15.

6 Biogenesis and Evolutionary Origin of Mitochondria Presence of mitochondrial DNA and of their own system of protein synthesis confers a relative autonomy to these organelles. Each mitochondrion becomes from a pre-existing mitochondrion through division. Even though some of the new components are synthesised inside the mitochondria, growth of mitochondria is ensured mainly by import of lipids and proteins from the cytosol. Transport of proteins into mitochondria is performed by two principal protein systems of independent transport, one located in the outer membrane (TOM), and the other in the inner membrane (TIM). These two transport systems may function independently but they also may be occasionally linked during the protein transport across the two mitochondrial membranes. The proteins to be imported have signal sequences either at the N-terminal end or inside the polypeptide chain, which target the proteins to their final destination. First of all, based on a matrix-location signal sequence the protein arrives into the matrix; if its destination is another compartment of the mitochondrion, the first signal sequence is removed and then a second signal sequence becomes active. The origin of this organelle is not well known yet. The presence of the circular DNA, the protein and ATP biosynthesis, the structure and the chemical composition similar to those of prokaryotes made the endosymbiotic theory the most accepted one. According to this hypothesis, a primitive mitochondrion evolved from a small aerobe bacterium that was incorporated by endocytosis in a bigger, anaerobe cell. In time, the invading bacterium lost some of its genes and became specialized in the ATP synthesis and also dependent of the host cell, which in turn supplied it the necessary metabolites. Because mitochondria are similar in all animal and plant organisms, the endocytosis event that led to the development of mitochondria is presumed to have occurred when oxygen entered the atmosphere in substantial amounts, before animals and plants separated. 15.7 Implications of Mitochondria in Pathology

Mitochondria are involved in two types of pathological processes: genetic mitochondrial diseases and other pathological processes. A. Genetic mitochondrial diseases. Mitochondrial diseases may be the result of mutations of mtDNA, defective transcription or translation of mitochondrial proteins encoded by nuclear genes, mutations in nuclear proteins that regulate the mitochondrial protein unfolding and passage through
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the mitochondrial membranes, etc. As a consequence, many mitochondrial proteins like components of the respiratory chain or ATP-synthase complex may undergo mutations. Generally, in this type of diseases, mitochondria are very large, but energetically inefficient they cannot supply enough ATP required by cells. The most sensitive tissues to defective mitochondrial functions are those that require high amounts of ATP: skeletal muscle, cardiac muscle, and central nervous system. For this reason, the genetic mitochondrial diseases are also called mitochondrial encephalomyopathies. They have the debut in young or adult patients. In other situations the liver and/or the kidney could be also affected, and the diseases are called mitochondrial cytopathies with debut in childhood. The diagnostic of such diseases is very difficult to be established and requests complex investigations: measurements of plasma concentrations of different substances (lactic acid), electron microscopy examination of mitochondria in biopsies, study of isolated mitochondria from biopsies for analysing the respiratory chain components, or sequencing the mitochondrial DNA. Each human cell has thousands copies of mitochondrial DNA (mtDNA). Both the normal and mutated mtDNA are inherited maternally. The expression of disease appears to be related to the amount of mutated mtDNA, but not all DNA molecules in a mitochondrion have mutations, and also not all mitochondria in a cell or in an organ have defective DNA. Thus by the processes of mitochondrial division and of cell division (during embryogenesis and during the adult life as well) could result both normal and affected cells. This aspect confers a high complexity to the mitochondrial diseases. Several examples of mitochondrial genetic diseases: Lebers disease (Lebers Hereditary Optic Neuropathy LHON): is an atrophy of optic nerve with debut at any age, which can lead to a rapid blindness (one of the risk factors is exposure to carbon monoxide, including by active or passive smoking). MERRF syndrome (Myoclonic Epilepsy with Ragged-Red Fibres): appears in childhood and is characterised by epilepsy and specific aspect of muscle fibres. MELAS syndrome (Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes) with debut between 5-15 years. Luft disease is produced by uncoupling of oxidative phosphorylation and consequently the energy resulted by cellular oxidations is released as heat. Symptoms are hyperthermia and intense sweat. B. Other pathological processes in which mitochondria are involved Nowadays it is considered that mitochondria are involved in all diseases and any cell gets sick without a proper source of energy. Moreover, because the mtDNA is not protected like the nuclear DNA, it is
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exposed to the action of reactive oxygen species (such as free radicals) produced in the mitochondrial matrix in redox reactions. The reactive oxygen species were proved to be involved in the pathogenesis of all diseases including those with a high mortality (cardiovascular diseases, cancer, and diabetes). Finally, mitochondria are involved in the first steps of programmed cell death apoptosis.

CHAPTER 16. Extracellular Matrix and Cell Adhesion

16.1 Structure-function relationship Extracellular matrix is a network of macromolecules that occupies intercellular space in tissues and that continues with glycocalyx. Some specialized structures are formed by the extracellular matrix: basal membranes, cartilages, tendons, and bones. Frequently the term of conjunctive tissue is used for the extracellular matrix together with the cells within it: fibroblasts, macrophages and mast cells. The conjunctive tissue is abundant in skin and in bones, and is present in a very few amount in brain. Functions of the extracellular matrix: a) stabilizes the physical structure of tissues; b) role in lubrication, attenuates mechanic shocks and provides tissue elasticity; c) role in cell to cell adhesion; d) influences shape of cells, migration and development of cells during embryogenesis; e) metabolic and nutritional role, because it facilitates the diffusion of nutrients; f) role in immunity; g) role in cellular differentiation and maintaining the characters of differentiated cell; the cultured undifferentiated cells remain undifferentiated in the absence of extracellular matrix; 16.2 Chemical composition and medical implications The extracellular matrix is a network of fibrous proteins immersed in hydrated polysaccharide gel. The proteins confer strength and elasticity, and the gel allows the diffusion of nutrients. All the components of the extracellular matrix are synthesized and secreted by the cells within it. The polysaccharides of the extracellular matrix are mucopolysaccharides or glycosaminoglycans. The glycosaminoglycans are attached on proteins and form proteoglycans that
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have random coiled conformations, and occupy large spaces being highly hydrated. These large molecules consist of about 90-95% glucides. The proteoglycans are degraded in lysosomes. The lack of a lysosomal enzyme involved in the degradation of mucopolysaccharides drives to their accumulation in organism and the mucopolysaccharides are eliminated in urine. These genetic lysosomal diseases are known as mucopolysaccharidoses such as syndromes Hurler, Hunter, Morquio, San Filippo. They are characterized by the accumulation of mucopolysaccharides in the conjunctive tissue from face and tongue driving to their swelling, hepato- and splenomegaly, skeletal abnormalities and mental retardation. The hyaluronic acid has functions in the embryo development and the healing of wounds. The most important proteins of the extracellular matrix are collagen, elastin, fibronectin, laminin and the cell adhesion molecules. Collagen is the most abundant protein in mammals (25% of all proteins). In consists of three polypeptide chains (!-chains) that form a triple helix. There are 7 types of !-chains and their combinations result in 5 different types of collagen molecules. The collagen molecule is synthesized in RER as !-prochains; three !-prochains associate and form the pro-collagen molecule, that follows the cellular secretion pathway, and outside the cell some peptides from the ends of molecules are removed, and in this way the tropocollagen is formed. The molecules arrange parallel and form covalent bonds, these are microfibrils; the microfibrils arrange in fascicles and form collagen fibrils. The fibrils arrange in fascicles and form the collagen fibers that are visible in the light microscope. The collagen molecules contain high amounts of glycine, and proline and also hydroxyproline and hydroxylysine. Stability of the molecule is given by hydrogen bonds between hydroxyproline and hydroxylysine. In deficit of vitamin C the proline residues are not hydroxylated. As consequence the collagen molecules are rapidly degraded and the fragility of skin and mucosa appears, in the disease named scorbute. This disease, in a mild form, is very frequent in spring and winter, especially in old people, that dont eat fresh fruits and vegetables. Elastin is a long molecule rich in proline and glycine (not hydroxylated) that is secreted in the extracellular space. It takes a random coiled shape, forming a network that changes its form depending on mechanical stress. Elastin is present in high amounts in skin, lungs, blood vessels etc. Molecules of cell to cell adhesion. Intercellular adhesion is measured through the force necessary to detach two cells. There are specific proteins of the intercellular adhesion. The molecules of cell adhesion are grouped in families. The selectins family is formed by proteins that link to glucidic groups of cells or extracellular matrix. The selectins have a transmembrane domain, a ctytosolic domain and an extracellular one. The later is very large and at its
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level the molecule interacts with other glucidic groups. The selectins specifically interacts with vegetal glycoproteins named lectins. The immunoglobulins family contains cell adhesion molecules that have an extracellular domain similar to that of antibodies. The cadherins family mediate interactions between cells of the same type. In the extracellular segment they have sites for calcium binding. There are cadherins in the enterocyte plasmalemma, the nervous system cells etc., especially in cytoplasmic plaques of spot desmosomes. Another family of adhesion molecules, independent of Ca, consists of transmembrane glycoproteins or linked to membrane by a phospholipid anchor, that mediate interactions between different types of cells. The integrins family mediate interactions between cells or cells and matrix components. They have a region with !-helix formed by hydrophobic aminoacids in the lipid bilayer, and two larger domains outside of membrane. The intracellular region is linked to cytoskeleton. Fibronectin is a glycoprotein of 220 kDa with 5% glucides, made of two liniar subunits linked at one of their extremities. It is located on the cell surface, in the intercellular space, in blood and other fluids. The cultured fibroblasts have fibronectin on their surface. The malignant fibroblasts have only a few fibronectin molecules and their adhesion is very weak; because of this, the malignant cells easily detach from primary tumours and invade the tissues, forming metastases. In cultures, the malignant cells weakly adhere to support and proliferate in more layers, not like the normal cells that proliferate in one layer and stop the multiplication in the moment of contact with the wall of flask. If fibronectin is added to malignant fibroblasts, they change their shape, become star-shaped and more adherent to support, but the malignant proliferation continues. 16.3 Basal lamina as specialization of the extracellular matrix Basal lamina is a specialized structure located under the epithelial cells, between two monocellular layers in the renal glomeruli, or surrounding individual cells muscle cells, adipose cells, Schwann cells. The basal lamina is secreted by the cells lying on it and contains collagen (type IV), proteglycans, laminin and fibronectin. Basal lamina has different functions: a) it is a semi-permeable filter in the renal glomerulus; in the diabetes and nephrotic syndrome the basal lamina is thickened; participates in the interactions of tissues with bacteria in pielonephrytis and other renal infections; b) it has a barrier function in epithelia, preventing their cross by fibroblasts but allowing macrophages and lymphocytes to pass;
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c) it has a role in regeneration of tissues after lesions, after destruction of muscles, nerves and epithelia. In the case of muscle cell the basal lamina has role in the reparation of the neuron-muscle junction and of axon.

CHAPTER 17. Cell Recognition

Cell recognition is the property of cells to specifically interact with each other. Cells are complex coordinated units that are integrated in more complex levels of organizations. During transfusions and transplants the foreign cells are recognized and destroyed by phagocytosis. The attachment of sperm to egg is a recognition process. If a chicken embryo is experimentally dissociated with trypsin into individual cells, after several hours the cells in suspension form an aggregate. When mesoderm and ectoderm cells are mixed, first of all a spherical cellular conglomerate is formed, and later (after 1-2 days) the cells separate and migrate, the mesoderm cells in interior, and the ectoderm cells in exterior. The normal cells in culture multiply in a monocellular layer and stop the proliferation when they reach the flask wall; this is called contact inhibition, and is a cellular recognition process. On the other hand, the malignant cells or the transformed cells dont display this phenomenon of contact inhibition. When are cultured in Petri flasks they continue to grow and divide even after a continuous cellular monolayer is formed, resulting in a multilayer culture. The malignant or transformed cells have another important property. They can be agglutinated by vegetal proteins called phytohaemaglutinins, process that indicate changes at the level of the membrane glycolipids and glycoproteins of the malignant or transformed cells.