobson R.C.J.1, 2, Codd R.3 and
Perugini M.A.1, 2
1
Department of Biochemistry and Molecular Biology, The University
of Melbourne, VIC 3010, Australia. 2Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, VIC 3010,
Australia. 3Department of Pharmacology, School of Medical Sciences,
The University of Sydney, NSW 2006, Australia.
Shewanella benthica (Sb) is a psychrophilic bacterium that inhabits cold
ocean environments. Studying enzymes from this bacterium thus offers
great potential to probe the molecular evolution of quaternary structure
of proteins, including the tetrameric bacterial enzyme dihydrodipicolinate
synthase (DHDPS). Recent studies show that tetramerisation of DHDPS
in mesophilic bacteria, such as Escherichia coli and Bacillus anthracis,
is essential for stability and also necessary for enzymatic activity. We
have cloned, expressed and purified milligram quantities of recombinant
DHDPS from Shewanella benthica (Sb-DHDPS). By employing a series
of enzyme kinetic, biophysical and structural analyses, we show that
DHDPS from the cold bacterium Shewanella benthica (Sb-DHDPS)
exists as an active dimer in solution and crystal form at biologically-
relevant temperatures (< 13 °C). However, Sb-DHDPS aggregates at
higher mesophilic-like temperatures (20-37°C) and is shown to possess
significantly less enzymatic activity relative to cooler temperatures. This
finding provides evidence for an ancestral dimer of DHDPS and offers
tremendous insight into the evolution of the quaternary structure of
bacterial dihydrodipicolinate synthase.
POSTERS WEDNESDAY & THURSDAYY
POS-WED-261
DEVELOPMENT OF A NEW SUITE OF POLYCYCLIC-
BASED CONJUGATES OF DESFERRIOXAMINE B FOR
IRON OVERLOAD DISEASE
Liu J.1, 2, Obando D.1, 2, Schipanski L.G.1, 2, Groebler L.K.1, 2, Witting P.K.1, 2,
Kalinowski D.1, 2, Richardson D.R.1, 2 and Codd R.1, 2
1
University of Sydney. 2Bosch Institute.
The hydrophilic nature of the ‘Gold standard’ for iron chelation therapy,
desferrioxamine B (DFOB), means that the drug is unable to cross
lipophilic cell membranes. This results in the retention of intracellular
pools of iron, leading to the continuous generation of reactive oxygen
species. Although DFOB coordinates Fe(III) effectively, the drug is not
orally available and has a short plasma half-life, which translates as
an arduous subcutaneous treatment regimen for iron overload patients.
The aim of this project is to use a semi-synthetic approach to modulate
the properties of DFOB. A library of DFOB conjugates has been
synthesised using peptide coupling methods [1]. All DFOB conjugates
maintained their iron binding abilities. Lipophilicity was determined as
log P values (octanol/water partition coefficient) as calculated from
RP-HPLC retention times. The compounds were more lipophilic than
DFOB (log P ~2.1). Iron(III)-loaded compounds had a shorter retention
time than the non-ferrated compound. This suggests that prior to metal
binding, the lipophilic conjugates may cross the lipid bi-layer of the cell,
bind to excess intracellular iron and become sufficiently hydrophilic to
allow for easy urinary excretion. This new class of iron chelating drugs
combines the affinity of DFOB to Fe(III) and the lipophilic nature of the
ancillary ligands, forming potentially superior iron chelators. 1.Liu, J.,
et al., Conjugates of Desferrioxamine B (DFOB) with Derivatives of
Adamantane or with Orally Available Chelators as Potential Agents for
Treating Iron Overload. J. Med. Chem., 2010. 53(3): p. 1370-1382.
POS-WED-263
OPTIMIZATION OF THE HORSERADISH
PEROXIDASE CATALYSED BIODEGRADATION OF
PENTACHLOROPHENOL
Martinez-Ruiz J.1, Tomas V.2, Martinez-Gutierrez R.3, Garcia-Canovas F.1
and Tudela J.1
1
GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz), Dept.
Bioquimica y Biologia Molecular-A, Facultad de Veterinaria, Universidad
de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de Quimica
Analitica, Facultad de Quimica, Universidad de Murcia. 3NOVOZYMES
SPAIN S.A. (www.novozymes.com), Madrid, Spain.
Pentachlorophenol (PCP CAS Registry No. 87-86-512) is a major industrial
and agricultural biocide that has been used primarily as a wood preservative.
Its worldwide usage, up to 90000 tons, and relative stability make PCP a
ubiquitous environmental pollutant that has contaminated air, soil, and water
and resulted in persistent, widespread exposure through drinking water and
food. The major target organs for PCP toxicity include the liver, kidneys,
hematopoietic system, pulmonary system, and central nervous system.
Horseradish peroxidase (HRP, EC 1.11.1.7) is an enzyme with Fe(III) in the
hemin group of its active site, and with two Ca(II) cations in the proximal
and distal dominia of HRP. There are few studies about the enzymatic
biodegradation of PCP by HRP, mainly focused on the identification of
2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ) and other oxidation products,
by MS and NMR. The aim of this work is the kinetic optimization of the
assay conditions for this enzymatic reaction, with determination of the PCP
breakdown by HPLC-DAD (Agilent 1200 UHPLC-RR). The enzymatic
activity and stability, lead to the best assay conditions for the uniexponential
biodegradation in 10 minutes of 260 micromolar PCP, which are 90 nM HRP,
130 micromolar hydrogen peroxide and 10 mM pH 4.0 sodium citrate buffer
at 25 ºC. The stoichiometry PCP:H2O2 = 2:1 was established. HRP shows
low affinity towards PCP, but generates in each catalytic cycle two molecules
of PCP-radical, which evolves generating several biodegradation products,
as reported by other authors. Experimental data were fitted by non linear
regression to equations of the kinetic analysis for the reaction mechanism
proposed. This work has been partially supported by grants from several
Spanish organizations. Projects BIO2009-12956 (MICINN, Madrid), BIO-
BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca,
CARM, Murcia). Predoctoral fellowship JMR BES-2007-16208 (FPI, MICINN,
Madrid).
Page 256 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-262
PAIN PHENOTYPING OF INTERSTITIAL CYSTITIS/
PAINFUL BLADDER SYNDROME PATIENTS TO TARGET
BIOMARKER STUDIES
Mayer R.
University of Rochester,Rochester, NY, USA.
Introduction: Interstitial Cystitis/ Painful Bladder syndrome is a chronic
debilitating idiopathic condition characterized by urinary frequency and
discomfort with bladder filling. Previous reports utilizing biomarkers to
characterize patients have been problematic due to likely heterogeneity.
Attempting to sub classify these patients may allow planning of future
molecular studies leading to effective therapy. We compared a cohort
of patients using pain severity scores to evaluate differences as a guide
for future investigations. Methods: The records of 186 patients with IC
who completed an intake form were reviewed under approval by our
institutional human subjects review board. Patients with mild pain score
0-2/10 (n=75) were compared patients with severe pain 8-10/10 ( n=32
) with SPSS software / chi square analysis (p< 0.05)used to determine
associations of significance. Results: There was no difference between
the groups with age or duration of symptoms. Anxiety and irritable
bowel syndrome were relatively frequent in both groups but were not
significantly different. Severe pain patients did report a higher frequency
of pelvic surgery, UTI, dysmenorrhea and/ or endometriosis in addition
to increased frequency of migraine and asthma suggestive of systemic
hypersensitivity. A history of abuse and increased contemporary
psychological stress were also more frequent in patients with severe
pain. Conclusions: Patients with Painful Bladder syndrome/Interstitial
Cystitis show differences in pain severity which correspond to different
patterns of clinical history. Estrogen related mechanisms of pain,
signaling pathways common to asthma, migraine, and anxiety/coping
may be fruitful targets of molecular research especially for those in
severe pain.
POS-THU-264
THE PLANT DEFENSIN NAD1 ACTS SYNERGISTICALLY
WITH FUNGICIDES TO INHIBIT THE GROWTH OF
FUNGI IN VITRO
McKenna J.A.1, Van Der Weerden N.L.2, Anderson M.A.2 and Heath R.L.1
1
School of Botany, The University of Melbourne, VIC 3010 Australia.
2
Department of Biochemistry, La Trobe University, VIC 3086 Australia.
Plant defensins are small, basic, cysteine-rich proteins that are members
of the innate plant defence mechanism. The plant defensin NaD1 was
isolated from the floral tissues of ornamental tobacco (Nicotiana alata)
and inhibits a range of filamentous fungi in vitro. NaD1 inhibits fungal
growth by the destabilising and permeabilising the fungal plasma
membrane. Members of the triazole and strobilurin families are highly
valued as agricultural fungicides. Together these classes of fungicide
are the dominant form of chemical protection of crop plants worldwide.
Strobilurins inhibit fungal growth by blocking mitochondrial respiration
while triazoles inhibit growth by blocking the production of ergosterol.
We have demonstrated that combinations of NaD1 and fungicides act
synergistically to inhibit the in vitro growth of F. graminearum and F.
oxysporum, dramatically reducing the concentration of fungicide and
NaD1 required for fungal control.
POSTERS WEDNESDAY & THURSDAY
POS-WED-265
REMAZOL BRILLIANT BLUE ROYAL
BIODEGRADATION CATALYSED BY HORSERADISH
PEROXIDASE
Mendez P.1, Parra M.1, Tomas V.2, Martinez-Gutierrez R.3,
Garcia-Canovas F.1 and Tudela J.1
1
GENZ-Grupo de Investigacion Enzimologia (www.um.es/
genz), Dept. Bioquimica y Biologia Molecular-A, Facultad de
Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia,
Spain. 2Departamento de Quimica Analitica, Facultad de Quimica,
Universidad de Murcia. 3NOVOZYMES SPAIN S.A. (www.novozymes.
com), Madrid, Spain.
Remazol Brilliant Blue Royal (RBBR, Reactive Blue 19, C.I. 61200) is an
anthraquinone dye related with the reactive dyes group, which attains
the 12 % of worldwide production. It has been estimated that 4 % of dyes
are discharged into the environment, with harmful effects on biodiversity
and humans. Horseradish peroxidase (HRP, EC 1.11.1.7) is an enzyme
with Fe(III) in the hemin group of its active site. There are few studies
about the biodegradation of RBBR by oxidative enzymes, mainly focused
on structural identification of their oxidation products. The aim of this
work is the kinetic optimization of assay conditions for the enzymatic
oxidation of RBBR by hydrogen peroxide with HRP as biocatalyst, with
separation and determination of RBBR by HPLC-DAD (Agilent 1200
UHPLC-RR) and by UV-visible spectrophotometry. The absorbance
decrease at 595 nm was recorded in scan (Perkin Elmer Lambda-35)
and kinetic (Molecular Devices SpectraMax-340PC) assays. The
enzymatic activity and stability, lead to the best assay conditions for the
uniexponential biodegradation in 20 minutes of 50 micromolar RBBR,
which are 30 nM HRP, 250 micromolar hydrogen peroxide and 10 mM
pH 5.0 sodium citrate buffer at 25 ºC. The stoichiometry RBBR:H2O2
= 1:1 was established. HRP generates in each catalytic cycle two
molecules of RBBR-radical, which react between them with regeneration
of one molecule of RBBR, to originate only one molecule of a transient
oxidation product, which evolves towards further products, as reported
by other authors. Experimental data were fitted by non linear regression
to equations of kinetic analysis for the reaction mechanism proposed.
This work has been partially supported by grants from several Spanish
organizations. Projects BIO2009-12956 (MICINN, Madrid), BIO-BMC
06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca,
CARM, Murcia). Predoctoral fellowship MPC 09378/FPI/08 (Fundacion
Seneca, CARM, Murcia).
POS-WED-267
INVESTIGATION OF MHC HAPLOTYPING WITH
PEPTIDE NUCLEIC ACIDS USING QUANTITATIVE REAL
TIME POLYMERASE CHAIN REACTION
Murphy N.M., Pouton C.W. and Irving H.R.
Monash Institute of Pharmaceutical Sciences, Monash University, 381
Royal Parade, Parkville, VIC 3052, Australia
This study reports on the modelling and assessment of a novel
haplotyping strategy using peptide nucleic acids (PNAs) to physically
separate haploid material in a diploid DNA sample. Alleles of the human
leukocyte antigen DRB1 gene were targeted by two biotinylated PNAs
synthesised to target the HLA-DRB1*01 and DRB1*03 alleles containing
either Alexa Fluor 488 or Alexa Fluor 532 respectively. Stocks of two
plasmids lines with the second exon of the HLA-DRB1*01 and the
DRB1*03 alleles were generated using the pDONR 201 Gateway
system. PNAs were hybridised to DNA and incubated on a neutravidin
bound 96-well microplate (Pierce). Samples were washed to remove
unbound PNA and any DNA remaining in solution, and fluorescence
levels were monitored on an Envision 2101 microplate fluorometer.
Following elution, allele-specific quantitative real-time polymerase
chain reaction (qRT-PCR) was used to determine the level of specific
and non-specific binding. Non-biotinylated PNAs were eluted following
a single wash once the first supernatant was removed. Biotinylated
samples remained bound to the plate and decreased in fluorescence
by ~ 2 % with each succeeding wash. The [Alexa Fluor 488]-PNA*01-
Biotin probe hybridised with the HLA-DRB1*01 plasmid construct had
a yield significantly higher than controls (P < 0.05). The [Alexa Fluor
532]-PNA*03-Biotin probe hybridised with the HLA-DRB1*03 plasmid
had a significantly higher yield than controls (P < 0.01). In conclusion, the
haplotyping model assay presented provides a foundation for creating a
haplotyping assay by hybridising PNAs to genomic DNA and physically
separating the chromosomes.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 257
POS-THU-266
ROSMARINIC ACID OXIDATION AND ITS ANTIOXIDANT
CAPACITY
Munoz-Munoz J.L.1, Garcia-Molina F.1, Garcia-Molina M.1, Ros E.1,
Varon R.2, Tudela J.1, Garcia-Canovas F.1 and Rodriguez-Lopez J.N.1
1
GENZ: Grupo de Investigación de Enzimología, Departamento de
Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad
de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de
Química-Física. Escuela de Ingenieros Industriales de Albacete.
Universidad de Castilla la Mancha. Avda. España s/n. Campus
Universitario, E-02071, Albacete, Spain.
Rosmarinic acid is a fundamental compound of Rosmarinus officinalis.
In this work, this compound has been kinetically characterized as
substrate of polyphenol oxidase and peroxidase (PPO and POD). In
addition, polyphenol oxidase inactivation under aerobic conditions
(suicide inactivation) has been studied. However, under anaerobic
conditions, polyphenol oxidase does not inactivate by this compound.
The chemical structure of rosmarinic acid predicts its capacity to act as
antioxidant, which has been characterized as a primary and secondary
antioxidant capacity. Prooxidant capacity of this compound has been
determined. This work has been partially supported by grants from
several Spanish organizations. Ministerio de Educación y Ciencia
(Madrid, Spain) Project BIO2009-12956, from the Fundación Séneca
(CARM, Murcia, Spain) Projects 08856/PI/08 and 08595/PI/08, from
the Consejería de Educación (CARM, Murcia, Spain) BIO-BMC 06/01-
0004 and from the Consejería de Salud y Bienestar Social de la Junta
de Comunidades de Castilla La Mancha, Project FISCAM PI-2007/53.
JLMM has a fellowship from the Ministerio de Educación y Ciencia
(Madrid, Spain) Reference AP2005-4721. FGM has a fellowship from
Fundación Caja Murcia (Murcia, Spain).
POS-THU-268
DEVELOPMENT OF A MHC CLASS II PEPTIDE BINDING
PREDICTION METHOD WITH HIGH-POPULATION
COVERAGE FOR EFFICIENT VACCINE DESIGN
Oyarzun P.1, 2, Ellis J.1 and Kobe B.1
1School of Chemistry and Molecular Biosciences, Institute for Molecular
Biosciences and Centre for Infectious Disease Research, The University
of Queensland, Australia. 2Universidad San Sebastian, Chile.
Vaccines have being historically obtained from living attenuated
organisms. Nowadays, the interest is turning to peptide vaccines
rationally designed using immunoinformatics. The peptides are small
fragments of antigenic proteins containing a molecular structure
(epitope) that is presented by MHC molecules on antigenic presenting
cells (APC) to T cell receptors (lymphocytes), potentially triggering an
immune response. Exogenous peptides bind MHC class II proteins
through a number of interactions with amino acids lying along a highly
polymorphic groove, leading to over 1200 MHC class II alleles. This high
variability pose serious difficulties for vaccine design. We developed
the method Predivac for prediction of peptide binding to MHC class II
proteins. The method is based on the so-called specificity-determining
residues (SDRs), which contact the side chains of the peptide ligand
in the binding groove. A similar approach has being used by our
group for prediction of kinase substrates (Predikin). The analyses
of crystal structures revealed that the SDRs are highly conserved
throughout the MHC class II molecules. This allowed us to establish
a predictive correlation between SDR patterns and binding specificity
for experimentally characterized and non-characterized alleles. The
bioinformatics tool is being written in Perl. Thus far, a database has
being constructed including high-affinity binding peptides for 28 MHC
class II alleles (DRB allotype). The database accounts for 500 alleles
for which binding predictions are possible. The performance of the
method was assessed by leave-one-out cross-validation in terms of the
area under ROC curve (AROC). The AROC values range from 0.647
to 0.938, which is comparable or better than available state-of-the-
art methods. Our method will help designing vaccines that effectively
protect genetically diverse human populations.
POSTERS WEDNESDAY & THURSDAY
POS-WED-269
INDIGO CARMINE BIODEGRADATION CATALYSED BY
METHYLSYRINGATE-LACCASE MEDIATOR SYSTEM
Parra M.1, Jimenez L.1, Tomas V.2, Martinez-Gutierrez R.3,
Garcia-Canovas F.1 and Tudela J.1
1
GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz), Dept.
Bioquimica y Biologia Molecular-A, Facultad de Veterinaria, Universidad
de Murcia, E-30100 Espinardo, Murcia, Spain. 2Departamento de
Quimica Analitica, Facultad de Quimica, Universidad de Murcia.
3
NOVOZYMES SPAIN S.A. (www.novozymes.com), Madrid, Spain.
Indigo Carmine (IC) is a pH and redox indicator used as denim dye and
in the manufacturing of capsules. IC is harmful to the respiratory tract
if swallowed, and irritant to the skin and eyes. Laccase (EC 1.10.3.1)
catalyses the oxidation by molecular oxygen of substrates up to radical
products. The broad substrate selectivity and the high oxidation potential
of LAC, have lead to be used in biotechnological applications. This
selectivity is enhanced by the use of mediators, true substrates of LAC
which radical products oxidize molecules that are not direct substrates
of the enzyme. There are few studies about the biodegradation of IC by
oxidative enzymes, mainly focused on the identification of the oxidation
product, isatin-5-sulfonic acid (ISA), by MS and NMR. The aim of this
work is the kinetic optimization of the assay conditions for the oxidation
of IC by a methylsyringate-laccase mediator system as biocatalyst, with
separation and determination of IC and ISA by HPLC-DAD and by UV-
visible spectrophotometry. The enzymatic activity and stability, lead to the
best assay conditions for the biodegradation in 10 minutes of 98 micromolar
IC, which are 63 nM LAC, 6 micromolar methylsyringate and 10 mM pH
5.0 sodium acetate buffer at 25 ºC. The reaction mechanism consists of
the enzymatic oxidation of the mediator to a radical, coupled to the non
enzymatic oxidation of IC by methylsyringate-radical, with regeneration of
the mediator. A kinetic equation is proposed to explain the experimental
data and to predict the kinetic behaviour of the methylsyringate-laccase
mediator system. This work has been partially supported by grants from
several Spanish organizations. Projects BIO2009-12956 (MICINN,
Madrid), BIO-BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08
(Fundacion Seneca, CARM, Murcia). Predoctoral fellowship MPC 09378/
FPI/08 (Fundacion Seneca, CARM, Murcia).
POS-WED-271
THE AGILENT TECHNOLOGIES SURESELECT TARGET
ENRICHMENT SYSTEM FOR NEXT-GENERATION
SEQUENCING : ENRICHMENT AND DEPLETION
APPLICATIONS FOR TRANSCRIPTOME ANALYSIS
Roberts D.1, Happe S.2, Lin E.1, Pabon C.1, Giuffre A.2,
Fullmer-Smentek S.1, McInnes R.3, Joshi S.2, Novak B.1 and Leproust E.1
1
Agilent Technologies, Santa Clara, California. 2Agilent Technologies,
Cedar Creek, Texas. 3Agilent Technologies, Melbourne, Victoria.
The discovery of rare polymorphisms, structural variants, and novel
transcripts has been accelerated dramatically by next-generation
sequencing technologies. However, it remains cost-prohibitive to
sequence entire genomes in large cohort studies. To enable larger
sample sets, Agilent Technologies has developed the SureSelect
platform, allowing focused analyses on specific genomic loci at
considerable cost savings. Agilent is continuing to expand its portfolio to
increase the number of applications available to users. We demonstrate
high performance of DNA capture of several focused areas as measured
by capture efficiency, uniformity, reproducibility, and SNP detection.
We also describe performance of new SureSelect panels such as the
mouse exome. We then describe the adaptation of SureSelect to whole
transcriptome analysis applications and introduce methods to deplete
highly abundant transcripts such as ribosomal RNAs. Finally, we reveal
the use of custom libraries to interrogate specific subsets of transcripts
for abundance and variation. SureSelect applications will continue to
expand as users harness the power of next-generation sequencing.
Page 258 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-270
DELIVERY OF MULTIPLE PROTEINS USING MULTI-
GENE EXPRESSION VEHICLES
Poon S.1, Connelly A.A.1, Rainone M.1, Guarino R.2, McKenna J.A.1,
Carroll K.T.2, Anderson M.A.2 and Heath R.L.1
1
School of Botany, The University of Melbourne, VIC 3010.
2
Department of Biochemistry, La Trobe University, VIC 3086.
We have developed Multi-Gene Expression Vehicles (MGEVs) based
on the modification of multi-domain proteinase inhibitors from Nicotiana
alata. The N. alata proteinase inhibitors (NaPIs) are produced as
precursor proteins which form a circular conformation via disulfide
linkage between the amino- and carboxy- terminal peptide domains.
Proteolytic processing of the precursor protein occurs in a linker
region between each domain resulting in the release of mature 6 kDa
proteinase inhibitors which have inhibitory activity against chymotrypsin
or trypsin. We have produced MGEVs which are either circular or
linear in conformation. Insertion of additional proteins such as plant
defensins or different PIs, or the replacement of the native NaPI
domains has allowed us to produce precursors that contain from two
to potentially eight individual proteins. We demonstrate the expression
and processing of multiple proteins from single MGEV constructs using
stable and transient gene expression systems and show that MGEVs
are a promising strategy for protein stacking in transgenic plants.
POS-THU-272
VITAMIN D INTERACTIONS WITH P-GLYCOPROTEIN
EXPRESSION AND FUNCTION IN INTESTINAL LS174T
CELLS
Kota B.P.1, Tran V.H.1, Allen J.2 and Roufogalis B.D.1
1
Faculty of Pharmacy, University of Sydney, NSW 2006. 2Centenary
Institute, University of Sydney, NSW, 2006.
Intestinal P-glycoprotein limits bioavailability of drugs by inhibiting
absorption of orally administered drugs from the intestine. Vitamin D
receptor (VDR) is a transcriptional factor that mediates induction of
P-glycoprotein (P-gp). The aim of the current study was to investigate
the role of VDR on P-gp expression and function in LS174T intestinal
cells. In addition, the effect of ketokonazole, a pregnane X receptor
(PXR) inhibitor, on VDR mediated P-gp expression and function was
examined to determine the nature of the interaction between VDR
and PXR mediated P-gp regulation. Intestinal LS174T cells were
treated with 1α-25(OH)2D3 (DHC), the active metabolite of Vitamin D3.
DHC was found to significantly increase P-gp expression in LS174T
cells after two days of treatment. The increase in P-gp expression
correlated with decreased accumulation of Rh123 (used to monitor
P-gp function) in LS174T cells. DHC treatment reduced the cytotoxic
effects of colchicine, a P-gp substrate (determined by MTS cytotoxicity
assay). Interestingly, ketoconazole, in combination with DHC, enhanced
P-gp protein expression above that of DHC alone. However, the effect
on P-gp expression by KTZ and DHC treatment was also observed in
PXR deficient Caco-2 cells, and was most likely due to the observed
enhanced VDR protein expression. We conclude that DHC increases
P-gp expression and reduces cytotoxicity in an intestinal cell line, which
can be useful for in vitro drug pharmacokinetic studies. The interaction
observed between ketaconozole and DHC may have implications in
decreasing oral drug bioavailability of P-gp substrates.
POSTERS WEDNESDAY & THURSDAY
POS-WED-273
GROWTH HORMONE GENE MANIPULATION IN
CHANNA STRIATUS
Shrestha T.R.1 and Majumdar K.C.2
1
Tribhuvan University, Department of Zoology, Bhaktapur M. Campus,
Bhaktapur, Nepal. 2Centre for Cellular and Molecular Biology, Uppal
Road, Hyderabad-500007, India.
The GH gene manipulation of Channa striatus was undertaken in order
to engineer autotransgenic construct to produce transgenic fish. Out
of 32 different species of Channa present in the world, this is the first
report on molecular biological study of GH gene and three constitutive
promoters (histone 3, β actin and myosin) of five Channa species, viz, C.
striatus, C.punctatus, C. gachua, C.marulius and C. diplogramma. For
autotransgene constructs, the three constitutive promoters were isolated,
cloned, sequenced and characterized from C. striatus. The functional
assays of these promoters were done in vivo in zebrafish by monitoring
the green fluorescence. The cDNA and chromosomal growth hormone
genes including 5’ and 3’UTR regions were isolated, cloned, sequenced
and characterized from pituitary and blood of Channa striatus by using
RACE and PCR strategies. Thus GH cDNA transcript has 848bp with
a coding region of 615bp, 54bp of 5UTR and 159bp of 3’ UTR regions
whereas total GH gene has 2089 including 5’ and 3’ UTR regions. In all
these species the GH gene has six exons and five introns and is single
copy. The two autotransgene constructs of C. striatus, CSH3-GHcDNA
and CSβ-GHcDNA were transferred into the eggs of H. fossilis and P.
suchi through sperm electroporation. Detection of transgene, CSH3-
GHcDNA by PCR in 18 months old putative transgenic H. fossilis and P.
suchi revealed their presence in 66.6% and 61.1% individual response
respectively. The growth analysis in our preliminary study revealed that
transgenic fishes grew faster than non-transgenic individuals confirming
the functionality of the transgenic DNA prepared from Channa striatus.
POS-WED-275
ANTIMICROBIAL PEPTIDES PRODUCED BY PEPSIN
DIGESTION OF THE FRUIT PROTEIN OF BRUCEA
AMARISSIMA DESV
Sornwatana T.1, Roytrakul S.2, Wetprasit N.3 and Ratanapo S.4
1
Biochemistry department, faculty of science, Kasetsart U., Bangkok,
Thailand 10900. 2Genome institute, National Center for Genetic
engineer and Biotechnology, National Science and Technology
Development Agency(NSTDA), Pathumthani, Thailand 12120.
3
Biotechnology department, faculty of science, Ramkhamhaeng
U., Bangkok, Thailand 10240. 4Biochemistry department, faculty of
science, Kasetsart U., Bangkok, Thailand 10900.
Peptides exhibiting antimicrobial activity has gained scientific interest as
components in functional food for their potential in disease prevention
and health improvement. In this study, antimicrobial active peptides
encrypted in the dried fruit protein of Brucea amarissima Desv was
released by in vitro digestion with pepsin for 12 h at enzyme/protein ratio
of 1:25. The hydrolysate was purified by reverse phase HPLC followed
by LC-MS analysis. The antimicrobial activity of each purified fraction
was performed by agar dilution technique. Two potent peptide fractions
with 2418.076 m/z and 2022.462 m/z showed broad-spectrum inhibition
against 7 strains of human-pathogenic bacteria and a fungi with the
most potent inhibitory activity against Streptococcus pyogenes at MIC
value of 2.6 μg/ml. S. pyogenes is the cause of many important human
diseases ranging from superficial skin infections to life-threatening
systemic disease. The result suggested that the pepsin digestion of the
fruit protein of B. amarissima generated bioactive peptides which might
provide health benefits in reducing the risk of developing the disease
caused by S. pyogenes.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 259
POS-THU-274
PRODUCTION OF NON-NATURAL SIDEROPHORES
FROM SHEWANELLA PUTREFACIENS AND
ALTEROMONAS HALOPLANKTIS
Soe C.Z.1, 2 , Pakchung A.A.H.1, 2 and Codd R.1, 2
1
Universiity of Sydney. 2Bosch Institute.
Iron is a vital element for life, being necessary in metalloproteins,
such as cytochromes and ribonucleotide reductase, which are key to
fundamental cellular processes. In oxic, aqueous environments, the
majority of Fe(III) exists in the form of insoluble oxyhydroxides (Ksp ~
10 -39). Microorganisms have evolved different strategies to overcome
this problem. Under iron-deprived conditions, selected bacteria
produce iron-chelating molecules called ‘siderophores’, classified as
hydroxamate-, catacholate- or mixed- type chelates. Polyamines, such
as putrescine, cadaverine and spermidine, are fundamental precursors
of siderophore assembly pathways. The current research is focused
on the macrocyclic dihydroxamate-based siderophores, due to their
potential as therapeutic lead compounds as anti-tumour and anti-
infective agents. The known members of this class are bisucaberin,
alcaligin and putrebactin isolated from Alteromonas haloplanktis,
Bordetalla pertussis and Shewanella putrefaciens, respectively. Based
on the genetic data on bacterial siderophore biosynthesis, this present
study will explore the potential of S. putrefaciens and A. haloplanktis as
biosynthetic machines to produce novel siderophores via augmenting
culture media with non-natural substrates. Bacterial cultures were
grown in the presence of a range of diamine precursor substrates and
ornithine decarboxylase (ODC) inhibitors for 6 d at room temperature.
Optical density (OD) and the Chrome Azurol S (CAS) assay were used for
siderophore detection and monitored at 600 and 630 nm, respectively.
The novel siderophores will be isolated from the culture supernatant
using chromatographic steps and the structure will be determined using
HPLC/MS and NMR spectroscopy.
POS-THU-276
BACILLUS CEREUS WHICH CAN GROW IN THE
PRESENCE OF BENZENE
Tsugeno Y.
Department of Environmental Management, School of Health
Sciences, University of Occupational and Environmental Health,
Japan.
The environment where the higher organism cannot live is called
“Extreme environment”, and the microorganism that can be grown
under such an extreme environment is said, “Extremophile” usually.
Most organic solvents are generally biotoxic and prohibit growth of
microorganisms even at low concentrations. Thus, interest stems from
the fact that many organic solvents are classified as environmental
pollutants and their degradation by microorganisms is the focus of the
emerging bioremediation industry. I isolated the benzene-resistant
bacterium from Japanese soil by selecting for microbial growth in a
nutrient medium containing benzene. To isolate benzene-resistant
microorganisms,a small amount of soil was suspended in sterile water
and 0.2ml of this suspension transferred to test tubes containing 5ml
of a nutrient medium comprising 0.1% glucose, 0.25% yeast extract
and 0.5% polypeptone, at pH7.0. Benzene was then added to a final
concentration of 5%, 15% and 30% (v/v), the test tubes were sealed
with a cap, and the cultures incubated at 37°C for 120h in a test tube
shaker. I report here the discovery of microorganisms which is capable
of growing in all media culture containing of 5%,15% and 30% benzene.
Cell growth was determined by measuring the optical density at 660 nm.
16S rRNA gene sequencing date of microorganisms indicated Bacillus
cereus, Bacillus mycoides,and Bacillus thuringiensis respectively at
the possibility of 99%. In addition, Bacillus cereus strain isolates were
identified by morphological, physiological and biochemical analysis, and
representative above-mentioned strains were identified at the molecular
level using 16S rRNA sequence analysis. This time, I succeeded in the
discovery of “Extremophile” that was able to be grown under benzene
that was the organic solvent.
POSTERS WEDNESDAY & THURSDAY
POS-WED-277
EXPRESSION ANALYSIS OF MINCLE ON HUMAN
PERIPHERAL BLOOD CELLS
Vijayan D.1, 4 , Radford K.2, Bellette B.1, Beckhouse A.1, Ashman R.3,
Nair R.4 and Wells C.1
1
Eskitis Institute for Cell and Molecular Therapies, Griffith University,
Australia. 2Mater Medical Research Institute, Australia. 3University of
Queensland, Australia. 4School of Dentistry and Oral Health, Griffith
University, Australia.
C-type lectins play key roles in antigen recognition, uptake and initiation
of signalling cascades that influence the balance between tolerance
and immunity. Mincle, a class II C-type lectin receptor is induced in
mouse macrophages responding to Candida albicans. Mice lacking
Mincle are susceptible to oral and systemic Candida infections.
Understanding the role of Mincle in different cell compartments will
provide important information about how the body recognises and
fights fungal infection. In the present work, we aim to determine the
cellular distribution of Mincle on human peripheral blood. These findings
form preliminary investigations into examining the role of this lectin in
patients with chronic oral candidiasis. Blood from healthy Red Cross
volunteers were analyzed by flow cytometry for the presence of Mincle
on leukocyte populations. Mincle expression was identified on human
monocytes, granulocytes, circulating dendritic cells and lymphocytes.
Mincle expression on monocytes directly correlated to its expression on
macrophages differentiated subsequently in culture. Mincle is expressed
on a wide range of immune cell types, and is particularly abundant on
professional antigen presenting cell including B-lymphocytes. This
study provides a first essential description of the distribution of a major
fungal receptor, which provides further insight into the molecular basis
for defence against chronic oral infections.
POS-WED-279
IMPLEMENTATION OF SPLICE SWITCHING THERAPIES
FOR DUCHENNE MUSCULAR DYSTROPHY
Wilton S.D. and Fletcher S.
CNND University of Western Australia, 35 Stirling Highway, Crawley,
WA 6009.
The exquisitely precise and co-ordinated process of gene transcript
splicing, that is, intron removal and joining of exons, not only greatly
increases genetic plasticity through alternative splicing, but also offers
a point of therapeutic intervention for some genetic disorders. Protein
truncating mutations in the 2.4Mbp, 79 exon dystrophin gene lead to
Duchenne muscular dystrophy (DMD), the most common and severe
form of childhood muscle wasting. The size and complexity of the
dystrophin gene poses major challenges to gene replacement therapy
for DMD, but antisense oligomer induced exon skipping has progressed
from a concept demonstrated in vitro to clinical trials in just over a
decade. Proof-of-concept was demonstrated by restoration of localised
dystrophin synthesis after injection of a morpholino oligomer, designed
to excise exon 51 from the dystrophin mRNA, into the extensior digitorum
brevis of non-ambulant DMD patients with a common genomic deletion
sub-type. Preliminary results from a systemic antisense oligomer, dose-
escalating study over 12 weeks indicate that the compound is well-
tolerated and showed that dystrophin synthesis was restored in patient
muscle. Specific skipping of dystrophin exon 51 will only be relevant to
about 10% of DMD individuals, however, we have developed a panel of
oligomers to skip different dystrophin exons and allow treatment for other
dystrophin mutations as a form of personalized genetic therapy. We now
report that subtle DNA changes in the target exon influence the exon
skipping efficiency of splice switching oligomers, further emphasizing
the individual nature of this potential treatment. DMD is regarded as
an orphan disease, and additional stratification of patients according to
the nature of their primary gene lesion will pose great challenges to the
application of this therapy.
Page 260 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-278
DEVELOPMENT OF A LUCIFERASE REPORTER CGMP
RESPONSIVE PROMOTER SYSTEM SUITABLE FOR
USE IN PLANT PROTOPLASTS
Wheeler J.I. and Irving H.R.
Monash Institute of Pharmaceutical Sciences.
cGMP is a second messenger molecule associated with plant
responses to hormones, light, nitric oxide and both biotic and abiotic
stress. Currently cGMP levels can be assessed using antibody kits
designed for mammalian cells or mass spectrometry analysis. Maathuis
(2006) used micro-array analysis of Arabidopsis roots to identify genes
that are differentially expressed 90 mins after a 30 min exposure to
membrane permeable cGMP. Three genes that were up-regulated were
NTL1 (At1g33440), CHX21 (At2g31910) and SOS3 (At5g24270). We
have shown that these three genes are also expressed in Arabidopsis
leaves and protoplasts derived from mesophyll cells. Promoters of
these genes were cloned into the pLUCTRAP3 vector (Calderron-
Villalobos et al., 2006) to generate luciferase fusions. Results of cGMP
induced expression of each promoter luciferase clone in freshly isolated
Arabidopsis mesophyll protoplasts show slight variations in luciferase
expression after cGMP treatment. The NLT1 promoter fragment contains
the mammalian cGMP responsive element (RE) identified by Hum et
al (2004). We are currently inserting four additional cGMP RE into the
NTL1 promoter with the aim of enhancing the promoter response to
cGMP. Bastian and colleges (2010) identified a common GA responsive
element (GARE) in the promoters of genes that are up regulated in
response to cGMP and down regulated in a GA insensitive mutant. We
have inserted six copies of the GARE into the NTL1 promoter fragment
and report here the effect on luciferase activity in our protoplast system.
The development of a cGMP sensitive luciferase reporter system would
then allow us to assess receptors such as AtBRI1, AtPSK1, AtRPK
and AtPepR1 with guanyl cyclase activity by measuring differences
in luciferase activity. Maathuis (2006) The Plant Journal 45: 700-711.
Calderron-Villalobos et al. (2006) Plant Physiology 141: 3-14. Hum
et al. (2004) Hypertension 43: 1270-1278. Bastian et al. (2010) Plant
Signalling and Behaviour 5: 1-9.
POS-THU-280
STRESS INDUCIBLE EXPRESSION OF TADREB4
INCREASES DROUGHT TOLERANCE OF TRANSGENIC
WHEAT AND PREVENTS UNDESIRABLE CHANGES IN
PLANT DEVELOPMENT
Yadav M.K.1, 2 , Eini O.1, Nataliya K.1, Tatiana P.1, Bazanova N.1, Singh R.1,
Ismagul A.1, Eliby S.1, Langridge P.1 and Lopato S.1
1
ACPFG, University of Adelaide Waite Campus Urrbrae Adelaide SA
5064. 2SVP University of Agriculture & Technology Modipuram Meerut
INDIA 250110.
Wheat is the most widely grown crop in the world covering around 16%
of the arable lands and staple food of about 35% of the world population.
Global environmental warming, followed by increasing drought threaten
the world’s food supply. One of the sustainable and economically viable
solutions for increasing crop stability and productivity is crop genetic
improvement for higher tolerance to abiotic stresses by the introgression
of agronomically important genes. Transcription factors (TFs) have been
shown to mediate stress signal transduction and response to different
stresses in plants. The full length cDNA of a new drought response
element binding factor, designated TaDREB4, was isolated in the Y1H
screen of the cDNA library prepared from flag leaves and spikes of
drought tolerant wheat cultivar subjected to drought/heat stress. The
DRE TACCGACT was used as bait. With the aim to increase drought
tolerance, we over-expressed TaDREB4 in transgenic barley and wheat.
Transgenic plants with strong constitutive over-expression of these factor
lead to little negative changes in growth and flowering time. However,
expression of this factor under stress inducible ZmRab17 promoter
shows no undesirable phenotypes in wheat plants. Transgenic wheat
T1 plants in the first experiment showed increased drought tolerance at
the seedling stage as compared with control plants and quick recovery
after re-watering. However, no frost tolerance improvement has been
demonstrated for barley plants with constitutive expression of TaDREB4
in several independent experiments. Analysis of the expression of
potential downstream genes is currently in progress.
POSTERS WEDNESDAY & THURSDAY
POS-WED-281
PLANT-BASED APPROACH TOWARDS
UNDERSTANDING THE INFLUENZA A VIRUS
ANTIGENIC DRIFT
Yamato H., Cueno M.E., Omagari K. and Okamoto T.
Nagoya City University, Nagoya City, Aichi, Japan.
Influenza A virus has been an elusive human pathogen mainly due
to its ability to rapidly mutate and evade antibodies specific for its
attachment protein, the hemagglutinin (HA). Current influenza vaccine
strategies target the HA protein, however, annual mutations within the
HA region occurs allowing the new viral strain to be elusive to previously
made vaccines. Several attempts had been made to understand the
HA antigenic drift in hopes to predict the next influenza strain and
develop appropriate vaccines in advance. To date, this approach has
been unsuccessful. In this study, we determined the possible role of
the conserved H1N1 HA1 and HA2 nucleotide regions in antigenic drift.
Codon-optimized FLAG-HA1-HA2-GUS genes were fused accordingly.
H1N1 HA1 and HA2 conserved regions were determined through
multiple sequence alignment. The gene construct was ligated into pBI121
expression vector, bombarded into leaf calli and grown in appropriate
growth medium. Immunoblot assays using antibodies against FLAG,
the universal epitope found in HA2 and GUS were used. Using FLAG
antibody, we detected a 36 kDa protein expressed during the callus
stage and two proteins, 36 and 30 kDa, expressed from transgenic
tomato leaves. Interestingly, when we used a GUS antibody to confirm
fusion protein expression, we only detected the 36 kDa protein from
callus and none form the transgenic leaves. In addition, when we used
the universal epitope antibody, no band was detected in both callus
and leaves. We verified the sequence of the gene fusion expressed in
tomato and found variations from the original sequence emphasizing
the significance of the HA conserved regions in causing antigenic drift.
POS-WED-283
ADIPOSE-DERIVED MESENCHYMAL STEM CELLS
ENHANCE TISSUE REGENERATION BY INDUCING
GROWTH OF BLOOD VESSELS AND NERVES
Kalinina N.I., Lopatina T.V., Efimenko A.Y.U., Parfyonova E.V. and
Tkachuk V.A.
Faculty of Fundamental Medicine, Lomonosov Moscow State
University.
Transplantation of adipose-derived stem cells (ASCs) induces tissue
regeneration by accelerating the growth of blood vessels and nerve
sprouts. Previously we demonstrated that ASCs promote the formation
and growth of blood vessels by producing angiogenic growth factors
as well as enhancing vessel maturation. However mechanisms of their
action on the growth of nerve fibers are only partially understood. Here
we show that transplantation of ASCs stimulates a repair of motor and
sensory nerves as well as induces nerve sprouts growth in matrigel
implants. Transcriptome analysis has revealed that ASCs express
mRNAs for growth factors and extracellular matrix proteins specific for
the outgrowth of nerve sprouts and myelination. Exposure of ASCs to
a combination of retinoic acid with 5-azacytidine up-regulates BDNF
production in these cells as well as their ability to induce nerve fiber
growth in matrigel implants. BDNF neutralizing antibodies have
abrogated stimulatory effect of ASCs on the growth of nerve sprouts.
These data suggest that ASCs induce nerve repair and growth via
BDNF production and this stimulatory effect can be further enhanced by
cell exposure to neural differentiation medium prior to transplantation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 261
POS-THU-282
NMDA RECEPTOR ANTAGONIST ADMINISTRATION
LEADS TO DOWN-REGULATION OF DISC1
EXPRESSION AND OVER MIGRATION OF NEWLY
GENERATED NEURONS IN THE ADULT HIPPOCAMPUS
Namba T.1, 2 , Uchino S.2, Kohsaka S.2 and Kaibuchi K.1
1
Dept. of Cell Pharmacology, Nagoya Univ. Grad. Sch. of Med., Aichi,
Japan. 2Dept. of Neurochemistry, National Institute of Neuroscience,
Tokyo, Japan.
New neurons are continuously generated throughout life in the
hippocampus. Newly generated neurons migrate short distance and
are integrated into pre-existing neuronal circuit. However, the extra
cellular signals which regulate the neuronal migration are not fully
understood. Here we report that NMDA receptor (NMDA-R) signaling is
involved in cellular migration and proper positioning of newly generated
neurons in the adult hippocampus. An intraperitoneal injection with
NMDA-R antagonists into adult male mice caused aberrant positioning
of doublecortin-positive immature neurons. BrdU labeling analysis
suggested that NMDA-R antagonist caused an over migration of newly
generated neurons. Using a quantitative real-time PCR analysis, we
examined the expression of Disrupted-In-Schizophrenia 1 (DISC1)
that has been identified as a candidate susceptibility gene for major
psychiatric disorders such as schizophrenia, because it has been known
that DISC1 is one of the important molecules regulating migration of
newly-generated neurons in the adult hippocampus. The expression
of DISC1 in the dentate gyrus was reduced by NMDA-R antagonist-
injection in a dose-dependent manner. In contrast, the expression of
the DISC1-interacting molecules, Lis1, NDEL1 and Girdin, was not
affected by the NMDA-R antagonist-injection. Furthermore, the over
migration phenotype by NMDA-R antagonists was partially rescued by
lentiviral-mediated DISC1 expression. These results suggest that the
NMDA-R signaling affects the migration of newly generated neurons via
regulating the expression of DISC1.
POS-THU-284
HYPEROSMOTIC AND OXIDATIVE STRESS INDUCES
INTRACELLULAR CA2+- DEPENDENT APOPTOSIS
THROUGH THE CATEPSIN B AND CASPASE 3-MEDIATED
PATHWAYS IN SKIN EPIDERMAL HUMAN CELL LINE
Silva R.A.1, Machado D.1, Souza A.C.S.1, Shishido S.M.1, Ferreira C.V.1,
Paredes-Gamero E.J.2 and Justo G.Z.1, 2
1
Departamento de Bioquímica, IB, UNICAMP, Campinas, SP.
2
Disciplina de Biologia Molecular, Departamento de Bioquímica,
UNIFESP, São Paulo, SP.
The epidermis is confronted with multiple environmental stressors that
affect its redox state and osmotic balance. One of the main factors by
which hyperosmotic and oxidative stress influence skin condition is the
modulation of intracellular Ca2+.Increase in cytosolic Ca2+ is an essential
element in the control of many physiological processes. However,
sustained increases in Ca2+ concentration may contribute to oxidative
stress and activation of different cell death mechanisms. Several
other events are related to the increase in Ca2+, including regulation
and activation of a number of enzymes such as Ca2+/phospholipid-
dependent protein kinase C (PkCs) and proteases. The aim of this study
is to examine the involvement of calcium homeostasis in the apoptotic
death induced by hyperosmotic and oxidative stress in human skin
epidermal cell line, HaCaT cells. This study shows that sorbitol and
H2O2 induce keratinocyte death, displaying an IC50 of 1,0 mol/L and
2.0 mmol/L, respectively. The increase of cytosolic Ca2+ induced the
production of reactive oxygen species (ROS). PepChip and Western
blot analysis demonstrated that the activity of different Ca2+-dependents
PKCs was modulated by hyperosmotic and oxidative stress. It was also
observed permeabilization of the lysosomal-membrane followed by
release of cathepsin B into the cytosol. Activation of caspase 3 was also
detected. Our findings clearly indicate that apoptosis occurs in a time
and dose-dependent manner and is the predominant type of cell death,
as proven by increase of Annexin V positively stained cells. Therefore,
our research suggests that control of calcium homeostasis may be a
key factor in protecting skin against environmental stress.
POSTERS WEDNESDAY & THURSDAY
POS-WED-285
ATF3 IS REQUIRED FOR TLR MEDIATED SURVIVAL
SIGNALLING IN MURINE BONE MARROW DERIVED
MACROPHAGES VIA ITS REPRESSION OF PRO-
APOPTOTIC BAK AND BAX
Thompson M.R., Xu D. and Williams B.R.G.
Monash Institute of Medical Research - Centre for Cancer Research.
Macrophages play an essential role in innate immune responses by
producing pro-inflammatory mediators and through phagocytic clearance
of pathogens or apoptotic cells. Activated macrophages, are bestowed
with resistance to apoptosis, allowing prolonged function in inflammatory
responses. However, excessive macrophage activation contributes
significantly to chronic inflammatory diseases. The toll-like receptor
(TLR) family are key regulators of innate immune responses through their
ability to induce pro-inflammatory cytokine production and elicit survival
responses. Recently the adaptive response gene, activating transcription
factor 3 (ATF3), has been shown to be induced by TLR signalling. Following
its induction, ATF3 is able to negative regulate TLR signalling through its
ability to repress transcription of pro-inflammatory cytokines, including IL-6
and IL-12p40. ATF3 has also been suggested to play various roles in the
regulation of cell cycle progression and apoptosis, so we were interested in
investigating whether ATF3 could elicit similar responses in macrophages
following TLR stimulation. Here we show that TLRs elicit survival of bone
marrow derived macrophages (BMM), in the absence of colony stimulating
factor 1 (CSF-1). Importantly, the induction of survival by TLRs in ATF3 -
/-
BMMs is significantly reduced. Compared to WT BMMs, ATF3 -/- BMMs
fail to down regulate apoptosis in response to LPS. In contrast, cell cycle
transition did not change between the genotypes, indicating that it is a defect
in the apoptotic response that contributes to decreased overall survival of
ATF3 -/- BMMs. In this regard, we demonstrate that ATF3 lies downstream
of c-Jun N-terminal kinase (JNK), an important regulator of apoptosis,
following TLR activation and that induction of ATF3 by TLR signalling allows
for the transcriptional repression of pro-apoptotic mediators BAK and BAX.
The deregulation of BAK and BAX in ATF3 -/- BMMs may be responsible for
skewed responses towards apoptosis. Therefore ATF3 is as an important
factor for the maintenance of innate immune homeostasis.
POS-WED-287
ANALYSIS OF SHARPIN MUTANT MICE AND THE ROLE
OF SHARPIN IN THE TNF RECEPTOR SUPER-FAMILY
PATHWAYS
Gangoda L.R.1, 2 , Hasbold J.3, Gerlach B.4, Carter H.1, Silke J.1 and
Vaux D.L.1
1
Department of Biochemistry, La Trobe University, Kingsbury Drive,
Bundoora, VIC 3086, Australia. 2Cooperative Research Centre for
Biomarker Translations (CRC), La Trobe University Bundoora, VIC,
3086 Australia. 3Walter & Eliza Hall Institute of Medical Research, 1G
Royal Parade, Parkville, Victoria 3050, Australia. 4Tumour Immunology
Unit, Imperial College London, London, UK.
Sharpin (Shank-associated RH domain-interacting protein) is a 45 kDa
protein abundantly expressed in brain, spleen and lung. Sharpin binds
to the HOIL-1 and HOIP, which are two components of the TNF-RSC
(Tumour necrosis factor receptor signaling complex) and regulate their
activity. It is also associated with the CD40 receptor signaling complex
where it is recruited and function together with HOIL-1 and HOIP
to enhance the downstream signaling which results in activation of
many genes leading to proper development of lymphoid tissues, B cell
activation, proliferation, survival and up-regulation of surface markers
involved in antigen presentation. The research aims are to discover
the role of Sharpin in the TNF-R1 and CD40 signaling pathways and
its putative role in other TNF superfamily (TNFSF) signaling pathways
such as BAFF and APRIL mediated pathways. Mice with spontaneously
occurring chronic proliferative dermatitis mutation (cpdm) of the Sharpin
gene have a phenotype that is entirely consistent with deregulated
TNF signaling, characterized histologically by severe inflammation and
defects in secondary lymphoid organ development.TNFSF signaling
impacts many human diseases, including tumorigenesis and auto-
immune diseases. Therefore defining the role of sharpin in these
signaling pathways could eventually lead to therapeutic applications.
Ultimately cpdm mice will also provide a unique opportunity to study the
development of lymphoid tissues and signals that regulate the humoral
response.
Page 262 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-286
PHENOTYPIC ANALYSIS OF SOYBEAN ROOT AND
NODULE TISSUES AFTER RNAI INDUCED SILENCING
OF THE MEMBRANE BOUND TRANSCRIPTION
FACTOR GMSAT1
Mohammadi-Dehcheshmeh M.1, Ebrahimie E.2, Chiasson D.1,
Tyerman S.D.1 and Kaiser B.N.1
1
Plant Research Center, School of Agriculture, Food and Wine, Waite
Campus, University of Adelaide, Australia. 2Department of Crop
Production & Plant Breeding, College of Agriculture, Shiraz University,
Iran.
GmSAT1 is membrane bound basic helix-loop-helix transcription factor
(TF) expressed in soybean nodules and localized to the peribacteroid
membrane surrounding nitrogen-fixing bacteroids and cell membrane
(Kaiser et al 1998). GmSAT1 was initially characterized as a putative
ammonium transporter based on its ability to complement growth of an
ammonium transport deficient yeast mutant 26972c. However, recent
studies have demonstrated GmSAT1 behaves as a TF in both yeast and
in soybean (Loughlin et al unpublished results). We are examining the
functional activity of GmSAT1 using a reverse genetics approach where
SAT1 has been silenced using RNAi technology in Agrobacterium
rhizogenes induced adventitious hairy-roots and nodules. We have
further developed this protocol using a combination of in vitro and in
vivo conditions where the efficiency of hairy root transformation has
been greatly improved. Analysis of silenced tissues reveals the loss of
SAT1 impacts upon root nodule development resulting in predominantly
ineffective white nodules. Moreover, light microscopy studies have also
indicated that silencing of SAT1 influences the vascular cylinder of
the root where xylem vessel and endosperm development are altered
relative to the roots containing the empty vector control. These studies
will provide insight into the overall function of GmSAT1 in both roots and
nodules and its relationship to the development and or maintenance of
the nitrogen-fixation symbiosis in soybean.
POS-THU-288
RECEPTOR INTERACTING PROTEIN KINASE 1 -
INTERACTIONS IN TNF RECEPTOR 1 SIGNALLING
Evans J.M., Gentle I.E., Vaux D.L. and Silke J.
La Trobe University, Bundoora.
Receptor interacting protein kinase 1 (RIPK1) is the founding member
of the RIP kinase family. It has multiple roles, and can promote both
cell survival and cell death following ligation of tumour-necrosis factor
receptor 1 (TNFR1). RIPK1 has been reported to interact with many
proteins, including TNFR1, TNF Receptor 1-associated death domain
protein (TRADD), TNFR-associated factor 2 (TRAF2) and 5 (TRAF5),
and another RIP kinase family member, RIPK3. Putative TRAF2
binding regions of Mus musculus (mm) RIPK1 were characterised. The
EE354AA mutant appeared to bind mmTRAF2 less efficiently than wild-
type RIPK1, however this finding was not consistent. The DE367AA
putative TRAF2 binding mutant was able to bind mmTRAF2 readily,
indicating this mutation did not cause a loss of function. A form of
RIPK1 lacking its death-domain (ΔDD RIPK1) retained the ability to bind
TRAF2, indicating that this region is not required for TRAF2 binding.
RIPK1-/- murine embryonic fibroblasts (MEFs) were reconstituted with
wild-type RIPK1, the putative TRAF2 binding mutants, or the ΔDD
RIPK1. Although the cells bearing the EE354AA mutant responded less
to TNF-α+cycloheximide (CHX) than MEFS with wild-type RIPK1, MEFs
bearing either TRAF2 binding mutant behaved the same as those with
wild-type RIPK1 in response to treatment with TNF-α and the Smac
mimetic drug, Compound A (CA). RIPK1-/- cells reconstituted with the
ΔDD RIPK1 responded the same way as RIPK1-/- cells in the presence
of TNF-α+CHX or TNF-α+CA, indicating that RIPK1 needs the death
domain for its functionality under these conditions. Cells reconstituted
with a RIPK1 mutant that could not to bind RIPK3 acted similarly to those
reconstituted with wild-type RIPK1 in response to both TNF-α+CHX
and TNF-α+CA, indicating that engagement of RIPK3 by RIPK1 is not
necessary for responses to TNF-α under those conditions.
POSTERS WEDNESDAY & THURSDAY
POS-WED-289
CELLULAR INHIBITOR OF APOPTOSIS PROTEINS AND
MOUSE EMBRYONIC FIBROBLAST PROLIFERATION
Rickard J.A.1, 2 , Moulin M.1, Feltham R.1, Anderton H.1, Vaux D.L.1, 2 and
Silke J.1, 2
1
Department of Biochemistry, La Trobe University, Kingsbury Drive,
Bundoora, VIC, 3086 Australia. 2Cooperative Research Centre for
Biomarker Translations (CRC), La Trobe University Bundoora, VIC,
3086 Australia.
Genetic deletion or drug-induced degradation of cIAP (cellular inhibitor of
apoptosis protein) 1 and 2 has been shown to increase murine embryonic
fibroblast (MEF) proliferation. Multiple myeloma has been found to select
for biallelic deletions of the BIRC2/BIRC3 locus encoding cIAPs which
may promote oncogenesis due to loss of inhibition of nuclear factor
kappa B signalling. This phenomenon is cell and cancer type specific
as cIAPs are over-expressed or implicated in the development of
other cancers. cIAP antagonism holds chemotherapeutic promise with
pre-clinical testing of novel compounds that induce cIAP degradation
underway. Characterisation of pro-proliferative effects of cIAP loss may
yield mechanistic insights into cancers like multiple myeloma whilst
concurrently having implications for treatment of other cancers with
IAP antagonists. This study shows wild type but not cIAP1/2 double
knockout (DKO) SV40T transformed MEFs proliferate more rapidly when
treated with a novel Smac (second mitochondria-derived activator of
caspases) mimetic compound that induces cIAP degradation (p ≤ 0.01).
TNF-receptor associated factor (TRAF) 2 and/or 5 may be important
for this increase in proliferation which is intriguing since a lack of
cIAP1 and TRAF5 interaction was found using co-immunoprecipitation
experiments. Reconstitution of cIAP DKO MEFs with over-expressed
levels of cIAP1 slowed proliferation by an average of 44% (p ≤ 0.01),
however no significant reduction was seen with wild type levels of
reconstitution. This study provides additional evidence that cIAP loss
increases SV40T MEF proliferation and establishes methods that may
be used to further characterise this phenomenon.
POS-WED-291
IN VIVO DYNAMICS OF HUNTINGTIN PROTEIN
AGGREGATION
McCoey J.M.1, 2 , Wong Y.Q.1, 2, Hatters D.M.2 and Quinn L.M.1
1
Department of Anatomy and Cell Biology, University of Melbourne.
2
Bio21 Molecular Science and Biotechnology Institute, Department of
Biochemistry and Molecular Biology, University of Melbourne.
Huntington’s disease occurs due to mutation that leads to the expansion
of a poly-glutamine sequence in the Huntingtin protein. In the disease
state, the mutant Huntingtin protein coalesces into large intracellular
protein agglomerates, known as inclusions, through a largely unknown
mechanism. Although inclusion formation is a key feature of the disease, it
is not understood how the Huntingtin protein is trafficked and aggregates
in cells. In particular, the identity of the cytoskeletal proteins controlling
inclusion formation and trafficking has remained poorly defined. We
have developed an in vivo Drosophila model to investigate aggregate
formation in live cells. Using time-lapse confocal microscopy, we have
observed the dynamic process of actin-cytoskeleton interactions with
aggregate formation directly in living tissue. We aim to use this model
combined with the powerful genetic tools available in Drosophila to
investigate the role of cytoskeletal proteins in the aggregation pathway
of Huntingtin and how this relates to cellular dysfunction.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 263
POS-THU-290
IMPROVEMENT OF WHEAT AND BARLEY TOLERANCE
TO DROUGHT BY MODULATED EXPRESSION OF
TRANSCRIPTION FACTORS
Eini O.1, Pyvovarenko T.1, Singh R.1, Eliby S.1, Sivasankar S.2,
Simmons C.2, Williams B.3, Tingey S.3, Langridge P.1 and Lopato S.1
1
Australian Centre for Plant Functional Genomics, University of
Adelaide, Waite Campus, Urrbrae, SA-5064, Australia. 2Pioneer Hi-
Bred, a DuPont Business, Johnston, IA 50131. 3DuPont Crop Genetics
Research, Wilmington, DE 19880.
Wheat and barley grown in Australia are subjected to intermittent periods
of drought from winter through spring that can significantly affect yield.
The expression of numerous plant genes involved in plant protection
and adaptation is regulated by drought stress. Promoter regions of
stress-inducible genes contain cis-acting elements involved in stress-
responsive gene expression. These elements act as binding sites for
transcription factors (TFs). The aim of our work was to identify and
isolate drought related transcription factors using cis-acting elements as
bait and to improve plant tolerance to drought by modulated expression
of selected stress-inducible transcription factors from drought-tolerant
varieties. We have prepared 12 different yeast expression cDNA
libraries using leaf, spike, developing grain and root tissues collected
from unstressed and drought, drought/heat, cold/frost stressed wheat,
barley and maize plants. These libraries were used to support yeast
one-hybrid (Y1H) and two-hybrid (Y2H) screens. More than 20 different
known and computer predicted cis-elements were used to prepare bait
yeast strains, over 10 of these elements were successfully used in the
Y1H screen. About 35 drought related TFs from 6 different families were
cloned either in the Y1H screen using cis-elements as baits or in the
Y2H screen using known TFs as baits. Selected candidates were over-
expressed under constitutive and stress-inducible promoters in wheat,
barley and maize varieties. Analysis of the developmental phenotypes,
drought tolerance and grain yields under drought conditions are
currently in progress.
POS-THU-292
DECIPHERING THE STEPS INVOLVED IN THE
TRAFFICKING OF PFEMP1, A KEY VIRULENCE
PROTEIN IN THE MALARIA PARASITE PLASMODIUM
FALCIPARUM
Mwesigye E.E.1, McMillan P.J.1, Maco B.1, Hanssen E.2 and Tilley L.1
1
Department of Biochemistry and Centre of Excellence for Coherent
X-ray Science, La Trobe University, VIC, Australia. 2Bio21 Institute,
Melbourne University, VIC, Australia.
The malaria parasite Plasmodium falciparum is responsible for one of
the most prevalent infectious diseases world wide. The most severe
symptoms of malarial infection are caused by the adhesion of parasite
infected erythrocytes to endothelial cells in the circulatory system.
The adhesion phenotype is a result of the parasite exporting proteins
into, and trafficking proteins across the erythrocyte cytoplasm into
the erythrocyte membrane. However, as the mature erythrocyte has
no trafficking machinery, the parasite must therefore develop its own
trafficking network in the erythrocyte cytoplasm. Maurer’s clefts (MCs)
are parasite derived membranous organelles present in the erythrocyte
cytoplasm and are known to be involved in trafficking of the major
virulence factor PfEMP1 as well as other proteins to the erythrocyte
membrane. Despite their importance in virulence, the mechanisms by
which MCs are formed and traffic PfEMP1 are not well understood.
Using highly synchronised cultures we will investigate the sequential
steps involved in the formation of Maurer’s cleft and PfEMP1 trafficking
to the surface of the erythrocyte. We will use P. falciparum transfectants
expressing Maurer’s cleft proteins (tagged with fluorescent proteins) to
visualise and investigate the processes involved in protein trafficking in
vitro. By combining immunofluorescence, live cell confocal microscopy
and immunoelectron microscopy we hope to gain a better understanding
of the interactions between these trafficking organelles and their cargo
proteins over the 48 hour asexual life cycle of the parasite.
POSTERS WEDNESDAY & THURSDAY
POS-WED-293
MYOSIN II AND RAB6 ARE INVOLVED IN GOLGI
VESICLE FORMATION/RELEASE AND CISTERNAL
MAINTENANCE
Micaroni M.1, Storrie B.2, Morgan G.P.1, 3 and Marsh B.J.1, 3, 4
1
Institute for Molecular Bioscience, The University of Queensland, St
Lucia, Brisbane, QLD, 4072, Australia. 2Department of Physiology &
Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR
72205, USA. 3Centre for Microscopy & Microanalysis, The University of
Queensland, St Lucia, Brisbane, QLD 4072, Australia. 4ARC Centre of
Excellence in Bioinformatics, The University of Queensland, St Lucia,
Brisbane, QLD 4072, Australia.
Bi-directional protein transport between spatially and functionally distinct
compartments of the biosynthetic/secretory pathway in mammalian
cells requires the formation and fission of membrane carriers from the
compartment where they originate, their processive movement along
cytoskeletal filaments and the subsequent docking and fusion of the
carrier with the compartmental membrane at their destination. Many of
these critical steps in protein transport are tightly coordinated by small
Rab GTPases and their cognate effectors, which together act to modulate
the specificity, directionality and efficiency of membrane trafficking
events. The Golgi-associated GTPase Rab6 has been shown to play
an important role in regulating membrane traffic at the Golgi to maintain
cisternal homeostasis, and a wide variety of proteins have been identified
as specific Rab6 effectors. Most recently, Miserey-Lenkei et al. (Nat Cell
Biol, 2010, 10.1038/ncb2067) have demonstrated that myosin II also acts
as a Rab6 effector and contributes to its localization at the Golgi as well as
regulating the fission of Rab6-positive carriers; siRNA-mediated depletion
of either protein was reported to yield a similar Golgi phenotype whereby
the fission of Rab6-positive carriers was inhibited. Here, we report that
selective depletion of Rab6 or myosin II manifests in distinctly different
structural phenotypes at the Golgi; silencing of both proteins impairs the
trafficking of anterograde as well as retrograde Golgi traffic. Whereas Rab6
knockdown results in the significant accumulation of vesicular traffic at the
Golgi and an increase in cisternal number, size and uniformity, myosin II
depletion induces fragmentation of the Golgi ribbon per se accompanied
by a significant inhibition of vesicle fission from Golgi cisternal membranes.
These results suggest that rather than acting as a direct effector of Rab6,
myosin II and Rab6 have related but not mutually dependent roles in Golgi
vesicle formation/release and cisternal maintenance.
POS-WED-295
ROLE OF CYSTEINE PROTEASES IN THE MECHANISM
OF ACTION OF THE ANTICONVULSANTS
LEVETIRACETAM AND CARBAMAZEPINE AND
THE CALPAIN INHIBITOR CALPASTATIN IN
PENTENYLTETRAZOL-KINDLED RATS
Mansour M.1, Albaker A.1 and Alhaider A.2
1
King Saud University-College of Pharmacy-Pharmacology. 2King
Saud University-College of Medicine-Pharmacology.
Atypical antiepileptic drugs like levetiracetam (LEV) shows antiepileptic
activity in animals and human and their actions do not seem to be similar
to the traditional antiepileptic drugs. The present study investigated
the possible role of cysteine proteases in the mechanism of action of
levetiracetam and the traditional antiepileptic carbamazepine (CBZ) in
kindled rats and the role of these enzymes in seizure. Materials and
Methods: The effect of increasing doses of CBZ (50, 100, and 200 mg
kg-1, p.o), LEV (13, 27, and 54 mg kg-1, p.o) and the calpain inhibitor
calpastatin (CS) (1.59, 3.18 and 6.36 mg kg-1, i.p) on calpain, caspase
3 and cathepsin B were studied in normal and in kindled rats. Results:
Seizure induced by pentylenetetrazole increased the level of calpain,
caspase 3 and cathepsin B (8.44+/-1.4 vs. 40.42+/-0.47 μmol min-1
mg-1 protein, P<0.05), (87.50+/-0.36 vs. 495.91+/-3.51 µmol min-1 mg-1
protein, P<0.05) and (88.89+/-0.38 vs. 500.66+/-2.51 µmol min-1 mg-1
protein, P<0.05), respectively. Two weeks pretreatment and treatment
of fully kindled rats with CBZ and LEV produced a significant inhibition
(P<0.05) in the level of these cysteine proteases in a dose dependent
manner besides CS returned calpain activity values to the level in
saline treated group (p < 0.05). Conclusions: These results indicate
that both antiepileptic drugs carbamazepine, levetiracetam and the
calpain inhibitor calpastatin act partially through the inhibition of these
proteases and also that their antagonism by calpastatin might has an
important role in the treatment of epilepsy.
Page 264 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-294
TBC1D24 REGULATES NEURONAL DIFFERENTIATION:
CELLULAR MECHANISMS BEHIND A NOVEL GENETIC
CAUSE OF FOCAL EPILEPSY AND INTELLECTUAL
DISABILITY
Jolly L.A.1, Corbett M.A.1, Bahlo M.2, Afawi Z.3, Gardner A.E.4, Oliver K.5,
Shalata A.6, Korczyn A.D.7, Samuel S.F.5 and Gecz J.1, 4, 8
1
Genetics and Molecular Pathology, SA Pathology, Adelaide, Australia.
2
Walter and Eliza Hall Research Institute, Melbourne, Australia.
3
Department of Neurology, Tel-Aviv Sourasky Medical Center, Tel-Aviv,
Israel. 4Women’s and Children’s Health Research Institute, Adelaide,
Australia. 5Epilepsy Research Centre, University of Melbourne, Austin
Health, West Heidelberg, Australia. 6Ginatuna Association, Sakhnin
city, West Galilee, Israel. 7Sieratzki Chair of Neurology, Tel-Aviv
University, Tel-Aviv, Israel. 8Department of Paediatrics, The University
of Adelaide, Adelaide, Australia.
We have characterised a novel syndrome of focal epilepsy, dysarthria
and mild to moderate intellectual disability in a consanguineous Arab-
Israeli family and mapped the gene to a 3.2 Mb interval within 16p13.3.
We used the new technology of targeted sequence enrichment and
massively parallel sequencing of the entire linkage interval to identify
a pathogenic mutation in the uncharacterised TBC1D24 gene. This
gene encodes a TBC domain and is predicted to function as a Rab
GTPase activator. Consistent with the clinical features, we show that
murine Tbc1d24 mRNA is expressed in neuronal populations. Further
analysis revealed that the mutant TBC1D24 protein could be stably
expressed, and like the wild-type protein, was found enriched at peri-
nuclear and growth-cone structures of cultured hippocampal neurons.
We discovered novel roles for TBC1D24 in the regulation of neuronal
morphology, with over-expression promoting neurite aborisation, axonal
growth and ectopic axonal specification. That these effects were not
observed upon over-expression of the mutant protein suggests a loss-
of-function. These cell culture data not only support the pathogenesis
of the TBC1D24 mutation, but also identifies candidate processes of
neuronal differentiation disrupted in patients.
POS-THU-296
SONIC HEDGEHOG AND NOTCH SIGNALING CAN
COOPERATE TO REGULATE NEUROGENIC DIVISIONS
OF NEOCORTICAL PROGENITORS
Dave R.K., Ellis T., Toumpas M.C., Robson J.P. and Wainwright B.J.
1The Institute for Molecular Bioscience, The University of Queensland,
Brisbane, QLD, Australia.
Hedgehog (Hh) signaling is crucial for the generation and maintenance
of both embryonic and adult stem cells, thereby regulating development
and tissue homeostasis. In the developing neocortex, Sonic Hedgehog
(Shh) regulates neural progenitor cell proliferation. During neurogenesis,
radial glial cells of the ventricular zone (VZ) are the predominant
neocortical progenitors that generate neurons through both symmetric
and asymmetric divisions. Despite its importance, relatively little is
known of the molecular pathways that control the switch from symmetric
proliferative to differentiative/neurogenic divisions in neural progenitors.
Here, we report that conditional inactivation of Patched1, a negative
regulator of the Shh pathway, in Nestin positive neural progenitors of the
neocortex leads to lamination defects due to improper corticogenesis
and an increase in the number of symmetric proliferative divisions of the
radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated
a concomitant upregulation of Hes1 and Blbp, downstream targets of
Notch signaling. The Notch signaling pathway plays a pivotal role in
the maintenance of stem/progenitor cells and the regulation of glial
versus neuronal identity. To study the effect of Notch signaling on Hh-
activated neural progenitors, we inactivated both Patched1 and Rbpj,
a transcriptional mediator of Notch signaling, in Nestin positive cells of
the neocortex. Our data indicate that by mid neurogenesis (embryonic
day 14.5), attenuation of Notch signaling reverses the effect of Patched1
deletion on neurogenesis by restoring the balance between symmetric
proliferative and neurogenic divisions. Hence, our results demonstrate
that correct corticogenesis is an outcome of the interplay between the
Hh and Notch signaling pathways.
POSTERS WEDNESDAY & THURSDAY
POS-WED-297
MICRORNAS ARE REQUIRED FOR THE PROPER
CORTICAL LAYERING OF THE ANTERIOR ZONE
CEREBELLUM
Constantin L., Dave R.K. and Wainwright B.J.
Institute for Molecular Bioscience, University of Queensland, St Lucia,
Queensland, Australia, 4072.
Multiple studies have implicated microRNAs (miRNAs) as important
regulators of cerebellar function. For example, they have been shown
to modulate the pathogenesis of neurodegenerative diseases such as
spinocerebellar ataxia type 1, act as tumour suppressors in malignant
tumours of the cerebellum (medulloblastoma) and their loss in Purkinje
cells has been shown to lead to neuronal degeneration. Canonical miRNA
processing is regulated by Dicer, which cleaves precursor miRNAs into
mature double-stranded RNA duplexes. In order to address the role of
miRNAs in cerebellar granule cell precursor (GCP) development, we
ablated mature miRNA production in GCPs by conditionally inactivating
Dicer1 via the GCP-specific Math1-Cre-recombinase transgene. Here,
we show that loss of Dicer1 function in GCPs resulted in a cortical
layering defect in the anterior zone of the cerebellum, due to an increase
in GCP differentiation rates during early and mid postnatal cerebellum
development. The increase in GCP differentiation rates led to a reduction
in mature granule neurons in the anterior zone only, and mice presented
with a high-frequency tremor. These findings have identified a novel role
for miRNAs in the compartmentalization of the mammalian cerebellum.
POS-WED-299
CONSTRUCTION OF DENGUE VIRUS-2 DOMAIN III
ENVELOPE PROTEIN
Ahmad Rashidi H.S., Yusof R. and Abdul Rahman N.
Drug Design and Development Research Group, University of Malaya,
50603, Kuala Lumpur, Malaysia.
The dengue virus, a flavivirus belonging to the Flaviridae family is a
single stranded RNA positive-strand virus that causes dengue fever
(DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome
(DSS). It is currently one of the leading public health threats throughout
tropical and subtropical regions and the existence of four closely
related, but antigenically distinct serotypes (DEN-1, 2, 3, 4) has been
the major factor that has hampered vaccine development efforts. The
envelope (E) protein domain III of dengue virus has been proposed to
be the possible binding sites on the post-fusion E trimer to host cell
and acts as an attractive target for prevention of infection or inhibition
of virus spread in an ongoing infection. Thus, we aim to construct a
clone of domain III envelope protein of dengue virus serotype-2
(DEN2EIII) to study the interaction of this domain to host cell surfaces.
The construction of recombinant DEN2EIII was prepared through
directional cloning into pQE9 and plasmid harboring DEN2EIII-pQE9
was transformed into Escherichia coli [strain XL-1]. Bacterial cells grown
in LB medium were induced with isopropyl-β-D-thiogalactopyranose
(IPTG) to allow high level expression of N-terminal hexahistidine-tagged
recombinant proteins and these proteins were then subjected to an
affinity purification using Ni charged resin column. The integrity of the
EIII protein was confirmed by performing Western blot analysis using
a monoclonal antibody against the DEN2 envelope protein and the
functional expressed EIII protein will be further employed in studying the
involvement of domain III in binding to the host cell.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 265
POS-THU-298
CONSTITUTIVE REDUCTION IN NEURITE GROWTH
BY TRPM2 UNDERLIES THE ETIOLOGY OF BIPOLAR
DISORDER
Oh U.1, 2 and Jang Y.1
1
Seoul National University, College of Pharmacy. 2Seoul National
University, Department of Molecular Medicine & Biopharmaceutical
Sciences.
Genetic linkage studies strongly suggest that TRPM2, a Ca2+-
permeable cation channel is associated with a mental disorder, but
the nature of this linkage is not known. Here we show that TRPM2
negatively regulates neurite growth. Knock-down of TRPM2 or
treatment with its pharmacological inhibitors markedly increased
neurite growth, whereas its up-regulation had a suppressive effect.
Furthermore, lysophosphatidic acid, a well established suppressor
of neurite growth, was found to act by enhancing TRPM2 activity.
More importantly, lithium, which is currently used to treat the mental
disorder, blocked the lysophosphatidic acid-induced activation of
TRPM2. One putative single nucleotide polymorphism of TRPM2 in
the mental disorder patients showed an augmented TRPM2 activity,
and led to the constitutive suppression of neurite growth. In addition,
amphetamine-induced hyperactivity, an animal model for the mental
disorder, was markedly reduced by chronic TRPM2 inhibitor treatment
in mice. Furthermore, the reduction by lithium of amphetamine-induced
hyperactivity was completely eliminated in TRPM2 deficient mice.
These results strongly suggest that prolonged suppression of neurite
growth by hyperactive TRPM2 mutant accounts for the pathology of the
mental disease. Furthermore, these results provide the link between the
mental disease and TRPM2 and delineate the mechanism underlying
the therapeutic effect of lithium.
POS-THU-300
SILENCING SUPPRESSION IN THE VIRAL FAMILY
LUTEOVIRIDAE
Fusaro A.F.1, Correa R.L.3, Kawchuk L.4, Jackson C.S.1, McLean H.1,
Vaslin M.F.S.3 and Waterhouse P.M.1, 2
1
University of Sydney, Australia. 2CSIRO- Plant Industry, Australia.
3
Federal University of Rio de Janeiro, Brazil. 4Agriculture and Agri-
Food Canada, Canada.
Gene silencing is a conserved mechanism for controlling gene expression
and in plants is associated with viral defense. Sequence specificity is
provided by small RNAs (sRNAs) made by the RNase III Dicer enzyme
that bind to effector proteins called Argonautes (AGO). The proliferation
and diversification of silencing-related genes during evolution allowed
the specialization of small RNA-directed pathways in controlling
different biological fates, like development, chromatin structure and
defense against viruses and repetitive sequences. In the model plant
Arabidopsis thaliana there are 4 Dicer-like (DCL1 to DCL4) and 10 AGO
(AGO1 to AGO10) genes, giving rise to at least five silencing pathways.
Exogenous invading sequences, like viruses, can be degraded by
DCL4, DCL2 and DCL3, giving rise to 21-, 22- and 24-nucleotide short
interfering RNAs (siRNAs), respectively. Virus-derived siRNAs can
both drive viral RNA degradation through AGO1-containing complexes
or recruit RNA-dependent RNA polymerase proteins for de novo
dsRNA synthesis. Viruses, as a counter defense, developed diverse
and unrelated strategies to block the plant RNA degradation machinery.
Binding to long or short dsRNAs is a common strategy for silencing
suppression and it seems to have evolved independently many times.
A different and curious mode of silencing suppression is observed in
some members of the viral family Luteoviridae. The family is composed
by positive single-stranded RNA viruses that are divided in three
different genera: Luteovirus, Polerovirus and Enamovirus. The P0 gene
from viruses belonging to the Polerovirus group encodes an F box-like
protein that is able to suppress silencing by targeting Argonaute proteins
for degradation. Luteovirus and Polerovirus are similar in their biology,
but surprisingly, the Luteovirus completely lack a P0 gene. Here we
explored the existence of different modes of silencing suppression in
the family Luteoviridae.
POSTERS WEDNESDAY & THURSDAY
POS-WED-301
A SENSITIVE ASSAY TO QUANTITATE O-GLYCANS IN
CRUDE SAMPLES
Bhavanandan V.P., Abdul-Rahman P.S. and Hashim O.H.
Department of Molecular Medicine and University of Malaya Centre
for Proteomics Resaerch, Faculty of Medicine, University of Malaya,
50603 Kuala Lumpur, Malaysia.
Mucins are large glycoproteins containing many clustered glycosylated
serines and threonines in tandem repeat regions. Mucins and mucin-
type glycoproteins, collectively referred to as O-glycans, are implicated
in many important biological functions and pathological conditions,
specifically malignancy. It is well established that in cancer patients a
repertoire of mucins synthesized by the tumor cells enters the circulation
by active secretion and by shedding during cell turnover. Currently there
are no reliable and sensitive methods to measure the total O-glycan
content particularly of crude samples such as tissue extracts or body
fluids. One of the reasons for this is the heterogeneous nature of the
saccharides in O-glycans. However, in contrast to the high variability of
the peripheral saccharides in O-glycans the sugar-amino acid linkage is
a unique and constant feature. Previously, we had developed an assay
based on the specific chemistry of the GalNAc-Ser/Thr linkages for
the estimation of O-glycans (Bhavanandan et al, Anal. Biochem.188,
142-148). However, the sensitivity of the colorimetric assay is limited.
Therefore, to measure trace amounts of O-glycans in samples we
have developed an alternate assay. The first step of this assay involves
the removal of the peripheral sugars of O-glycans by chemical and or
enzymatic techniques. In the second step the exposed linkage GalNAc
residues are estimated by competitive enzyme-linked lectin assay to
provide a measure of the total O-glycan content of the sample. The
assay was evaluated using porcine gastric mucin, bovine submaxillary
mucin, fetuin and orosomucoid as well as normal human serum spiked
with these glycoconjugates. [Supported by grant RG210/10HTM from
the University of Malaya].
POS-WED-303
DECURSIN INHIBITS HISTONE DEACETYLASE
ACTIVITY BY ACTIVATING SPHINGOSINE KINASE
TYPE II
Shin, K.O., Kim, S.M., Seo, C.H., Lee, E.S. and Lee, Y.M.
College of Pharmacy, CBITRC, Chungbuk National University,
Chongju, Korea.
Inhibition of angiogenesis is an attractive approach for the treatment of
angiogenic diseases, such as cancer. Sphingosine-1-phosphate(S1P) is
one of the most important activators of angiogenesis. The pyranocoumarin
compound decursin isolated from the herb, Angelica gigas, are known
to possess potent anti-inflammatory activities. However, little is known
about their antiangiogenic activity Here, we show the antiangiogenic
effects of decursin using in vitro assays. Decursin inhibited sphk -induced
angiogenic processes in vitro, including proliferation, migration of
human umbilical vein endothelial cells(HUVEC). The sphk type1 activity
in HUVEC treated with decursin for 24hr was significantly decreased
(43%) compared to control group. However, sphk type 2 activity was
mildly increased (1.6 fold) in HUVECs. According to the increase of a
sphk2, the histone deacetylase is inhibited by the accumulated S1P. Our
data suggests that decursin might be a potent anti-angiogenic agent
regulating sphk type1 and sphk type2 signaling pathway. “This work was
supported by the grant of the Korean Ministry of education science and
technology” ( The Regional Core Research Program / Chungbuk BIT
Research-Oriented University consortium).
Page 266 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-302
INHIBITORY EFFECTS OF PES3520 ON PDGF-BB-
INDUCED PROLIFERATION OF VSMCS THROUGH
BLOCKING PDGF-RB PHOSPHORYLATION
Park, E.S.1, Lim, Y.2, Tudev, M.1, Yoo, H.S.1 and Yun, Y.P.1
1
College of Pharmacy, CBITRC, Research Center of Bioresource and
Health,. 2Research Institute of Veterinary Medicine, Chungbuk National
University, Korea.
The abnormal proliferation of vascular smooth muscle cells (VSMCs) in
arterial wall is an important pathogenic factor for vascular disorders such
as atherosclerosis and restenosis after angioplasty. The present study
was designed to investigate the inhibitory effects of PES3520 on FBS-
and PDGF-BB-induced VSMC proliferation as well as their molecular
mechanisms. PES3520 significantly inhibited the proliferation of FBS-
and PDGF-BB-stimulated VSMCs and suppressed the DNA synthesis.
In accordance with these findings, PES3520 blocked of the FBS- and
PDGF-BB-induced progression through G0/G1 phase of the cell cycle
in synchronized cells. PES3520 also decreased the expressions of
cyclin-dependent kinase (CDK)2, cyclinE, CDK4 and cyclinD1 as well
as retinoblastoma (Rb) protein and proliferative cell nuclear antigen
(PCNA) from PDGF-BB-stimulated VSMCs. Preincubation of VSMCs
with PES3520 significantly inhibited the PDGF-BB-induced extracellular
signal-regulated kinase 1/2 (ERK1/2), Akt as well as phospholipase C
(PLC)-γ1 activation. Consisted with these findings, PES3520 at the
dosage of 10, 20 and 40 μM suppressed the PDGF-Rβ phosphorylation
induced by PDGF-BB in a concentration-dependent manner. These
results suggest that the inhibitory effect of PES3520 on the FBS-
and PDGF-BB-induced proliferation of VSMCs may be mediated by
blocking PDGF-Rβ phosphorylation. Thus, PES3520 may be a potential
antiproliferative agent for the therapy of atherosclerosis and angioplasty
restenosis.
POS-THU-304
ROLE OF ANTI-APOPTOTIC BCL-2 FAMILY MEMBER
MCL-1 IN LYMPHOMA DEVELOPMENT
Grabow S.1, 2, 3, Kelly P.N.1, Bouillet P.1 and Strasser A.1
1
The Walter and Eliza Hall Institute of Medical Research, Australia.
2
University of Melbourne, Department of Medical Biology, Australia.
3
Cancer Therapeutics CRC, Australia.
The transcription factor Myc is mainly involved in the control of cell
growth, proliferation, differentiation and, under conditions of stress
(e.g. cytokine deprivation) also apoptosis. Myc function is abnormally
increased in ~70% of human cancers. Over-expression of c-myc
under control of the IgH enhancer Eµ causes abnormal accumulation
of pro-B and pre-B cells during the pre-leukaemic state and ultimately
results in monoclonal pre-B or B lymphoma in all animals. Myc-induced
lymphomagenesis is impeded by apoptotic death of pre-leukaemic
pre-B/B cells and, accordingly, over-expression of anti-apoptotic Bcl-2
greatly accelerates tumorigenesis in this model. We are interested in
determining which pro-survival Bcl-2 family members are essential for
Eμ-myc induced lymphoma development. Previous studies using gene-
targeted mice have shown that, surprisingly, endogenous Bcl-xL, but not
Bcl-2, is required for Eµ-myc-induced B lymphomagenesis. It remains,
however, unclear whether Bcl-xL is the sole pro-survival Bcl-2 family
member required for tumorigenesis in this experimental model. Mcl-1,
another pro-survival member of the Bcl-2 family, is highly expressed
during the early stages of B lymphopoiesis (HSC, CLP, pro-B), and we
therefore decided to examine its role in pre-B/B lymphoma development.
Mice that are deficient in Mcl-1 die around embryonic day 4 prior to
implantation. We therefore generated gene-targeted mice in which the
mcl-1 gene has been flanked with loxP sites to facilitate its deletion by
Cre recombinase in a lineage-specific manner. We introduced onto the
mcl-1loxP background a CD19-Cre transgene, in which allows specific
deletion of loxP flanked genes in B lymphoid cells, and the Eμ-myc
transgene to promote lymphoma development. Analysis of Eμ-myc/
CD19-Cre/mcl-1loxP/loxP mice will provide valuable insight into the
role of Mcl-1 in the survival of pre-leukaemic B cell progenitors and
lymphoma development.
POSTERS WEDNESDAY & THURSDAY
POS-WED-305
NRF2 DYSFUNCTION IN PATIENT-DERIVED CELL
LINES IN A NEW MODEL OF SPORADIC PARKINSONS
DISEASE
Cook A.L.1, 2 , Vitale A.M.2, Ravishankar S.2, Matigian N.2, Mellick G.D.2,
Wells C.A.2, Mackay-Sim A.2 and Wood S.A.2
1
School of Human Life Sciences, University of Tasmania, Launceston,
Tasmania, Australia. 2National Centre for Adult Stem Cell Research,
Griffith University, Brisbane, Queensland, Australia.
We recently reported a novel patient-derived cellular model of Parkinsons
disease generated from biopsies of the olfactory mucosa, the organ of
smell in the nose. Olfactory neurosphere-derived (hONS) cells from
Parkinsons disease patients had reduced metabolic functions already
associated with disease, including reduced levels of the important
antioxidant molecule glutathione. Transcriptomic analysis of patient
and Control hONS cells identified the signalling pathway surrounding
NRF2, the major transcriptional regulator of the antioxidant response,
as being highly differentially expressed in Parkinsons disease. We have
shown that activation of the NRF2 pathway in Parkinsons disease hONS
cultures restored metabolic function to Control levels, thereby validating
NRF2 as a genuine therapeutic target. Paradoxically, transcriptomic
analysis of hONS cells after NRF2 pathway activation revealed increased
dysregulation of the NRF2 pathway in treated Patient-derived cells. We
are now investigating the functional consequences of dysregulated
NRF2 activation in Patient-derived hONS cells. Specifically, we
are pursuing the recently described link between NRF2 and p62, an
important regulator of autophagy, a cellular process implicated in many
neurodegenerative diseases. Our results demonstrate that Patient cells
can respond to NRF2 activation but that this may elicit a different cellular
outcome than that of Control cells. This may be an important factor when
developing new therapeutics.
POS-WED-307
NEURODEVELOPMENTAL PATHWAYS ALTERED
IN A NOVEL, PATIENT-DERIVED CELL MODEL OF
SCHIZOPHRENIA
Fan Y.1, Abrahamsen G.1, Mills R.2, Cooper-White J.2, McGrath J.J.1, 2
and Mackay-Sim A.1
1
National Centre for Adult Stem Cell Research, Griffith University,
Brisbane, Qld, Australia. 2The University of Queensland, Brisbane,
Qld, Australia.
Schizophrenia is considered a disease of brain development, based
on post-mortem brain and epidemiological analyses. Genetic and
environmental risk factors are tested in animal models but their clinical
relevance is not clear. Our novel model uses patient-derived cells from
the olfactory mucosa, the organ of smell, which regenerates throughout
life from a neural stem cell. Olfactory neurosphere-derived (hONS)
cells were generated from biopsies of olfactory mucosa from healthy
controls and patients with schizophrenia. Gene expression analysis
identified 1700 genes whose expression was dysregulated in 9 hONS
cell lines from schizophrenia patients compared to 9 control cell lines.
Among the down-regulated genes were RGS4, RLN, ERBB3 and
GSK3b, which are all down-regulated in post-mortem brain in patients.
Pathway analysis identified core neurodevelopmental functions that were
dysregulated in schizophrenia including cell cycle and focal adhesion.
Subsequent functional studies demonstrated a faster rate of proliferation
in schizophrenia hONS cells due to altered cell cycle regulation. After
cell cycle synchronisation the schizophrenia cells had earlier and larger
increases in cyclin D, cyclin A, and phospho-ERK expression preceding
S-phase and a larger proportion of cells entering S-phase in the first
30 hr after synchronisation. Focal adhesion kinase protein levels were
significantly lower in the schizophrenia hONS cells and numerous genes in
this signalling pathway had altered mRNA expression. This was associated
with significantly lower adhesion of the schizophrenia cells to fibronectin
and collagen substrates. In addition, schizophrenia hONS cells migrated
twice as quickly as control hONS cells. This difference was eliminated by
inhibition of focal adhesion kinase. This novel approach identifies for the
first time significant alterations in neurodevelopmental signalling pathways
in patient-derived cells in schizophrenia.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 267
POS-THU-306
HOMOZYGOUS FRAME SHIFT MUTATION IN ECM1
GENE IN TWO SIBLINGS WITH LIPOID PROTEINOSIS
Azhar A.1, Samdani A.J.2, Shahid S.M.1, Nawab S.N.1, Sheikh R.1,
Qader S.A.1 and Ismail M.3
1
The Karachi Institute of Biotechnology & Genetic Engineering
(KIBGE), University of Karachi, Karachi. 2Department of Dermatology,
Jinnah Postgraduate Medical Center, Karachi. 3Institute of Biomedical
& Genetic Engineering (IBGE), Islamabad, Pakistan.
The extracellular matrix protein 1 (ECM1) is a glycoprotein, expressed
in skin and other tissues. Loss-of-function mutation in ECM1 causes
a rare, autosomal recessive disorder, Lipoid Proteinosis (LP). LP is
presented by varying degrees of skin scars, beaded papules along the
eyelid margins, variable signs of hoarseness and respiratory disorders.
Over 250 cases of this disorder have been described in the literature,
but occurrence of LP in siblings is very rare. In this study, two siblings of
a Pakistani family are reported. Two (12 and 9-years-old) sisters were
presented with scaly itchy lesions on the whole body, hoarse voice and
macroglossia. Their deceased father had similar clinical manifestations
but mother and younger brother were unaffected. Blood samples from
affected and clinically unaffected family members were collected with
informed consent. The coding region of ECM1 gene containing 10 exons
were amplified and sequenced. Both the affected siblings were shown
to have homozygous frame shift mutation by deletion of the nucleotide
T at 507, codon 169, exon 6. This resulted in a frame shift from codon
169 and the appearance of stop codon at 177. The normal ECM1 protein
contains 540 amino acids. The frame shift mutation has resulted in
appearance of a mutated protein containing 176 amino acids. A case
of homozygous 62-bp insertion in ECM1 causing LP has been reported
in a Pakistani family. The current study presents a novel homozygous
frame shift mutation supporting an unusual function of ECM1 protein
and broadens the spectrum of disease-linked mutations in this rare
case of genodermatosis in this region.
POS-THU-308
APOPTOTIC GENES EXPRESSION IN MICE
HEPATOCYTES DURING MALARIA INFECTION
Alkahtani S.
King Saud University.
Apoptotic genes expression in mice hepatocytes during malaria
infection Saad Alkahtani Department of Biology, Teachers College, King
Saud University, Riyadh, Saudi Arabia. Abstract: Malaria represents a
major global health problem by causing multi-cellular dysfunctions in
the infected host. The aim of the present study was to investigate the
Apoptotic gene expression in liver of mice during malaria disease by
mRNA expression of three genes involved in apoptosis; Bax, Bcl-2 and
Caspase-3 and other parameters at different time points after infection
with Plasmodium chabaudi in the liver cells of female C57BL/6 mice.
Mice were injected intraperitoneally (ip) with 106 P. chabaudi-infected
erythrocytes and then scarified at days (0, 1, 4 and 8 respectively).
Quantitative real time PCR and immunoblotting were used to quantify
apoptotic genes and protein kinetic, respectively. The levels of Bax,
Bcl-2 and Caspase-3 were significantly (P<0.05) increased only at
days 1 and 8 compared with day 0 in both the liver cells and protein.
The levels of parasitemia was significantly (P<0.01) increased at day
8 compared with day 0 in the blood. These results have shown that P.
chabaudi induced apoptosis in the liver cells. Thus, this study suggest
that the induced apoptotic gene expression was due to the out come
of malaria.
POSTERS WEDNESDAY & THURSDAY
POS-WED-309
LILRA2 SELECTIVELY REGULATES LPS-MEDIATED
CYTOKINE PRODUCTION AND PHAGOCYTOSIS IN
PERIPHERAL BLOOD MONOCYTES
Lu H.K.1, Endoh Y.1, Huynh O.1, Mitchell A.1, Hampartzoumian T.1,
Geczy C.1, Bryant K.1, Arm J.P.2, Borges L.3 and Tedla N.1
1
Inflammation and Infection Research Center, School of Medical
Sciences, University of New South Wales, NSW, Australia. 2Harvard
Medical School, Boston, MA. 3Amgen, Seattle, WA, USA.
The activating immunoglobulin-like receptor A2 (LILRA2) is expressed
on the surface of cells that play key role in innate immune responses such
as monocytes, macrophages and neutrophils. LILRA2 cross-linking on
these cells induces pro-inflammatory cytokine production. However,
whether the functions of this molecule differ to the effects of the primary
receptors involved in the innate immunity including responses to LPS
remain unknown. In this study, we show optimal activation of monocyte
by LILRA2 cross-linking failed to induce IL-12 and MCP-1 production that
were strongly up regulated by LPS. Interestingly, LILRA2 cross-linking
on monocytes induced a similar level of IL-6, IL-8, G-CSF and MIP-1α
but lower levels of TNFα, IL-1β, IL-10 and IFNγ to those stimulated with
LPS. In addition, cross-linking of LILRA2 on monocytes significantly
decreased phagocytosis of IgG-coated micro-beads, while had no
effect on phagocytosis of Escherichia coli. Unexpectedly, simultaneous
co-stimulation of monocytes through LILRA2 and LPS, or pre-activation
of monocytes via LILRA2 followed by LPS treatment led to significantly
lower levels of TNFα, IL-1β and IL-12 production as compared to LPS
alone. However, there was no effect on the levels of IL-6, IL-10 and IFNγ
production. Furthermore, LILRA2 cross-linking on monocytes caused
significant down-regulation of TLR4 and CD14 mRNA and protein,
indicating LILRA2-mediated suppression of LPS responses might be in
part through regulation of these receptors. Taken together, we provide
evidence that LILRA2 strongly and selectively regulates LPS-mediated
monocyte activation and IgG-dependent phagocytosis.
POS-WED-311
DIFFERENTIAL METHYLATION OF HNRNP A2/B1
PARALOGS DETECTED IN NEURONAL CELLS AND
CELL LINES BY MASS SPECTROMETRY
Friend L.R.1, Landsberg M.J.2, Nouwens A.S.1, Rothnagel J.A.1 and
Smith R.1
1
School of Chemistry and Molecular Biosciences, The University of
Queensland. 2Institute for Molecular Biosciences, The University of
Queensland.
The heterogeneous nuclear ribonucleoprotein paralogs, A1, A2/B1
and A3 (hnRNPs A/B) share a high degree of sequence similarity and
may have closely related roles in processes such as RNA packaging,
alternative splicing, telomere maintenance and cytoplasmic trafficking.
However, in a previous study, the hnRNPs A/B were shown to exhibit
different patterns of sub-nuclear localization, suggesting that these
paralogs have diverged to perform functionally distinct roles. Additional
functional diversity is created by post-translational modifications, coupled
with the generation of alternatively spliced isoforms. In the work reported
here, we have isolated hnRNP A2 using pulldowns on immobilized
RNA-trafficking elements or telomeric ssDNA repeat oligonucleotides,
followed by reverse-phase HPLC. We investigated hnRNP A2 post-
translational modifications in native rat brain, rat B104, human HeLa
and human SH-SY5Y transformed cells using mass spectrometry
and Edman degradation. Our results show that hnRNP A2 is NG,NG -
dimethylated on a single arginine residue, Arg254, encoded within the
alternatively spliced exon 9. This is in contrast to hnRNP A1, which has
four of its five RGG motifs NG,NG -dimethylated on the arginine residues.
In native rat brain, Arg254 is almost completely dimethylated, while in
rat and human cell lines, a greater proportion of this arginine residue is
either unmethylated or monomethylated. This conserved modification of
Arg254 in hnRNP A2, suggests that hnRNP A2 is functionally regulated
by dimethylation in a manner that distinguishes it from hnRNP A1.
Page 268 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-310
ATRX IS A SERTOLI CELL SURVIVAL FACTOR
AND REGULATOR OF SPERMATOGENESIS VIA
INTERACTION WITH ANDROGEN RECEPTOR
Bagheri-Fam S.1, Argentaro A.1, Svingen T.2, Combes A.2, Koopman P.2
and Harley V.R.1
1
Prince Henry’s Institute of Medical Research, Melbourne, VIC
Australia. 2Institute of Molecular Bioscience, UQ, Brisbane, QLD,
Australia.
The ATR-X syndrome, (α-thalassemia mental retardation, X-linked) is a
developmental disorder affecting males. ATR-X syndrome individuals
display a range of genital abnormalities, including small testes.
ATRX shows nucleosome remodeling activity in vitro, suggesting an
involvement in gene regulation but no target genes have been defined.
We inactivated Atrx specifically in testicular Sertoli cells (ScAtrxKO
mice) from embryonic day 14.5. Fluorescence-based three-dimensional
modeling at E17.5 revealed that testis cord volume in ScAtrxKO mice is
~30% of wildtype, with discontinuous or isolated testis cords apparent.
Sertoli cell apoptosis is increased 10-fold with no difference in Sertoli
cell proliferation. One third of ScAtrxKO tubules contain germ cells
arrested in late meiosis or at the round spermatid stage. Sertoli cells,
upon androgen stimulation of the androgen receptor (AR) transcribe
genes necessary for the completion of germ cell meiosis and spermatid
development, such as Rhox5. While gene expression of AR itself was
unchanged in ScAtrxKO testes, expression of three AR-dependent
genes including Rhox5 was significantly down-regulated. Moreover
ATRX and AR proteins interact in vivo and in the TM4 Sertoli cell line to
co-operatively activate the Rhox5 promoter. In summary, ATRX plays an
important role in Sertoli cell survival during fetal testis development and
in adult testis function, where ATRX protein interacts with AR to regulate
the transcription of AR-dependent genes that control spermatogenesis,
such as Rhox5, the first ATRX target gene identified.
POS-THU-312
STRUCTURAL/FUNCTIONAL PROPERTIES OF
ISOLATED HUR-HNRNP COMPLEXES OF MAMMALIAN
CELL ORIGIN
Papadopoulou C., Ganou V., Patrinou-Georgoula M. and Guialis A.
Institute of Biological Research and Biotechnology, National Hellenic
Research Foundation, Athens, GREECE.
Post-transcriptional processes that include splicing, polyadenylation,
transport, stability and translation of mRNA are important events in
regulating gene expression in higher eukaryotes. These are brought
about by the extensive network of interactions among a large number
of RNA-binding proteins (RBPs) operating within diverse multifactorial
RNP complexes. We have recently focused on RBP proteins; hnRNPs
having a major role in RNA splicing, and HuR an AU-rich element-
binding protein (ARE-BP) with a so far established role in stability/
translation of target mRNAs. Both hnRNPs and HuR have a major
nucleoplasmic localization and ability for nuclear/ cytoplasmic shuttling.
Recently we reported on an extensive range of hnRNP-HuR interactions
within immunopurified hnRNP and mRNP complexes (Papadopoulou et
al., Biochem Biophys Acta, 2010,1804:692-703). We have now extended
our study in directly immunoselected HuR complexes from HeLa
nuclear extracts. With respect to associated protein species, these
HuR complexes were strikingly similar with immunopurified hnRNPs
via the anti-hnRNP C1/C2 (4F4) antibody. The authenticity of the HuR-
associated proteins was tested using a mouse embryonic cell line
(MEF) completely devoid of HuR. Presently, we combine biochemical
analysis with confocal microscopy on intact as well as on nuclear matrix
preparation of HeLa cells to look at cellular localization of HuR and of
selected hnRNP proteins under several growth conditions. In particular,
results concerning the functional implications of the identified HuR-
hnRNP interactions under stress conditions, as well as under inhibition
of protein phoshorylation, will be presented.
POSTERS WEDNESDAY & THURSDAY
POS-WED-313
CELL POLARITY COMPLEXES REGULATE GROWTH
VIA THE SALVADOR/WARTS/HIPPO (SWH) PATHWAY
Parsons L.M., Grzeschik N.A., Allott M.L., Harvey K.F. and
Richardson H.E.
Peter MacCallum Cancer Centre, 2 St Andrew’s Place East Melbourne,
Victoria 8006, Australia.
Inactivating mutations in Drosophila tumour-suppressor genes result
in tissue overgrowth. It has long been recognized that loss-of-function
mutations in the junctional-scaffold neoplastic tumour suppressor gene,
lethal-2-giant-larvae (lgl) result in cellular overproliferation, ultimately
resulting in the development of lgl- tumours. We have shown that in
the developing Drosophila eye, loss of lgl activity results in ectopic
proliferation and suppression of developmental cell death (apoptosis),
without loss of cell polarity. In this system, targets of the SWH
pathway are increased upon lgl inactivation. This occurs by decreased
phosphorylation and re-localization of the SWH pathway transcriptional
activator, Yorkie (Yki). A critical role for the SWH pathway and Yki activity
in the development of lgl- tumours is supported by genetic evidence that
shows Yki is required for the development of lgl- tumour phenotypes.
The SWH pathway is proposed to modulate Yki activity by two distinct
mechanisms: 1) Fat/Dachs proteins which regulate the level of Warts
protein (Warts kinase is responsible for phosphorylating Yki and inhibiting
its activity) and 2) Ex protein which binds Yki, sequestering it within
the cell cortex thus preventing it from entering the nucleus. We have
genetic and biochemical evidence that lgl activity does not regulate Yki
activity by regulating Warts protein levels. Our immuno-histochemistry
data shows that a second kinase (Hippo, Hpo) that also regulates Yki
activity is mis-localised in lgl mutant tissue. Thus, we propose a novel
mechanism whereby the apical-basal polarity regulator Lgl regulates the
SWH pathway by correctly localizing components of the SWH pathway.
These data provide the first mechanistic links between the neoplastic
and hyperplastic tumour suppressor pathways in Drosophila.
POS-WED-315
BETA-SITOSTEROL REGULATES ADIPOCYTE GENES
INVOLVED IN GLUCOSE UPTAKE, ADIPOGENESIS AND
LIPID MOBILIZATION
Kanthimathi M.S., Chai J.W. and Kuppusamy U.R.
Department of Molecular Medicine, Faculty of Medicine, University of
Malaya, 50603 Kuala Lumpur, Malaysia.
The nutraceutical benefits of β-sitosterol (SIT) are well documented. The
present study investigated the in vitro effects of SIT on adipogenesis,
glucose transport and lipid mobilization in rat adipocytes. Primary
cultures of rat preadipocytes and differentiated adipocytes were used in
this study. Glucose uptake was measured by the uptake of radio-labeled
glucose. Adipogenesis and lipolysis were measured by oil-red-O and
glycerol quantification methods, respectively. The expression of protein
kinase B (Akt), glucose transporter 4 (GLUT4), hormone sensitive lipase
(HSL) and phosphatidylinositol-3-kinase (PI3K) genes in SIT-treated
adipocytes were assessed by real-time reverse transcription polymerase
chain reaction (RT-PCR). The data showed that SIT induced glucose
uptake in adipocytes. It also stimulated adipogenesis in differentiating
preadipocytes. Interestingly, although SIT displayed general insulin-
mimetic activity by stimulating glucose uptake and adipogenesis, it also
induced lipolysis in adipocytes. Furthermore, the SIT-induced lipolysis
was not attenuated by insulin and co-incubation of SIT with epinephrine
improved epinephrine-induced lipolysis. GLUT4 gene expression
was highly down-regulated in SIT-treated adipocytes, compared to
insulin-treated adipocytes, which was up-regulated. Insulin- and SIT-
treated adipocytes showed similar levels of Akt, HSL and PI3K gene
down-regulation. These observations suggest that the elevation of
glucose uptake in SIT-treated adipocytes was unrelated to de novo
synthesis of GLUT4 and that the SIT-induced lipolysis is associated
with the down-regulation of Akt and PI3K genes. The unique effects
of SIT on the regulation of glucose uptake, adipogenesis and lipolysis
in adipocytes show that it has potential to be utilized in diabetes and
weight management.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 269
POS-THU-314
QUINIC ACID METABOLISM IN KIWIFRUIT
Marsh K.B.1, Boldingh H.2 and Laing W.1
1
Plant and Food Research, Private Bag 92-169, Auckland, 1142, NZ.
2
Plant and Food Research Ruakura, Private Bag 3123, Hamilton 3249,
NZ.
Kiwifruit are unique in containing high concentrations of quinic acid (1-
2% w/w), which contribute to the flavour, and health-giving properties
of the fruit. In a study of quinic acid storage and metabolism in three
kiwifruit species (Actinidia chinensis, A. deliciosa and A. arguta),
quinic acid accumulation was shown to occur principally in the early
stages (<60 days after anthesis; (DAA)) of fruit development. We
studied the genes responsible for quinic acid metabolism, including
DAHP (Dehydroquinate synthase) and two iso-enzymes of a DHQ (bi-
functional dehydroquinate dehydratase/shikimate dehydrogenase) by
quantitative reverse transcriptase PCR and by over-expression of ESTs
in recombinant systems. RT-qPCR results for DHQ synthase showed
that there was a high level of expression in the early season, which was
sustained through the mid-season. A kiwifruit EST that had homology to
chloroplastic isoforms of SDH showed an induction in the middle to late
season but could not explain the high shikimate dehydrogenase activity
in the early season. The activity in the early season (<30 DAA) may
have been due to the expression of a cytosolic isoform of the enzyme.
This suggests that dehydroquinate is a key substrate, particularly for a
bifunctional dehydroquinate dehydratase /shikimate dehydrogenase at
the branch point in the shikimate and quinate pathways. We have been
studying the genes that control the synthesis of Dehydroquinate using a
recombinant DAHP synthase, and a DHQ synthase, and also attempting
to recombinantly express cytosolic and chloroplastic versions of the
bifunctional dehydroquinate dehydratase/shikimate dehydrogenase.
POS-THU-316
A CRITICAL ROLE FOR THE PROTEIN
PHOSPHATASE 2A B’α REGULATORY SUBUNIT IN
DEPHOSPHORYLATION OF SPHINGOSINE KINASE 1
Pitman M.R.1,3, Barr R.K.1,3, Magarey A.M.1, Moretti P.A.B.1 and
Pitson S.M.1, 2
1
Molecular Signalling Laboratory, Centre for Cancer Biology, SA
Pathology, Frome Road, Adelaide, South Australia 5000, Australia.
2
School of Molecular and Biomedical Science, University of Adelaide,
Adelaide, South Australia 5005, Australia. 3These authors contributed
equally to this work.
Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling
that has gained recent attention as a potential target for anti-cancer
therapies. SK1 activity, subcellular localisation and oncogenic function
are regulated by phosphorylation and dephosphorylation at Ser225.
ERK1/2 have been identified as the protein kinases responsible for
phosphorylation and activation of SK1. Conversely, dephosphorylation
and deactivation of SK1 occurs by protein phosphatase 2A (PP2A).
Active PP2A, however, is a heterotrimer, composed of tightly
associated catalytic and structural subunits that can interact with
an array of regulatory subunits which are critical for determining
holoenzyme substrate specificity and subcellular localization. Thus,
PP2A represents a large family of holoenzyme complexes with different
activities and diverse substrate specificities. To date the regulatory
subunit essential for targeting PP2A to SK1 has remained undefined.
Here, we demonstrate a critical role for the B’α (B56α/PR61α/PPP2R5A)
regulatory subunit of PP2A in SK1 dephosphorylation. B’α was found to
interact with the C-terminus of SK1, and reduce SK1 phosphorylation
when overexpressed, while having no effect on upstream ERK1/2
activation. Furthermore, siRNA-mediated knockdown of B’α increased
SK1 phosphorylation, activity and membrane localisation of endogenous
SK1. Thus, the PP2A-B’α holoenzyme appears to function as an
endogenous regulator of SK1.
POSTERS WEDNESDAY & THURSDAY
POS-WED-317
SUMOYLATION OF IGF-1R IS MEDIATED THROUGH
SUMO-E3 LIGASE RANBP2
Sjostrom S.1, Warsito D.1, Sehat B.1, 2 and Larsson O.1
1
Karolinska Institute, Department of Oncology-Pathology, CCK, R8:04,
SE-17176 Stockholm, Sweden. 2McGill University, Goodman cancer
center, 1160 Ave Des Pine Quest,Montreal, QC H3A 1A3, Canada.
The insulin-like growth factor-1 receptor (IGF-1R) is a cell surface
receptor belonging to a class of receptors known as receptor tyrosine
kinase (RTK). It is known that the IGF-1R is vastly expressed in
malignant tissues and plays a crucial role in growth and survival of
cancer cells. Inhibition of IGF-1R has been demonstrated to cause
apoptosis. However, in many cases patients treated with drugs aimed to
inhibit the IGF-1R have developed resistance against the drug, and the
explanation behind this is not fully understood.Recently, our laboratory
reported that IGF-1 stimulation promotes SUMO-1 modification of the
IGF-1R, followed by nuclear translocation of the receptor. Nuclear IGF-
1R associates with enhancer-like elements of the genomic DNA and
increases transcription. Malignant cells, e.g. breast cancer cells, have
more than a 20-fold increase in nuclear IGF-1R comparing to normal
cells, but the total amount of IGF-1R is only 5-fold increased in malignant
cells. In this present study we show that the IGF-1R is co-localized
with Importinβ, which is known to transport proteins into the nucleus
through the nuclear pore complex (NPC). Further, we found that IGF-
1R interacts with RanBP2, a SUMO-E3 ligase enzyme, also located
at the NPC. By over-expressing the 32 kDa active SUMO-E3 ligase
fragment of RanBP2, we can show that there is enhanced SUMOylation
of the IGF-1R, confirming that RanBP2 is a significant part of the IGF-1R
SUMOylation chain. Further investigations in how the IGF-1R enters the
nucleus would provide novel drug targets and enable the development
of novel cancer therapies.
POS-WED-319
A NOVEL 4,6-DISUBSTITUTED
2,2-DIMETHYLCHROMAN INDUCES APOPTOSIS IN
HUMAN CERVICAL CARCINOMA HELA CELL VIA
INHIBITION OF DNA SYNTHESIS
Zhang X., Hwang J. and Kim D.-K.
Department of Biomedicinal Chemistry, Inje University, 607 Aubang-
dong, Gimhae 621-749, Korea.
1-((3S,4R)- 4-(2-Ethoxy- 4-methyl-1H-pyrrol-1-yl)-3-hydroxy-2,2-
dimethylchroman-6-yl)-3-phenylurea (S24), a novel 4,6-disubstituted
2,2-dimethylchroman was synthesized and its apoptotic activity
was investigated on the in vitro growth of human cervical carcinoma
HeLa cells. Significantly decreased rates of proliferation and viability
(IC50 ~150 μM) as well as evidence of apoptosis were observed with
S24. Cell morphological changes observed under light microscopy
confirmed apoptosis occurrence. The results from Annexin V/PI dual
staining and the cell cycle arrest assay indicated that S24 induced an
earlier apoptosis of HeLa cells in a time-dependent manner. S24 also
induced the activation of a protease cascade involving caspase-9 and
-3 by western blotting assay. Furthermore, [3H]thymidine incorporation
and flow cytometry analysis revealed that S24 treatment decreased
DNA synthesis and arrested the cells at the G0/G1phase of the cell
cycle. In addition, the very sensitive alkaline microgel electrophoresis
technique (comet assay) was used for estimation of the S24-induced
DNA single- and double-strand breaks. Taken together, we suggest that
S24-induced apoptosis through caspase pathway and the apoptotic
effect may correlate with its inhibition of DNA synthesis.
Page 270 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-318
EVALUATION OF P16 PROTEIN AS A BIOMARKER IN
PENILE CARCINOMA
Kagohara L.T.1, Guimaraes G.C.2, Lopes A.2, Cunha I.W.1, Coudry R.A.1
and Soares F.A.1
1
Hospital A.C. Camargo - Anatomic Pathology Department. 2Hospital
A.C. Camargo - Pelvic Surgery Department.
Penile carcinoma is an uncommon disease and the most frequent
subtype is squamous cell carcinoma. This cancer represents less than
1% of all cases in developed countries. However, in some regions of
developing countries it represents 20% of all male cancers. The main
risk factors associated to this tumor are poor hygienic habits, low quality
of life, HPV infections and high number of sexual partners. Very little
is known about penile carcinoma, including its pathogenesis, etiology
and gene expression patterns. To identify the role of the protein p16 in
penile carcinoma progression, we evaluated its expression pattern by
immunohistochemistry in 301 samples displayed in tissue microarrays.
This protein is known to have reduced levels in many tumor types,
is associated to patient’s clinical outcome and is involved in the
tumorigenesis process. The p16 protein has an important role in the
cell cycle control. It is a cyclin-dependent kinase (CDK) inhibitor that
inhibits retinoblastoma protein phosphorilation. The stained slides were
analyzed at the ACIS-III (DAKO) equipment, which digitalizes images
and quantifies frequency and intensity based on the brown areas of the
samples. We observed loss of expression of p16 in 65.1% of the cases.
The statistical analysis showed an association between the presence
of protein p16 and low histologic grade tumors, while loss of expression
of p16 is associated to presence of perineural and vascular invasion.
These results indicate that p16 loss of expression is an important event
in penile carcinoma tumorigenesis and it may be a biomarker for this
tumor type as perineural and vascular invasion are clinical markers of
bad prognosis.
POS-THU-320
TISSUE MICROARRAY STUDY OF MATRIX
METALLOPROTEINASES IN SOFT TISSUE SARCOMAS
Muto N.H.1, Cunha I.W.1, Carvalho K.C.2, Malagoli R.1, Buim M.E.C.1,
Nascimento C.F.1, Ferreira S.S.1 and Reis L.F.L.3
1
Hospital AC Camargo, Sao Paulo, Brazil. 2Faculdade de Medicina -
USP, Sao Paulo, Brazil. 3Hospital Sirio Libanes, Sao Paulo, Brazil.
Soft tissue tumors are a heterogeneous group of mesenchymal tumors
with variable clinical behavior and histological presentation. Tumor
size, histologic grade, depth, and status of surgical margins have been
identified as prognostic factors. However, these prognostic variables do
not explain biologic differences in aggressiveness between sarcomas.
Given that human soft tissue sarcoma is a highly lethal malignancy
in which control of metastasis determines survival, a marker of
more aggressive and metastatic behavior could be valuable. Matrix
metalloproteinases (MMPs) are zinc-dependent endopeptidases that,
collectively, are capable of degrading almost all ECM components.
MMPs can contribute to local and distant tumor invasion through
degradation of basement membrane and stromal invasion. In this study
we investigated the immunohistochemical expression of a panel of
MMPs (-2, -9, -13 and -19) in 3 tissue microarrays (TMAs) containing
a total of 266 sarcoma samples, including 118 sunovial sarcomas,
37 liposarcomas, 45 pleomorphic sarcomas, 18 leiomyosarcomas,
12 myxoid type sarcomas, 5 malignant peripheral nerve sheet tumor,
6 fibrosarcoma, 18 fibromatoses, 7 benign tumors and 20 other type
sarcomas. Images were digitalized and quantified using ACIS® III
Tissue Microarray Analysis (DAKO, CA) and data were statistically
analyzed using the software PRISM 4. In general, we found a higher
expression of MMP-2 and -9 in synovial sarcomas compared to other
samples, and a higher expression of MMP-13 and -19 in synovial and
pleomorphic sarcomas. Finnancial support: CEPID-FAPESP.
POSTERS WEDNESDAY & THURSDAY
POS-WED-321
INDUCTION OF APOPTOSIS AND DNA DAMAGE BY AN
ANALOGUE OF METHYL JASMONATE
Zhao J.1, Zhang X.1, Kang S.1, Hwang J.1, Jung J.2 and Kim D.-K.1
1
Department of Biomedicinal Chemistry, Inje University, 607 Aubang-
dong, Gimhae 621-749, Korea. 2College of Pharmacy, Busan National
University, Busan 609-735, Korea.
The current study was undertaken to investigate the effects of methyl
5-chloro-4, 5-didehydrojasmonate (J7), an analogue of methyl
jasmonate, on the in vitro growth of human cervical carcinoma HeLa
cells. Significantly decreased rates of viability (IC50 ~15 μM) as well
as evidence of apoptosis were observed with J7. Cell morphological
changes observed under light microscopy confirmed apoptosis
occurrence. The results from Annexin V-FITC/PI double staining and
the cell cycle arrest assay further supported that J7 was able to induce
apoptosis of HeLa cell and influence cell cycle process resulting in S
phase arrest. By Western blot assay, a decrease in Bcl-2 level followed
by an increase of a protease cascade involving caspase-9 and -3 was
observed. Furthermore, an activation of SAPK/JNK and p38 MAPK was
also detected. The single-cell gel electrophoresis (comet assay) and
[3H]dTTP incorporation assay indicated that J7 induced DNA damage
and inhibited DNA replication. Taken together, our studies suggest
that J7 induces HeLa cell apoptosis though activation of both caspase
pathway and the sub-MAPK pathways. Moreover, the apoptotic effect
is associated with DNA damage. Therefore, J7 may be a candidate
compound to be developed into an anticancer agent.
POS-WED-323
ACTIN FIBERS AND FIBRONECTIN NETWORKS
REMODELING DURING EARLY ADIPOCYTE
DIFFERENTIATION
Nobusue H.1, Oki Y.1, Shimizu T.2, Onishi N.2, Saya H.2 and Kano K.1
1
Laboratory of Cell and Tissue Biology, College of Bioresource Sciences,
Nihon University. 2Division of Gene Regulation, Institute for Advanced
Medical Research, Keio University School of Medicine.
Adipocyte differentiation at an early stage is directly regulated by gene
expression of a master regulator such as peroxisome proliferator-activated
receptor-γ2 (PPARγ2). During adipogenesis, extracellular matrix (ECM)
remodeling defines important events of the differentiation process. This is
characterized by conversion from the fibronectin (FN)-rich stromal matrix
of a preadipocyte to the basement membrane of an adipocyte. Adipocyte
differentiation has been reported to result in the conversion of filamentous
actin from stress fibers and lamellipodia to cortical actin structures.
However, the order of the following events has not been clarified: (a)
PPARγ2 expression, (b) ECM remodeling, and (c) actin cytoskeleton
remodeling. In this study, we aimed to elucidate the remodeling of the
actin cytoskeleton and ECM during early adipocyte differentiation. Before
adipogenic induction, preadipocytes showed fibroblastic morphology with
a well-developed actin stress fiber pattern and formed FN networks in
connection with their actin cytoskeltons. After differentiation for 24 h, the
actin fibers were disassembled and disorganized, and as a result, the FN
networks were also disorganized. After 48 h of differentiation, the fibers
disappeared and a cortical actin structure was formed. Coincidentally,
the FN networks degraded and disappeared. After 60 h of differentiation,
lipid droplets accumulated in the cytoplasm and laminin-8 expression was
detected on the plasma membrane. Matrix metalloproteinase (MMP)-2, a
catabolic enzyme essential for ECM remodeling, was activated after 48
h of adipocyte differentiation, whereas MMP-9 was not detected. And
PPARγ2 increased significantly after 48 h of differentiation. Furthermore,
the remodeling of the actin fibers and the FN networks occurred after 24 h
of differentiation even when depleted expression of PPARγ was achieved
by retrovirus-mediated shRNA. These results indicate that remodeling of
the actin fibers and the FN networks precedes PPARγ2 expression during
adipocyte differentiation, and therefore, we suggest that this remodeling
acts as a trigger for adipocyte differentiation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 271
POS-THU-322
COPY NUMBER VARIATION IN INDIVIDUALS WITH
CLINICAL DIAGNOSIS OF LI-FRAUMENI OR LI-
FRAUMENI LIKE SYNDROME AND WITHOUT
GERMLINE ALTERATIONS IN TP53 GENE
Silva A.G.S.1, Achatz M.I.1, Brentani R.1, Rosenberg C.2 and Krepischi A.C.1
1
Hospital A.C. Camargo, Sao Paulo, Brazil. 2Laboratory of Human
Genetics, Department of Genetics and Evolutionary Biology,
University of Sao Paulo, Brazil.
Li-Fraumeni syndrome (LFS) is an autosomal dominantly inherited
disorder characterized by a remarkably increased risk of early-
onset and multiple cancers, including breast, brain tumors and other
neoplasms. Constitutional copy number variation of DNA segments
(CNV) are likely associated to cancer, either as a susceptibility factor
or as a result of a genetic instability. It has been recently reported that
individuals carrying TP53 mutations show about 3-fold increase in CNV
frequency compared to wild type individuals, raising the possibility that
the number of CNVs could be larger in individuals with high cancer risk.
We used a 180K oligoarray platform from Agilent to estimate the CNV
frequency in 63 affected and unrelated patients with clinical diagnosis of
Li-Fraumeni and Li-Fraumeni-like syndrome. We compared the resulting
frequencies between these individuals to a population based control
group (n=100). We have defined as rare genomic alterations those
located in coding regions and covered by ≤3 individual CNVs described
in DGV (Database of Genomic Variants). The initial results point to a
slight increase in the average frequency of CNVs per individual in the
cancer patients samples (9.3) when compared to the control sample
(6.8). Although the sample is still small, the category so-called rare
alterations are approximately twice as frequent among cancer patients
(average of 1.36±1.69 per individual) than among controls (average of
0.42±0.67 per individual). Some of the genes encompassed by these
CNVs have been previously implicated in tumors, but not reported in
germline.
POS-THU-324
A WINDOW OF OPPORTUNITY: THE MECHANISM OF
PERFORIN AND GRANZYME SYNERGY
Lopez J.A., Trapani J.A. and Voskoboinik I.
Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne,
VIC 3002.
Perforin is a pore forming member of the Membrane-Attack-Complex-
Perforin (MACPF) family of proteins. It is stored in the secretory granules
of cytotoxic lymphocytes (CLs) represented by natural killer cells and
cytotoxic T lymphocytes. CLs seek out and bind to virally infected or
transformed target cells and form a transient immunological synapse.
Formation of the synapse results in cell polarisation and exocytosis of
secretory granules containing monomeric perforin molecules together
with pro-apoptotic serine proteases (granzymes) into the synaptic cleft.
At the target cell, perforin facilitates the delivery of granzymes into the
cytosol to initiate cell apoptosis. The precise mechanism of perforin-
mediated granzyme delivery has remained elusive. To investigate the
mechanism of perforin mediated granzyme delivery, we have performed
a detailed kinetic analysis of perforin function on target cells. Sublytic
doses of perforin were shown to elicit a transient calcium influx into target
cells. Restoration of intracellular calcium concentrations was attributed
to the induction of a membrane repair response by the target cell.
Inhibition of membrane repair through temperature restriction resulted
in a block in intracellular calcium restoration and the induction of cell
death through cell lysis/necrosis. Membrane repair of perforin pores
formed at the plasma membrane resulted in a rapid loss of synergy with
granzymes. Collectively, these data suggest that perforin acts primarily
at the plasma membrane of target cells to facilitate granzyme entry,
leading to cell death through apoptosis. Concomitantly, the repair of
perforin lesions acts to prevent cell death through necrosis.
POSTERS WEDNESDAY & THURSDAY
POS-WED-325
CONSERVED EXPRESSION OF VASA IN MARSUPIALS
AND MONOTREMES
Hickford D.1, Frankenberg S.1, Pask A.2, Shaw G.1 and Renfree M.B.1
1
The University of Melbourne, Australia. 2The University of
Connecticut, USA.
VASA is a widely-used marker of germ cells across all animal phyla,
but little is known of its distribution and expression in non-eutherian
mammals (marsupials and monotremes). VASA was cloned from a
marsupial, the tammar wallaby, and a monotreme, the platypus. Tammar
VASA has 21 exons encoding a protein with 726 amino acids; platypus
VASA has 22 exons encoding 743 amino acids. VASA protein sequence
is highly conserved among mammals, especially within the DEXDc
and HELICc domains, which contain nine conserved sequence motifs.
The N terminus of VASA is less conserved and the human, mouse, rat,
wallaby and platypus all have at least two transcripts for VASA, one
encoding a full length protein and the other missing an exon within the
N terminus. The exon missing varies between species. Curiously, the
platypus has an extra exon not present in other mammals. In contrast to
eutherians, wallaby germ cells do not express VASA protein until ~4-5
days after they reach the gonads. In females, germ cells remain VASA-
positive throughout their life cycle and in some oocytes VASA appears
to be concentrated in a distinct paranuclear patch, as occurs in humans
but not mice. In both wallaby and platypus adult testes, VASA protein is
present in spermatocytes and round spermatids, with weaker staining
in spermatogonia and elongating spermatids and none in spermatozoa.
The spermatocytes of both species exhibited distinct VASA-positive
granules within their cytoplasm, similar to in eutherians. In summary,
the high sequence conservation and similarity in VASA distribution in
marsupials and monotremes suggest conservation of the role of VASA
in germ cell development across all mammals.
POS-WED-327
THE ROLE OF NEDD4 IN EMBRYONIC BLOOD AND
LYMPHATIC VASCULAR DEVELOPMENT
Secker G., Kazenwadel J., Boase N., Kumar S. and Harvey N.
Division of Haematology, Centre for Cancer Biology, SA Pathology,
Adelaide, Australia.
The cardiovascular system is the first organ network to develop in
the vertebrate embryo. Assembly of the initial blood vascular plexus
occurs via the aggregation of endothelial progenitor cells and is
known as vasculogenesis, while the subsequent sprouting growth and
remodeling of the primary vascular network is known as angiogenesis.
Lymphatic vessels are a critical, but often overlooked component of
the cardiovascular system. These specialised vessels return interstitial
fluid and protein to the bloodstream, absorb lipids from the digestive
tract and traffic cells of the immune system. Formation of the lymphatic
vasculature is initiated once the major arteries (dorsal aortae) and veins
(cardinal veins) have been established, originating from the cardinal
veins following the onset of expression of the Prox1 transcription factor
in a polarized population of venous endothelial cells. Our current work is
focused on the identification of genes and molecular events critical for
generation of the vasculature during embryogenesis. In particular, we
are investigating the role of Nedd4, an E3 ubiquitin ligase, in this process.
Ubiquitination of a protein can lead to its degradation, stabilisation,
changes in subcellular localization or modification of biochemical
properties. As such, protein modification by ubiquitination has crucial
roles in regulating many signalling pathways and misregulation of this
process is associated with numerous human pathologies. Here we
present data that demonstrates loss of Nedd4 function leads to impaired
development of the blood and lymphatic vasculature. Future studies aim
to define the mechanism by which Nedd4 regulates signalling involved
in blood and lymphatic vascular development.
Page 272 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-326
EPIGENETIC REGULATION OF OCT4 AND PDX-1 IN
MOUSE EMBRYONIC STEM CELLS AND MIN6 CELLS
Wong J.C.Y.1, Jack M.M.2 and O’Neill C.1
1
Kolling Institute of Medical Research, The University of Sydney,
Sydney, Australia. 2Department of Endocrinology, Royal North Shore
Hospital, Sydney, Australia.
The differentiation of embryonic stem cells (ESCs) to insulin producing
β-cells involves a complex series of transcriptional changes such as
repression of pluripotency associated with the Oct4 gene and activation
of Pdx-1, a critical transcription factor that is considered the earliest
specific marker of pancreatic endoderm. These changes may be
regulated by epigenetic factors such as DNA methylation and covalent
histone modifications. This study aimed to determine the role of these
mechanisms in the regulation of Oct4 and Pdx-1 in the D3 mouse ESC
line in comparison to the insulinoma cell line, MIN6. The presence of
histone modifications was examined by chromatin immunoprecipitation
assays using anti-histone antibodies and DNA extracts from D3 and
MIN6 cells. Specific modifications at the Oct4 and Pdx-1 promoter were
then quantitated by real-time PCR. The DNA methylation status of CpG
rich areas of Oct4 and Pdx-1 was determined by bisulfite sequencing.
The study demonstrated that Oct4 was hypomethylated in D3 and
hypermethylated in MIN6. The histone marks found to be present also
corresponded to the Oct4 expression status in each cell line. In contrast,
Pdx-1 was hypomethylated in both D3 and MIN6 despite it being
repressed in ESCs. D3 cells also possessed a bivalent domain of both
the active H3K4Me3 and repressive H3K27Me3 histone marks at the
Pdx-1 promoter. Together, the results suggest a means by which Pdx-1
is maintained at a transcription-ready state in ESCs. It also provides
further insight into identifying the mechanisms that are necessary to
activate and repress the key homeotic genes that are required for β-cell
specification.
POS-THU-328
IDENTIFICATION OF EPIGENETIC COMPONENTS
REQUIRED FOR CELL-TO-CELL MOVEMENT OF AN
RNA SILENCING SIGNAL IN ARABIDOPSIS
Searle I.R.1, 2 , Smith L.2 and Baulcombe D.C.2
1
Research School of Biology, ANU, Canberra. 2Department of Plant
Sciences, University of Cambridge, UK.
RNA silencing is a sequence-specific RNA degradation process
conserved in fungi, animals and plants that is associated with cell-to-
cell movement of a mobile silencing signal. We developed a cell-to-cell
movement of RNA silencing system in Arabidopsis using an inverted
repeat transgene under the control of a phloem-specific promoter
such that the spread of RNA silencing was manifested in regions
around veins. We have previously identified epigenetic components;
Polymerase IV, RNA-Dependent-RNA-polmyerase 2, and CLSY (a
SNF2chromatin remodelling factor) that are required for mobile RNA
silencing. In addition we also identified a putative histone H3 lysine 4
trimethyl demethylase, JMJ14, that is required for mobile RNA silencing.
In addition to an effect on mobile silencing the jmj14 mutants also had
reduced CHH DNA methylation, increased abundance of endogenous
transposon transcripts and they flowered earlier than wild type. We
placed the activity of JMJ14 at a downstream point in RNA silencing
pathways because the subcellular locations of upstream components
RNA-dependent RNA polymerase (RDR2) and Argonaute (AGO4)
were not perturbed in jmj14 mutants. These results illustrate the
potential for a link between RNA silencing and demethylation of histone
H3 trimethylysine. We propose that JMJ14 acts downstream of the
Argonaute effector complex to demethylate histone H3 lysine 4 trimethyl
residues at the target of RNA silencing.
POSTERS WEDNESDAY & THURSDAY
POS-WED-329
PAP SIGNALS FROM THE CHLOROPLAST REGULATE
EXORIBONUCLEASES, GENE SILENCING AND
STRESS RESPONSES IN ARABIDOPSIS
Crisp P.A.1, Estavillo G.M.1, Pornsiriwong W.1, Wirtz M.2, Carrie C.3,
Hell R.2, Whelan J.3 and Pogson B.J.1
1
ARC Centre of Excellence in Plant Energy Biology,The Australian
National University, Canberra, ACT, Australia, 0200. 2University of
Heidelberg, Heidelberg Institute for Plant Sciences, Im Neuenheimer
Feld 360, 69120 Heidelberg, Germany. 3ARC Centre of Excellence in
Plant Energy Biology,The Universuty of Western Australia, Crawley,
6009, WA, Australia.
Chloroplasts regulate nuclear gene expression during plant development
and in response to environmental stresses via retrograde signalling
pathways. Here we report a novel chloroplast-nucleus signal that
regulates RNA metabolism and gene silencing in the nucleus. In a
genetic screen to identify components of the chloroplast stress signalling
pathways we isolated the Arabidopsis mutant altered APX2 expression
8 (alx8) [1]. The alx8 mutant exhibits constitutive up-regulation of stress
inducible genes, including APX2 and ELIP2, and drought tolerance.
alx8 is a mutation in the SAL1 phosphatase [2], which we now show to
be localised to the chloroplast, not the nucleus as recently proposed
[3]. We have demonstrated that the loss of chloroplastic SAL1 leads to
a 10 fold elevation of its substrate, 3-phosphoadenosine 5-phosphate
(PAP), which is an inhibitor of 5’-3’ exoribonucleases (XRNs) in yeast
[4]. Building on an earlier report linking SAL1 to the activity of the
exoribonucleases in plants [5], we show that stress-inducible genes
elevated in the SAL1 knockouts – alx8 and fiery1 – are similarly up-
regulated in the xrn2,xrn3 double mutant. Furthermore, we show that
the alx8 mutation restores sensitivity to ABA in stomata and seeds of
abi1 and ost1 (ABA insensitive mutants). Thus, these results implicate
exoribonucleases in stress response and gene regulation, and establish
a compelling link between chloroplast and stomatal signalling, RNA
metabolism and gene expression. [1] Rossel et al. (2006) Plant Cell
Environ. [2] Wilson, Estavillo et al. (2009) Plant J. [3] Kim and von Arnim
(2009) Plant J. [4] Dichtl et al. (1997) EMBO. [5] Gy et al. (2007) Plant
Cell. Research supported by the ARC Centre for Plant Energy Biology
(CE0561495); and GRDC Scholarship (GRS184).
POS-WED-331
FIELD EVALUATION OF TRANSGENIC PERENNIAL
RYEGRASS (LOLIUM PERENNE L.) PLANTS FOR
ENHANCED FRUCTAN ACCUMULATION
Dimech A.M.1, 3, Badenhorst P.E.2, 3, Panter S.1, 3, Liu Z.1, 3, Ramage C.1, 3,
Stewart A.V.4, Smith K.F.2, 3, Mouradov A.1, 3, 5 and Spangenberg G.C.1, 3, 5
1
Department of Primary Industries, Victorian AgriBiosciences Centre,
Bundoora, Victoria 3083, Australia. 2Department of Primary Industries,
Hamilton Centre, Hamilton, Victoria 3300, Australia. 3Molecular Plant
Breeding Co-operative Research Centre, Bundoora, Victoria 3083,
Australia. 4PGG Wrightson Seeds, Lincoln, New Zealand. 5La Trobe
University, Bundoora, Victoria 3086, Australia.
Fructans, a class of water-soluble storage carbohydrates, play an
important role in the forage quality of pasture grasses, providing an
energy source for livestock. Up to 279 independent transformation
events in elite perennial ryegrass (Lolium perenne L.) germplasm were
evaluated for increased fructan accumulation and enhanced biomass
yield under field conditions in Hamilton, Victoria, between September
2008 and January 2010. Chimeric fructosyltransferase genes, Lp1-SST
and Lp6G-FFT, were expressed in transgenic plants as single genes
or as a Lp1-SST::Lp6G-FFT translational fusion under the control of
constitutive and light-regulated promoters. Quantitative real-time PCR
was used to measure transgene expression and High-Performance
Anion-Exchange Chromatography was used to determine fructan
accumulation throughout the growing season. Multiple independent
transformation events were identified with fructan levels in whole
tillers and leaf blades that were up to 80% higher than levels in
corresponding tissues of non-transgenic plants of the same genotype.
Elite transformation events were selected based on enhanced biomass
production, low transgene copy number and consistently high fructan
accumulation for transgenic germplasm development.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 273
POS-THU-330
MECHANISMS OF SMALL, NON-CODING RNA-
DIRECTED GENE SILENCING IN FISH
McFarlane L.J., Fitzner A., Koopman P. and Wilhelm D.
Institute for Molecular Bioscience; The University of Queensland,
Brisbane, Australia.
RNA interference (RNAi) has emerged as a powerful platform technology
for investigating gene function. Although widely used in the major model
organisms, the use of RNAi in teleost fish has proven unreliable. We
are exploring the reasons for this unreliability. Using a comparative
genomics approach, we have identified that the teleost clade has,
uniquely among the vertebrates, undergone an expansion of the AGO
protein sub-family, the key effector proteins of RNAi and other small
non-coding RNA regulatory pathways. Although previous studies have
suggested that the vertebrate AGO homologues are largely redundant,
our approach identified numerous features that suggest these proteins
may have specialised functions. To explore their function, we have
isolated the five teleost Ago genes from the zebrafish Danio rerio for
use in a variety of assays. We have also developed an in vitro system
for biochemically dissecting the RNAi response to determine whether
it is analogous with the other more well-characterised model systems.
Our results will illuminate the mechanisms of small RNA-directed gene
regulation in the teleosts, as well as identifying potential strategies to
enable the use of RNAi in fish.
POS-THU-332
REGULATION OF THE HUMAN CHOP GENE
PROMOTER BY THE STRESS RESPONSE
TRANSCRIPTION FACTOR ATF5 VIA THE AARE1 SITE
IN HUMAN HEPATOMA HEPG2 CELLS
Yamazaki T., Ohmi A., Kurumaya H., Kato K., Abe T., Okuyama R.,
Umemura M., Kaise T., Takahashi S. and Takahashi Y.
The Laboratory of Environmental Molecular Physiology, School of Life
Science, Tokyo University of Pharmacy and Life Sciences, Hachioji,
Tokyo 192-0392, Japan.
Activating transcription factor (ATF) 5 is a member of the cAMP response
element binding protein (CREB)/ATF family of transcription factors.
We have shown that ATF5 is a stress response transcription factor
that responds to amino acid limitation, arsenite exposure, or cadmium
exposure. In this study, we used a transient transfection system to
express ATF5 and showed that ATF5 activates the CCAAT/enhancer-
binding protein (C/EBP) homologous protein (CHOP) gene promoter
in human hepatoma, HepG2, cells. Both deletion analysis and point
mutations of the promoter revealed that amino acid response element
(AARE) 1 is responsible for ATF5-dependent promoter activation.
Furthermore, the existence of either AARE1 or activating protein-1
(AP-1) is sufficient for transcriptional activation of the CHOP gene
promoter by arsenite exposure, although complete induction requires
the existence of both elements. We also demonstrated that knockdown
of ATF5 reduced arsenite-induced CHOP protein expression. These
results demonstrated that besides the AP-1 site, the AARE1 site is also
important for arsenite-induced CHOP gene promoter activation. Taken
together, these results suggested that the CHOP gene is a potential
target for ATF5, and that ATF5 raises the arsenite-induced CHOP gene
expression level via the AARE1 site in HepG2 cells.
POSTERS WEDNESDAY & THURSDAY
POS-WED-333
INVESTIGATING THE ROLE OF KLF3 IN
ERYTHROPOIESIS AT THE PHYSIOLOGICAL AND
MOLECULAR LEVEL
Norton L.J.1, Mak K.1, Funnell A.P.W.2, Pearson R.C.M.1 and Crossley M.1
1
School of Biotechnology and Biomolecular Sciences, University
of New South Wales, Australia. 2School of Molecular and Microbial
Biosciences, University of Sydney, Australia.
Development is governed by transcription factors which orchestrate
the ordered expression of genes. A major family of transcription factors
are the Krüppel like factors (Klfs) which play a role in a wide variety of
processes including stem cell biology, adipogenesis, skin development
and erythropoiesis. Klf1 was the first Klf to be discovered and is a
master regulator of erythropoiesis and driver of β-globin expression.
Klf1 has been shown to induce the expression of a related family
member, Klf3, which indicates a possible role for this transcription factor
in erythropoiesis. This research aims to investigate the role of Klf3 in
erythropoiesis in vivo through the investigation of a Klf3 knock out
mouse model. An examination of the peripheral blood of the Klf3 KO has
revealed extensive defects in haematopoiesis. In particular, we have
observed a significant increase in the number of reticulocytes, indicative
of anaemia. In agreement with this, the spleen of the KO mouse is
enlarged and tissue sections have revealed a dramatic increase in
the presence of red pulp. Flow cytometry has confirmed noticeable
compensatory erythropoiesis in the spleens of the KO mouse. We have
also used flow cytometry to study erythroid differentiation in the fetal
liver and adult bone marrow and have observed impaired development.
Taken together, this evidence supports a role for Klf3 in normal red
blood cell development. We have now collected and purified erythroid
tissue from the Klf3 KO mouse at various time points and are using a
microarray approach to determine the role of Klf3 in erythropoiesis at
the molecular level.
POS-WED-335
WASH-DEPENDENT ACTIN DRIVES VESICLE
NEUTRALISATION: A REQUIREMENT FOR
EXOCYTOSIS
Carnell M.J. and Insall R.
The Beatson Institute for Cancer Research.
WASH, a recently identified member of the WASP protein family, is an
activator of actin polymerisation around endocytic vesicles. Utilising
the model organism Dictyostelium discoideum we demonstrate a novel
role for actin in controlling vesicle identity. Here we show that WASH
is essential for the formation of actin-coats around late endocytic
compartments. The generation of WASH null cells has revealed that
these actin-coats are essential for driving lysosome neutralisation,
and that failure to do so results in a complete block in the exocytosis
of indigestible material. Our data suggests an important role for both
WASH and actin in vesicle maturation by driving the removal of the
H+ V-ATPase from internal vesicles prior to fusion with the plasma
membrane.
Page 274 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-334
DOES THE EXTRACTION OF CELL CONTENTS DURING
DIGESTION OF GRASSES BY THE AUSTRALIAN
PLAGUE LOCUST OCCUR VIA PLASMODESMATA?
Armour W.J., Clissold F.J., Barton D.A., Overall R.L. and Simpson S.J.
School of Biological Sciences, The University of Sydney, Sydney, NSW
Australia.
Barbehenn (2005) has proposed nutrients are extracted from plant
tissue via plasmodesmata during digestion by insects. We investigated
this in the Australian plague locust (Chortoicetes terminifera, hereafter
locusts) and its two natural plant hosts the C4 grasses, Dactyloctenium
radulans and Astrebla lappacea. Typically C4 grasses have large,
chloroplast rich, bundle sheath (BS) cells that appear to remain intact
during digestion by grasshoppers. We found many plasmodesmata
pitfields between BS cells and the surrounding ring of cells of both
grasses when stained using aniline blue. We investigated if locusts could
use these plasmodesmata for nutrient extraction. Leaf fragments taken
from locusts four min after ingestion had almost no callose when stained
with aniline blue. Thus to test if locust gut enzymes can degrade callose,
transverse sections of both grasses were induced to form callose by
plasmolysis and then treated with phosphate buffered saline (PBS) or
locust enzymes. As there was no significant change in the number of
BS cells with callose at plasmodesmata in either treatment after 30 min
it suggests locust enzymes cannot degrade callose. We hypothesised
plant tissue ingested by locusts had almost no callose because the
cells were dying. We tested membrane integrity of BS cells in ingested
tissue by loading leaves with the membrane impermeant dye, carboxy-
fluorescein (CF). After feeding, leaf fragments taken from locusts four
min after ingestion had less than 6% of BS cells retaining CF compared
to 70% for controls, suggesting most BS cells lost membrane integrity
during ingestion. Thus it appears nutrient extraction via plasmodesmata
is unlikely as the cells die early during ingestion.
POS-THU-336
EXENDIN-4 ENHANCES ADULT NEUROGENESIS
AFTER FOCAL CEREBRAL ISCHEMIA IN MICE
Kang H.M., Jin J. and Park C.
Department of Anatomy and Neurobiology, Biomedical Science
Institute, School of Medicine, Kyung Hee University, Seoul 130-701,
Republic of Korea.
Cerebral ischemia induces neurogenesis. Neurogenesis occurs
mainly in two area of adult mammalian brain including of humans : the
subgranular zone (SGZ) of the dentate gyrus (DG) and the anterior
part of the subventricular zone (SVZ), along the ventricle. In addition
newly generated neurons in the SVZ migrated to the ischemic boundary
region. Several factors regulated proliferation, migration, differentiation
of neuronal precursor cells. Exendin-4 is peptide hormone, stable
analog of glucagon-like peptide-1 (GLP-1), and that selectively binds
GLP-1 receptor. Recently, It has been demonstrated that GLP-1 receptor
promotes conservation dopaminergic neurons in rodent Parkinson’s
diseases model, Exendin-4 markedly reduced cortical infarction induced
by transient Middle Cerebral Artery occlusion (MCAo). However,
Exendin-4 is still unknown whether it is enhanced neurogenesis. In
this study, we used photothrombotic focal ischemia model in mice
and observed that systemic administration of Exendin-4 significantly
increased not only 5-bromo-2-deoxyuridine (BrdU), proliferation marker,
but also Ki-67, necessary cellular marker for proliferation in SVZ.
Furthermore immature neurons marker, doublecortin (DCX), increased
in ischemic penumbra region. These findings suggest that Exendin-4
may be increases proliferation of neural stem cells through activation of
GLP-1 receptor.
POSTERS WEDNESDAY & THURSDAY
POS-WED-337
HYDROXYSTEROID DEHYDROGENASE
TRANSFORMATIONS OF 5β-SCYMNOL
AND IDENTIFICATION OF OXOSCYMNOL
TRANSFORMATION PRODUCTS BY LCMS
Macrides T.A.1, Hodges L.D.1, Wynne P.M.2, Glowacki L.L.1, Kalafatis N.1
and Wright P.F.A.3
1
Natural Products Research Group, RMIT University, Bundoora, VIC.
3083 Australia. 2Shimadzu Scientific Instruments (Oceania) Pty Ltd, Mt
Waverley, VIC. 3149 Australia. 3Applied & Nutritional Toxicology Key
Centre, RMIT University, Bundoora, VIC. 3083 Australia.
Our interest in 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,24,26,27-
hexol] stems from the therapeutic potential of this natural marine shark
bile alcohol. However, the xenobiotic transformation of scymnol in
mammals is unknown. Hydroxysteroid dehydrogenase (HSD) reactions
are fundamental to the in vivo transformations of mammalian bile acids,
and the presence of 3α-, 7α- and 12α-hydroxyls on scymnol support an
investigation of scymnol oxidation products in HSD reactions. Mixtures
of scymnol, respective HSD enzyme and NAD+ cofactor were incubated
for 5 min (25 °C, pH 8.9) before the addition of methanol. Precipitated
protein was removed by centrifugation, and the supernatant analysed for
scymnol metabolites by means of a Zorbax SB-C18 column using either
an Agilent 1090M liquid chromatograph interfaced with a Thermo Quest
LCQ Duo mass spectrometer, or an Agilent 1050 liquid chromatograph
interfaced with a Thermo Quest LCQ Classic mass spectrometer. The
authentic scymnol standard yielded a protonated molecular ion at m/z
469 Da, and higher mass adduct ions attributed to M+NH4+, M+CH3OH2+
and M+CH3COOH2+. 3-Oxoscymnol [(24R)-(+)-5β-cholestan-3-one-
7α,12α,24,26,27-pentol, m/z 467 Da, relative retention time 0.89] was
identified as the principle molecular species of scymnol in the reaction
with 3α-HSD pure enzyme. 7-Oxoscymnol [(24R)-(+)-5β-cholestan-7-one-
3α,12α,24,26,27-pentol, m/z 467 Da, RRT 0.79] and 12-oxoscymnol [(24R)-
(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol, m/z 467 Da, RRT 0.81]
were similarly identified as principle molecular species in the respective
7α-HSD and 12α-HSD reactions. Confirmation that 5β-scymnol is an
oxidative substrate for steroid-metabolising enzymes was made possible
by the use of sophisticated LCMS techniques that will likely provide the
basis for further exploration of scymnol as a therapeutic compound.
POS-WED-339
NEUROPROTECTIVE EFFECTS OF ETHYL PYRUVATE
ON PARKINSON’S DISEASE MODEL
Choi J.S. and Jeong J.W.
Department of Anatomy and Neurobiology, Biomedical Science
Institute, School of Medicine, Kyung Hee University, Seoul 130-701,
Republic of Korea.
Ethyl pyruvate (EP), a simple derivative of endogenous pyruvate, has an
anti-inflammatory function. Recently, the protective neurological effects
of EP have been reported in cell culture and animal models of neurological
diseases. The present study investigates the protective effects of EP on
dopaminergic cell death in Parkinson’s disease models. The selective
death of dopaminergic neurons in substantia nigra was prevented by EP
in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. EP also
suppressed the 1-methyl-4-pyridinium-induced cell death of SH-SY5Y
cells and restored the phosphorylation of extracellular signal-regulated
kinase. Thus, EP has neuroprotective effects of EP in Parkinson’s
disease and its related signaling pathways.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 275
POS-THU-338
CHARACTERIZING THE CONFORMATION OF
MONOMERIC HUNTINGTIN
Scicluna B.J.1, 2 , Ramdzan Y.M.1, 2, Mok Y.F.1, 2, Mulhern T.D.1, 2 and
Hatters D.M.1, 2
1
Department of Biochemistry & Molecular Biology, The University
of Melbourne, VIC 3010, Australia. 2Bio21 Molecular Science &
Biotechnology Institute, The University of Melbourne, VIC 3010,
Australia.
Huntington’s disease is one of nine neurodegenerative disease caused
by the expansion of a polyglutamine (polyQ) region. Recent research
has suggested that the expanded polyQ causes the huntingtin monomer
to misfold into abnormal collapsed structures that are toxic to the cells.
Using small angle x-ray scattering (SAXS) and sedimentation velocity
(SV) analysis, we determined the size and shape of the huntingtin
monomer and compared non-pathogenic forms (25Q) with pathogenic
forms (46Q). Both 25Q and 46Q huntingtin species had similar size
and radius of gyration, despite 46Q being 21 residues longer than
25Q, which suggests the polyQ region is unusually collapsed. Shape
reconstruction from SAXS data of 25Q and 46Q huntingtin exon 1
fusions with MBP (N-terminal) and Cerulean (C-terminal) depicted
both huntingtin regions as extended rod-like shapes, likely due to the
averaging of heterogeneous mixtures of structures with a length of ~65
Å. Shape heterogeneity of monomeric huntingtin exon 1 fusions was
also observed by SV analysis, which is consistent with the monomer
existing as multiple rigid structures.
POS-THU-340
THE ROLE OF PLZF IN SHAPING ONCOGENIC
CHARACTERISTICS OF CANCER CELLS
Suliman B.A.1, 2 , Xu D.2 and Williams B.R.G.2
1
College of Applied Medical Sciences, Taibah University, Madinah,
Saudi Arabia. 2Monash Institute of Medical Research, Monash
University, Melbourne, Australia.
Despite the advances over the past decade in our comprehension
of cancer cell biology and the role played by the accumulation of
different genetic mutations in numerous proto-oncogenes and tumour-
suppressor genes resulting in aggressive activation of many key
signalling pathways that delineate the nature of cancerous cells, it is still
unclear how transformed cells acquire certain oncogenic characteristics,
including enhanced proliferation, lack of contact inhibition, anchorage-
independent cell growth, and low apoptotic potential, which are common
features linked to the cell’s cytoskeleton. PLZF is a transcriptional
repressor belonging to POZ-Krupple (POK) family of transcription
factors with critical roles on oncogenesis development and stem cell
maintenance. Microarray data suggest that PLZF expression is low
or completely lost in many cancer cell lines. PLZF expression is also
associated with growth inhibition and cell cycle arrest through its
ability to repress the expression of a number of growth promoting and
proto-oncogenic genes. Recently, we found that overexpression of
PLZF in PC3 (prostate cancer cell line with a high metastatic potential)
diminished their motility, cytoskeleton integrity and viability. These
results may indicate a potential connection between PLZF induced
changes to cytoskeleton architecture and PLZF tumour suppression
in oncogene-driven cellular transformation. Cytoskeleton remodelling
by PLZF will be further characterized during epithelial to mesenchymal
transition (EMT) in prostate and other cancer cells to understand the
important roles played by PLZF in precluding the provoked proliferation
and invasive nature of these cancers.
POSTERS WEDNESDAY & THURSDAY
POS-WED-341
TRPC6 WAS NEUROPROTECTIVE IN CEREBRAL
ISCHEMIA
Du W.L., Huang J.B., Yao H.L., Zhou K.C. and Duan B.
Institute of Neuroscience, Chinese Academy of Sciences.
Neurons are the most vulnerable cells upon ischemic insults. Preservation
of neuronal survival is thus important for prevention of ischemic brain
injury. We report that the transient receptor potential canonical (TRPC)
6 was neuroprotective in focal cerebral ischemia. TRPC6 protein level in
the neurons was greatly reduced in ischemia. This downregulation was
specific for TRPC6 in the members of TRPC subfamily and mediated
by calpain proteolysis. A peptide that contains a sequence spanning
calpain cleavage site on TRPC6 protein specifically blocked TRPC6
degradation. Moreover, this peptide was neuroprotective against
ischemic insults in both cultured neurons and animals. The level of
phosphorylated cAMP-response element binding protein (pCREB) was
maintained by the peptide, suggesting that the CREB signaling pathway
was involved in the neuroprotective effect. Our results indicated that
suppression of calpain-mediated TRPC6 degradation preserved
neuronal survival and prevented ischemic brain damage.
POS-WED-343
ANTIPROLIFERATIVE ACTIVITY AND
PROTEOMIC ANALYSIS OF GENE EXPRESSED IN
HEPATOCELLULAR CARCINOMA (HEPG2) CANCER
CELL LINES TREATED WITH PHYTIC ACID
Saad N.1, Norhaizan M.E.2, 3, Hairuszah I.1, 2, Sabariah A.R.1, 4 and
Shafie N.H.2
1
UPM-MAKNA Cancer ResearchLaboratory, Institute of
Bioscience,Universiti Putra Malaysia. 2Laboratory of Molecular
Biomedicine, Institute of Bioscience, Universiti Putra Malaysia.
3
3Department of Nutrition and Dietetics, Faculty of Medicine and
Health Sciences, Universiti Putra Malaysia. 4Department of Pathology,
Faculty of Medicine and Health Sciences,Universiti Putra Malaysia.
Phytic acid or IP6 is a naturally occurring polyphoshorylated
carbohydrate, present ubiquitous in plants and animals. Phytic acid is
not only a natural antioxidant, but may also be the precursor/storage of
intracellular inositol phosphates, important for various cellular functions
and potential as anticancer compound. A prominent anticancer action
of phytic acid has been demonstrated both in vivo an in vitro in a
variety of tumor types, possibly via inhibition of tumor cell growth and
differentiation. In this study, the growth inhibitory effect of phytic acid
extracted from rice bran on human hepatocellular carcinoma (HepG2)
has been tested. Phytic acid proved to induce growth inhibition and
differentiation in HepG2. A dose and time dependent growth inhibition
of phytic acid was observed as tested by MTT assay. Analysis of flow
cytometry was performed for the analysis of cell cycle and apoptosis.
Treatment of phytic acid against HepG2 also resulted in cell cycle arrest.
Besides, the induction of early apoptosis in a dose- and time dependent
manner was confirmed using an annexin V-based assay. In a PCR
analyses, phytic acid confirmed the upregulated proapoptotic genes and
downregulated antiapoptotic genes. In conclusion, with taken together
results from our findings, phytic acid may be an excellent candidate for
adjuvant chemotherapy and prevention of cancer.
Page 276 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-342
STREPTOCOCCUS PNEUMONIAE: CHOOSING RATIONAL
TARGETS FOR STRUCTURE DETERMINATION
Riboldi-Tunnicliffe A.
Australian Synchrotron.
Streptococcus pneumoniae (the pneumococcus) is a significant pathogen, responsible
for high levels of morbidity and mortality worldwide. Most affected by this organism are
children with immature immune systems, the elderly and individuals with compromised
immune systems, such as those with diabetes or acquired immunodeficiency syndrome
[1, 2]. As a human pathogen S. pneumoniae is the most common bacterial cause of
acute respiratory infection and otitis media and is estimated to cause 3 million deaths in
children from pneumonia, bacteraemia or meningitis every year [3] accounting for 9% of
deaths in underdeveloped countries [4]. These diseases are not limited to the developing
world, despite the availability of a vaccine and a wide range of antibiotics, S. pneumoniae
remains one of the top ten causes of death in the US, where one third of isolates are
resistant to penicillin [5]. Since 1990 the resistance to penicillin has increased from 1-5%
(isolated strains) to 25-80%, with many strains having resistance to multiple antibiotics
[6, 7]. Streptococcus pneumoniae is a bacterium commonly found in the nasopharynx
(back of the nose) of 30 –50% of individuals, albeit asymptomatically. Occasionally S.
pneumoniae will spread from the nasopharynx of a colonised person into other parts
of the body and cause diseases including otitis media (ear infection), sinusitis (sinus
infection) and pneumonia (lung infection) [1]. In addition S. pneumoniae can sometimes
get into normally sterile places in the body such as the blood, where it causes sepsis
and/or bacteraemia, or the lining of the brain and spinal cord, where it causes meningitis.
The gene regulatory mechanisms involved in the transition between, and survival in,
these distinct host niches are poorly understood [8]. Modulating bacterial virulence
provides an array of molecular processes such as adherence factors, toxins and lytic
enzymes, which can be targeted to provide new therapies for disease prevention. This
proposal aims to provide a thorough understanding of the essential underlying molecular
mechanisms contributing to Streptococcus virulence and survival. Use of this systematic
functional and structural approach provides a first step to the characterization of new
therapeutic molecular targets for drug discovery. 1.Musher, D.M., Clin Infect Dis, 1992.
14: p. 801-7. 2.AlonsoDeVelasco, E., A.F. Verheul, J. Verhoef, and H. Snippe, Microbiol
Rev, 1995. 59: p. 591-603. 3.Greenwood, B., Philos Trans R Soc Lond B Biol Sci, 1999.
354: p. 777-85. 4.Poland, G.A., Vaccine, 1999. 17: p. 1674-9. 5.Thornsberry, C., et al.,
and D.F. Sahm, Antimicrob Agents Chemother, 1999. 43: p. 2612-23. 6.Tomasz, A., N
Engl J Med, 1995. 333: p. 514-5. 7.Doern, G.V., A.B. Brueggemann, H. Huynh, and E.
Wingert, Emerg Infect Dis, 1999. 5: p. 757-65. 8.LeMessurier, K.S., A.D. Ogunniyi, and
J.C. Paton, Microbiology, 2006. 152: p. 305-11.
POS-THU-344
CHARACTERISATION OF THE MECHANISM BY WHICH
THE BTB/POZ ZINC FINGER PROTEIN, ABRUPT
MEDIATES TUMOURIGENESIS IN DROSOPHILA
Turkel N.1, 2, 3, Brumby T.1, 2, Goulding K.1, Bolden J.1, Martin-Blanco E.3
and Richardson H.1, 2
1
Cell Cycle and Development, Peter MacCallum Cancer Centre,
Melbourne, VIC, Australia. 2Anatomy and Cell Biology, University of
Melbourne, Melbourne, VIC, Australia. 3Instituto de Biología Molecular
de Barcelona, Consejo Superior de Investigaciones Científicas, Parc
Científic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain.
Loss of function of the apico-basal cell polarity regulator, Scribble
(scrib-) in patches of cells (clones) leads to over-proliferation, rounding-
up of cells and multi-layering in the epithelium of 3rd instar larval eye
imaginal discs. However overgrowth of the scrib- clones is prevented by
Jun kinase (JNK) signalling induced by the surrounding wild type tissue.
Oncogenic versions of Ras(RasACT) or Notch(NotchACT) cooperates with
scrib- to block the apoptotic activity of JNK resulting in invasive tumours.
In a genetic screen, abrupt was identified as an oncogene, that when
over-expressed, cooperates with scrib- to promote tumourigenesis in
a similar manner of RasACT or NotchACT. Abrupt encodes BTB/POZ Zinc
Finger domain protein. Mammalian homologs of BTB/POZ Zn finger
domain proteins are implicated in cancer. We predict that identifying
the oncogenic mechanism of Abrupt will increase our understanding
of how this class of oncogenes can contribute to tumourigenesis
in mammals. Here, we show that over-expression of abrupt in scrib-
clones blocks JNK-mediated cell death and results in tumours that
massively overgrow and invade the adjacent brain lobes. Blocking JNK
signalling in the tumour can prevent invasion, but does not inhibit tumour
overgrowth. We found that atypical protein kinase C (aPKC) signaling
is responsible for overgrowth in abrupt scrib- tumours. In addition,
expression array and chromatin immunoprecipitation coupled with high-
throughput sequencing have been applied and revealed that abrupt act
as an oncogene by promoting the retention of a progenitor-like state in
antenna and eye disc.
POSTERS WEDNESDAY & THURSDAY
POS-WED-345
BIOFILM FORMATION AS CHARACTER TO SELECT
WINE SACCHAROMYCES CEREVISIAE STRAINS
Sidari R.1 and Howell K.S.2
1
Department of Scienze e Tecnologie Agro-Forestali e Ambientali,
Mediterranea University of Reggio Calabria, Via Feo di Vito, Reggio
Calabria, Italy. 2Department of Agriculture and Food Systems,
Melbourne School of Land and Environment, University of Melbourne,
Parkville, Victoria, Australia.
The wine yeasts Saccharomyces cerevisiae are selected according
to both technological and quality parameters to obtain starters with
oenological characters useful for winemaking. Searching for strains with
an ideal combination of oenological characteristics as well as the use of
novel oenological traits are two aspects of a continuous process toward
the improvement of winemaking. Biofilm growth describes the ability
of microorganisms to grow and behave as a multi-cellular community
with altered metabolism and reduced susceptibility to chemicals and
antiseptics. Early work on S. cerevisiae has identified the Flo11p as
protein involved in biofilm formation to various surfaces. The ability
to form biofilm is a character well studied in yeasts of medical interest
while very little fundamental research has occurred in wine yeasts.
Their ability to form biofilm has significant biotechnological relevance;
from attachment of yeasts to grape berries in the vineyard, to adhesion
and contamination of different surfaces in the winery. The present work
aimed to study the behaviour of 50 wine S. cerevisiae strains, already
selected for the common oenological characters, toward the aptitude
to form biofilm on plastic surfaces. This to give a contribution to the
knowledge of the biodiversity in the specie S. cerevisiae but chiefly to
propose the biofilm formation as a new possible character of selection
due the fact that yeast strains able to form biofilm could remain attached
to the various surfaces in winery becoming a problem to the management
of the winemaking in purity during years.
POS-WED-347
EFFECTS OF PROGESTERONE ON CELL GROWTH OF
T CELL LEUKEMIA CELLS
Kon A., Sato M. and Toyoda H.
Dept. of Clin. Mol. Genet., School of Pharm., Tokyo Univ. of Pharm.
and Life Sci.,1432-1 Horinouchi,Hacioji,Tokyo,Japan.
Progesterone (Pg) has been used for the treatment of patients with
breast and uterine cancer. It is also known to show antileukemic effects.
However, detailed mechanisms are yet to be understood. It has been
reported that the long-term survival rate of children and adult of acute
lymphoblastic leukemia (ALL) is 80% and 40%, respectively. A serious
side effect of current chemotherapy including bone marrow depression
is an important issue to improve quality of life (QOL) of patients. In order
to understand mechanisms involved in antileukemic effects, two cell
lines derived from Jurkat were used to study the effects of Pg on cell
proliferation. Two cell lines used in this study were E6-1 (parental cell
line) and A3 (sensitive to Fas-mediated apoptosis). We have previously
demonstrated that the antiproliferative effects of Pg was most prominent
in A3; an increase of cells in S-phase and decrease of those in G2/M
accompanied with the increase of cells in subG1 phase; and a high-
molecular weight DNA fragmentation was observed. In this study,
more than 20% of A3 cells showed Annexin-positive compared with
those of 8% of E6-1 cells. No DNA fragmentation was observed on an
agarose gel electrophoresis, but TUNEL positive cells were detected
only microscopically. An increased activity of caspase-3, -8 and -9 was
observed in A3 compared with that in E6-1. Furthermore, our results
demonstrated that membrane progesterone receptor α was activated
by Pg. Our results demonstrated that Pg administration inhibited cell
growth of T-cell derived leukemic cells mediated through membrane
progesterone receptor α, following membrane destruction, activation of
caspase-8, caspase-9 and caspase-3 along with high-molecular weight
DNA fragmentation.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 277
POS-THU-346
REGULATION OF THE ESSENTIAL M. TUBERCULOSIS
PEPTIDOGLYCAN-PRECURSOR FLIPPASE
Gee C.L.1, 4 , Papavinasasundaram K.G.3, Blair S.R.1, Baer C.E.1,
Falik A.M.2, King D.S.2, Sassetti C.M.2 and Alber T.1
1
Department of Molecular and Cell Biology, QB3 Institute, University
of California, Berkeley, CA 94720-3220. 2Howard Hughes Medical
Institute, University of California, Berkeley, CA 94720-3200.
3
Department of Molecular Genetics and Microbiology, University of
Massachusetts Medical School, 55 Lake Avenue North, Worcester,
MA 016551. 4Australian Synchrotron, 800 Blackburn Rd, Clayton, Vic
3168 Australia.
Receptor Ser/Thr kinases control broad aspects of physiology in bacteria,
but little is known about how the kinases regulate cellular pathways.
Among the Mycobacterium tuberculosis (Mtb) proteins essential in
an animal model is the flippase that delivers the peptidoglycan (PG)
precursor, lipid II, to the cell surface for incorporation into the cell wall.
In mycobacteria and several other actinomycetes, the flippase has
accessory domains, which presumably add functionality or regulate
the activity. We found that the Ser/Thr protein kinase, PknB, efficiently
phosphorylates the intracellular pseudokinase domain of the Mtb
flippase, and this single modification creates a binding site for the
forkhead associated (FHA) domain protein FhaA in vitro and in vivo.
To define the mechanisms of recognition, we determined the crystal
structures of the flippase extracellular domain and the intracellular
pseudokinase alone and in combination with FhaA. The extracellular
accessory domain is homologous to a galactose binding domain, while
the intracellular domain has a highly diverged, inactive protein-kinase
fold. These results suggest support a model in which extracellular PG
regulates PG synthesis by controlling assembly of a protein complex
containing the lipid II flippase.
POS-THU-348
SYNERGISTIC CYTOCIDAL EFFECTS OF DELPHINIDIN
WITH ARSENITE ON HUMAN LEUKEMIA CELL LINES
Yoshino Y., Yuan B., Okuzumi S., Takemae F. and Toyoda H.
Dept. of Clin. Mol. Genet., Tokyo Univ. of Pharm. and Life Sci., 1432-1
Horinouchi, Hachioji, Tokyo, Japan.
Arsenic trioxide (arsenite, As III ) is effective in the treatment of acute
promyelocytic leukemia (APL). Recently, it is demonstrated that As
III
induces cell death in malignant cells other than APL. However, the
relatively low sensitivity of most malignant cells to As III is shown,
suggesting that it may require higher and more toxic doses to show clinical
efficacy. Therefore, we initiated to seek reagents that exert synergistic
effects with As III to increase the efficacy of As III and reduce its dosage to
clinical achievable concentrations. Delphinidin, a major anthocyanidin
present in many pigmented fruits and vegetables, possesses cytocidal
effects on various tumor cells including acute myeloid leukemia and solid
tumor cells. Moreover, normal cells show low sensitivity to delphinidin-
induced cytotoxicity. Our study demonstrated that co-treatment with
delphinidin and As III induced synergistic cell death in As III -resistant
leukemic cell line, HL-60, but not in human peripheral blood mononuclear
cells (PBMCs) from healthy donors. Annexin V staining assay showed
that co-treatment with As III and delphinidin induced apoptosis along
with necrosis to HL-60. Importantly, the synergistic effect of As III and
delphinidin was selectively toxic for leukemia cells, and no combination
effect was observed in human PBMCs from healthy donors. Our data
suggested that delphinidin may extend the therapeutic spectrum of As III
in the treatment of malignant cells that are less sensitive to As III .
POSTERS WEDNESDAY & THURSDAY
POS-WED-349
PUTATIVE CIS-REGULATORY ELEMENTS IN HIGHLY
EXPRESSING SPERM CELL GENES OF RICE
Sharma N.1, Russell S.D.2, Bhalla P.L.1 and Singh M.B.1
1
Plant Molecular Biology and Biotechnology Laboratory, Melbourne
School of Land and Environment, University of Melbourne, Parkville,
Victoria 3010, Australia. 2Department of Botany and Microbiology,
University of Oklahoma, Norman, OK 73019 USA.
Male germ line augmentation in angiosperms is initiated via asymmetric
division within pollen grains, following which the smaller cell becomes
totally encased within a much larger vegetative cell. The generative cell
subsequently divides to give rise to two non-motile diminutive sperm cells
that take part in subsequent developmental advances. The sperm cells
have been very difficult to investigate because of their presence within
the confines of the vegetative cell. Recent availability of techniques for
the isolation of rice sperm cells and the fully annotated rice genome
sequence has allowed the transcriptional repertoire of sperm cells to
be characterized. Microarray gene expression data has identified a set
of genes that show unique or highly preferential expression in sperm
cells. This information has made it feasible to identify cis-regulatory
elements (CREs) that are conserved in sperm expressed genes and
are putatively associated with control of cell specific expression. The
aim of our study was to identify CREs associated with rice sperm cell
specific gene expression data. Our approach involves analysing one
Kb upstream regions of top 40 sperm cell co-expressed genes for
over-represented conserved and novel motifs. Analysis of upstream
sequences with SIGNALSCAN program on PLACE database, MEME
and Mclip tool has identified combinatorial sets of known CREs with
two novel motifs putatively associated with co-expression of sperm cell
specific genes. These motifs represent likely targets of transcriptional
factors regulating sperm cell gene expression and can then be used to
design experimental verification of regulatory elements.
POS-WED-351
MOLECULAR CHARACTERIZATION OF THE
DUPLICATED TAUROCYAMINE KINASE FROM
SCHISTOSOMA MANSONI
Laurent S.1, Awama A.1, Salvador A.2, Gouet P.3 and Marcillat O.1
1
Université Lyon 1, Institut de Chimie et Biochimie Moléculaires et
Supramoléculaires -UMR CNRS 8246. 2Université Lyon 1, Laboratoire
des Sciences Analytiques (LSA), UMR 5180 CNRS. 3Université Lyon 1,
Institut de Biologie et Chimie des Protéines -UMR CNRS 5086.
Guanidino kinases (GK) catalyze the reversible transfer of a phosphoryl
group between ATP and a guanidino compound. These enzymes,
named according to their specificity towards their guanidino substrate
–e.g. creatine kinase, arginine kinase, taurocyamine kinase (TK)-, play
a prominent role in energy management in a wide range of eukaryotic
cells. Schistosoma mansoni (Sm) is a human parasite responsible for
schistosomiasis, a tropical disease that affects about 200 million people
worldwide. Sm cercariae express a duplicated TK in which the two
structural domains (1-363 and 364-716) show high structure similarity
(53% sequence identity). SmTK has an original U-shaped structure,
in which the C-terminal side of the first domain interacts with the
N-terminal side of the second domain. This arrangement differs from
the classical banana-shape observed in dimeric and octameric GKs,
in which pairs of monomers show a twofold rotation axis. In spite of
this difference, each domain shows the characteristic GK fold with a
first α-helical sub-domain (about 100 amino acids) and a second larger
one (260 amino acids) with an anti-parallel β-sheet and long α-helices.
The enzyme shows its highest activity using taurocyamine (120U/mg)
although it is also able to use other guanidino substrates like guanidino
propionate. Schistosoma mansoni cercariae contain both taurocyamine
and guanidino propionate indicating that both phosphotaurocyamine
and phosphoguanidino propionate can form in vivo. The functional
implications of the structure will be presented.
Page 278 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010
POS-THU-350
A BACTERIAL ELONGATION FACTOR G HOMOLOG
EXCLUSIVELY FUNCTIONS IN RIBOSOME RECYCLING
IN THE SPIROCHAETE BORRELIA BURGDORFERI
Suematsu T.1, Yokobori S.2, Morita H.3, Yoshinari S.1, Ueda T.3, Kita
K.1, Takeuchi N.3 and Watanabe Y.1
1
Grad. Sch. of Medicine, University of Tokyo. 2Dept. Mol. Biol., Tokyo
University of Pharmacy and Life Sciences. 3Grad. Sch. of Frontier
Sciences, University of Tokyo.
Translation elongation factor G (EF-G) in bacteria plays two distinct
roles in different phases of the translation system. EF-G catalyzes the
translocation of tRNAs on the ribosome in the elongation step, as well as
the dissociation of the post-termination state ribosome into two subunits
in the recycling step. In contrast to this conventional view, it has very
recently been demonstrated that the dual functions of bacterial EF-G
are distributed over two different EF-G paralogs in human mitochondria.
In the present study, we show that the same division of roles of EF-G is
also found in bacteria. Two EF-G paralogs are found in the spirochaete
Borrelia burgdorferi, EF-G1 and EF-G2. We demonstrate that EF-
G1 is a translocase, while EF-G2 is an exclusive recycling factor. We
further demonstrate that B. burgdorferi EF-G2 does not require GTP
hydrolysis for ribosome disassembly, provided that translation initiation
factor 3 (IF-3) is present in the reaction. These results indicate that
two B. burgdorferi EF-G paralogs are close relatives to mitochondrial
EF-G paralogs rather than the conventional bacterial EF-G, in both their
phylogenetic and biochemical features.
POS-THU-352
IDENTIFICATION OF BENZOPYRAZINE DERIVATIVES
AS A NOVEL CLASS OF GRB7 ANTAGONISTS
Ambaye N.D., Gunzburg M., Wilce M.C.J. and Wilce J.A.
Department of Biochemistry and Molecular Biology, Monash
University, VIC 3800, Australia.
Growth factor receptor-bound protein-7 (Grb7) is an adapter protein that
functions as a downstream effector of growth factor mediated signal
transduction. Over-expression of Grb7 has been implicated in a variety
of tumors such as breast, blood, pancreatic, esophageal and gastric
carcinomas. Inhibition of Grb7 has been shown to reduce the migratory
and proliferative potential of these cancers, making it an attractive
therapeutic target. Starting with a known peptide antagonist, the
present work reports the application of a succession of computational
ligand design tools comprising a ligand shape based similarity search,
molecular docking and a 2D-similarity search to identify small molecular
antagonists of the Grb7-SH2 domain from the NCI chemical database.
Binding to the Grb7-SH2 domain was then experimentally tested using
melting point shift assays and isothermal titration calorimetry. Overall,
a total of 10 benzopyrazine based small molecular antagonists were
identified with affinity for the Grb7-SH2 domain. Representative
compounds tested using ITC were revealed to possess moderate binding
affinity in the low micromolar range. It is expected that the identified
antagonists will be useful additions to further explore the functional and
signaling studies of Grb7 protein.
POSTERS WEDNESDAY & THURSDAY
POS-WED-353
BIOINFORMATIC ANALYSIS OF SHEEP
CORNEODESMOSIN (CDSN) AND ALPACA AGOUTI
SIGNALLING PROTEIN (ASIP)
Bottomley S., Subramaniam N.S., Feeley N.L., Morgan E.F., Munyard K.A.
and Groth D.M.
School of Biomedical Sciences. Faculty of Health Sciences. Curtin
University. Western Australia.
There has been an exponential increase in protein sequence information
but a corresponding lack of experimental or structural information. This
has contributed to an information gap. Single Nucleotide polymorphisms
(SNPs), in particular, are the most common sequence variation with
non-synonymous SNPs (nsSNPs) most likely affecting protein structure
and function. It would be very time consuming, difficult, and costly to
experimentally verify the impact of all nsSNPs on protein structure or
function. Consequently, bioinformatic methods for predicting potential
effects of nsSNPs, or predicting the structure or function of a newly
discovered protein sequence, are potentially useful ways to fill the
information gap. In this case study we used freely available, and recently
developed, bioinformatic methods to predict the potential effect of
nsSNPs and aspects of protein structure and function in sheep CDSN
and alpaca ASIP. Bioinformatic analyses included protein secondary
structure, N-terminal signal peptides, transmembrane helices, mutation
analysis, and tertiary structure. The sheep CDSN and alpaca ASIP
proteins offered a convenient way to compare and contrast the various
bioinformatics methods because they differed in their structure, role,
cellular location, and knowledge base. The results suggest that the
application of bioinformatic methods can provide useful value-added
annotation of newly discovered protein sequences but can also provide
some contradictory or ambiguous results. Final interpretation of the
results of bioinformatic methods will necessarily involve a subjective
assessment based on knowledge of the underlying bioinformatics
algorithms, the biology of the protein, and the existing literature.
POS-WED-355
CROSSLINKED SEED POLYSACCHARIDES FOR
ONE-STEP PURIFICATION OF GALACTOSE-BINDING
LECTINS
Teixeira D.M.A.1, Braga R.C.1, Gadelha P.1, Reicher F.2, Moreira R.A.3
and MonteiroMoreira A.C.O.3
1
Universidade Federal do Ceara. 2Universidade Federal do Parana.
3
Universidade de Fortaleza.
Lectins, a special class of proteins with the particular property of interact,
specifically and reversibly, with carbohydrates and glycoconjugates,
without any modification in their covalent structure, can bind to cell
surfaces producing a series of modifications on their physiology. This
interaction is dependent on the OH orientation/substitution in the C2, C3
and C4 of the sugar ring and, thus, can be grouped in Glu-, Man-, Gal-,
GlNAc-, GalNAc-binding lectins, and some of them show an even higher
specificity, binding to only one of the anomers. Affinity chromatography
on sugar containing matrices is the best way to isolate lectins whose
specificity dictates the choice of ligand. While commercially available
matrices present a mixture of the two anomers, natural glycoconjugates
synthesized via enzyme driven reactions, presents only one anomer.
This is the case of the endospermic galactomannans [alfa-(1-6)-D-
galactose-substituted-mannan] and seed galactoxyloglucans [beta-(1-
6)-D-galactose-substituted-xyloglucan]. 12 seed galactomannans and
5 seed xyloglucan were cross-linked by alkaline epichlorohydrin. After
exhaustive washing, the cross-linked polysaccharides were packed
in a column, to which the excess pure alfa- and beta-linking lectins
were applied. After elution of the non-retained proteins, the column
was washed with 0.1 M D-galactose. When Artocarpus integrifolia and
Artocapus incisa alfa-D-galactose-binding seed lectins were applied to
these columns, only the galactomannan columns were able to retain
them. When, on the other hand, the Abrus precatorius beta-D-galactose-
binding seed lectin was retained only in the xyloglucan columns. These
results suggest that these natural polysaccharides can, thus, be used as
affinity matrices, not only for the isolation of lectins but, more important,
also to determine their fine anomeric specificity. Supported by: FINEP,
CNPq, RENORBIO, FUNCAP.
OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010 Page 279
POS-THU-354
RHEOLOGICAL BEHAVIOR OF DAIRY DESSERTS
PREPARED WITH CAESALPINIA PULCHERRIMA SEED
GALACTOMANNAN
Arcanjo S.R.S.1, Holanda M.L.2, Beserra J.F.3, Oliveira A.M.C.3, Paula
R.C.M.2, MonteiroMoreira A.C.O.3 and Moreira R.A.3
1
Universidade Federal do Piaui. 2Universidade Federal do Ceara.
3
Universidade de Fortaleza.
Seed galactomannans, having a polymeric main chain of (1 →
4)-linked beta-D-mannopyranosyl residues, substituted at O-6 by an
alfa-D-galactopyranosyl units, are widely used in the food industry as
thickening and stabilizing agents. They have lower cost as compared
with traditional thickening agents and their synergistic interactions
raised their industrial importance. This study evaluated the rheological
behavior of dairy desserts prepared with Caesalpinea pulcherrima seed
galactomannan blended with starch and milk. From a basic mixture (A)
of milk (16.37%), sugar (13.35%), starch (3.35%) and galactomannan
(1.71%), different formulations, (B) galactomannan (0.49%), (C) starch
(0.65%), (D) sugar (6.63%) and (E) milk (9.63%) were used. The curves
obtained at the shear rate: 0.08 to 300 s-1, at 25 °C were used to calculate
the flow behavior index (n) and consistency (k). The formulations were
adjusted to the Ostwald model-de-Waele (R 2 = 0.97-0.99) exhibiting
pseudoplastic behavior with n varying from 0.12 to 0.17. The formulation
A had k=102.3 Pa sn while the formulations C, D and E showed k values
equal to 83.6, 151.5 and 223.4 Pa sn, respectively. Lower concentrations
of starch, milk and sugar, caused positive synergism in C and a negative
interference of the polymers of milk (protein) and sugar in E and D,
respectively, in starch and galactomannan interaction when compared
to A. Given these results we may suggest that the Caesalpinea
pulcherrima galactomannan shows a pseudoplastic behavior, and
variability of their consistency indices in the presence of biopolymers
such as starch and protein. Supported by: FINEP, CNPq, RENORBIO,
FUNCAP.
POS-THU-356
MOLECULAR CLONING AND CHARACTERIZATION
OF STRICTOSIDINE SYNTHASE OF MITRAGYNA
SPECIOSA
Zainal Z.1, 2 , Jumali S.S.1, Baharum S.N.1, Mohd Said I.1 and Ismail I.1
1
Institute of System Biology, Universiti Kebangsaan Malaysia. 2School
of Biosciences and Biotechnology, Universiti Kebangsaan Malaysia.
Mitragynine is one of the most dominant indole alkaloids present in
Mitragyna speciosa leaves, found in most species of Rubiaceae. This
alkaloid is believed to be synthesized via condensation of the amino acid
derivative tryptamine and secologanine by the action of Strictosidine
synthase (STR). The cDNA clone encoding STR from M. speciosa
was cloned through RT-PCR. The clone is a full length cDNA with a
size of 1257 bp contained an open reading frame of 1056 bp starting
from base pair 19 to 1077 and termed as STRMs1. Sequence analyses
showed that STRMs1 (accession HM543187) had high homology with
other STRs from some TIA-producing plants. The deduced acid amino
sequence is 352 residues with a predicted molecular weight of 30 kDa
and isoelectric point of 5.04. Southern blot performed showed that only
one copy of STR is present in the genome of M. speciosa. Expression
pattern on different tissues were tested using RT-PCR revealed that
beside leaf, the expression was also detected in roots, stem and flower.
Expression profiles under plant defense signal using salicylic acid
(SA) was investigated on leaf tissues and the results showed that the
transcript of STRMs1 were detected before and after treatment with
salicylic acid. Result obtained from phylogenetic analysis suggested
that STRMs1 is the most evolved protein among other STRs. However,
the 3D prediction of STRMs1 showed that there are alpha helices and
beta propeller structures which remained conserved to other STRs.
POSTERS WEDNESDAY & THURSDAY

POS-WED-357
Synergistic effects of ascorbic acid (AA)
and thiazolidinedione (TZD) on secretion of
high molecular weight adiponectin
Webster, J., Rose, F., Richards, A. and Whitehead, J.
Mater Medical Research Institute and University of Queensland

Adiponectin is an insulin-sensitizing/anti-inflammatory adipokine that

circulates in a variety of stable multimers (low and high molecular
weight (LMW and HMW)). A characteristic of obesity and type 2
diabetes (T2D) is hypoadiponectinaemia, which reflects a specific loss
of the more metabolically active HMW form. The efficacy of the TZD
class of insulin-sensitizing drugs correlates with increases in HMW
adiponectin. Formation of HMW adiponectin is dependent on post-
translational modifications including hydroxylation of conserved lysine
residues, by Lysyl Hydroxylase (LH). Low levels of AA, a co-substrate
for LH, are observed in obesity and T2D raising the possibility that
reduced AA may contribute to the specific loss of HMW adiponectin.
Our aim was to determine if AA supplementation, alone/or in
combination with TZD, would increase production of HMW adiponectin.
Human SGBS adipocytes were cultured -/+ AA -/+ TZD for 24 h. AA
supplementation increased secretion of HMW adiponectin (1.7 fold)
without affecting adiponectin (mRNA) expression or total adiponectin
secretion. TZD significantly increased adiponectin expression (3 fold)
and total adiponectin secretion (1.4 fold), however it did not increase
HMW secretion. In combination, AA + TZD increased adiponectin
expression (3 fold) and secretion of total (2 fold) and HMW (5 fold)
adiponectin. Similar beneficial effects were observed in cells treated
with the pro-inflammatory cytokine TNFα. We conclude that AA
supplementation can increase secretion of HMW adiponectin and that
this effect is synergistic with TZD. Our findings raise the possibility
that differences in AA levels may contribute to (i) the variability in
adiponectin multimer profiles and (ii) the efficacy of TZD in humans
and (iii) establish a platform for clinical trials.

Page 280 OzBio2010 Combined Conference s Melbourne s September 26 - October 1, 2010