Cell Adaptations to Stress (Final.pdf)

29 CELL ADAPTATIONS TO STRESS ILOs By the end of this lecture, students will be able to 1. Compare adaptive responses as regards Etiology, pathogenesis, and morphology. 2. Classify adaptive responses into physiological or pathological and reversible or irreversible....

29 CELL ADAPTATIONS TO STRESS ILOs By the end of this lecture, students will be able to 1. Compare adaptive responses as regards Etiology, pathogenesis, and morphology. 2. Classify adaptive responses into physiological or pathological and reversible or irreversible. 3. Relate adaptive responses to most common corresponding clinical conditions. Adaptations are reversible changes in the size, number, phenotype, metabolic activity, or functions of cells in response to changes in their environment. Adaptations may take several distinct forms; hypertrophy, hyperplasia, atrophy and metaplasia. 1. Cells capable of division may respond to stress by undergoing both hyperplasia and hypertrophy, whereas nondividing cells as myocardial fibers increase tissue mass due to hypertrophy only. Hypertrophy and hyperplasia may coexist, occur due to the same triggers and both contributing to increased organ size. 2. If the limits of adaptive responses are exceeded or if cells are exposed to damaging insults, deprived of critical nutrients, or compromised by mutations that affect essential cellular functions, a sequence of events follows that is termed cell injury, whether reversible or irreversible. 1. Hypertrophy: Hypertrophy is an increase in the size of cells that results in an increase in the size of the affected organ. The hypertrophied organ has no new cells, just larger cells. Pathogenesis: The most common stimulus for hypertrophy of skeletal and cardiac muscle is increased workload. Muscle cells respond by synthesizing more protein, increasing production of growth factors and genetic modulation of some muscle proteins, leading to increasing the number of myofilaments per cell and increasing the amount of force each myocyte can generate and thus the strength and work capacity of the muscle as a whole. Hypertrophy can be classified into; physiologic (due to increased functional demand) or pathologic (due to stimulation by hormones and growth factors). Physiologic hypertrophy. Example; Uterine hypertrophy during pregnancy: The massive physiologic growth of the uterus during pregnancy; is stimulated by estrogenic hormone signalling through oestrogen receptors that eventually result in increased synthesis of smooth muscle proteins and an increased cell size. The bulging muscles of bodybuilders engaged in “pumping iron” result from enlargement of individual skeletal muscle fibers in response to increased workload and increased cellular demand. Page 1 of 3  Pathologic hypertrophy. The striated muscle cells in the heart and skeletal muscles have only a limited capacity for division and respond to increased metabolic demands mainly by undergoing hypertrophy. Example; Concentric left ventricular hypertrophy of heart in response to pressure overload due to increased peripheral resistance to cardiac pumping of blood. this occurs in case of systemic hypertension, or aortic valve disease. 2- Hyperplasia Hyperplasia is an increase in the number of cells in an organ or tissue in response to a stimulus. Hyperplasia can only take place if the tissue contains cells capable of dividing, thus increasing the number of cells. It can be physiologic or pathologic. Mechanism of Hyperplasia Hyperplasia is the result of growth factor–driven proliferation of mature cells and activation of signalling pathways that stimulate cell proliferation by increasing output of new cells from stem cells. Physiologic hyperplasia occurs whenever there is a need to increase functional capacity of hormone sensitive organs, or when there is need for compensatory increase after damage or resection. 1. Hormonal hyperplasia: the proliferation of the glandular epithelium of the female breast at puberty and during pregnancy, and lactation usually accompanied by enlargement (hypertrophy) of the glandular epithelial cells. 2. Compensatory hyperplasia: liver regeneration usually occurs in individuals who donate one lobe of the liver for transplantation, the remaining cells proliferate so that the organ soon grows back to its original size. 3. The bone marrow hyperplasia: in response to a deficiency of mature blood cells in the setting of blood donation, chronic bleeding, acute blood loss, haemolysis or high altitudes. Pathologic hyperplasia. Most forms of pathologic hyperplasia are caused by excessive or inappropriate actions of hormones or growth factors acting on target cells. Endometrial and Breast hyperplasia, under the effect of increased estrogen [whether due to excessive hormone production by a tumor of due to exogenous drug intake], a common cause of abnormal uterine bleeding and breast mass, respectively. NOTE; both may turn malignant. Benign prostatic hyperplasia: in response to hormonal stimulation by imbalanced estrogen and androgens in old aged males. 1. Atrophy Atrophy is a reduction in the size of an organ or tissue due to a decrease in cell size and number. Atrophy can be physiologic or pathologic. Mechanisms of Atrophy 1- Decreased protein synthesis Page 2 of 3  2- Increased protein degradation 3- Increased autophagy with presence of intracytoplasmic autophagic granules, containing debris from degraded organelles. Physiologic atrophy occurs during normal development as atrophy of embryonic structures, as notochord and thyroglossal duct during fetal development and uterine atrophy after menopause or reduction of uterine size after delivery. Pathologic atrophy may be local or generalized. Example; 1. Disuse atrophy; caused by decreased workload, e.g., following complete bed rest due to fractured bone with prolonged immobilization. It is reversible once activity is resumed. 2. Loss of innervation (denervation atrophy). Damage to the nerves leads to atrophy of the muscle fibers supplied by those nerves since metabolism and function of skeletal muscle are dependent on its nerve supply. (Irreversible) 3. Diminished blood supply. chronic ischemia as a result of slowly developing arterial occlusion results in tissue atrophy. Example is senile atrophy of brain, and renal atrophy mainly because of reduced blood supply as a result of atherosclerosis. (Irreversible) 4. Inadequate nutrition. Profound protein-calorie malnutrition (marasmus) is associated with the utilization of skeletal muscle proteins as a source of energy after other reserves such as adipose stores have been depleted: Cachexia. 5. Loss of endocrine stimulation. Postmenopausal loss of estrogen stimulation results in atrophy of the endometrium, vagina, and breast. The prostate atrophies following chemical or surgical castration (e.g., for treatment of prostate cancer). 6. Pressure atrophy. Prolonged tissue compression causes atrophy. 2. Metaplasia Metaplasia is a reversible change in which one differentiated\mature cell type (epithelial or mesenchymal) is replaced by another mature cell type. It occurs often in response to chronic irritation, or when one cell type is sensitive to a particular stress. These cells are replaced by another resistant cell type that is better able to withstand the adverse environment. Mechanisms of Metaplasia, it results from stimulation and reprogramming of local tissue stem cells or colonization by differentiated cell populations from adjacent sites. Examples; Squamous metaplasia of respiratory bronchial epithelium, gall bladder \ gland duct epithelium under chronic irritation by smoking or stones or due to Vit A deficiency. Squamous metaplasia of transitional epithelium of urinary bladder under chronic irritation by stone or bilharziasis eggs. Columnar cell \intestinal metaplasia of squamous esophageal epithelium under irritation by acidic gastric juice Page 3 of 3  30 REVERSIBLE RESPONSE TO INSULTS AND CYTOPLASMIC ACCUMULATES ILOs By the end of this lecture, students will be able to 1. Emphasis on the sequence of events in cell injury and cell death [adaptation response, reversible and irreversible cellular injury]. 2. Discuss reversible subcellular changes in case of mild to moderate cell injury 3. Identify cut-off subcellular changes that transform a reversible cell injury to an irreversible change. 4. Discuss biochemical mechanisms and morphological alterations associated with necrosis. REVERSIBLE CELL INJURY Reversible cell injury is characterized by functional and structural alterations in early stages or mild forms of injury, which are correctable if the damaging stimulus is removed. Two features are consistently seen in reversibly injured cells; 1- HYDROPIC CHANGE; Earliest alterations in reversible injury in response to non-lethal injury, include; A. Generalized swelling of the cell and its organelles B. Blebbing of the plasma membrane C. Detachment of ribosomes from the endoplasmic reticulum (ER) D. Clumping of nuclear chromatin. 2- FATTY CHANGE; Occurs in organs that are actively involved in lipid metabolism (e.g., liver). It results when toxic injury disrupts metabolic pathways and leads to rapid accumulation of triglyceride filled-vacuoles within cytoplasm. Cellular swelling\hydropic change \ vacuolar degeneration. Causes. Oxygen deficiency, which interferes with mitochondrial oxidative phosphorylation. Radiation and toxins leading to mitochondrial damage. Both lead to ATP depletion. Pathogenesis. Results from influx of water within cytoplasm caused by failure of the ATP-dependent Na+-K+ plasma membrane pump, which in turn occur as a result of ATP depletion. Morphology Grossly, the affected organ becomes pale, with increased turgor, and increased weight. Microscopic features, Small clear vacuoles may be seen within the cytoplasm (representing distended ER). The cytoplasm appears red (eosinophilic) when stained with hematoxylin and Page 1 of 3  Ultrastructure features, Loss of plasma membrane structures; cilia and microvilli, and develops cytoplasmic “blebs” at apical cell surfaces. Mitochondria, ER and Golgi complex are swollen or dilated. Accumulation of myeline figures; phospholipid-derived from damaged cell membranes. 2+ Proteins and Ca precipitate in the cytosol and in organelles, especially mitochondria leading to mitochondrial densities. 2-Steatosis (Fatty Change),abnormal intracellular fat accumulation Means excessive, abnormal accumulations of triglycerides within parenchymal cells. Causes; Alcohol abuse, diabetes mellitus, obesity, toxins, protein malnutrition, and anoxia. Location: often seen in the liver (fatty liver) because it is the major organ involved in fat metabolism, as well as the cardiac muscle, skeletal muscle, and kidney. Best example is fatty change of the liver. Morphology: Grossly, a yellow, greasy, soft organ. Histologically, intracellular clear fat vacuoles, the nucleus is shifted against the cell membrane. Irreversible cell injury\ CELL DEATH There are two principal types of cell death, necrosis and apoptosis, which differ in their mechanisms, morphology, and roles in physiology and disease. Lately, autophagy is becoming the third mode of cell death. Necrosis is almost always pathological and is associated with severe mitochondrial damage with depletion of ATP and rupture of lysosomal and plasma membranes. On the other hand, apoptosis is a physiological process, that may have some pathological aspects, and it has different pathways Necrosis is the consequence of severe injury that irreparable damages so many cellular components that the cell simply “falls apart.” It is usually associated with severe mitochondrial damage with depletion of ATP and rupture of lysosomal and plasma membranes. Causes: Loss of oxygen supply (ischemia) Exposure to toxins Microbial infection Burns and trauma Other forms of chemical and physical injury Pathogenesis: Mitochondrial damage with production of reactive oxygen species (ROS) leading to peroxidation of phospholipids in cell membranes and organelles membranes, nuclear damage and ATP depletion. Page 2 of 3  Leakage of lysosomal enzymes into the cytoplasm due to severe damage to cell membranes → digest the cell → denaturation of cellular proteins. Damaged plasma membrane →Leakage of cellular contents into the extracellular space → elicit a host reaction (inflammation). Leakage of intracellular proteins; as damage-associated molecular patterns (damps); ATP (released from damaged mitochondria), uric acid (a breakdown product of DNA) → recognized by macrophages → trigger phagocytosis of the debris as well as the production of cytokines that induce inflammation. Inflammatory cells produce more proteolytic enzymes, and the combination of phagocytosis and enzymatic digestion usually leads to clearance of the necrotic cells. MORPHOLOGY Cytoplasmic changes Increased eosinophilia in H&E stains, due to the loss of cytoplasmic RNA (which bind the blue dye hematoxylin) and accumulation of denatured cytoplasmic proteins (which bind the red dye eosin). Glassy homogeneous appearance as a result of the loss of glycogen particles. Moth -eaten and vacuolated due to enzymatic digestion of cell’s organelles By electron microscopy, necrotic cells are characterized by discontinuities in plasma and organelle membranes, marked dilation of mitochondria with the appearance of large amorphous densities, intracytoplasmic myelin figures, amorphous debris, and aggregates of fluffy material representing denatured protein. Nuclear changes appear in one of three patterns, all due to breakdown of DNA. Karyolysis, the basophilia of the chromatin, may fade, reflecting loss of DNA because of enzymatic degradation by endonucleases. A second Pyknosis, (also seen in apoptotic cell death) nuclear shrinkage and chromatin condenses into a dense, shrunken basophilic mass. Karyorrhexis, a pyknotic nucleus undergoes fragmentation, then totally disappears Within 1 or 2 days. Page 3 of 3  31 IRREVERSIBLE RESPONSE TO CELL INJURY: “NECROSIS\ APOPTOSIS” ILOs By the end of this lecture, students will be able to 1. Correlate different patterns of necrosis as coagulative, liquefactive, caseous, gangrenous, fatty fibrinoid necrosis to corresponding clinical situations. 2. Evaluate mechanisms of apoptosis in physiological and pathological removal of dead cells in relation to some clinical conditions. 3. Differentiate between morphological and diagnostic features of apoptosis vs necrosis. Biochemical changes in necrosis and its clinical applications Value of early biochemical testing in diagnosis of cell necrosis Necrosis is early associated with leakage of intracellular proteins through damaged plasma membranes into the circulation. This set the biochemical basics of early detection of tissue –specific cell injury by blood testing. It usually occurs very early before ultrastructural, histological or gross morphological changes. Examples; Damaged cardiac muscle cells express cardiac-specific variants of the contractile protein troponin. Damaged Liver cells express transaminases. Damaged Bile duct cells express alkaline phosphatases. Phenomena consistently characterize irreversibility; The inability to reverse mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even after resolution of the original injury Profound disturbances in membrane integrity with leakage of lysosomal enzymes. Nuclear changes. Patterns of necrosis 1. Coagulative necrosis is a form of necrosis in which the architecture of dead tissue is preserved for a span of at least some days. Caused due to ischemia or impairment of arterial supply. The localized area of coagulative necrosis is called an infarct. Pathogenesis The injury denatures structural proteins and enzymes and so blocks the proteolysis of the dead cells leading to intensely eosinophilic cells with distinct cellular outlines and indistinct or reddish nuclei that persist for days or weeks. The necrotic cells are broken down by the Page 1 of 4 action of lysosomal enzymes derived from infiltrating leukocytes, followed by removal of cell debris by phagocytosis. Clinical examples; Necrosis of myocardial cells [ myocardial infarction under the effect of ischemia due to inadequate blood supply by narrowed \occluded coronary arteries in context of atherosclerosis], and kidney [ renal infarction in similar condition to the heart]. 2. Liquefactive necrosis; characterized by release of hydrolytic enzymes that digest the dead cells, resulting in transformation of the tissue into a viscous liquid. Clinical examples; Abscess with pus formation Focal bacterial or fungal infections that stimulate the accumulation of leukocytes and the liberation of digestive enzymes transforming the necrotic material into creamy yellow pus containing dead tissue cells, and bacteria. Hypoxic death of the central nervous system cells often manifests as liquefaction of the necrosed tissue for unknown reasons. 3. Gangrenous necrosis a clinical terminology describing necrosis with superadded putrefaction infection. It is usually applied to an organ, that has lost its arterial blood supply and has undergone necrosis (typically coagulative necrosis) involving multiple tissue planes (giving rise to so-called gangrene e.g., dry gangrene of foot due to diabetes mellitus and wet gangrene of small intestine due to mesenteric vascular occlusion). 4. Caseous necrosis, It combines features of both coagulative and liquefactive necrosis, and is encountered in foci of tuberculous infection. Occurs in reaction of lipid content of cell wall of these organisms. Grossly, it resembles dry cheese and is soft, granular and yellow. On microscopic examination, the necrotic area appears as a structureless collection of fragmented or lysed cells and amorphous granular debris enclosed within a distinctive inflammatory border; this appearance is characteristic of a focus of inflammation known as a granuloma. 5. Fat necrosis death of fat cells under effects of lytic enzymes. Fat necrosis occurs at fat-rich locations in the body. Fat necrosis: occurs in two forms: a. Traumatic fat necrosis, which occurs after a severe injury to tissue with high fat content, such as the breast. b. Enzymatic mesenteric fat necrosis, which is a complication of acute pancreatitis, with diffusion of proteolytic and lipolytic pancreatic enzymes into inflamed tissue and literally digest the parenchyma. Gross examination, visible chalky-white areas (fat saponification), which enable the surgeon and the pathologist to identify the underlying disorder. Fate of necrosis Within the living patient most necrotic cells and their contents disappear due to enzymatic digestion and phagocytosis of the debris by leukocytes. Page 2 of 4  If necrotic cells and cellular debris are not promptly destroyed and reabsorbed, they provide a nidus for the deposition of calcium salts and other minerals and thus tend to become calcified, dystrophic calcification. Apoptosis: Cell proliferation and cell death are usually balanced in all the normal tissues of multicellular organisms. Apoptosis is programmed cell death, induced by a tightly regulated suicide program. Cells destined to die will activate intrinsic enzymes that degrade the cells’ genomic DNA and nuclear and cytoplasmic proteins. Apoptosis affects only single cells or few cells. Mechanism of Apoptosis Apoptosis occurs in two broad contexts as part of normal physiologic processes, and as a pathophysiologic mechanism of cell loss in many different diseases. It occurs in a cascade of molecular events that include the activation of caspases initiation phase followed by an execution phase [when the enzymes cause cell death]. Apoptosis in physiological conditions; Cells undergo apoptosis because they are deprived of necessary survival signals, such as growth factors and interactions with the extracellular matrix, or they receive pro-apoptotic signals from other cells or the surrounding environment. During Embryogenesis (for normal development) ; it is critical for involution of primordial structures and remodelling of maturing tissues. Example; The formation of the digits during embryogenesis in the foetus occurs by the apoptosis of interdigital tissues. Hormone-dependent involution of tissues; During Menstrual Cycle, the sloughing off the inner lining of the uterus (the endometrium) after withdrawal of oestrogens and progesterone, ovarian follicular atresia in menopause, and regression of the lactating breast after weaning. Cell turnover in proliferating cell populations to maintain a constant cell number, such as immature lymphocytes in the bone marrow and thymus. Elimination of potentially harmful \ dangerous cells as Virus infected cells [eliminated by Cytotoxic T cells] and cells with DNA damage that fail to repair by p53 activation, then, they will undergo apoptosis to protect cells from risk of mutations. Autoreactive T cells: Autoreactive T cells in the thymus are killed by apoptosis. Page 3 of 4 Apoptosis in pathological conditions The main purpose of apoptosis is to eliminate cells that are injured beyond repair without eliciting a host reaction, thus limiting collateral tissue damage. Dysregulated (“too little or too much”) apoptosis may prolong the survival or reduce the turnover of abnormal cells, Such abnormally accumulated cells may lead to: 1. Tumorigenesis: under normal situations, radiation and cytotoxic drugs can damage DNA, either directly or via production of free radicals. If repair mechanisms cannot correct the damage, the cell triggers intrinsic mechanisms that induce apoptosis as a protective effect by preventing the survival of cells with DNA mutations that can lead to malignant transformation. On the other hand, In case of mutated apoptosis genes; carcinogenic agents can damage p53 tumor suppressor gene that lead to inhibition of apoptosis and immortalization of abnormal cells and development of malignant cells. 2. Accumulation of abnormal proteins. Cell death triggered by improperly folded intracellular proteins and the subsequent endoplasmic reticulum response → Neurodegenerative disorders. 3. Autoimmune Diseases; A decrease in apoptosis of self-reactive immune cells can lead to the development of autoimmune diseases. 4. Infectious conditions; elimination of viral infected cells (e.g., hepatitis), is mediated by apoptosis induced mechanisms. Dysregulation of this pathway , will fail the body ‘s defensive mechanisms. Morphologic features of apoptosis include: Involves single isolated cells or small clusters of cells within a tissue. Lack of inflammatory response Blebbing of plasma membrane Cytoplasmic and cellular shrinkage Chromatin condensation and fragmentation Budding of cell and Fragmentation into apoptotic bodies (membrane-bound segments) Phagocytosis of apoptotic bodies by adjacent healthy cells or macrophages. Page 4 of 4  8 Nucleus and phases of cell cycle ILOs By the end of this lecture, students will be able to 1. Correlate structure of different components to the nucleus to its function. 2. Differentiate between functional forms of the chromatin. 3. Interpret structural organization of the chromosome. 4. Discuss the nuclear and cellular changes during the phases of the cell cycle The Cell Nucleus: Introduction The nucleus contains a blueprint for all cell structures and activities, encoded in the DNA of the chromosomes. It also contains the molecular machinery to replicate its DNA and to synthesize and process the three types of RNA; ribosomal (rRNA), messenger (mRNA), and transfer (tRNA). (Are there DNA in the cell outside the nucleus?) The nucleus does not produce proteins; the numerous protein molecules needed for the activities of the nucleus are imported from the cytoplasm. Structure of the nucleus as seen by LM The nucleus frequently appears as a rounded or elongated structure, usually in the center of the cell (Figure.1A). Its main components are the nuclear envelope, chromatin, nucleolus, and nuclear matrix (Figure.1B). The size and morphological features of nuclei in a specific normal tissue tend to be uniform. In common hematoxylin and eosin-stained preparations; the nucleus, however, appears intensely stained dark blue or black. (Why?) Ultrastructure of the nucleus Three components are recognized: 1. Nuclear Envelope Electron microscopy shows that the nucleus is surrounded by two parallel membranes separated by a narrow space called the perinuclear cisterna. Together, the paired membranes and the intervening space make up the nuclear envelope. (Fig 1B) Polyribosomes are attached to the outer membrane, showing that the nuclear envelope is a in continuity with the endoplasmic reticulum. (Why?) At sites at which the inner and outer membranes of the nuclear envelope fuse, there are gaps, the nuclear pores (Figure 1B), that provide controlled pathways between the nucleus and the cytoplasm. Because the nuclear envelope is impermeable to ions and molecules of all sizes, the exchange of substances between the nucleus and the cytoplasm is made only through the nuclear pores. Ions and Page 1 of 5 molecules with a diameter up to 9 nm pass freely through the nuclear pore without consuming energy. But molecules and molecular complexes larger than 9 nm are transported by an active process, mediated by receptors, which uses energy from adenosine triphosphate (ATP). 2. Chromatin o Chromatin is composed mainly of coiled strands DNA bound to basic proteins (histones). o The basic structural unit of chromatin is the nucleosome (Figure 2), which consists of a core of four types of histones, wrapped around DNA base pairs. (What is the role of histones?) o Linker DNA; An additional DNA segment forms a link between adjacent nucleosomes, and another type of histone is bound to this DNA. This organization of chromatin has been referred to as "beads-on-a-string." Nonhistone proteins are also associated with chromatin, but their arrangement is less well understood. o Functional forms of Chromatin; in nondividing nuclei, is in fact the chromosomes in a different degree of uncoiling. According to the degree of chromosome condensation, two types of chromatin can be distinguished with both the light and electron microscopes (Figure 3). Heterochromatin (Gr. heteros, other, + chroma, color), which is electron dense, appears as coarse granules in the electron microscope and as basophilic clumps in the light microscope. It represents the inactive form of chromatin and acts as a reserve in less active cells. Euchromatin is the less coiled portion of the chromosomes, visible as a finely dispersed granular material in the electron microscope and as lightly stained basophilic areas in the light microscope. It represents the active form of chromatin and more abundant in active cells. The proportion of heterochromatin to euchromatin accounts for the light-to-dark appearance of nuclei 0in tissue sections as seen in light and electron microscopes. The intensity of nuclear staining of the chromatin is frequently used interpret the functional state of the nucleus. (How?) Figure 2 Nucleosome structure Page 2 of 5  Figure 3 Electron micrograph of a nucleus showing the heterochromatin (HC) and euchromatin (EC). Unlabeled arrows indicate the nucleolus-associated chromatin around the nucleolus (NU). Arrowheads indicate the perinuclear cisterna. Underneath the cisterna is a layer of heterochromatin, the main component of the so-called nuclear membrane seen under the light microscope. X 26,000. Careful study of the chromatin of mammalian cell nuclei reveals a heterochromatin mass that is frequently observed in female cells but not in male cells. This chromatin clump is the sex chromatin and is one of the two X chromosomes present in female cells. The X chromosome that constitutes the sex chromatin remains tightly coiled and visible, whereas the other X chromosome is uncoiled and not visible. Evidence suggests that the sex chromatin is genetically inactive. The male has one X chromosome and one Y chromosome as sex determinants; the X chromosome is uncoiled, and therefore no sex chromatin is visible. In human epithelial cells, sex chromatin appears as a small granule attached to the nuclear envelope. The cells lining the internal surface of the cheek are frequently used to study sex chromatin. Blood smears are also often used, in which case the sex chromatin appears as a drumstick-like appendage to the nuclei of the neutrophilic leukocytes. 3. Nucleolus o The nucleolus is a spherical structure (Figure 17-5) that is rich in rRNA and protein. It is usually basophilic when stained with hematoxylin and eosin. o Significance of the nucleolus; it contains DNA that codes for rRNA (type of RNA present inside ribosomes). The nucleolus is the site of synthesis of ribosomal subunits (to be explained later). Ribosomal proteins, synthesized in the cytoplasm, become associated with rRNAs in the nucleolus; ribosome subunits then migrate into the cytoplasm. Heterochromatin is often attached to the nucleolus (nucleolus-associated chromatin), but the functional significance of the association is not known. The rRNAs are synthesized and modified inside the nucleus. In the nucleolus they receive proteins and are organized into small and large ribosomal subunits, which migrate to the cytoplasm through the nuclear pores. (Figure 17-4) Page 3 of 5  4. Nucleoplasm: The protoplasm within the nucleus, consisting of a fluid portion, a proteinaceous matrix, and various ribonucleoproteins particles. Nuclear & cellular changes during cell cycle: Cell cycle The cell cycle is a series of events within the cell that prepare the cell for dividing into two daughter cells. Phases of the cell cycle: I- Interphase; long period of time during which the cell increases its size and content and replicates its genetic material. It includes three stages: a) G1 (gap) phase, synthesis of macromolecules essential for DNA duplication begins & cell growth. b) S (synthetic) phase, DNA is duplicated. c) G2 phase, the cell undergoes preparations for mitosis. II- Mitosis, a shorter period of time during which the cell divides its nucleus and cytoplasm, giving rise to two daughter cells. The cell cycle may be thought of as beginning at the conclusion of the telophase stage in mitosis, after which the cell enters interphase. (Figure 6) The interphase Gap 1 Daughter cells formed during mitosis enter the G1 phase. During this phase, the cells synthesize RNA, regulatory proteins essential to DNA replication, and enzymes necessary to carry out these synthetic activities. Thus, cell growth occurs restoring cell size to normal. The centrioles (a cell organelle involved in cell division) begin to duplicate themselves, a process that is completed by the G2 phase. S Phase During the S phase, the synthetic phase of the cell cycle, the genome is duplicated. The cell now contains twice the normal complement of its DNA. Autosomal cells contain the diploid amount of DNA before the synthetic (S) phase of the cell cycle that becomes doubled after S phase in preparation for cell division. Page 4 of 5 G2 Phase The gap 2 phase (G2 phase) is the period between the end of DNA synthesis and the beginning of mitosis. During the G2 phase, the RNA and proteins essential to cell division are synthesized, the energy for mitosis is stored. Duplication of centrioles and formation of the needed microtubules are completed. DNA replication is analyzed for possible errors, and any of these errors is corrected. Cells that become highly differentiated (What is meant by differentiation?) after the last mitotic event may stop to undergo mitosis either permanently (e.g., neurons, muscle cells) or temporarily (e.g., peripheral lymphocytes) and return to the cell cycle at a later time. Cells that have left the cell cycle are said to be in a resting stage, the G0 (outside) phase, or the stable phase. Figure 6. The cell cycle in actively dividing cells. Nondividing cells, such as neurons, leave the cycle to enter the G0 phase (resting stage). Other cells, such as lymphocytes, may return to the cell cycle. Page 5 of 5  13 Cytoskeletal microtubules & Microfilaments & motility ILOs By the end of this lecture, students will be able to 1. Correlate the molecular organization of microtubules to its dynamic nature. 2. Interpret structural adaptation of MT to their function. 3. Correlate the relevant motor proteins in cell trafficking to MT. 4. Appraise the importance of microtubules as a target for drug action. 5. Correlate molecular structure of actin to its function. Cytoskeleton The cytoplasm of animal cells contains a cytoskeleton, an intricate three-dimensional meshwork of protein filaments that are responsible for the maintenance of cell shape (Fig 1). Additionally, the cytoskeleton is an active participant in cellular motion, whether of organelles or vesicles within the cytoplasm, regions of the cell, or the entire cell. The cytoskeleton has three components: thin filaments (microfilaments), intermediate filaments, and microtubules. Fig. 1 The cytoskeleton Microtubules Microtubules (MTs) are long, straight, hollow structures that act as intracellular pathways. The centrosome is a region close to the nucleus that houses the centrioles, as well as several hundred ring-shaped γ-tubulin ring complex molecules that act as nucleation sites for microtubules (fig. 2). Microtubules are dynamic structures that frequently change their length by undergoing growth spurts and then becoming shorter; both processes occur at the plus ends (oriented away from the nucleus), so that the average half-life of a microtubule is only about 10 minutes (fig. 2-B). Molecular structure of MTS Each microtubule consists of 13 parallel protofilaments composed of heterodimers of the globular 1  polypeptide α- and β-tubulin subunits. Fig. 2 Molecular structure of MTs A B Functions of MTs Provide rigidity and maintain cell shape. Regulate intracellular movement of organelles and vesicles. Establish intracellular compartments. Provide the capability of ciliary and flagellar (tail of the sperm) motion. During cell division, rapid polymerization of existing, as well as, new microtubules is responsible for the formation of the mitotic spindle. Clinical correlation The dynamic process of microtubule formation is disrupted by some drugs which can have different clinical effects on cellular functions e.g. Colchicine: By binding to the tubulin molecules in leukocytes, colchicine prevents its polymerization into microtubules. It thus interferes with leukocyte migration, phagocytosis and further release of inflammatory mediators, and this is the basis for its anti-inflammatory effect in acute gouty arthritis (Joint inflammation due to precipitation of urate crystals). Microtubule-associated proteins Microtubule-associated proteins are motor proteins that assist in the translocation of organelles and vesicles inside the cell. 2  Their primary functions are to prevent depolymerization of microtubules and to assist in the intracellular movement of organelles and vesicles. Movement along a microtubule occurs in both directions and is toward both the plus end and the minus end. The two major families of microtubule motor proteins, the MAPs dynein and kinesin, bind to the microtubule as well as to vesicles and organelles In the presence of ATP, dynein moves the vesicle toward the minus end of the microtubule. Kinesin effects vesicular (and organelle) transport in the opposite direction, toward the plus end. Centrioles Centrioles are small, cylindrical structures composed of nine microtubule triplets; they constitute the core of the microtubule organizing center or the centrosome. They are paired structures, arranged perpendicular to each other, and are located in the microtubule organizing center, the centrosome, in the vicinity of the Golgi apparatus. Functions of the centriole The centrosome assists in the formation and organization of microtubules as well as in its self- duplication before cell division. During cell division, centrioles are responsible for the formation of the spindle apparatus. Additionally, centrioles are the basal bodies that guide the formation of cilia and flagella (motile cell processes). 3  Fig. 5 Cilia & Flagellum Actin Filaments (microfilaments) Thin filaments (microfilaments) are composed of two chains of globular subunits (G-actin) coiled around each other to form a filamentous protein, F-actin. Thin filaments are 6-nm thick and possess a faster-growing plus end and a slower-growing minus end. Functional forms of actin 1. Contractile bundles: Their actin filaments are arranged loosely, parallel to each other, with the plus and minus ends alternating in direction. They form cleavage furrows (contractile rings) during mitotic division. Movement of organelles and vesicles within the cell. Cellular activities, such as exocytosis and endocytosis, as well as the extension of filopodia and cell migration (fig. 6-D). 2. Gel-like networks: provide the structural foundation of much of the cell cortex (fig. 6-C). 3. Bundles: form the core of microvilli (apical cell projections) (fig. 6-A). 4. Focal points: points of contact between the cell and the extracellular matrix (fig 6-B). 4  Fig. 6- Functional forms of actin A B Focal points F C D 5  14 Cell Cycle control & Mitosis ILOs By the end of this lecture, students will be able to 1. Interpret the significance of different phases of the cell cycle. 2. Correlate the phases of mitosis to normal and abnormal division outcomes. Cell cycle control The capability of the cell to begin and advance through the cell cycle is governed by the presence and interactions of a group of related proteins known as cyclins, with specific cyclin-dependent kinases (CDKs). Thus: Cyclin D, synthesized during early G1 phase, binds to CDK4 as well as to CDK6. Additionally, in the late G1 phase cyclin E is synthesized and binds to CDK2. These three complexes, through other intermediaries, permit the cell to enter and progress through the S phase. Cyclin A binds to CDK2 and CDK1 and these complexes permit the cell to leave the S phase and enter the G2 phase and induce the formation of cyclin B. Cyclin B binds to CDK1, and this complex allows the cell to leave the G2 phase and enter the M phase.(Figure 23-2) Fig 23-2. Control of the cell cycle 1 Fate of cyclins Once the cyclins have performed their specific functions, they enter the ubiquitin- proteasome pathway, where they are degraded into their component molecules. Cell cycle check points The cell also employs quality control mechanisms, known as checkpoints, to safeguard against early transition between the phases. These checkpoints ensure the meticulous completion of essential events, such as adequate cell growth, correct DNA synthesis, and proper chromosome segregation, before permitting the cell to leave its current phase of the cell cycle. The cell accomplishes such delays in the progression through the cell cycle by activating inhibitory pathways and/or by suppressing activating pathways. (check the details in Figure 23-3) Factors stimulating the cell to enter the cycle: The triggers inducing the cell to enter the cell cycle may be: (1) A mechanical force (e.g., stretching of smooth muscle), (2) Injury to the tissue (e.g., ischemia), and (3) Cell death. All of these incidents cause the release of ligands by signaling cells in the involved tissue. Frequently, these ligands are growth factors that indirectly induce the expression of proto-oncogenes, genes that are responsible for controlling the proliferative pathways of the cell. Clinical Hint Oncogenes and cancer: Normal cell proliferation and differentiation are controlled by a group of genes called protooncogenes; altering the structure or expression of these genes promotes the production of tumours. The expression of proto-oncogenes must be very strictly regulated to prevent unwanted and uncontrolled cell proliferation. Mutations in proto-oncogenes that enable the cell to escape control and divide in an uncontrolled way are responsible for many cancers. Such mutated proto-oncogenes are known as oncogenes. Altered oncogene activity can be induced by a change in the DNA sequence (mutation), an increase in the number of genes (gene amplification), or gene rearrangement. The Chromosomes Chromosomes are chromatin fibers that become so condensed and tightly coiled during mitosis and meiosis so that they are visible with the light microscope. 2  Each chromosome is formed of two sister chromatids attached at a point called the centromere. As the cell leaves the interphase stage and prepares to undergo mitotic or meiotic activity, the chromatin fibers are extensively condensed to form chromosomes, carrying the duplicated DNA (In which phase of the cell cycle?) Fig. 23-3. Structure of the chromosome 3 Types of cells in the human body 1. Somatic cells They are the body cells that are produced by mitosis and contain 46 chromosomes (diploid number), representing 23 homologous pairs of chromosomes. One member of each of the chromosome pairs is derived from the maternal parent; the other comes from the paternal parent. Of the 23 pairs, 22 are called autosomes; the remaining pair, which determines gender, are the sex chromosomes. The sex chromosomes of the female are two X chromosomes (XX); those of the male are the X and Y chromosomes (XY). Only one of the two X chromosomes in female somatic cells is transcriptionally active. The inactive X chromosome, randomly determined early in development, remains inactive throughout the life of that individual. The number of chromosomes in somatic cells is specific for the species and is called the genome, the total genetic makeup. 2. Germ cells They are either the sperm in male or ovum in the female and produced by meiosis. They contain 23 chromosomes (haploid number) and one sex chromosome either X or Y. Mitosis (M) occurs at the conclusion of the G2 phase and thus completes the cell cycle. Mitosis is the process whereby the cytoplasm and the nucleus of the cell are divided equally into two identical daughter cells. First, the nuclear material is divided in a process called karyokinesis, followed by division of the cytoplasm, called cytokinesis. The process of mitosis is divided into four distinct stages: prophase, metaphase, anaphase, and telophase Mitosis Mitosis is the process of cell division that results in the formation of two identical daughter cells. I- Prophase During prophase, the chromosomes condense, and the nucleolus disappears. Each chromosome consists of two parallel sister chromatids. As chromosomes condense, the nucleolus disappears. The centrosome also divides into two regions, each half containing a pair of centrioles and a microtubule-organizing center (MTOC), which migrate away from each other to opposite poles of the cell. 4  Fig. 23-4 Centrosome structure & prophase From each MTOC, astral rays and spindle fibers develop, giving rise to the mitotic spindle apparatus. It is thought that the astral rays (microtubules that radiate out from the pole of the spindle) may assist in orienting the MTOC at the pole of the cell. Those microtubules that attach to the centromere region of the chromosome are the spindle fibers, which assist in directing the chromosome alignment on cell equator and later migration of chromatids to the cell pole. In the absence of centrioles, the microtubule-nucleating material is dispersed within the cytoplasm with the result that astral rays and spindle fibers do not form properly, and mitosis does not proceed in the appropriate manner. (How is related to the cell cycle?) Spindle fibers bind to the kinetochore in preparation for chromatid migration to effect karyokinesis. II- Metaphase The nuclear envelope disappears early at this phase. The newly duplicated chromosomes align themselves on the equator of the mitotic spindle, being directed by the spindle fibers, and become maximally condensed. Each chromatid is oriented parallel to the equator, and spindle microtubules are attached to its kinetochore (a protein on the centromere that helps in movement of chromatids), radiating to the spindle pole (fig. 22-5). 5  Fig. 23-5. Metaphase & kinetochore III- Anaphase During anaphase, the sister chromatids separate and begin to migrate to opposite poles of the cell, and a cleavage furrow begins to develop (fig. 22-6). The spindle/kinetochore attachment site leads the way, with the arms of the chromatids simply moving along, contributing nothing to the migration or its pathway. In late anaphase, a cleavage furrow begins to form at the plasmalemma, indicating the region where the cell will be divided during cytokinesis. Figure 23-6 Anaphase & telophase IV- Telophase Telophase, the terminal phase of mitosis, is characterized by cytokinesis, reconstitution of the nucleus and nuclear envelope, disappearance of the mitotic spindle, and unwinding of the chromosomes into chromatin. 6. The chromosomes uncoil and become organized into heterochromatin and euchromatin of the interphase cell. Nucleolus reappears. Cytokinesis is the division of the cytoplasm into two equal parts during mitosis. The cleavage furrow continues to deepen (fig. 22-6). The remaining microtubules are surrounded by a contractile ring, composed of actin and myosin filaments attached to the plasma membrane. Constriction of the ring helps in separation of the two new cells. Each daughter cell resulting from mitosis is identical in every respect, including the entire genome, and each daughter cell possesses a diploid (2n) number of chromosomes. Clinical correlations Understanding of mitosis and the cell cycle has greatly aided cancer chemotherapy, making it possible to use drugs at the time when the cells are in a particular stage of the cell cycle. For example, vincristine and similar drugs disrupt the mitotic spindle, arresting the cell in mitosis. Colchicine , another plant alkaloid that produces the same effect, has been used extensively in studies of individual chromosomes and karyotyping. 7  17 GENE EXPRESSION 2. RNA TRANSLATION AND GENETIC CODE ILOs By the end of this lecture, students will be able to 1. Discuss the rules of genetic code 2. Correlate the function of different RNAs to the process of translation 3. Describe the process of translation 4. Interpret role of translation and post translational modification in health and disease What is translation? It is the translation of the nucleotide sequence of a mRNA (Codons) into an amino acid sequence of a protein, in order to synthesize proteins. Each codon consists of a sequence of 3 nucleotides i.e. it is a triplet code. Collection of these codons makes up the genetic code. Protein biosynthesis is called translation because it involves translation of information from the 4- letter language and structure of nucleic acid into the 20-letter language and structure of proteins Requirements of translational process m RNA as a carrier of genetic information. tRNA as an adapter molecule, which recognizes an amino acid on one end and its corresponding codon on the other end. At least one specific type of tRNA is required for each amino acid. Ribosomes as the molecular machine coordinating the interaction between mRNA, tRNA, the enzymes and the protein factors required for protein synthesis. Genetic code (Figure 1) It is the relationship between nucleotide sequence in DNA or mRNA AND amino acids in a polypeptide chain. Each amino acid can be specified by more than one codon. There is one start codon: AUG (METHIONINE) There are 3 STOP codons (UAA, UAG, UGA) The genetic information along mRNA is read from 5’ to 3’ direction. Figure 1.genetic code 1 Characteristics of genetic code 1- Genetic code is degenerate i.e. multiple codons can code for the same amino acid except tryptophan and methionine.(both are coded by only one codon) Wobble theory: The 3rd nucleotide in a codon is less important than the other two in determining the specific amino acid to be incorporated. (i.e if an amino acid has several codons, all such cosons will usually have the first 2 letters in common but the third is different, which means the 3 rd is of less importance) 2- Genetic code is unambiguous i.e. each codon specifies no more than one amino acid. 3- Genetic code is non-overlapping and Commaless meaning that the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time without any punctuation between codons. For example, AGCUGGAUACAU is read as AGC UGG AUA CAU. 4-Genetic code is universal i.e. the same code words are used in all organisms (pro- and eukaryotes) Protein Biosynthesis stages 1- Initiation 2- Elongation 3- Termination Stage 1: Initiation For initiation of protein biosynthesis, there must be:- - tRNA - rRNA - mRNA - Eukaryotic initiation factors (eIFs). - GTP, ATP and different amino acids. In this stage, The 80 S eukaryotic ribosome is dissociated into 40 S and 60 S subunits. eIF – 3 and eIF-1 bind to 40 S subunit thus preventing re-association between the 2 subunits. GTP and eIF-2 bind, in addition to binding of mRNA (accompanied by hydrolysis of ATP to ADP+Pi)and Methionine– tRNA (a tRNA specifically involved in binding to the initiation codon AUG). This This is followed by re-association of both ribosomal units, with dissociation of initiation factors and hydrolysis of GTP. This is termed the initiation complex. (Can you enumerate its components?) (Figure2) Figure 2. Initiation of translation 2  N.B: t RNA charging (Figure 3) It means recognition and attachment of the specific amino acid to the 3` hydroxyl adenosine terminus (to the sugar) of tRNA in an ester linkage. Figure 3.tRNA charging Stage 2: Elongation It is a cyclic process involving 3 steps I. Binding of aminoacyl – tRNA to the A site The ribosome has three binding sites for tRNA molecules: the A, P, and E sites.(Figure 4) In the complete 80S ribosome subunit, A site is free (N.B. A=aminoacyl binding site) Binding of aminoacyl t-RNA to A site needs activation of aminoacyl tRNA by binding of eukaryote elongation factor - 1 (e EF-1) and GTP. When aminoacyl tRNA binds to A site, GTP is hydrolysed and e EF-1 is released. Figure 4..Binding sites of tRNA in ribosome N.B: Anticodon: Each tRNA molecule contains a three-base nucleotide sequence, the anticodon, which pairs with a specific codon on the mRNA within the ribosome. This codon specifies which aminoacid will be inserted in the growing peptide sequence. The first codon on mRNA always codes for methionine. II. Peptide bond formation (Figure 5) The alpha amino group of the new amino acid carried by t-RNA in the A site attacks the carboxylic group of the peptidyl-tRNA in the P site. This reaction needs peptidyl transferase enzyme (RIBOZYME) The reaction results in attachment of the growing peptide chain to the tRNA in the A site. III. Translocation (Figure 5) Upon removal of the peptide from t-RNA in the P site, the discharged t-RNA quickly dissociates. eEF-2 and GTP are responsible for translocation of the newly formed peptidy t-RNA from A site to P site 3  The A site is now free to receive a new aminoacyl-RNA Stage 3: Termination (Figure 6) After many cycles of elongation, the non-sense or stop codon of mRNA (UAA, UAG or UGA) appears in the A site. Normally, there is no tRNA with an anticodon capable of recognizing such a termination signal. Releasing factors (eRFS) can recognize the termination signals in the A site Releasing factors(eRFs), GTP and peptidyl transferase promote the hydrolysis of the bond between the peptide chain and t-RNA at P site 80S subunit dissociates and all the factors , tRNA , mRNA, GDP and Pi are released Figure 5. Process of elongation Figure 6.Termination of translation N.B. The formation of one peptide bond requires energy resulting from hydrolysis of 4 high energy phosphate bonds:- Charging of tRNA with amino acyl moiety requires hydrolysis of an ATP to an AMP. (2 high energy bonds) The entry of amino tRNA into the A site requires one GTP hydrolysis to GDP. The translocation of the newly formed peptidyl – tRNA in the A site into the P site results in hydrolysis of one GTP to GDP N.B. Errors in translation will result in faulty proteins, which will be either targeted for degradation in proteasome or will be non- functioning or abnormally functioning. Protein maturation Aim: Activation of protein to a functional form 4  Localization in subcellular compartment Secretion from the cell Protein maturation involves the following: I- Protein folding: Folding to 3D structures, aided by molecular chaperone. (Refer to protein structure lecture). Misfolded proteins are targeted for destruction II- Post – translational processing: a- Proteolysis: It means removal of amino terminal, carboxy terminal or internal sequences. Examples: 1-Removal of amino terminal methionine residues 2-Removal of signal peptides by signal peptidases (signal peptides help translocate the proteins to its final destination and hence are removed after protein transport) (figure 7) b- Modifications of individual amino acids Figure 7.Proteolysis Hydroxylation: of proline and lysine for collagen synthesis Phosphorylation of serine, threonine or tyrosine (Important in cell signaling) γ-carboxylation of glutamic acid in prothrombin and osteocalcin (this helps Ca 2+ binding which is necessary for blood clotting and bone ossification in both proteins respectively) c- Addition of certain groups Glycosylation: Attachment of CHO side chain to form glycoproteins. Glycosylation can help stabilize glycoproteins against degradation or provide proper conformation for protein function. CHO can be either N linked( to asparagine), or O-linked( to serine and threonine) Acylation: Addition of fatty acids to various amino acid side chains. Acylation has several functional effects on proteins, especially to help anchor them to membranes. Clinical implications: I) Many antibiotic work by altering the translation of bacterial DNA and are generally classified as bacterial protein synthesis inhibitors as Tetracyclines and Macrolides like Erythromycin. II) Some toxins can cause death by inhibiting eukaryotic translation: Shiga toxin / ricin (produced by E-coli bacteria and causes bloody diarrhea) inhibits tRNA binding by acting on 60S subunit Diphtheria toxin (produced by bacteria and causes difficult breathing, heart failure, paralysis, and even death) inhibits translocation through binding to Eukaryotic- EF-2 5  21 Autophagy, Lysosomes, Peroxisomes & cell inclusions ILOs By the end of this lecture, students will be able to 1. Explain the role of autophagy as a cellular sink 2. Describe the origin of lysosomal enzymes and their function in health and disease. 3. Predict the role especially of autolysosome and phagolysosome in physiological conditions 4. Connect the structure of proteasome to its degrative function. 5. Predict the role of peroxisomes in cell adaptation to patterns of stress. 6. Correlate the types of cytoplasmic inclusions to patterns of cell activity 7. Justify the impact of its derangement on cellular health. 1. Lysosomes Lysosomes are membrane bounded cell organelles that have an acidic pH and contain hydrolytic enzymes. It contains at least 40 different types of acid hydrolases, such as sulfatases, proteases, nucleases, lipases that are active in acidic pH. These enzymes are manufactured in the same steps of protein synthesis following the same steps in rER, packed in Golgi complex and released in vesicles from trans Golgi network. Lysosomes receive contents to be digested from late endosomes. Lysosomes aid in digesting phagocytosed microorganisms, cellular debris, and cells but also excess or senescent organelles, such as mitochondria and RER. The various enzymes digest the engulfed material into small, soluble end products that are transported by carrier proteins in the lysosomal membrane from the lysosomes into the cytosol andare either reused by the cell or exported from the cell into the extracellular space. Transport of Substances into Lysosomes Substances destined for degradation within lysosomes reach these organelles in one of three ways: through phagosomes, pinocytotic vesicles, or autophagosomes. 1- Phagosomes: Phagocytosed material, contained within phagosomes, moves toward the interior of the cell. The phagosome joins either a lysosome or a late endosome. The hydrolytic enzymes digest most of the contents of the phagosome, especially the protein and carbohydrate components. Lipids, however, are more resistant to complete digestion, and they remain enclosed within the spent lysosome, now referred to as a residual body. (Fig. 1) 1  Fg 1. Pathways of intracellular digestion by lysosomes 2- Autophagy: The term autophagy is derived from the Greek word meaning 'self- devouring'. Senescent organelles such as mitochondria or the RER, need to be degraded. The organelles in question become surrounded by elements of the endoplasmic reticulum and are enclosed in vesicles called autophagosomes. Fate of autophagosomes: These structures fuse either with late endosomes or with lysosomes and share the same subsequent fate as the phagosome. (Fig. 1) Autophagy is a self-digesting mechanism responsible for removal of long-lived proteins, damaged organelles, and malformed proteins during biosynthesis by lysosome. Significance of autophagy Regulation of diverse cellular functions including growth, differentiation, response to nutrient deficit and oxidative stress, cell death, and macromolecule and organelle turnover. Mechanism Autophagosome formation is regulated by dozens of “autophagy-related genes” called Atgs. Mutation leads to formation of a double-membrane vesicle, which encapsulates cytoplasm, malformed proteins, long-lived proteins, and organelles and then fuses with lysosomes for degradation. 2  Autophagy Regulation Autophagy is activated in response to diverse stress and physiological conditions. For example, food deprivation, hyperthermia, and hypoxia, which are known as major environmental modulators of ageing, are also conditions that induce autophagy. Figure 2 - Stages of autophagy Autophagy and Diseases Autophagy is important in normal development and responds to changing environmental stimuli. On starvation, autophagy is greatly increased, allowing the cell to degrade proteins and organelles and thus obtain a source of macromolecular precursors, such as amino acids, fatty acids, and nucleotides, which would not be available otherwise. Autophagy roles in cancer are a topic of intense debate. In one hand, autophagy has an anticancer role. On the other hand, when tumor cells are starved due to limited angiogenesis, autophagy stops them from dying. Autophagy is important in numerous diseases, including bacterial and viral infections, neurodegenerative disorders, several myopathies, and cardiovascular diseases. 3 Autophagy and weight loss A type of intermittent fasting is used to stimulate autophagy and to 'trick' one's metabolism into working longer hours and burning more fat. Notably, pharmacological stimulation of autophagy can reduce both weight gain and obesity-associated alterations upon hypercaloric regimens usage. Proteasomes Proteasomes are small organelles composed of protein complexes (proteases) that are responsible for proteolysis (protein breakdown) of malformed and ubiquitin-tagged proteins. Proteasomes monitor the protein content of the cell to ensure degradation of unwanted proteins, such as excess enzymes and other proteins that become unnecessary to the cell after they perform their normal functions, and malformed proteins. Protein encoded by virus should also be destroyed. The process of cytosolic proteolysis is carefully controlled by the cell, and it requires that the protein be recognized as a potential candidate for degradation. This recognition involves ubiquination, a process whereby several ubiquitin molecules (a 76-amino acid long polypeptide chain) are attached to the candidate protein using ATP. Once a protein has been marked, it is degraded by proteasomes. (Fig 2) During proteolysis, the ubiquitin molecules are released and re-enter the cytosolic pool to be re used. Fig. 3. The structure and function of the proteasome 4 Protein degradation by proteasomes in health and disease Proteins destined for degradation are labeled with ubiquitin through covalent attachment to a lysine side chain. The amino acid composition at the amino terminus determines how quickly the protein will be ubiquinated and thus the half-life of the protein. Some proteins have very long half-lives, such as the crystallins in the lens of the eye; these proteins do not turn over significantly during the human life span. Because they were synthesized largely in utero, about half the crystallins in the adult lens are older than the person. Other proteins have half-lives of 4 months (proteins such as hemoglobin that last as long as the red blood cell), or the half-life can be very short, such as for ornithine decarboxylase, which has a half-life of 11 minutes. The half-lives of proteins is influenced by the amino (N)-terminal residue, the so- called N-end rule. Destabilizing N-terminal amino acids (causing short half-life) include arginine and acetylated alanine. In contrast, serine is a stabilizing amino acid. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences) are rapidly ubiquinated and degraded and, therefore, have short half-lives Poly-ubiquination, which increases the rate of turnover/degradation of a protein, occurs by successive addition of free ubiquitin to that which is already bound to the protein. Failure of degradation of misfolded proteins by proteasomes, can lead to accumulation of abnormal proteins and development of certain diseases such as Alzheimer’s disease and Creutzfeldt–Jakob disease (Mad-cow disease). Peroxisomes Peroxisomes are small membrane bounded, self-replicating organelles. They contain more than 40 oxidative enzymes, especially urate oxidase, and D- amino acid oxidase that contain oxidative enzymes. Peroxisomes function in the catabolism of long-chained fatty acids (beta oxidation), forming acetyl coenzyme A (CoA) as well as hydrogen peroxide (H2O2) by combining hydrogen from the fatty acid with molecular oxygen. Similar to mitochondria, peroxisomes increase in size and undergo fission to form new peroxisomes; however, they possess no genetic material of their own. Inclusions Inclusions are non-living components of the cell that do not possess metabolic activity and are not bounded by membranes. The most common inclusions are glycogen, lipid droplets, pigments, and crystals. 5  1. Glycogen Glycogen is the most common storage form of glucose in human and is especially abundant in cells of muscle and liver. It appears in electron micrographs as clusters, or rosettes, of β particles (and larger α particles in the liver) that resemble ribosomes, located in the vicinity of the SER. On demand, enzymes responsible for glycogenolysis degrade glycogen into individual molecules of glucose. 2. Lipids Lipids, triglycerides in storage form, not only are stored in specialized cells (adipocytes) but also are located as individual droplets in various cell types, especially hepatocytes. Lipids are considered as potential source of energy within the cells. 3. Pigments It could be natural pigments as haemoglobin of red blood cells, melanin in the skin and hair and a yellow-to-brown pigment, lipofuscin in the long-lived cells, such as neurons and cardiac muscle. Tattoos is the injection of ink intracellular that could be phagocytosed by macrophages leading to its permanent effect. Fig. 4 Types of inclusions A. TEM of glycogen ganules in rosettes B. Lipid droplets in fat cell Clinical hint: abnormal accumulations I. Lipids 1-Steatosis (Fatty Change) Means excessive, abnormal accumulations of triglycerides within parenchymal cells due to alcohol abuse, diabetes mellitus, obesity, toxins, protein malnutrition, and anoxia 2- Cholesterol and Cholesterol Esters as in atherosclerosis. 6  I. Proteins as inAlzheimer disease. II. Glycogenas in Diabetes mellitus and Glycogen storage diseases. III. Pigments; Exogenous Carbon (coal dust),The most common air pollutant in urban areas. Its accumulation could lead to Anthracosis occurs in heavy smokers, and coal mines workers with accumulation of carbon pigment within lungs and regional lymph nodes. Endogenous Pigments Lipofuscin Patients with severe malnutrition& Cancer cachexia. Melanin: Hyperpigmentation generalized due to excessive sun exposure or localized as in benign (nevus) and malignant cutaneous tumors. Hypopigmentation Generalized as in albinism or localized as in vitiligo (autoimmune disorder). 7 26 Cytoskeleton & intercellular junctions ILOs By the end of this lecture, students will be able to 1. Correlate stable nature of IM to its supportive role in the cell. 2. Discuss the significance of cellular specificity of the IM in diagnosis of tumors origin. 3. Deduce structural adaptation of the cell junction to its function. 4. Interpret the impact of molecular structure abnormality on tissue integrity. Intermediated Filaments These rope-like intermediate filaments are constructed of tetramers of rod-like proteins that are tightly bundled into long helical arrays (Fig 1). Fig 1. Molecular structure and organization of Intermediated filaments The individual subunit of each tetramer differs considerably for each type of intermediate filament. The categories of intermediate filaments include keratins, desmin, vimentin, glial fibrillary acidic protein, neurofilaments, and nuclear lamins. Page 1 of 4  Examples of Intermediate Filaments Found in Eukaryotic Cells. Filament Type Cell Type Examples Keratins Epithelium Both keratinizing and nonkeratinizing epithelial cells Desmin Muscle Striated and smooth muscle (except vascular smooth muscle) Gilial fibrillary acidic Glial cells Astrocytes proteins Neurofilaments Neurons Nerve cell body and processes The intercellular junctions These are specialized attachment areas (cell junctions) present on cells that are in close contact with each other. Morphologically, they are classified into three types: a) Zonula (belt-like) junctions completely encircle the cells. b) Fascia (sheet- like) junctions form broad areas of contact between cells. c) Macula (disc-like) junctions are like spot welds on the cell surface. According to their function, the common cell junctions are: 1. Occluding junctions: function in joining cells to form an impermeable barrier, preventing material from taking an intercellular route in passing across the cellular sheath. Page 2 of 4  2. Anchoring junctions (zonula and macula adherens) function in maintaining cell-to-cell or cell-to-basal lamina adherence, thus provide mechanical stability in tissues subjected to mechanical pressure. 3. Communicating junctions: function in permitting movement of ions or signaling molecules between cells, thus coupling adjacent cells both electrically and metabolically. According to their molecular structure, they include: I- Zonula occludens (tight junctions): They form a "belt-like" junction that encircles the entire circumference of the cell. It is established by fusion of the outer layers of the cell membranes of the two cells in an interrupted pattern, where fusion is represented by focal points. (Fig. 1) At the fusion sites, transmembrane junctional proteins called claudins and occludins bind to each other, thus forming a seal occluding the intercellular space. A third transmembrane protein called cadherin reinforces the other two types. (Fig 2) II- Zonula adherentes The intercellular space of 15 to 20 nm between the outer leaflets of the two adjacent cell membranes is occupied by the extracellular moieties of cadherins. (Fig 2) These Ca2+-dependent integral proteins of the cell membrane are transmembrane linker proteins. Their intracytoplasmic aspect binds to a specialized region of the cell web, specifically a bundle of actin filaments. III- Desmosomes (macula adherentes) These "spot weld"-like junctions that are randomly distributed along the lateral cell membranes of adjacent cells. (Fig 1) Disk-shaped attachment plaques are located opposite each other on the cytoplasmic aspects of the plasma membranes of adjacent epithelial cells. (Fig 2) Each plaque is composed of a series of attachment proteins. Intermediate filaments of cytokeratin are observed to insert into the plaque, where they make a hairpin turn, then extend back out into the cytoplasm. IV- Gap junctions (Nexus) Gap junctions are built by six closely packed transmembrane channel-forming proteins (connexins) that assemble to form channel-structures called connexons, aqueous pores through the plasma membrane that juts out about 1.5 nm into the intercellular space. (Fig 2A) Page 3 of 4 When a connexon of one plasma membrane is in register with its counterpart of the adjacent plasma membrane, the two connexons fuse, forming a functional intercellular hydrophilic communication channel. Page 4 of 4 32 Meiosis ILOs By the end of this lecture, students will be able to 1. Correlate chromosomal changes to different phases of meiosis. 2. Interpret role of crossing over in genetic variations. 3. Interpret the different types of chromosomal abnormalities. 4. Differentiate between aneuploidy and polyploidy. 5. Differentiate between balanced and unbalanced karyotypes. 6. Correlate the phenotypic outcome with types of chromosomal aberrations. Meiosis Meiosis is a special type of cell division resulting in the formation of gametes (spermatozoa or ova) whose chromosome number has been reduced from the diploid (2n) to the haploid (1n) number. Meiosis begins at the conclusion of interphase in the cell cycle. It produces the germ cells- the ova and the spermatozoa. This process has two crucial results: 1. Reduction in the number of chromosomes from the diploid (2n) to the haploid (1n) number, ensuring that each gamete carries the haploid amount of DNA and the haploid number of chromosomes. 2. Recombination of genes, ensuring genetic variability and diversity of the gene pool Meiosis is divided into two phases: I- Meiosis I, or reductional division (first event): Homologous pairs of chromosomes line up, members of each pair separate and go to opposite poles, and the cell divides; thus, each daughter cell receives half the number of chromosomes (haploid number). II- Meiosis II, or equatorial division (second event): The two chromatids of each chromosome are separated, as in mitosis, followed by migration of the chromatids to opposite poles and the formation of two daughter cells. These two events produce four cells (gametes), each with the haploid number of chromosomes and haploid DNA content. Page 1 of 3  In gametogenesis, when the germ cells are in the S phase of the cell cycle preceding meiosis, the amount of DNA is doubled to 4n but the chromosome number remains at 2n (46 chromosomes). Meiosis I 1. Prophase I: It begins after the DNA has been doubled to 4n in the S phase. Prophase of meiosis I lasts a long time. Homologous pairs of chromosomes approximate each other and condense. The most significant event in prophase I is formation of chiasmata (crossing over sites) as random exchange of genetic material occurs between homologous chromosomes. 2. Metaphase I: is characterized by lining up of homologous pairs of chromosomes, each composed of two chromatids, on the equatorial plate of the meiotic spindle. 3. Anaphase I: Homologous chromosomes migrate away from each other, going to opposite poles. 4. Telophase I: The chromosomes reach the opposing poles, nuclei are re-formed and cytokinesis occurs, giving rise to two daughter cells. Each cell possesses 23 chromosomes, the haploid (1n) number, but because each chromosome is composed of two chromatids, the DNA content is still diploid. Figure 2. Phases of meiosis d; double amount of DNA, s; haploid amount of DNA Page 2 of 3 Meiosis II (equatorial division) occurs without DNA synthesis and proceeds rapidly through four phases and cytokinesis to form four daughter cells each with the haploid chromosome number It is subdivided into prophase II, metaphase II, anaphase II, telophase II, and cytokinesis The chromosomes line up on the equator, the kinetochores attach to spindle fibers, followed by the chromatids migrating to opposite poles, and cytokinesis divides each of the two cells. Outcome of meiosis II: 1. Results in a total of four daughter cells from the original diploid germ cell. Each of the four cells contains a haploid amount of DNA and a haploid chromosome number. 2. The cells are genetically distinct because of reshuffling of the chromosomes and crossing over. Thus, every gamete contains its own unique genetic complement. Page 3 of 3  7 Dynamics of Drug Actions ILOs By the end of this lecture, students will be able to: 1. Appraise the importance of efficacy versus potency in therapeutic selection. 2. Compare the quantitative distinction in response of different drugs when either acting on the same receptors or on different ones. 3. Explain the importance of potentiation & antagonism in fields of therapy. 4. Predict relative drug safety and drugs to be monitored upon analysing the quantal dose-frequency curves considering its effective and toxic responses. 5. Appraise implications of variation of drug response, in fields of therapy. GRADED DOSE-RESPONSE CURVE IS USED FOR:  Quantitative Comparison of Effect of Different Drugs Acting on SAME RECEPTOR: E A B  Comparing agonistic action of B, C, D, E, F to the full agonist “A” C as shown in Figure 1 which reveals: a. Drugs B, C, E are of same efficacy as “A” i.e., Full Agonists. D F b. It also reveals that potency of E> “A” while “A”>B>C in potency. c. Drugs D & F have less efficacy than “A” i.e., Partial Agonists. D>F in efficacy, while F> D in potency.  Comparing the effect of addition of another drug to “A” a. If this drug causes a slope shift to the left “like the effect of E”: it is called “ POTENTIATION “. b. If this drug causes a slope shift to the right“like the effect of B” it is called “ANTAGONISM”.  Comparing the effect of addition of an antagonist to “A” as shown in Figure 2 which reveals: a. If it causes a right parallel shift and appears to decrease potency of an agonist as in “B” and can be overcome by increasing concentration of the agonist, it is a Competitive Reversible Antagonist. b. If it causes a nonparallel shift to the right and appears to decrease efficacy of an agonist as in “C” and cannot be overcome by increasing concentration of the agonist, it is either a Competitive Irreversible Antagonist or a Non- Competitive Antagonist. A B Competitive – Potency Antagonism Efficacy  Quantitative Comparison of Effect of Different Drugs C Irreversible - Acting on DIFFERENT RECEPTORS: Competitive Antagonism Non--Competitive Antagonism Fig 1: Comparing effects of Different Agonists. Fig 2: Dose-Response-Curve of Different Antagonists  Comparing the action of drugs, A, B, C, D, on different receptors, shown in Figure 3 reveals: They can vary in efficacy; Drug B >A >D >C in efficacy. They could not be compared in potency as they do not act on same receptor. N.B. If one drug acting on a receptor increases the action of another drug acting on a different receptor; this is termed “SYNERGISM” or “ SUMMATION” , the new curve induced by both drugs will be more efficacious than that of the first drug alone. This is to differentiate from the forestated “POTENTIATION”, where the new curve induced by both drugs will be of more potency than that of the first drug alone. N.B. The Graded-Dose-Response-Curve gives information about the relation of drug concentration/dose in a particular tissue or whole body, but it does not reflect the relation between the drug dose and the proportion of population that therapeutically responded or that developed side effects. Alternatively, a QUANTAL DOSE-RESPONSE-CURVE (figure 4) has become of major clinical importance in justifying that. It is quantal because for any individual in the population the response is always all or none, i.e., - Therapeutically [a drug for sleep; induce sleep or not / a drug lowering cholesterol; dropped it to target level or not] - Adversely, e.g., hypoglycaemia, hepatic injury, hypertension, etc. or not]. QUANTAL DOSE-RESPONSE CURVE IS USED FOR:  Predicting the relative DRUG SAFETY by: 1. Determining from this dose-response-frequency curve: Median-Effective-Dose, ED50: the drug dose that induces a specific therapeutic response in half the population. Median-Toxic -Dose, TD50: the drug dose that induces a special (adverse) toxic response in half the population. 2. Calculating the relative measure of drug safety, termed “THERAPUTIC INDEX” [TI] = TD50 / ED50 whereby if: TI is low  drug is = not safe, as Digoxin. TI is high  drug is = safe, as Penicillin (regarding the high doses).  Determining Drugs that need THERAPEUTIC MONITORING: In clinical practice, determination of blood drug concentration is recommended for certain therapeutics. This is termed Therapeutic Drug Monitoring and is indicated when a drug has narrow therapeutic window, i.e., when the difference between Fig 3: Comparable Dose-Response of Different Fig. 4: Quantal Dose-Response-Curve the dose causing Drugs acting on different receptors.  toxicity and therapeutic effect is very small, i.e., unsafe drugs as Warfarin. Drugs with wide therapeutic window, are safe and do not need monitoring as Ampicillin as shown in Figure 5. Fig. 5: Narrow versus Wide Therapeutic Window of drugs. VARIATION IN DRUG RESPONSE In certain instances, the response of drugs may become reduced, increased, or altered.  If responsiveness to a drug becomes REDUCED gradually, in consequence to repeated administration, this is “TOLERANCE”. It indicates a need to increase the dose of a drug, to maintain the attained response. It could be caused by down regulation of receptors, or decrease in response effectiveness. “TACHYPHYLAXIS” is an acute rapidly developed tolerance, when doses of a drug are repeated in quick succession. N.B. “REFRACTORINESS” signifies the loss of therapeutic efficacy of a drug. “RESISTANCE” signifies the complete loss of effectiveness to antibiotics or anticancer…etc.  If responsiveness to a drug becomes INCREASED: as the exaggeration in vasodilatation produced by Nitrates when it induces syncope; this is “HYPER-SUSCEPTIBILITY” (DRUG INTOLERANCE).  If responsiveness to a drug becomes ALTERED:  When an abnormal response to a therapeutic dose of a drug develops due to a genetic defect, this is “IDIOSYNCRASY” as with Sulphonamide developing haemolytic anaemia in patients with glucose-6- phosphate deficiency.  When an immune response develops due to formation of antigen-antibody reaction, this is “HYPERSENSITIVITY REACTION” as with Penicillin developing skin reaction, bronchial asthma, or even anaphylaxis.  When an adaptive state develops to repeated drug administration and upon its cessation, withdrawal manifestations appear, this is “DEPENDENCE” as with Habituation; developing to Nicotine in Cigarettes or Cannabis or as Physical Dependence “Addiction”; developing to Diazepam or Morphine.  27 Drugs Affecting Mediators & Transmitters of Communication ILOs By the end of this lecture, students will be able to 1. Deduce the therapeutic utility of drugs modulating some mediators. 2. Predict how modulation of transmitters can serve in control of many diseases From previous lectures, it was clear that signals of communication can PASS LOCALLY from the signaling cell to the target cell by Direct Contact, or by acting in an Autocrine or Paracrine manner. When that Paracrine Communication occurs between a. Neurons, i.e., then their action occurs via the synapse at the SYNAPTIC CLEFT. b. A nerve and a skeletal muscle, i.e., then their action occurs via MOTOR END PLATE The mediator then is considered a NEUROTRANSMITTERS. N.B. Some of these local mediators can still find access and exert also systemic effects. If a signal of communication passes directly to blood and is CONVEYED SYSTEMICALLY to distant targets via the circulation, then that signal is considered a HORMONE and its signaling cell belongs to an endocrine gland. Examples of different NON-ENDOCRINE MEDIATORS are shown in figure 1: Fig. 1: Different types of non-endocrine mediators ATP & ADP; Adenosine tri- & di-phosphate - NO; Nitric Oxide - AT; Angiotensin - ET; Endothelin - NPY; Neuropeptide Y - ANP; Atrial natriuretic peptide - VIP; Vaso-active intestinal peptide In many disease states, such mediators may be upregulated/increased, downregulated/ suppressed or malfunctioning. That is why we use DRUGS accordingly to reverse the situation and set balance back to normal. N.B. DRUG EXAMPLES ONLY WRITTEN IN PURPLE MUST BE KNOWN BY THEIR NAMES Example of such drugs and their modality of action in some diseases will be stated. ____________________ Page 1 of 3  DRUGS that suppress mediators or block their action, if found in excess, in some diseases is achieved by: ▪ Inhibiting their synthesis ▪ Blocking their receptor interaction by receptor antagonists ▪ Increasing their degradation ▪ Inhibiting their Synthesis as: Using Angiotensin Converting Enzyme Inhibitors [Ramipril] to suppress increased Angiotensin-II [AgII] level to lower the blood pressure when treating hypertension or manifestations of heart failure. ▪ Blocking their Receptors by Receptor Antagonists as: Using the Angiotensin-1 Receptor Blockers [Valsartan] to block the effect of Angiotensin-II; thus, helping in treatment of hypertension and heart failure. ▪ Increase their degradation: Using the Cholinesterase Reactivators [Oximes] to set the enzyme Acetylcholinesterase [Ach-E] active again in order to degrade excess Acetylcholine (ACh) at the synaptic cleft during insecticide (organophosphorus) poisoning. ____________________ DRUGS that increase mediators or mimic their action if found suppressed or malfunctioning in some diseases by: ▪ Giving the mediator itself or its analogues ▪ Giving a drug that can mimic the mediator’s action ▪ Stimulating mediator synthesis ▪ Increasing mediator release from stores ▪ Decreasing mediator breakdown ▪ Inhibiting the uptake of the mediator ▪ Activating the receptors’ interaction by a receptor agonist: ▪ Giving the mediator itself or its analogues: Using an Insulin Analogue to overcome the existing reduction in insulin release & action in cases of diabetes. ▪ Giving a drug that can mimic the mediator’s action: Using Nitric Oxide [NO] donners as organic nitrates [Nitroglycerine], which can cause venous and arterial dilatation that is demanded for treatment of angina pectoris and relieving symptoms of heart failure. ▪ Stimulating mediator synthesis: Using the beta 2-adrenoreceptor [2-ADR] blocker [Nebivolol] to activate Nitric Oxide Synthase that will increase NO production and lower the blood pressure when treating hypertension or manifestations of heart failure. Page 2 of 3  ▪ Increasing mediator release from stores: Using the Dual Acting Sympathomimetics [Pseudoephedrine] that (beyond its direct action) can indirectly release catecholamines as epinephrine (adrenaline) from stores to activate alpha 1-adrenoreceptor [1-ADR] causing vasoconstriction, thus used as nasal drops in flue remedy. ▪ Decreasing mediator breakdown: Using a Reversible Anticholinesterase Inhibitor [Ach-E Is] [Neostigmine] that will prevent degradation; so, will increase ACh at the motor end plate to increase skeletal muscle contraction in cases of myasthenia gravis. ▪ Inhibiting the uptake of the mediator: Using Selective Serotonin Reuptake Inhibitors [SSRIs] [Fluoxetine] to prevent 5-HT presynaptic re-uptake; so, increases 5-HT within the synaptic cleft that is needed to elevate mood and treat depression. ▪ Activating the receptors’ interaction by a receptor agonist: Using the beta 1-adrenoreceptor [1-ADR] Agonist [Dobutamine] to activate the cardiac muscle; like that induced by epinephrine (adrenaline) to treat cardiogenic shock. Page 3 of 3 49 Chromosomal Aberration (Numerical & Structural) ILOs By the end of this lecture, students will be able to 1. Interpret the different types of chromosomal abnormalities. 2. Differentiate between aneuploidy and polyploidy. 3. Differentiate between balanced and unbalanced karyotypes. 4. Correlate the phenotypic outcome with types of chromosomal aberrations. Numerical aberrations Euploidy: The normal number of chromosomes for a species. In humans, the euploid number of chromosomes is 2n (46) in somatic cells and n (23) in gametes. Types of numerical chromosomal abnormalities: A. Polyploidy: It is a condition in which the chromosome number is a simple multiple of a haploid chromosome set. Triploidy:  69 chromosome with XXX or XXY or XYY; there are 3 copies for each chromosome (3n) (Figure 1).  Caused by failure of reduction division in meiosis in an ovum or sperm.  Alternatively it can be caused by fertilization of an ovum by two sperm: this is known as dispermy.  Usually results in early spontaneous miscarriage.  Example: Partial vesicular mole occurs when triploidy results from an additional set of paternal chromosomes, in which placenta is severely malformed and appears as bunch of small vesicles, and the embryo is abnormal. Figure 1. Karyotype from products of conception showing triploidy. Tetraploidy:  92 chromosome with XXX or XXYY; There are 4 copies for each chromosome (4n)  Occurs due to failure of the first cleavage zygotic division resulting in doubling of the chromosome numbers immediately after fertilization (4n).  Very rare and always lethal. Page 1 of 5 B. Aneuploidy It is an abnormal chromosome number due to an extra or missing chromosome but does not involve the whole chromosome set. When the abnormality involves the autosome the abnormal phenotype is more severe than involvement of sex chromosomes. Aneuploidy is caused by: 1. Non-disjunction: failure of homologous chromosomes segregation during meiosis I or failure of segregation of sister chromatids during meiosis II or mitosis. Mostly, it is due to the aging effect on the primary oocyte in old age mothers. 2. Anaphase lag: Failure of chromosome or chromatid to be incorporated into one of the daughter nuclei following cell division, as a result of delayed movement (lagging) during anaphase. Trisomy:  It is the presence of three copies instead of two for an autosome or sex chromosome in an otherwise diploid cell (2n+1) (Figure 2).  The karyotype is describes as (47, sex chromosomes, + the number of the chromosome with an extra copy). Ex: Down syndrome male karyotype (47, XY,