SBMA1101 Cell Biology & Genetics Past Paper PDF

SCHOOL OF BIO AND CHEMICAL ENGINEERING DEPARTMENT OF BIOMEDICAL ENGINEERING UNIT – I -Cell Biology & Genetics – SBMA1101 1 CELL STRUCTURE Cells are the building blocks of life. A cell is chemical system that is able to maintain its structure and reproduce. Cells are t...

SCHOOL OF BIO AND CHEMICAL ENGINEERING DEPARTMENT OF BIOMEDICAL ENGINEERING UNIT – I -Cell Biology & Genetics – SBMA1101 1 CELL STRUCTURE Cells are the building blocks of life. A cell is chemical system that is able to maintain its structure and reproduce. Cells are the fundamental unit of life. All living things are cells or composed of cells. Although different living things may be as unlike as a violet and an octopus, they are all built in essentially the same way. The most basic similarity is that all living things are composed of one or more cells. This is known as the Cell Theory. Our knowledge of cells is built on work done with microscopes. English scientist Robert Hooke in 1665 first described cells from his observations of cork slices. Hooke first used the word "cell". Dutch amateur scientist Antonie van Leeuwenhoek discovered microscopic animals in water. German scientists Schleiden and Schwann in 1830's were first to say that all organisms are made of one or more cells. German biologist Virchow in 1858 stated that all cells come from the division of pre-existing cells. The Cell Theory can be summarized as:  Cells are the fundamental unit of life - nothing less than a cell is alive.  All organisms are constructed of and by cells.  All cells arise from preexisting cells. Cells contain the information necessary for their own reproduction. No new cells are originating spontaneously on earth today.  Cells are the functional units of life. All biochemical processes are carried out by cells. Groups of cells can be organized and function as multicellular organisms  Cells of multicellular organisms can become specialized in form and function to carry out subprocesses of the multicellular organism. Cells are common to all living beings, and provide information about all forms of life. Because all cells come from existing cells, scientists can study cells to learn about growth, reproduction, and all other functions that living things perform. By learning about cells and how they function, we can learn about all types of living things. Classification of cells: All living organisms (bacteria, blue green algae, plants and animals) have cellular organization and may contain one or many cells. The organisms with only one cell in their body are called unicellular organisms (bacteria, blue green algae, some algae, Protozoa, etc.). The organisms having many cells in their body are called multicellular organisms (fungi, most plants and animals). Any living organism may contain only one type of cell either A. Prokaryotic cells; B. Eukaryotic cells. The terms prokaryotic and eukaryotic were suggested by Hans Ris in the 1960’s. This classification is based on their complexity. Further based on the kingdom into which they may fall i.e the plant or the animal kingdom, plant and animal cells bear many differences. These will be studied in detail in the upcoming sections PROKARYOTIC CELLS Prokaryote comes from the Greek words for pre-nucleus. Prokaryotes: i. One circular chromosome, not contained in a membrane. ii. No histones or introns are present in Bacteria; both are found in Eukaryotes and Archaea. iii. No membrane-bound organelles. (Only contain non membrane-bound organelles). iv. Bacteria contain peptidoglycan in cell walls; Eukaryotes and Archaea do not. v. Binary fission. 2  Size, Shape, and Arrangement of Bacterial Cells. i. Average size of prokaryotic cells: 0.2 -2.0 µm in diameter 1-10 µm (0.001 – 0.01 mm) [book says 2 – 8 µm] in length. 1. Typical eukaryote 10-500 µm in length (0.01 – 0.5 mm). 2. Typical virus 20-1000 nm in length (0.00000002 – 0.000001 m). 3. Thiomargarita is the largest bacterium known. It is about the size of a typed period (0.75 mm). 4. Nanoarchaeum is the smallest cell known. It is at the lower theoretical limit for cell size (0.4 µm). ii. Basic bacterial shapes: 1. Coccus (sphere/round). 2. Bacillus (staff/rod-shaped). 3. Spirilla (rigid with a spiral/corkscrew shape). a. Flagella propel these bacteria. 4. Vibrio (curved rod). 5. Spirochetes (flexible with a spiral shape). Axial filaments (endoflagella) propel these bacteria. iii. Descriptive prefixes: 1. Diplo (two cells). 2. Tetra (four cells). 3. Sarcinae (cube of 8 cells). 4. Staphylo (clusters of cells). 5. Strepto (chains of cells). iv. Unusual bacterial shapes: 1. Star-shaped Stella. 2. Square/rectangular Haloarcula. v. Arrangements: 1. Pairs: diplococci, diplobacilli 2. Clusters: staphylococci 3. Chains: streptococci, streptobacilli. vi. Most bacteria are monomorphic. They do not change shape unless environmental conditions change. vii. A few are pleomorphic. These species have individuals that can come in a variety of shapes ULTRA STRUCTURE OF PROKARYOTIC CELLS: Fig 1.1 Structure of Prokaryotic cell 3 Structures External to the Prokaryotic Cell Wall. a. Glycocalyx (sugar coat). i. Usually very sticky. ii. Found external to cell wall. iii. Composed of polysaccharide and/or polypeptide. iv. It can be broken down and used as an energy source when resources are scarce. v. It can protect against dehydration. vi. It helps keep nutrients from moving out of the cell. 1. A capsule is a glycocalyx that is neatly organized and is firmly attached to the cell wall. a. Capsules prevent phagocytosis by the host’s immune system. 2. A slime layer is a glycocalyx that is unorganized and is loosely attached to the cell wall. b. Extracellular polysaccharide (extracellular polymeric substance) is a glycocalyx made of sugars and allows bacterial cells to attach to various surfaces.Prokaryotic Flagella. i. Long, semi-rigid, helical, cellular appendage used for locomotion. ii. Made of chains of the protein flagellin. 1. Attached to a protein hook. iii. Anchored to the cell wall and cell membrane by the basal body. iv. Motile Cells. 1. Rotate flagella to run and tumble. 2. Move toward or away from stimuli (taxis). a. Chemotaxis. b. Phototaxis. c. Axial Filaments (Endoflagella). i. In spirochetes: 1. Anchored at one end of a cell. 2. Covered by an outer sheath. 3. Rotation causes cell to move like a corkscrew through a cork. d. Fimbriae. i. Shorter, straighter, thinner than flagella. ii. Not used for locomotion. iii. Allow for the attachment of bacteria to surfaces. iv. Can be found at the poles of the cell, or covering the cell’s entire surface. v. There may be few or many fimbriae on a single bacterium. e. Pili (sex pili). i. Longer than fimbriae. ii. Only one or two per cell. iii. Are used to transfer DNA from one bacterial cell to another, and in twitching & gliding motility. IV. The Prokaryotic Cell Wall. a. Chemically and structurally complex, semi-rigid, gives structure to and protects the cell. b. Surrounds the underlying plasma membrane. 4 c. Prevents osmotic lysis. d. Contributes to the ability to cause disease in some species, and is the site of action for some antibiotics. e. Made of peptidoglycan (in bacteria). i. Polymer of a disaccharide. 1. N-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM). ii. Disaccharides linked by polypeptides to form lattice surrounding the cell. Fig. iii. Penicillin inhibits this lattice formation, and leads to cellular lysis. f. Gram-positive cell walls. Fig. i. Many layers of peptidoglycan, resulting in a thick, rigid structure. ii. Teichoic acids. 1. May regulate movement of cations (+). 2. May be involved in cell growth, preventing extensive wall breakdown and lysis. 3. Contribute to antigenic specificity for each Gram-positive bacterial species. 4. Lipoteichoic acid links to plasma membrane. 5. Wall teichoic acid links to peptidoglycan. g. Gram-negative cell walls. i. Contains only one or a few layers of peptidoglycan. 1. Peptidoglycan is found in the periplasm, a fluid-filled space between the outer membrane and plasma membrane. a. Periplasm contains many digestive enzymes and transport proteins. ii. No teichoic acids are found in Gram-negative cell walls. iii. More susceptible to rupture than Gram-positive cells. iv. Outer membrane: 1. Composed of lipopolysaccharides, lipoproteins, and phospholipids. 2. Protects the cell from phagocytes, complement, antibiotics, lysozyme, detergents, heavy metals, bile salts, and certain dyes. 3. Contains transport proteins called porins. 4. Lipopolysaccharide is composed of: a. O polysaccharide (antigen) that can be used to ID certain Gram- negative bacterial species. b. Lipid A (endotoxin) can cause shock, fever, and even death if enough is released into the host’s blood. h. Gram Stain Mechanism. i. Crystal Violet-Iodine (CV-I) crystals form within the cell. ii. Gram-positive: 1. Alcohol dehydrates peptidoglycan. 2. CV-I crystals cannot leave. iii. Gram-negative: 1. Alcohol dissolves outer membrane and leaves holes in peptidoglycan. 2. CV-I washes out. 3. Safranin stains the cell pink. iv. Table 1, pg. 94, compares Gram-positive and Gram-negative bacteria. i. Damage to Prokaryotic Cell Walls. i. Because prokaryotic cell walls contain substances not normally found in animal 5  cells, drugs or chemicals that disrupt prokaryotic cell wall structures are often used in medicine, or by the host to combat the bacteria. 1. Lysozyme digests the disaccharides in peptidoglycan. 2. Penicillin inhibits the formation of peptide bridges in peptidoglycan. ii. A protoplast is a Gram-positive cell whose cell wall has been destroyed, but that is still alive and functional. (Lost its peptidoglycan). iii. A spheroplast is a wall-less Gram-negative cell. (Lost its outer membrane and peptidoglycan). iv. L forms are wall-less cells that swell into irregular shapes. They can live, divide, and may return to a walled state. v. Protoplasts and spheroplasts are susceptible to osmotic lysis. vi. Gram-negative bacteria are not as susceptible to penicillin due to the outer membrane and the small amount of peptidoglycan in their walls. vii. Gram-negative bacteria are susceptible to antibiotics that can penetrate the outer membrane (Streptomycin, chloramphenicol, tetracycline). V. Structures Internal to the Cell Wall. a. Plasma Membrane (Inner Membrane). a. Phospholipid bilayer lying inside the cell wall. 1. The phospholipid bilayer is the basic framework of the plasma membrane. 2. The bilayer arrangement occurs because the phospholipids are amphipathic molecules. They have both polar (charged) and nonpolar (uncharged) parts with the polar “head” of the phospholipid pointing out and the nonpolar “tails” pointing toward the center of the membrane, forming a nonpolar, hydrophobic region in the membrane’s interior. b. Much of the metabolic machinery is located on the plasma membrane. Photosynthesis, aerobic cellular respiration, and anaerobic cellular respiration reactions occur here. This means that there is a surface area to volume ratio at which bacteria reach a critical size threshold, beyond which bacteria can’t survive. i. Thiomargarita (0.75 mm) is the largest known bacterium and is larger than most eukaryotic cells. It has many invaginations of the plasma membrane, which increases it surface area relative to its volume. c. Peripheral proteins. i. Enzymes. ii. Structural proteins. iii. Some assist the cell in changing membrane shape. d. Integral proteins and transmembrane proteins. i. Provide channels for movement of materials into and out of the cell. e. Fluid Mosaic Model. i. Membrane is as viscous as olive oil. ii. Proteins move to function. iii. Phospholipids rotate and move laterally. f. Selective permeability allows the passage of some molecules but not others across the plasma membrane. i. Large molecules cannot pass through. ii. Ions pass through very slowly or not at all. iii. Lipid soluble molecules pass through easily. iv.Smaller molecules (water, oxygen, carbon dioxide, some simple sugars) 6  usually pass through easily. g. The plasma membrane contains enzymes for ATP production. h. Photosynthetic pigments are found on in-foldings of the plasma membrane called chromatophores or thylakoids. Fig. 15. i. Damage to the plasma membrane by alcohols, quaternary ammonium compounds (a class of disinfectants) and polymyxin antibiotics causes leakage of cell contents. j. Movement of Materials Across Membranes. 1. Passive Processes: a. Simple diffusion: Movement of a solute from an area of high concentration to an area of low concentration (down its concentration gradient) until equilibrium is reached. b. Facilitated diffusion: Solute combines with a transport protein in the membrane, to pass from one side of the membrane to the other. The molecule is still moving down its concentration gradient. The transport proteins are specific. c. Osmosis. i. Movement of water across a selectively permeable membrane from an area of higher water concentration to an area of lower water concentration. ii. Osmotic pressure. The pressure needed to stop the movement of water across the membrane. iii. Isotonic, hypotonic, and hypertonic solutions. 2. Active Processes: a. Active transport of substances requires a transporter protein and ATP. The solute molecule is pumped against its concentration gradient. Transport proteins are specific. i. In group translocation (a special form of active transport found only in prokaryotes) movement of a substance requires a specific transport protein. 1. The substance is chemically altered during transport, preventing it from escaping the cell after it is transported inside. 2. This process requires high-energy phosphate compounds like phosphoenolpyruvic acid (PEP) to phosphorylate the transported molecule, preventing its movement out of the cell. b. Cytoplasm. i. Cytoplasm is the substance inside the plasma membrane. ii. It is about 80% water. iii. Contains proteins, enzymes, carbohydrates, lipids, inorganic ions, various compounds, a nuclear area, ribosomes, and inclusions. c. Nuclear Area (Nucleoid). i. Contains a single circular chromosome made of DNA. 1. No histones or introns in bacteria. 2. The chromosome is attached to the plasma membrane at a point along its length, where proteins synthesize and partition new DNA for division during binary fission. ii. Is not surrounded by a nuclear envelope the way eukaryotic chromosomes are. iii. Also contains small circular DNA molecules called plasmids. 1. Plasmids can be gained or lost without harming the cell. 2. Usually contain less than 100 genes. 3. Can be beneficial if they contain genes for antibiotic resistance, tolerance to toxic metals, production of toxins, or synthesis of enzymes. 4. They can be transferred from one bacterium to another. 7  5. Plasmids are used in genetic engineering. d. Ribosomes. i. Site of protein synthesis. ii. Composed of a large and small subunit, both made of protein and rRNA. iii. Prokaryotic ribosomes are 70S ribosomes. 1. Made of a small 30S subunit and a larger 50S subunit. iv. Eukaryotic ribosomes are 80S ribosomes. 1. Made of a small 40S subunit and a larger 60S subunit. v. Certain antibiotics target only prokaryotic ribosomal subunits without targeting eukaryotic ribosomal subunits. e. Inclusions. i. Reserve deposits of nutrients that can be used in times of low resource availability. ii. Include: 1. Metachromatic granules (volutin). Reserve of inorganic phosphate for ATP. 2. Polysaccharide granules. Glycogen and starch. 3. Lipid inclusions. 4. Sulfur granules. Energy reserve for “sulfur bacteria” that derive energy by oxidizing sulfur and sulfur compounds. 5. Carboxysomes. Contain an enzyme necessary for bacteria that use carbon dioxide as their only source of carbon for carbon dioxide fixation. 6. Gas vacuoles. Help bacteria maintain buoyancy. 7. Magnetosomes. Made of iron oxide, they serve as ballast to help some bacteria sink until reaching an appropriate attachment site. They also decompose hydrogen peroxide. f. Endospores. i. Resting Gram-positive bacterial cells that form when essential nutrients can no longer be obtained. ii. Resistant to desiccation, heat, chemicals, radiation. iii. Bacillus anthracis (anthrax), Clostridium spp. (gangrene, tetanus, botulism, food poisoning). iv. Sporulation (sporogenesis): the process of endospore formation within the vegetative (functional) cell. This takes several hours. 1. Spore septum (invagination of plasma membrane) begins to isolate the newly replicated DNA and a small portion of cytoplasm. This results in the formation of two separate membrane bound structures. 2. The plasma membrane starts to surround the DNA, cytoplasm, and the new membrane encircling the material isolated in step 1, forming a double-layered membrane-bound structure called a forespore. 3. Thick peptidoglycan layers are laid down between the two membranes of the forespore. 4. Then a thick spore coat of protein forms around the outer membrane of the forespore, which is responsible for the durability of the endospore. 5. When the endospore matures, the vegetative cell wall ruptures, killing the cell, and freeing the endospore. a. The endospore is metabolically inert, and contains the chromosome, 8  some RNA, ribosomes, enzymes, other molecules, and very little water. b. Endospores can remain dormant for millions of years. v. Germination: the return to the vegetative state. 1. Triggered by damage to the endospore coat. The enzymes activate, breaking down the protective layers. Water then can enter, and metabolism resumes. vi. Endospores can survive conditions that vegetative cells cannot: boiling, freezing, desiccation, chemical exposure, radiation, etc. EUKARYOTES: Fig. 1.2 Structure of Eukaryotic cell a. Make up algae, protozoa, fungi, higher plants, and animals. Flagella and Cilia. Rotate Cilia are numerous, short, hair-like projections extending from the surface of a cell. They function to move materials across the surface of the cell, or move the cell around in its environment. i. Flagella are similar to cilia but are much longer, usually moving an entire cell. The only example of a flagellum in the human body is the sperm cell tail. 1. Eukaryotic flagella move in a whip-like manner, while prokaryotic flagella 9 b. Cell Wall. i. Simple compared to prokaryotes. 1. No peptidoglycan in eukaryotes. a. Antibiotics that target peptidoglycan (penicillins and cephalosporins) do not harm us. ii. Cell walls are found in plants, algae, and fungi. iii. Made of carbohydrates. 1. Cellulose in algae, plants, and some fungi. 2. Chitin in most fungi. 3. Glucan and mannan in yeasts (unicellular fungi). c. Glycocalyx. i. Sticky carbohydrates extending from an animal cell’s plasma membrane. ii. Glycoproteins and glycolipids form a sugary coat around the cell—the glycocalyx— which helps cells recognize one another, adhere to one another in some tissues, and protects the cell from digestion by enzymes in the extracellular fluid. 1. The glycocalyx also attracts a film of fluid to the surface of many cells, such as RBC’s, making them slippery so they can pass through narrow vessels. d. Plasma Membrane. i. The plasma membrane is a flexible, sturdy barrier that surrounds and contains the cytoplasm of the cell. 1. The fluid mosaic model describes its structure. 2. The membrane consists of proteins in a sea of phospholipids. a. Some proteins float freely while others are anchored at specific locations. b. The membrane lipids allow passage of several types of lipid-soluble molecules but act as a barrier to the passage of charged or polar substances. c. Channel and transport proteins allow movement of polar molecules and ions across the membrane. ii. Phospholipid bilayer. 1. Has the same basic arrangement as the prokaryotic plasma membrane. iii. Arrangement of Membrane Proteins. 1. The membrane proteins are divided into integral and peripheral proteins. a. Integral proteins extend into or across the entire lipid bilayer among the fatty acid tails of the phospholipid molecules, and are firmly anchored in place. i. Most are transmembrane proteins, which span the entire lipid bilayer and protrude into both the cytosol and extracellular fluid. b. Peripheral proteins associate loosely with the polar heads of membrane lipids, and are found at the inner or outer surface of the membrane. 10  2. Many membrane proteins are glycoproteins (proteins with carbohydrate groups attached to the ends that protrude into the extracellular fluid). iv. Functions of Membrane Proteins. 1. Membrane proteins vary in different cells and function as: a. Ion channels (pores): Allow ions such as sodium or potassium to cross the cell membrane; (they can't diffuse through the bilayer). Most are selective—they allow only a single type of ion to pass. Some ion channels open and close. b. Transporters: selectively move a polar substance from one side of the membrane to the other. c. Receptors: recognize and bind a specific molecule. The chemical binding to the receptor is called a ligand. d. Enzymes: catalyze specific chemical reactions at the inside or outside surface of the cell. e. Cell-identity markers (often glycoproteins and glycolipids), such as human leukocyte antigens. f. Linkers: anchor proteins in the plasma membrane of neighboring cells to each other or to protein filaments inside and outside the cell. 2. The different proteins help to determine many of the functions of the plasma membrane. v. Selective permeability of the plasma membrane allows passage of some molecules. 1. Transport mechanisms: a. Simple diffusion. b. Facilitated diffusion. c. Osmosis. d. Active transport. (No group translocation in Eukaryotes). e. Vesicular Transport. i. A vesicle is a small membranous sac formed by budding off from an existing membrane. ii. Two types of vesicular transport are endocytosis and exocytosis. 1. Endocytosis. a. In endocytosis, materials move into a cell in a vesicle formed from the plasma membrane. b. Viruses can take advantage of this mechanism to enter cells. c. Phagocytosis is the ingestion of solid particles, such as worn out cells, bacteria, or viruses. Pseudopods extend and engulf particles. d. Pinocytosis is the ingestion of extracellular fluid. The membrane folds inward bringing in fluid and dissolved substances. 2. In exocytosis, membrane-enclosed structures called secretory vesicles that form inside the cell fuse with the plasma membrane and release their contents into the extracellular fluid. f. Cytoplasm. i. Substance inside the plasma membrane and outside nucleus. ii. Cytosol is the fluid portion of cytoplasm. iii. Cytoskeleton. 1. The cytoskeleton is a network of several kinds of protein filaments that extend throughout the cytoplasm, and provides a structural framework for the cell. 2. It consists of microfilaments, intermediate filaments, and microtubules. 11  a. Most microfilaments (the smallest cytoskeletal elements) are composed of actin and function in movement (muscle contraction and cell division) and mechanical support for the cell itself and for microvilli. b. Intermediate filaments are composed of several different proteins and function in support and to help anchor organelles such as the nucleus. c. Microtubules (the largest cytoskeletal elements) are composed of a protein called tubulin and help determine cell shape; they function in the intracellular transport of organelles and the migration of chromosome during cell division. They also function in the movement of cilia and flagella. iv. Cytoplasmic streaming. 1. Movement of cytoplasm and nutrients throughout cells. 2. Moves the cell over surfaces. g. Organelles. i. Organelles are specialized structures that have characteristic shapes and perform specific functions in eukaryotic cellular growth, maintenance, reproduction. 1. Nucleus. The nucleus is usually the most prominent feature of a eukaryotic cell. b. Most have a single nucleus; some cells (human red blood cells) have none, whereas others (human skeletal muscle fibers) have several in each cell. c. The parts of the nucleus include the: i. Nuclear envelope (a double membrane), which is perforated by channels called nuclear pores, that control the movement of substances between the nucleus and the cytoplasm. 1. Small molecules and ions diffuse passively, while movement of most large molecules out of the nucleus involves active transport. ii. Nucleoli function in producing ribosomes. d. Genetic material (DNA). Within the nucleus are the cell’s hereditary units, called genes, which are arranged in single file along chromosomes. Each chromosome is a long molecule of DNA that is coiled together with several proteins (including histones). 2. RIBOSOMES. a. Sites of protein synthesis. b. 80S in eukaryotes. i. Membrane-bound ribosomes found on rough ER. ii. Free ribosomes found in cytoplasm. c. 70S in prokaryotes. i. Also found in chloroplasts and mitochondria. 3. Endoplasmic Reticulum. a. The endoplasmic reticulum (ER) is a network of membranes extending from the nuclear membrane that form flattened sacs or tubules. b. Rough ER is continuous with the nuclear membrane and has its outer surface studded with ribosomes, which synthesize proteins. The proteins then enter the space inside the ER for processing (into glycoproteins or for attachment to phospholipids) and sorting, 12  and are then either incorporated into organelle membranes, inserted into the plasma membrane, or secreted via exocytosis. c. Smooth ER extends from the rough ER to form a network of membrane tubules, but it does not contain ribosomes on its membrane surface. In humans, it synthesizes fatty acids and steroids, detoxifies drugs, removes phosphate from glucose 6-phosphate (allowing free glucose to enter the blood), and stores and releases calcium ions involved in muscle contraction. 4. Golgi Complex. The Golgi complex consists of four to six stacked, flattened membranous sacs (cisterns). The cis (entry) face faces the rough ER, and trans (exit) face faces the cell’s plasma membrane. Between the cis and trans faces are the medial cisternae. b. The cis, medial, and trans cisternae each contain different enzymes that permit each to modify, sort, and package proteins received from the rough ER for transport to different destinations (such as the plasma membrane, to other organelles, or for export out of the cell). 5. Lysosomes. a. Lysosomes are membrane-enclosed vesicles that form from the Golgi complex and contain powerful digestive enzymes. b. Lysosomes function in digestion of substances that enter the cell by endocytosis, and transport the final products of digestion into the cytosol. c. They digest worn-out organelles (autophagy). d. They digest their own cellular contents (autolysis). e. They carry out extracellular digestion (as happens when sperm release lysosomal enzymes to aid in penetrating an oocyte). 6. Vacuoles. a. Space in the cytoplasm enclosed by a membrane called a tonoplast. b. Derived from the Golgi complex. c. They serve in the following ways: i. Temporary storage for biological molecules and ions. ii. Bring food into cells. iii. Provide structural support. iv. Store metabolic wastes. 7. Peroxisomes. a. Peroxisomes are similar in structure to lysosomes, but are smaller. b. They contain enzymes (oxidases) that use molecular oxygen to oxidize (remove hydrogen atoms from) various organic substances. 13  c. They take part in normal metabolic reactions such as the oxidation of amino and fatty acids. d. New peroxisomes form by budding off from preexisting ones. e. They produce and then destroy H2O2 (hydrogen peroxide) in the process of their metabolic activities. 8. Centrosomes. a. Centrosomes are dense areas of cytoplasm containing the centrioles, which are paired cylinders arranged at right angles to one another, and serve as centers for organizing microtubules and the mitotic spindle during mitosis. 9. Mitochondria. a. Found in nearly all eukaryotic cells. b. A mitochondrion is bound by a double membrane, with a fluid-filled space between called the intermembranous space. The outer membrane is smooth, while the inner membrane is arranged in folds called cristae. The mitochondrial matrix is found inside the inner mitochondrial membrane. c. The folds of the cristae provide a large surface area for the chemical reactions that are part of the aerobic phase of cellular respiration. These reactions produce most of a eukaryotic cell’s ATP, and the enzymes that catalyze them are located on the cristae and in the matrix. d. Mitochondria self-replicate using their own DNA and contain 70S ribosomes. They grow and reproduce on their own in a way that is similar to binary fission. Mitochondrial DNA (genes) is inherited only from the mother, since sperm normally lack most organelles such as mitochondria, ribosomes, ER, and the Golgi complex. Any sperm mitochondria that do enter the oocyte are soon destroyed. 10. Chloroplasts. a. Found only in algae and green plants. b. Contain the pigment chlorophyll and enzymes necessary for photosynthesis. c. Chloroplasts self-replicate using their own DNA and contain 70S ribosomes. They grow and reproduce on their own in a way that is similar to binary fission. VII. Endosymbiotic Theory. a. Large bacterial cells lost their cell walls and engulfed smaller bacteria. b. A symbiotic (mutualistic) relationship developed. i. The host cell supplied the nutrients. ii. The engulfed cell produced excess energy that the host could use. iii. The relationship evolved. c. Evidence: 14  i. Mitochondria and chloroplasts resemble bacteria in size and shape. 1. They divide on their own—independently of the host, and contain their own DNA (single circular chromosome). This process is nearly identical to binary fission seen in bacteria. 2. They contain 70S ribosomes. 3. Their method of protein synthesis is more like that of prokaryotes (no RNA processing). 4. Antibiotics that inhibit protein synthesis on ribosomes in bacteria also inhibit protein Difference among eukaryotic cells There are many different types of eukaryotic cells, though animals and plants are the most familiar eukaryotes, and thus provide an excellent starting point for understanding eukaryotic structure. Fungi and many protists have some substantial differences, however. Animal cell An animal cell is a form of eukaryotic cell that makes up many tissues in animals. Animal cells are distinct from other eukaryotes, most notably plant cells, as they lack cell walls and chloroplasts. They also have smaller vacuoles. Due to the lack of a cell wall, animal cells can adopt a variety of shapes. A phagocytic cell can even engulf other structures. There are many different types of cell. For instance, there are approximately 210 distinct cell types in the adult human body. Plant cell Plant cells are quite different from the cells of the other eukaryotic organisms. Their distinctive features are: A large central vacuole (enclosed by a membrane, the tonoplast), which maintains the cell's turgor and controls movement ofmolecules between the cytosol and sap A primary cell wall containing cellulose, hemicellulose and pectin, deposited by the protoplast on the outside of the cell membrane; this contrasts with the cell walls of fungi, which contain chitin, and the cell envelopes of prokaryotes, in which peptidoglycans are the main structural molecules The plasmodesmata, linking pores in the cell wall that allow each plant cell to communicate with other adjacent cells; this is different from the functionally analogous system of gap junctions between animal cells. 15  Plastids, especially chloroplasts that contain chlorophyll, the pigment that gives plants their green color and allows them to perform photosynthesis Bryophytes and seedless vascular plants lack flagellae and centrioles except in the sperm cells. Sperm of cycads and Ginkgoare large, complex cells that swim with hundreds to thousands of flagellae. Conifers (Pinophyta) and flowering plants (Angiospermae) lack the flagellae and centrioles that are present in animal cells. Structure and functions of cell organelles Mitochondria: The mitochondrion (plural mitochondria) is a double membrane-bound organelle found in most eukaryotic cells. The word mitochondrion comes from the Greek μίτος, mitos, i.e. "thread", and χονδρίον, chondrion, i.e. "granule" or "grain-like". Mitochondria range from 0.5 to 1.0 μm in diameter. A considerable variation can be seen in the structure and size of the organelle though. These, unless specifically stained, are not visible. These structures are described as "the powerhouse of the cell" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in other tasks, such as signaling, cellular differentiation, cell death, as well as maintaining the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders, cardiac dysfunction, and heart failure. A recent University of California study including ten children diagnosed with severe autism suggests that autism may be correlated with mitochondrial defects as well. Several characteristics make mitochondria unique. The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. For instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the 16 intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of protein have been identified from cardiacmitochondria, whereas in rats, 940 proteins have been reported. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes. Components of Mitochondria The first observations of intracellular structures that probably represent mitochondria were published in the 1840s. Richard Altmann, in 1894, established them as cell organelles and called them "bioblasts". The term "mitochondria" itself was coined by Carl Benda in 1898. Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900. Friedrich Meves, in 1904, made the first recorded observation of mitochondria in plants (Nymphaeaalba) and in 1908, along with Claudius Regaud, suggested that they contain proteins and lipids. Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.In 1913, particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called "grana". Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925, when David Keilin discovered cytochromes, that the respiratory chain was described. In 1939, experiments using minced muscle cells demonstrated that cellular respiration using one oxygen atom can form twoadenosine triphosphate (ATP) molecules, and, in 1941, the concept of the phosphate bonds of ATP being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann. In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known. The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria. Over time, the fractionation method was tweaked, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria. The first high-resolution micrographs appeared in 1952, replacing the Janus Green stains as the preferred way of visualising the mitochondria. This led to a more detailed analysis of the structure of the mitochondria, including confirmation that they were surrounded by a membrane. It also showed a second membrane inside the mitochondria that folded up in ridges dividing up the inner chamber and that the size and shape of the mitochondria varied from cell to cell. The popular term "powerhouse of the cell" was coined by Philip Siekevitz in 1957. 17 In 1967, it was discovered that mitochondria contained ribosomes. In 1968, methods were developed for mapping the mitochondrial genes, with the genetic and physical map of yeast mitochondria being completed in 1976 Mitochondrion ultrastructure A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which increase its surface area. While commonly depicted as an "orange sausage with a blob inside of it" (like it is here), mitochondria can take many shapes and their intermembrane space is quite thin. A mitochondrion contains outer and inner membranes composed of phospholipid bilayers andproteins. The two membranes have different properties. Because of this double- membraned organization, there are five distinct parts to a mitochondrion. They are: 1. the outer mitochondrial membrane, 2. the intermembrane space (the space between the outer and inner membranes), 3. the inner mitochondrial membrane, 4. the cristae space (formed by infoldings of the inner membrane), and 5. the matrix (space within the inner membrane). Mitochondria stripped of their outer membrane are called mitoplasts. Outer membrane The outer mitochondrial membrane, which encloses the entire organelle, is 60 to 75angstroms (Å) thick. It has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral membrane proteinscalled porins. These porins form channels that allow molecules of 5000 daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Mitochondrial pro-proteins are imported through specialised translocate complexes. The outer membrane also contains enzymes involved in such diverse activities as the elongation of fatty acids, oxidation of epinephrine, and the degradation of tryptophan. These enzymes include monoamine oxidase, rotenone-insensitive NADH-cytochrome c- reductase, kynurenine hydroxylase and fatty acid Co-A ligase. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death. The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is important in the ER-mitochondria calcium signaling and is involved in the transfer of lipids between the ER and mitochondria.Outside the outer membrane there are small (diameter: 60Å) particles named sub-units of Parson. 18 Intermembrane space The intermembrane space is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules, such as ions and sugars, in the intermembrane space is the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c. Inner membrane The inner mitochondrial membrane contains proteins with five types of functions: 1. Those that perform the redox reactions of oxidative phosphorylation 2. ATP synthase, which generates ATP in the matrix 3. Specific transport proteins that regulate metabolite passage into and out of the matrix 4. Protein import machinery 5. Mitochondrial fusion and fission protein It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two, and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn't contain porins, and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane, formed by the action of the enzymes of the electron transport chain. Cristae The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria, the area of the inner membrane is about five times as large as the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function. One recent mathematical modeling study has suggested that the optical properties of the cristae in filamentous mitochondria may affect the generation and propagation of light within the tissue. 19 Matrix The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle. Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins (see: protein biosynthesis). A published human mitochondrial DNA sequence revealed 16,569 base pairs encoding 37 genes: 22 tRNA, 2 rRNA, and 13 peptide genes. The 13 mitochondrial peptides in humans are integrated into the inner mitochondrial membrane, along with proteins encoded by genes that reside in the host cell's nucleus. Mitochondria-associated ER membrane (MAM) The mitochondria-associated ER membrane (MAM) is another structural element that is increasingly recognized for its critical role in cellular physiology and homeostasis. Once considered a technical snag in cell fractionation techniques, the alleged ER vesicle contaminants that invariably appeared in the mitochondrial fraction have been re-identified as membranous structures derived from the MAM—the interface between mitochondria and the ER. Physical coupling between these two organelles had previously been observed in electron micrographs and has more recently been probed with fluorescence microscopy. Such studies estimate that at the MAM, which may comprise up to 20% of the mitochondrial outer membrane, the ER and mitochondria are separated by a mere 10–25 nm and held together by protein tethering complexes Purified MAM from subcellular fractionation has be shown to be enriched in enzymes involved in phospholipid exchange, in addition to channels associated with Ca2+signaling. These hints of a prominent role for the MAM in the regulation of cellular lipid stores and signal transduction have been borne out, with significant implications for mitochondrial-associated cellular phenomena, as discussed below. Not only has the MAM provided insight into the mechanistic basis underlying such physiological processes as intrinsic apoptosis and the propagation of calcium signaling, but it also favors a more refined view of the mitochondria. Though often seen as static, isolated 'powerhouses' hijacked for cellular metabolism through an ancient endosymbiotic event, the evolution of the MAM underscores the extent to which mitochondria have been integrated into overall cellular physiology, with intimate physical and functional coupling to the endomembrane system. 20 Phospholipid transfer The MAM is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase on the ER face and phosphatidylserine decarboxylase on the mitochondrial face. Because mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a constant and well-regulated supply of phospholipids for membrane integrity. But mitochondria are not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolism. Such trafficking capacity depends on the MAM, which has been shown to facilitate transfer of lipid intermediates between organelles. In contrast to the standard vesicular mechanism of lipid transfer, evidence indicates that the physical proximity of the ER and mitochondrial membranes at the MAM allows for lipid flipping between opposed bilayers. Despite this unusual and seemingly energetically unfavorable mechanism, such transport does not require ATP. Instead, in yeast, it has been shown to be dependent on a multiprotein tethering structure termed the ER-mitochondria encounter structure, or ERMES, although it remains unclear whether this structure directly mediates lipid transfer or is required to keep the membranes in sufficiently close proximity to lower the energy barrier for lipid flipping. The MAM may also be part of the secretory pathway, in addition to its role in intracellular lipid trafficking. In particular, the MAM appears to be an intermediate destination between the rough ER and the Golgi in the pathway that leads to very-low-density lipoprotein, or VLDL, assembly and secretion. The MAM thus serves as a critical metabolic and trafficking hub in lipid metabolism. Calcium signaling A critical role for the ER in calcium signaling was acknowledged before such a role for the mitochondria was widely accepted, in part because the low affinity of Ca 2+ channels localized to the outer mitochondrial membrane seemed to fly in the face of this organelle's purported responsiveness to changes in intracellular Ca2+ flux. But the presence of the MAM resolves this apparent contradiction: the close physical association between the two organelles results in Ca2+ microdomains at contact points that facilitate efficient Ca 2+ transmission from the ER to the mitochondria. Transmission occurs in response to so-called "Ca2+ puffs" generated by spontaneous clustering and activation of IP3R, a canonical ER membrane Ca2+ channel. The fate of these puffs—in particular, whether they remain restricted to isolated locales or integrated into Ca2+ waves for propagation throughout the cell—is determined in large part by MAM dynamics. Although reuptake of Ca2+ by the ER (concomitant with its release) modulates the intensity of the puffs, thus insulating mitochondria to a certain degree from high 21 Ca2+ exposure, the MAM often serves as a firewall that essentially buffers Ca2+ puffs by acting as a sink into which free ions released into the cytosol can be funneled. This Ca 2+ tunneling occurs through the low-affinity Ca2+ receptor VDAC1, which recently has been shown to be physically tethered to the IP3R clusters on the ER membrane and enriched at the MAM. The ability of mitochondria to serve as a Ca2+ sink is a result of the electrochemical gradient generated during oxidative phosphorylation, which makes tunneling of the cation an exergonic process. But transmission of Ca2+ is not unidirectional; rather, it is a two-way street. The properties of the Ca2+ pump SERCA and the channel IP3R present on the ER membrane facilitate feedback regulation coordinated by MAM function. In particular, theclearance of Ca2+ by the MAM allows for spatio-temporal patterning of Ca2+ signaling because Ca2+ alters IP3R activity in a biphasic manner. SERCA is likewise affected by mitochondrial feedback: uptake of Ca2+ by the MAM stimulates ATP production, thus providing energy that enables SERCA to reload the ER with Ca2+ for continued Ca2+ efflux at the MAM. Thus, the MAM is not a passive buffer for Ca2+ puffs; rather it helps modulate further Ca2+signaling through feedback loops that affect ER dynamics. Regulating ER release of Ca2+ at the MAM is especially critical because only a certain window of Ca2+ uptake sustains the mitochondria, and consequently the cell, at homeostasis. Sufficient intraorganelle Ca2+ signaling is required to stimulate metabolism by activating dehydrogenase enzymes critical to flux through the citric acid cycle. However, once Ca2+ signaling in the mitochondria passes a certain threshold, it stimulates the intrinsic pathway of apoptosis in part by collapsing the mitochondrial membrane potential required for metabolism. Studies examining the role of pro- and anti-apoptotic factors support this model; for example, the anti-apoptotic factor Bcl-2 has been shown to interact with IP3Rs to reduce Ca2+ filling of the ER, leading to reduced efflux at the MAM and preventing collapse of the mitochondrial membrane potential post- apoptotic stimuli. Given the need for such fine regulation of Ca2+ signaling, it is perhaps unsurprising that dysregulated mitochondrial Ca2+ has been implicated in several neurodegenerative diseases, while the catalogue of tumor suppressors includes a few that are enriched at the MAM. Molecular basis for tethering Recent advances in the identification of the tethers between the mitochondrial and ER membranes suggest that the scaffolding function of the molecular elements involved is secondary to other, non-structural functions. In yeast, ERMES, a multiprotein complex of interacting ER- and mitochondrial-resident membrane proteins, is required for lipid transfer at the MAM and exemplifies this principle. One of its components, for example, is also a constituent of the protein complex required for insertion of transmembrane beta-barrel proteins into the lipid bilayer. However, a homologue of the ERMES complex has not yet been identified in 22 mammalian cells. Other proteins implicated in scaffolding likewise have functions independent of structural tethering at the MAM; for example, ER-resident and mitochondrial-resident mitofusins form heterocomplexes that regulate the number of inter-organelle contact sites, although mitofusins were first identified for their role in fission and fusion events between individual mitochondria. Glucose-related protein 75 (grp75) is another dual-function protein. In addition to the matrix pool of grp75, a portion serves as a chaperone that physically links the mitochondrial and ER Ca2+channels VDAC and IP3R for efficient Ca2+ transmission at the MAM. Another potential tether is Sigma-1R, a non-opioid receptor whose stabilization of ER- resident IP3R may preserve communication at the MAM during the metabolic stress response. Perspective The MAM is a critical signaling, metabolic, and trafficking hub in the cell that allows for the integration of ER and mitochondrial physiology. Coupling between these organelles is not simply structural but functional as well and critical for overall cellular physiology and homeostasis. The MAM thus offers a perspective on mitochondria that diverges from the traditional view of this organelle as a static, isolated unit appropriated for its metabolic capacity by the cell. Instead, this mitochondrial-ER interface emphasizes the integration of the mitochondria, the product of an endosymbiotic event, into diverse cellular processes. Organization and distribution Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. A single mitochondrion is often found in unicellular organisms. Conversely, numerous mitochondria are found in human liver cells, with about 1000–2000 mitochondria per cell, making up 1/5 of the cell volume. The mitochondrial content of otherwise similar cells can vary substantially in size and membrane potential, with differences arising from sources including uneven partitioning at cell divisions, leading to extrinsic differences in ATP levels and 23 downstream cellular processes. The mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum. Often, they form a complex 3D branching network inside the cell with the cytoskeleton. The association with the cytoskeleton determines mitochondrial shape, which can affect the function as well. Mitochondria in cells are always distributed along microtubules and the distribution of these organelles is also correlated with the endoplasmic reticulum. Recent evidence suggests that vimentin, one of the components of the cytoskeleton, is also critical to the association with the cytoskeleton. Function The most prominent roles of mitochondria are to produce the energy currency of the cell, ATP (i.e., phosphorylation of ADP), through respiration, and to regulate cellularmetabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs cycle. However, the mitochondrion has many other functions in addition to the production of ATP. Energy conversion A dominant role for the mitochondria is the production of ATP, as reflected by the large number of proteins in the inner membrane for this task. This is done by oxidizing the major products of glucose: pyruvate, and NADH, which are produced in the cytosol. This process of cellular respiration, also known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited, the glycolytic products will be metabolized by anaerobic fermentation, a process that is independent of the mitochondria. The production of ATP from glucose has an approximately 13-times higher yield during aerobic respiration compared to fermentation. Recently it has been shown that plant mitochondria can produce a limited amount of ATP without oxygen by using the alternate substrate nitrite. ATP crosses out through the inner membrane with the help of aspecific protein, and across the outer membrane via porins. ADP returns via the same route. Pyruvate and the citric acid cycle Main articles: Pyruvate dehydrogenase, Pyruvate carboxylase and Citric acid cycle Pyruvate molecules produced by glycolysis are actively transported across the inner mitochondrial membrane, and into the matrix where they can either be oxidized and combined with coenzyme A to form CO2, acetyl-CoA and NADH, or they can be carboxylated (by pyruvate carboxylase) to form oxaloacetate. This latter reaction” fills up” the amount of oxaloacetate in the citric acid cycle, and is therefore an anaplerotic reaction, increasing the cycle’s capacity to metabolize acetyl-CoA when the tissue's energy needs (e.g. in muscle) are suddenly increased by activity. 24 In the citric acid cycle, all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate) are regenerated during each turn of the cycle. Adding more of any of these intermediates to the mitochondrion therefore means that the additional amount is retained within the cycle, increasing all the other intermediates as one is converted into the other. Hence, the addition of any one of them to the cycle has an anaplerotic effect, and its removal has a cataplerotic effect. These anaplerotic and cataplerotic reactions will, during the course of the cycle, increase or decrease the amount of oxaloacetate available to combine with acetyl-CoA to form citric acid. This in turn increases or decreases the rate of ATP production by the mitochondrion, and thus the availability of ATP to the cell. Acetyl-CoA, on the other hand, derived from pyruvate oxidation, or from the beta- oxidation of fatty acids, is the only fuel to enter the citric acid cycle. With each turn of the cycle one molecule of acetyl-CoA is consumed for every molecule of oxaloacetate present in the mitochondrial matrix, and is never regenerated. It is the oxidation of the acetate portion of acetyl-CoA that produces CO2 and water, with the energy thus released captured in the form of ATP In the liver, the carboxylation of cytosolic pyruvate into intra-mitochondrial oxaloacetate is an early step in the gluconeogenic pathway, which converts lactate and de-aminatedalanine into glucose, under the influence of high levels of glucagon and/or epinephrine in the blood. Here, the addition of oxaloacetate to the mitochondrion does not have a net anaplerotic effect, as another citric acid cycle intermediate (malate) is immediately removed from the mitochondrion to be converted into cytosolic oxaloacetate, which is ultimately converted into glucose, in a process that is almost the reverse of glycolysis. The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process, produces reduced cofactors (three molecules of NADH and one molecule of FADH 2) that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP). NADH and FADH2: the electron transport chain 25 Diagram of the electron transport chain in the mitonchondrialintermembrane space The redox energy from NADH and FADH2 is transferred to oxygen (O2) in several steps via the electron transport chain. These energy-rich molecules are produced within the matrix via the citric acid cycle but are also produced in the cytoplasm byglycolysis. Reducing equivalents from the cytoplasm can be imported via the malate-aspartate shuttle system of antiporterproteins or feed into the electron transport chain using a glycerol phosphate shuttle. Protein complexes in the inner membrane (NADH dehydrogenase (ubiquinone), cytochrome c reductase, and cytochrome c oxidase) perform the transfer and the incremental release of energy is used to pump protons (H+) into the intermembrane space. This process is efficient, but a small percentage of electrons may prematurely reduce oxygen, forming reactive oxygen species such as superoxide. This can cause oxidative stress in the mitochondria and may contribute to the decline in mitochondrial function associated with the aging process. As the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established across the inner membrane. The protons can return to the matrix through the ATP synthase complex, and their potential energy is used to synthesize ATP from ADP and inorganic phosphate (Pi). This process is called chemiosmosis, and was first described by Peter Mitchell who was awarded the 1978 Nobel Prize in Chemistry for his work. Later, part of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their clarification of the working mechanism of ATP synthase. Heat production Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncouplingand is due to the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. The process is mediated by a proton channel called thermogenin, or UCP1. Thermogenin is a 33 kDa protein first discovered in 1973. Thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and decreases with age Storage of calcium ions The concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. Mitochondria can transiently store calcium, a contributing process for the cell's homeostasis of calcium. In fact, their ability to rapidly take in calcium for later release makes them very good "cytosolic buffers" for calcium. The endoplasmic reticulum (ER) is the most significant storage site of calcium, and there is a significant interplay between the mitochondrion and ER with regard to calcium. The calcium is taken up into the matrix by a calcium 26 uniporter on the inner mitochondrial membrane. It is primarily driven by the mitochondrial membrane potential. Release of this calcium back into the cell's interior can occur via a sodium- calcium exchange protein or via "calcium-induced-calcium-release" pathways. This can initiate calcium spikes or calcium waves with large changes in the membrane potential. These can activate a series of second messenger system proteins that can coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells. Ca2+ influx to the mitochondrial matrix has recently been implicated as a mechanism to regulate respiratorybioenergetics by allowing the electrochemical potential across the membrane to transiently "pulse" from ΔΨ-dominated to pH-dominated, facilitating a reduction of oxidative stress. In neurons, concominant increases in cytosolic and mitochondrial calcium act to synchronize neuronal activity with mitochondrial energy metabolism. Mitochondrial matrix calcium levels can reach the tens of micromolar levels, which is necessary for the activation of isocitrate dehydrogenase, one of the key regulatory enzymes of the Kreb's cycle. Additional functions Mitochondria play a central role in many other metabolic tasks, such as: Signaling through mitochondrial reactive oxygen species Regulation of the membrane potential Apoptosis-programmed cell death Calcium signaling (including calcium-evoked apoptosis Regulation of cellular metabolism Certain heme synthesis reactions Steroid synthesis. Hormonal signaling Mitochondria are sensitive and responsive to hormones, in part by the action of mitochondrial estrogen receptors (mtERs). These receptors have been found in various tissues and cell types, including brain and heart Some mitochondrial functions are performed only in specific types of cells. For example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. A mutation in the genes regulating any of these functions can result in mitochondrial diseases. Endoplasmic reticulum: Endoplasmic reticulum is a network of interconnected internal membranes generally, the largest membrane in a eukaryotic cell—an extensive network of closed, flattened membrane- bounded sacs called cisternae(Figure 3). The name “endoplasmic reticulum” was coined in 1953 by Porter, who had observed it in electron micrographs of liver cells. The endoplasmic reticulum has a number of functions in the cell but is particularly important in the synthesis of lipids, membrane proteins, and secreted proteins. 27 Figure 3: The Endoplasmic reticulum. Occurrence: The occurrence of the endoplasmic reticulum is in eukaryotic cells with variation in its position from cell to cell. The erythrocytes (RBC), egg and embryonic cells lack in endoplasmic reticulum. ER is poorly developed in certain cells as the RBC which produces only proteins to be retained in the cytoplasmic matrix (haemoglobin), although the cell may contain many ribosomes). The spermatocytes also have poorly developed endoplasmic reticulum. Morphology: The endoplasmic reticulum occurs in three forms: 1. Lamellar form or cisternae which is a closed, fluid-filled sac, vesicle or cavity is called cisternae; 2. Vesicular form or vesicle and 3.Tubularform 1. Cisternae: The cisternae are long, flattened, sac-like, unbranched tubules having diameter of 40 to 50 μm. They remain arranged parallely in bundles or stakes. RER mostly exists as cisternae which occur in those cells which have synthetic roles as the cells of pancreas, notochord and brain. 2. Vesicles: The vesicles are oval, membrane-bound vacuolar structures having diameter of 25 to 500 μm. They often remain isolated in the cytoplasm and occur in most cells but especially abundantintheSER. 3. Tubules: The tubules are branched structures forming the reticular system along with the cisternae and vesicles. They usually have the diameter from 50 to 190 μm and occur almost in all the cells. Tubular form of ER is often found in SER and is dynamic in nature, i.e., it is associated with membrane movements, fission and fusion between membranes of cytocavity network. Ultrastructure: The cavities of cisternae, vesicles and tubules of the endoplasmic reticulum are bounded by a thin membrane of 50 to 60 Aº thickness. The membrane of endoplasmic reticulum is fluid- mosaic like the unit membrane of the plasma membrane, nucleus, Golgi apparatus. The membrane of 28 endoplasmic reticulum remains continuous with the membranes of plasma membrane, nuclear membrane and Golgi apparatus. The cavity of the endoplasmic reticulum is well developed and acts as a passage for the secretory products. Palade in the year 1956 has observed secretory granules in the cavity of endoplasmic reticulum amking it rough in appearance. Sometimes, the cavity of RER is very narrow with two membranes closely apposed and is much distended in certain cells which are actively engaged in protein synthesis (acinar cells, plasma cells and goblet cells). The membranes of the endoplasmic reticulum contains many kinds of enzymes which are needed for various important synthetic activities. Some of the most common enzymes are found to have different transverse distribution in the ER membranes. The most important enzymes are the stearases, NADH-cytochrome C reductase, NADH diaphorase, glucose-6-phosphotase and Mg++ activated ATPase. Certain enzymes of the endoplasmic reticulum such as nucleotide diphosphate are involved in the biosynthesis of phospholipid, ascorbic acid, glucuronide, steroids and hexose metabolism. Types of endoplasmic reticulum: Agranular or smooth endoplasmic reticulum: ER with no studded ribosomes makes it smooth in appearance. The adipose tissues, brown fat cells and adrenocortical cells, interstitial cells of testes and cells of corpus luteum of ovaries, sebaceous cells and retinal pigment cells contain only smooth endoplasmic reticulum (SER). The synthesis of fatty acids and phospholipids takes place in the smooth ER. It is abundant in hepatocytes. Enzymes in the smooth ER of the liver modify or detoxify hydrophobic chemicals such as pesticides and carcinogens by chemically converting them into more water-soluble, conjugated products that can be excreted from the body. High doses of such compounds result in a large proliferation of the smooth ER in liver cells. Granular or rough endoplasmic reticulum: Ribosomesbound to the endoplasmic reticulum make it appear rough. The rough ER synthesizes certain membrane and organelle proteins and virtually all proteins to be secreted from the cell. A ribosome that fabricates such a protein is bound to the rough ER by the nascent polypeptide chain of the protein. As the growing polypeptide emerges from the ribosome, it passes through the rough ER membrane, with the help of specific proteins in the membrane. Newly made membrane proteins remain associated with the rough ER membrane, and proteins to be secreted accumulate in the lumen of the organelle. All eukaryotic cells contain a discernible amount of rough ER because it is needed for the synthesis of plasma membrane proteins and proteins of the extracellular matrix. Rough ER is particularly abundant in specialized cells that produce an abundance of specific proteins to be secreted. The cells of those organs which are actively engaged in the synthesis of proteins such as acinar cells of pancreas, plasma cells, goblet cells and cells of some endocrine glands are found to contain rough endoplasmic reticulum (RER) which is highly developed. Rough endoplasmic reticulum and protein secretion: George Palade and his colleagues in the 1960s were the first to demonstrate the role of endoplasmic reticulum in protein secretion. The defined pathway taken by secreted protein is: Rough ER - Golgi - secretory vesicles- cell exterior. The entrance of proteins into the ER represents a major branch point for the traffic of proteins within eukaryotic cells. In mammalian cells most proteins are transferred into the ER while they are being translated on membrane bound ribosomes. Proteins that are destined for secretion are then targeted to the endoplasmic reticulum by a signal sequence (short stretch of hydrophobic amino acid residues) at the amino terminus of the growing polypeptide chain. The signal sequence is K/HDEL which is Lys/His- Asp-Glu-Leu. This signal peptide is recognized by a signal recognition particle consisting of six polypeptides and srpRNA. 29 The SRP binds the ribosome as well as the signal sequence, inhibiting further translation and targeting the entire complex (the SRP, ribosome, and growing polypeptide chain) to the rough ER by binding to the SRP receptor on the ER membrane. Binding to the receptor releases the SRP from both the ribosome and the signal sequence of the growing polypeptide chain. The ribosome then binds to a protein translocation complex in the ER membrane, and the signal sequence is inserted into a membrane channel or translocon with the aid of GTP. Transfer of the ribosome mRNA complex from the SRP to the translocon opens the gate on the translocon and allows translation to resume, and the growing polypeptide chain is transferred directly into the translocon channel and across the ER membrane as translation proceeds. As translocation proceeds, the signal sequence is cleaved by signal peptidase and the polypeptide is released into the lumen of the ER. Smooth endoplasmic reticulum and lipid synthesis: Phospholipids are synthesized in the cytosolic side of the ER membrane from water-soluble cytosolic precursors. Other lipids that are synthesized in the ER are cholesterol and ceramide which is further converted to either glycolipids or sphingomyelin in the golgi apparatus. Smooth ER are also the site for the synthesis of the steroid hormones from cholesterol. Thus steroid producing cells in the testis and ovaries are abundant in smooth ER. Common functions of SER and RER: 1. The endoplasmic reticulum provides an ultrastructural skeletal framework to the cell and gives mechanical support to the colloidal cytoplasmic martix. 2. The exchange of molecules by the process of osmosis, diffusion and active transport occurs through the membranes of endoplasmic reticulum. The ER membrane has permeases and carriers. 3. The endoplasmic membranes contain many enzymes which perform various synthetic and metabolic activities and provides increased surface for various enzymatic reactions. 4. The endoplasmic reticulum acts as an intracellular circulatory or transporting system. Various secretory products of granular endoplasmic reticulum are transported to various organelles as follows: Granular ER- agranular ER - Golgi membrane- lysosomes, transport vesicles or secretory granules. Membrane flow may also be an important mechanism for carrying particles, molecules and ions into and out of the cells. Export of RNA and nucleoproteins from nucleus to cytoplasm may also occur by this type of flow. 5. The ER membranes are found to conduct intra-cellular impulses. For example, the sarcoplasmic reticulum transmits impulses from the surface membrane into the deep region of the muscle fibres. 6. The sarcoplasmic reticulum plays a role in releasing calcium when the muscle is stimulated and actively transporting calcium back into the sarcoplasmic reticulum when the stimulation stops and the muscle must be relaxed. The Golgi Complex: Processes and Sorts Secreted and Membrane Proteins The golgi complex was discovered by Camillo Golgi during an investigation of the nervous system and he named it the “internal reticular apparatus”.Functionally it is also known as the post office of the cell.Certain important cellular functions such as biosynthesis of polysaccharides, packaging (compartmentalizing) of cellular synthetic products (proteins), production of 30 exocytotic (secretory) vesicles and differentiation of cellular membranes, occurs in the Golgi complex or Golgi apparatus located in the cytoplasm of animal and plant cells. Occurrence: The Golgi apparatus occurs in all eukaryotic cells. The exceptions are the prokaryotic cells (mycoplasmas, bacteria and blue green algae) and eukaryotic cells of certain fungi, sperm cells of bryophytes and pteridiophytes, cells of mature sieve tubes of plants and mature sperm and red blood cells of animals. Their number per plant cell can vary from several hundred as in tissues of corn root and algal rhizoids (i.e., more than 25,000 in algal rhizoids, Sievers,1965), to a single organelle in some algae. In higher plants, Golgi apparatuses are particularly common in secretory cells and in young rapidly growing cells. In animal cells, there usually occurs a single Golgi apparatus, however, its number may vary from animal to animal and from cell to cell. Paramoeba species has two golgi apparatuses and nerve cells, liver cells and chordate oocytes have multiple golgi apparatuses, there being about 50 of them in the liver cells. Morphology The Golgi apparatus is morphologically very similar in both plant and animal cells. However, it is extremely pleomorphic: in some cell types it appears compact and limited, in others spread out and reticular (net-like). Its shape and form may vary depending on cell type. It appears as a complex array of interconnecting tubules, vesicles and cisternae. There has been much debate concerning the terminology of the Golgi’s parts. The simplest unit of the Golgi apparatus is the cisterna. This is a membrane bound space in which various materials and secretions may accumulate. Numerous cisternae are associated with each other and appear in a stack-like (lamellar) aggregation. A group of these cisternae is called the dictyosome, and a group of dictyosomes makes up the cell’s Golgi apparatus. All dictyosomes of a cell have a common function. The detailed structure of three basic components of the Golgi apparatus are as follows: 1. Flattened Sac or Cisternae Cisternae of the golgi apparatus are about 1 μm in diameter, flattened, plate-like or saucer- like closed compartments which are held in parallel bundles or stacks one above the other. In each stack, cisternae are separated by a space of 20 to 30 nm which may contain rod- like elements or fibres. Each stack of cisternae forms a dictyosome which may contain 5 to 6 Golgi cisternae in animal cells or 20 or more cisternae in plant cells. Each cisterna is bounded by a smooth unit membrane (7.5 nm thick), having a lumen varying in width from about 500 to 1000 nm. Polarity. The margins of each cisterna are gently curved so that the entire dictyosome of Golgi apparatus takes on a bow-like appearance. The cisternae at the convex end of the dictyosome comprise proximal, forming or cis-face and the cisternae at the concave end of the dictyosome comprise the distal, maturing or trans-face. The forming or cis face of Golgi is located next to either the nucleus or a specialized portion of rough ER that lacks bound ribosomes and is called “transitional” ER. Trans face of Golgi is located near the plasma membrane. This polarization is called cis-trans axis of the Golgi apparatus. 2. Tubules A complex array of associated vesicles and tubules (30 to 50 nm diameter) surround the 31 dictyosome and radiate from it. The peripheral area of dictyosome is fenestrated or lace-like in structure. 3. Vesicles The vesicles are 60 nm in diameter and are of three types: (i) Transitional vesicles are small membrane limited vesicles which are form as blebs from the transitional ER to migrate and converge to cis face of Golgi, where they coalasce to form new cisternae. (ii) Secretory vesicles are varied-sized membrane-limited vesicles which discharge from margins of cisternae of Golgi. They, often, occur between the maturing face of Golgi and the plasma membrane. (iii) Clathrin-coated vesicles are spherical protuberances, about 50 μm in diameter and with a rough surface. They are found at the periphery of the organelle, usually at the ends of single tubules, and are morphologically quite distinct from the secretory vesicles. The clathrin-coated vesicles are known to play a role in intra- cellular traffic of membranes and of secretory products. Figure :The Golgi complex. Functions: 1. Modifying, sorting, and packaging of macromolecules for cell secretion: The golgi complex is involved in the transport of lipids around the cell, and the creation of lysosomes.Proteins are modifiedby enzymes in cisternae by glycosylation and phosphorylation by identifying the signal sequence of the protein in question. For example, the Golgi apparatus adds a mannose-6-phosphate label to proteins destined for lysosomes. One molecule that is phosphorylated in the Golgi is Apolipoprotein, which forms a molecule known as VLDL that is a constituent of blood serum. The phosphorylation of these molecules is important to help aid in their sorting for secretion into the blood serum. 32 2. Proteoglycans and carbohydrate synthesis: This include the production of glycosaminoglycans (GAGs), long unbranched polysaccharides which the Golgi then attaches to a protein synthesised in the endoplasmic reticulum to form proteoglycans. 3. Golgi Functions in Animals: In animals, Golgi apparatus is involved in the packaging and exocytosis of the following: Zymogen of exocrine pancreatic cells; Mucus (a glycoprotein) secretion by goblet cells of intestine; Lactoprotein (casein) secretion by mammary gland cells (Merocrine secretion); Secretion of compounds (thyroglobulins) of thyroxine hormone by thyroid cells; Secretion of tropocollagen and collagen; Formation of melanin granules and other pigments; and Formation of yolk and vitelline membrane of growing primary oocytes. It is also involved in the formation of certain cellular organelles such as plasma membrane, lysosomes, acrosome of spermatozoa and cortical granules of a variety of oocytes. 4. Golgi Functions in Plants: In plants, Golgi apparatus is mainly involved in the secretion of materials of primary and secondary cell walls (formation and export of glycoproteins, lipids, pectins and monomers for hemicellulose, cellulose, lignin). During cytokinesis of mitosis or meiosis, the vesicles originating from the periphery of Golgi apparatus, coalesce in the phragmoplast area to form a semisolid layer, called cell plate. The unit membrane of Golgi vesicles fuses during cell plate formation and becomes part of plasma membrane of daughter Ribosome Ribosomes are the protein synthesis units of a cell described by G.E. Palade in 1952. They are complex of ribosomal RNA and various proteins. Their presence in both free and endoplasmis reticulum membrane attached form (rough endoplasmic reticulum) was confirmed by Palade and Siekevitz by the electron microscopy. We will have discussion about endoplasmic reticulum in this lecture after discussion about ribosome. Ribosomes are small, dense, rounded and granular particles of the ribonucleoprotein. As mentioned, they occur either freely in the matrix of mitochondria, chloroplast and cytoplasm or remain attached with the membranes of the endoplasmic reticulum. They occur in most prokaryotic and eukaryotic cells and provide a scaffold for the ordered interaction of all the molecules involved in protein synthesis. They are the most abundant RNA-protein complex in the cell, which directs elongation of a polypeptide at a rate of three to five amino acids added per second. Small proteins of 100–200 amino acids are therefore made in a minute or less. On the other hand, it takes 2–3 hours to make the largest known protein, titin, which is found in muscle and contains about 30,000 amino acid residues. Occurrence and distribution: The ribosomes occur in both prokaryotic and eukaryotic cells. In prokaryotic cells the ribosomes often occur freely in the cytoplasm or sometimes as polyribosome. In eukaryotic cells the ribosomes either occur freely in the cytoplasm or remain attached to the outer surface of the membrane of endoplasmic reticulum. The yeast cells, reticulocytes or lymphocytes, meristamatic plant tissues, embryonic nerve cells and cancerous cells contain large number of ribosomes which 33 often occur freely in the cytoplasmic matrix. Cells like the erythroblasts, developing muscle cells, skin and hair which synthesize specific proteins for the intracellular utilization and storage contain also contain large number of free ribosomes. In cells with active protein synthesis, the ribosomes remain attached with the membranes of the endoplasmic reticulum. Examples are the pancreatic cells, plasma cells, hepatic parenchymal cells, Nissls bodies, osteoblasts, serous cells, or the submaxillary gland, thyroid cells and mammary gland cells. Types of ribosomes: Ribosomes are classified into two types based on their sedimentation coefficient, 70S and 80S. S stands for Svedberg unit and related to sedimentation rate (sedimentation depends on mass and size). Thus, the value before S indicates size of ribosome. 70S Ribosomes: Prokaryotes have 70S ribosemes. The 70S ribosomes are comparatively smaller in size and have sedimentation coefficient 70S with molecular weight 2.7×10 6daltons. Electron microscopy measures the dimension of the 70S ribosomes as170 ×170 × 200 A o. They occur in the prokaryotic cells of the blue green algae and bacteria and also in mitochondria and chloroplasts of eukaryotic cells. 80S Ribosomes: Eukaryotes have 80S ribosomes. The 80S ribosomes have sedimentation coefficient of 80S and molecular weight 40 × 106daltons. The 80S ribosomes occur in eukaryotic cells of the plants and animals. The ribosomes of mitochondria and chloroplasts are always smaller than 80S cytoplasmic ribosomes and are comparable to prokaryotic ribosomes in both size and sensitivity to antibiotics. However their sedimentation values vary in different phyla, 77S in mitochondria of fungi, 60S in mitochondria of mammals and 60S in mitochondria of animals. Number of ribosomes: An E. coli cell contains 10,000 ribosomes, forming 25 per cent of the total mass of the bacterial cell. Whereas, mammalian cultured cells contain 10 million ribosomes per cell. Chemical composition: The ribosomes are chemically composed of RNA and proteins as their major constituents; both occurring approximately in equal proportions in smaller as well as larger subunit. The 70S ribosomes contain more RNA (60 to 40%) than the proteins (36 to 37%). The ribosomes of E. coli contain 63% rRNA and 37% protein. While the 80S ribosomes contain less RNA (40 to 44%) than the proteins (60 to 56%), yeast ribosomes have 40 to 44% RNA and 60 to 56% proteins; ribosomes of pea seedling contain 40% RNA and 60% proteins. There is no lipid content in ribosomes. Ribosomal RNAs: RNA constitutes about 60 percent of the mass of a ribosome. The 70S ribosomes contain three types of rRNA, viz., 23S rRNA, 16S rRNA, 5S rRNA. The 23S and 5S rRNA occur in the larger 50S ribosomal subunit, while the 16S rRNA occurs in the smaller 30S ribosomal subunit. Assuming an average molecular weight for one nucleotide to be 330 daltons, one can calculate the total number of each type of rRNA. Thus, the 23S rRNA consists of 3300 nucleotides, 16S rRNA contains 1650 nucleotides and 5S rRNA includes 120 nucleotides in it. The 80S ribosomes contain four types of rRNA, 28S rRNA (or 25-26 rRNA in plants, fungi and protozoa), 18S rRNA, 5S rRNA and 5.8S rRNA. The 28S, 5S and 5.8S rRNAs occur in the larger 60S ribosomal subnit, while the 18S rRNA occurs in the smaller 40S ribosomal subunit. About 60 per cent of the rRNA 34 is helical (i.e., double stranded) and contains paired bases. These double stranded regions are due to hairpin loops between complimentary regions of the linear molecule. The 28S rRNA has the molecular weight 1.6 × 106daltons and its molecule is double stranded and having nitrogen bases in pairs. The 18S rRNA has the molecular weight 0.6x10 6daltons and consists of 2100 nucleotides. The 18S and 28S ribosomal RNA contain a characteristic number of methyl groups, mostly as 2'-O-methyl ribose. The molecule of 5S rRNA has a clover leaf shape and a length equal to 120 nucleotides. The 5.8S rRNA is intimately associated with the 28S rRNA molecule and has, therefore, been referred to as 28S-associated ribosomal RNA (28S- ArRNA). The 55S ribosomes of mammalian mitochondria lack 5S rRNA but contain 21S and 12S rRNAs. The 21S rRNA occurs in larger or 35S ribosomal subunits, while 12S rRNAoccur in smaller or 25S ribosomal subunit. It is thought that each ribosomal subunit contains a highly folded ribonucleic acid filament to which the various proteins adhere. But as the ribosomes easily bind the basic dyes so it is concluded that RNA is exposed at the surface of the ribosomal subunits, and the protein is assumed to be in the interior in relation to non-helical part of the RNA. Ribosomal Proteins: A ribosome is composed of three (in bacteria) or four (in eukaryotes) different rRNA molecules and as many as 83 proteins, organized into a large subunit and a small subunit. The primary structure of several of these proteins has been elucidated. Most of the recent knowledge about the structure of ribosomal proteins has been achieved by dissociation of ribosomal subunits into their component rRNA and protein molecules. When both 50S and 30S ribosomal subunits are dissociated by centrifuging both of them in a gradient of 5 M cesium chloride, then there are two inactive core particles (40S and 23S, respectively) which contain the RNA and some proteins called core proteins (CP) at the same time several other proteins—the so-called split proteins (SP) are released from each particle.There are SP50 and SP30 proteins which may reconstitute the functional ribosomal subunit when added to their corresponding core. Some of the split proteins are apparently specific for each ribosomal subunit. The split proteins have been further fractionated and divided into acidic (A) and basic (B) proteins. According to Nomura (1968, 1973) and Garett and Wittmann (1973) each 70S ribosome of E. coli is composed of about 55 ribosomal proteins. Out of these 55 proteins, about 21 different molecules have been isolated from the 30S ribosomal subunit, and some 32 to 34 proteins from the 50S ribosomal subunit. Similar organization of ribosomal proteins and RNA is found in 80S Ribosomes. Different rRNA molecules evidently play a central role in the catalytic activities of ribosomes in the process of protein synthesis. Structure The ribosomes are oblate spheroid structures of 150 to 250 Ao in diameter. Each ribosome is porous, hydrated and composed of two subunits. One ribosomal subunit is large in size and has a domelike shape, while the other ribosomal subunit is smaller in size, occurring above the larger subunit and forming a cap-like structure. The small ribosomal subunit contains a single rRNA molecule, referred to as small rRNA. The large subunit contains a molecule of large rRNA and one molecule of 5S rRNA, plus an additional molecule of 5.8S rRNA in vertebrates. The lengths of the rRNA molecules, the quantity of proteins in each subunit, and consequently the sizes of the subunits differ in bacterial and eukaryotic cells. The assembled ribosome is 70S in bacteria and 35  80S in vertebrates. There are great structural and functional similarities between ribosomes from all species which is another reflection of the common evolutionary origin of the most basic constituents of living cells. The 70S ribosome consists of two subunits, 50S and 30S. The 50S ribosomal subunit is larger in size and has the size of 160 Ao to 180 Ao. The 30S ribosomal subunit is smaller in size and occurs above the 50S subunit like a cap. The 80S ribosome also consists of two subunits, 60S and 40S. The 60S ribosomal subunit is dome-shaped and larger in size. In the ribosomes which remain attached with the membranes of endoplasmic reticulum and nucleus, the 60S subunit remains attached with the membranes. The 40S ribosomal subunit is smaller in size and occurs above the 60s subunit forming a cap-like structure. Both the subunits remain separated by a narrow cleft. The two ribosomal subunits remain united with each other due to high concentration of the Mg++(.001M) ions. The actual three-dimensional structures of bacterial rRNAs from Thermusthermopolis recently have been determined by x-ray crystallography of the 70S ribosome. The multiple, much smaller ribosomal proteins for the most part are associated with the surface of the rRNAs. During translation, a ribosome moves along an mRNA chain, interacting with various protein factors and tRNAs and very likely undergoing large conformational changes. Despite the complexity of the ribosome, great progress has been made in determining the overall structure of bacterial ribosomes and in identifying various reactive sites. X-ray crystallographic studies on the T. thermophilus 70S ribosome, for instance, not only have revealed the dimensions and overall shape of the ribosomal subunits but also have localized the positions of tRNAs bound to the ribosome during elongation of a growing protein chain. In addition, powerful chemical techniques such as footprinting, have been used to identify specific nucleotide sequences in rRNAs that bind to protein or another RNA. Figure 1 illustrates the ribosomes. Figure :The detailed structure of a ribosome involved in protein synthesis. 36 Golgi bodies, The Golgi apparatus (/ˈɡoʊldʒiː/), also known as the Golgi complex, Golgi body, or simply the Golgi, is an organelle found in most eukaryotic cells. It was identified in 1897 by the Italian physician Camillo Golgi and named after him in 1898. Part of the cellular endomembrane system, the Golgi apparatus packages proteins into membrane- bound vesicles inside the cell before the vesicles are sent to their destination. The Golgi apparatus resides at the intersection of the secretory, lysosomal, and endocytic pathways. It is of particular importance in processing proteins for secretion, containing a set of glycosylationenzymes that attach various sugar monomers to proteins as the proteins move through the apparatus. Discovery Owing to its large size and distinctive structure, the Golgi apparatus was one of the first organelles to be discovered and observed in detail. It was discovered in 1898 by Italian physician Camillo Golgi during an investigation of the nervous system. After first observing it under his microscope, he termed the structure the internal reticular apparatus. Some doubted the discovery at first, arguing that the appearance of the structure was merely an optical illusion created by the observation technique used by Golgi. With the development of modern microscopes in the 20th century, the discovery was confirmed. Early references to the Golgi referred to it by various names including the "Golgi–Holmgren apparatus", "Golgi–Holmgren ducts", and "Golgi–Kopsch apparatus". The term "Golgi apparatus" was used in 1910 and first appeared in scientific literature in 1913. Subcellular localization Among eukaryotes, the subcellular localizat