Biochem Final.docx

Homework Questions for Ocular Fluids 1. Why is water a partially charged molecule? It has positively charged hydrogens and a negatively charged oxygen 2. What is solvation and how does water accomplish solvation? Solvation is the process in which a solvent surrounds and interacts with solute particles, facilitating their dissolution. Water accomplishes solvation by surrounding solute particles, utilizing its polar nature, forming hydrogen bonds, and disrupting intermolecular forces, making it a universal solvent 3. What are the kind of weak interactions that water enters into while solubilizing molecules? Hydrogen bonds, van der Waals forces, ionic bonds 4. What are hydrogen bonds and how does water accomplish solvation through hydrogen bonds? Hydrogen bonds are attractive forces between the hydrogen atom of one molecule and an electronegative atom (usually oxygen or nitrogen) of another, and water accomplishes solvation through hydrogen bonds by forming interactions between water molecules and solute particles, aiding in their dissolution 5. How does water interact with proteins? By creating weak bonds between the hydrogen of water and the amino group of a protein; these bonds are weak and numerous– easily formed, easily broken 6. Define pH and give its significance pH is the negative log of the hydrogen ion concentration; pH must be tightly controlled in order to preserve tissue structure and cell viability 7. How can pH be determined? In fluids- through a pH meter Mathematically- with the Henderson-Hasselbalch equation pH= pKa + log(salt)/(acid) 8. How does weak electrolyte function to regulate pH? Weak electrolytes are excellent buffers. They help to maintain pH by partially ionizing in solution and either taking up or donating hydrogen ions. 9. What is dissociation constant and how is it expressed mathematically? Dissociation constant: the ionization of weak electrolytes, determined by comparing the concentrations of the ionized to the nonionized forms Ka= [H+][A-] / [HA] 10. What is pka and what is its application? pKa is the negative log of the dissociation constant; Equal to pH value of a solution when it is 50% ionized Useful to determine the pH of a solution of weak acid 11. What is buffer capacity and what is it dependent on? Buffer capacity: the extent to which a buffer will absorb acid or base; Dependent on its concentration in solution 12. What is the range of buffer and how is it related to pka of the buffer? pH range over which a buffer can effectively neutralize an added acid or an alkaline Extends approximately 1 pH unit above and below the pKa of the buffer 13. What is Henderson-Hasselbalch equation and its applications? pH= pKa + log(salt)/(acid) Used for the preparation of buffers and for the formulation of drugs and drug vehicles that require buffers on topical instillation of a drug 14. How does phosphate buffer system function and what is its pka and buffer range? Already ionized electrolyte ionizes further by the release of a proton pKa of 6.86 and buffer range of 5.86-7.86 15. How does breathing rate influence bicarbonate buffer and what is the cause for respiratory alkalosis? CO2 can be removed with expired hair; Elevated breathing rate raises pH Respiratory alkalosis can result from hyperventilation 16. What are protein buffers and how are the properties of its constituents balanced? Protein buffers are molecules within proteins that help regulate and maintain a stable pH in biological systems by accepting or releasing protons Acidic properties are balanced by basic groups on the same protein 17. What are the ocular fluids found in the nonocular regions as well as those found within the eye? Aqueous fluid, vitreous, precorneal tears 18. What is the pH range of blood in the ocular globe? pH is 7.4 (range is 7.33 to 7.45) 18b. How does the partial pressure of blood in the ocular globe compare with that in arterial blood? Partial pressure of oxygen is much lower in ocular capillary bed (50 mmHg) compared with that in arterial blood (83-108 mmHg) 19. What is the role of albumin and globulin in blood? Albumin: protein that carries water-insoluble blood components Globulin: one of several related water-soluble proteins, some of which are involved in immunological functions 20. What is the role of calcium, potassium and hemoglobin in blood? Calcium: soluble ion with a myriad of functions, including blood clotting, enzyme activation, hormone activity, and muscle contraction Potassium: principal cation of intracellular fluid; very important for enzyme function Hemoglobin: protein that carries oxygen to cells 21. What is aqueous fluid and what are the mechanisms involved in its production? Aqueous fluid: controlled filtrate of blood produced by ciliary body Mechanisms involved in its production: diffusion, ultrafiltration, active secretion 22. What are the cells nourished by aqueous? Cells of corneal endothelium and epithelium, stromal keratocytes, lens 23. How does aqueous humor protect lens and corneal endothelium? Functions as a source of antioxidants for the lens and corneal endothelium 24. What is the role of IOP exerted by the aqueous humor? Maintains shape of the ocular globe and protects to some extent from physical shock 25. What are the two aspects by which aqueous differs from blood? Decreased protein component and no cellular components → enhances ability to transmit light Ascorbic acid concentration is higher → protects lens and cornea against oxidative damage 26. Why does aqueous have a reduced buffer capacity and how is the pH of aqueous maintained? Reduced buffer capacity due to loss of protein pH of aqueous maintained due to retention of sufficient phosphate and bicarbonate levels 27. How does the composition of vitreous humor change as a person ages? Ratio is initially 80% gel, 20% fluid Changes to 40% gel, 60% fluid with age 28. What are the reasons for liquefaction of vitreous and how does it affect the retina? Increase in proportion of fluid with age→ Breakdown of type II collagen→ Destabilization of the retinal surface→ Retinal detachment 29. How does vitreous humor help in absorption of shock? Helps to absorb shock due to exterior forces 30. What is the reason for the vitreous to have viscoelastic property? Due to proteoglycans (hyaluronic acid) and collagens 31. Why does vitreous have a high concentration of ascorbic acid compared to blood? Ascorbic acid acts as an antioxidant and the vitreous, being close proximity to the lens and retina, requires extra protection against oxidative damage. 32. How does the protein content and hyaluronic acid content of vitreous compare with that of blood? Protein and hyaluronic acid contents of vitreous are elevated 33. Why is the potassium content in vitreous less than that of blood? Few hyalocytes in the cortical region of the vitreous 34. Where is precorneal tear film present and what are its functions? A film between the inside of the lids and the cornea Functions include protecting the eye from microorganisms, lubricating the lid-cornea interface, and acting as a temporary depository for instillation of topical drugs 35. Why is the potassium content of tear film higher than that of blood? Potassium content of tear film is nearly 7x higher because of the KCl secretions for the lacrimal gland 36. Why does precorneal tear film not serve as a nutrient source for cornea and conjunctiva? Because the glucose concentration is less Homework Questions for Proteins part I What are proteins and what is the difference between protein and a peptide? Proteins: polymers of amino acids linked by peptide bonds; >10,000 D molecular weight Peptides: molecules with <10,000 D molecular weight How do amino acids differ from each other? What are the general functions of proteins? All amino acids have at least one carboxylic acid and one amino group attached to an adjacent carbon. They differ by groups that are attached to the alpha carbon What are the general functions of proteins? Mechanical support Regulate growth and differentiation Catalysts Transportation and storage Motion Nerve propagation and immune protection Intra and extracellular buffers What are the functions of proteins relevant to the eye? Support structure and clarity of cornea Variable light refraction of the lens Initiate transduction of light into electrical signal Generate IOP Lyse bacteria in precorneal tear film How are proteins classified? By function, location, size, and solubility How are proteins subclassified based on solubility? Give an example for each class. Water-soluble: blood plasma proteins such as albumin Lipid-soluble: intrinsic membrane proteins such as rhodopsin Insoluble: ECM proteins such as collagen What are the four basic structures of proteins? Primary, secondary, tertiary, quaternary What is the primary structure of a protein? Sequence of amino acids named left to right from the free amino group end (N-terminal end) What are the factors influencing the configuration and shape of a protein? Factors influencing configuration: bulkiness, changing density, hydrophobic regions of amino acids Factors influencing shape: hydrogen bonding, disulfide bonding within and between amino acid chains Name the four secondary structures of proteins Beta-pleated sheets, alpha-helices, random coils, beta-turns What are random coils and sheets? Irregular forms that connect other secondary structures What are beta-pleated sheets? Give an example of a protein in the eye with beta-pleated sheet structure. Beta-pleated sheet: parallel or antiparallel sequence of primary structures; held together by numerous hydrogen bonds Example of protein in eye with beta-pleated sheet structure: soluble lens crystallins What kind of proteins have alpha-helix structure? Name a protein in the eye with alpha-helix structure. Transmembrane proteins have alpha-helix structures. Protein in the eye with alpha-helix structure: rhodopsin What is a domain and a motif? Domain: subdivision of a tertiary structure; structural or functional part of one chain containing secondary structures Motif: simplified version of a domain What constitutes the tertiary structure of protein? Tertiary structure: entire shape or conformation of one polypeptide chain What is the quaternary structure of a protein comprised of? Entire confirmation of a protein; Consists of two or more polypeptide chains What are disulfide bonds and their role? Disulfide bonds: covalent interactions formed between the sulfur atoms of two cysteine residues Role of disulfide bonds/sulfur bridges: help in joining chains of immunoglobulin G How do proteins contribute to corneal deturgescence? Collagen fibrils resist swelling, endothelial cells pump ions to control fluid balance, proteoglycans maintain hydration What is the role of proteins with regard to generation of IOP? Na+K-ATPase is involved in the production of aqueous humor What are crystallins and where are they found in lens and give their role? Crystallins: a group of structural proteins that are water-soluble Found in the epithelial and fiber cells of the ocular lens They maintain the elongated shape of lens fiber cells and lens structure, and they also affect the refraction of light How are crystallins classified? Alpha-crystallins: alpha-A and alpha-B Beta-crystallins: beta-H and beta-L Gamma-crystallins: 6 subtypes from gamma-A to gamma-F What is the role of alpha-crystallins in lens and what is their influence on light scattering? Alpha-crystallins are molecular chaperones that help in the refolding of beta and gamma-crystallins. They help prevent crystallin aggregation, which causes light scattering (especially aggregation of beta-crystallin). What is the influence of acetylation on crystallins? Acetylation prevents cellular degradation of proteins. Upon synthesis, alpha and beta-crystallins are acetylated and stable. What are the reactions contributing to the alteration of primary structure of crystallins? Phosphorylation, incorporation of sugars (glycosylation- temporary and glycation- peremant), and deamidation and degradation of the polypeptide chain What are the features of beta-pleated structure of crystallins? There are rows of the same polypeptide chain, these rows run antiparallel to each other Chains are held together by hydrogen bonds There are a number of hydrophobic amino acids in each beta-pleated sheet What are the two amino acid sites found on gamma-crystallin susceptible for oxidation? Methionine (102) and cysteine (109) What are the structural features of gamma-crystallins? Two sets of sheets (motifs) are aligned in a V-shape Form a hydrophobic globular chain- domain What are the structural features of alpha-crystallins? Two bound rings of crystallin subunits laid on top of each other Large subunit spheres: hydrophilic C-terminal domains Small subunit spheres: hydrophobic N-terminal domains What is the quaternary structure of crystallin comprised of? Polypeptides assembled in various combinations; Referred to as aggregate What are the percentages of occurrence of different crystallins in lens? Alpha-crystallin: 35% Beta-crystallin: 55% Gamma-crystallin: 10% What is phosphorylation of crystallins? How does it influence the protein? Phosphorylation: when phosphate groups are added to serine amino acids The amount of phosphorylated chains of alpha-crystallin increases as lens fiber cells age This amplifies the negative charge and potential energy; Bad and unpreventable How are disulfide bonds formed in crystallins? What is the amino acid contributing to the disulfide bond formation? Formed by crosslinking between different crystallin polypeptides; Oxidative process Cysteine group is the amino acid group that contributes to the disulfide bond formation It is a potential source of molecular bridging or aggregation via disulfide bonding How does the molecular weight and cysteine content vary among different crystallins? Cysteine content is highest in gamma-crystallins and lowest in alpha-crystallins Molecular weight is highest in alpha-crystallins and lowest in gamma-crystallins What is the influence of deamidation on crystallins? Deamidation: loss of an amide group from asparagine and glutamine by oxidation Aspargine and glutamine are converted to their corresponding dicarboxylic acids Parts of lens aging process Where does peptide bond disruption occur and what is the enzyme mediating the reaction? Near the N-terminal region with beta-crystallins Near the C-terminal region with gamma-crystallins Enzyme mediating this reaction is endopeptidase What is the influence of solubility of crystallins on cataract formation? Cataractous lens - a large increase in concentration of insoluble proteins after age 50 Where are HM1 and HM2 aggregates found and what are their properties? Found in older lens Soluble crystallin complexes with molecular weight >1,000,000 Daltons What is the composition and properties of HM3 aggregate? Insoluble, solubilized by guanidium chloride– a denaturing agent Where are HM4 aggregates found? What is their influence on incident light and image quality? A mixture of crystallins as well as noncrystallin 43 k dalton protein Noncrystallin protein - inner face of the plasma membrane of lens fiber cells Mol wt - 2-33 million daltons Held together by disulfide bonds Associated with cortical cataracts Briefly explain the mechanism involved in cortical cataract. Growing HM fractions Form at the inner layer of the lens cell plasma membrane HM fractions continue to grow into cell cytoplasm (Senile cataracts) Formation of nuclear cataracts with nondisulfide bonds Formation of disulfide, covalent bonds Continued growth of protein complexes HM aggregates produce strain on the cell membrane Membrane ruptures Destruction of lens fiber cells Cellular debris What are the two oxidizable amino acids found in crystallins? What happens when these amino acids are exposed to oxidative environment? Methionine and cysteine Exposed cysteines oxidize and form disulfide bonds with other crystallins and membrane proteins What component of crystallin contributes to the yellow coloration found in nuclear cataracts? What range of UV wavelength induces cataract? Tryptophan 300-400 nm induces cataracts What is the role of anthranilate and beta-carboline found in lens? Substances found in lens that promote nondisulfide crosslinking What is the influence of 3-hydroxykynurenine on lens? These metabolites normally act as filters for UV light (300-400 nm) and protect the retina from UV damage Briefly explain the mechanism of nuclear cataract Tryptophan undergoes catabolism in lens epithelial and outer lens fiber cells Catabolism produces 3 hydroxykynurenine (3OHK) and its glucose bound analogue These metabolites normally act as filters for UV light (300-400 nm) - protect the retina from UV damage N-terminal domain of a-crystallins – involved in formation of colored compounds associated with nuclear cataracts What is the colored complex formed on metabolism of 3OHK? What is its influence on cataract? 3OHK following oxidation can form reactive intermediates These intermediates covalently bind with lens proteins 3OHK - oxidized to an o-quinonimine to form quinilinobenzoxamine(QBA), a colored product QBA: Proposed covalent crosslink for nuclear cataracts Formation can occur at the N-terminus of crystallins ***************** Homework Questions for Proteins Part II What is Rhodopsin and its role in the retina? Rhodopsin is an intrinsic membrane protein that initiates the process of phototransduction It plays a role in visual sensation at low light levels Where is Rhodopsin found in photoreceptors? Mainly in the photoreceptor discs Found to a lesser extent in the plasma membranes of rod outer segments How is the N-terminal of rhodopsin oriented and why is this orientation necessary? N-terminal end faces either: Intradiscal space on the disc membrane IPM (interphotoreceptor matrix) on the plasma membrane Orientation is necessary for the protein to maintain its functional role What is the role of the C-terminal of rhodopsin? C-terminal contains hydroxy amino acids, which can be phosphorylated What is the role of the alpha-helical structure of rhodopsin on its association with the membrane? Helices impart a strong association of the protein with the lipids that make up the membrane Where and in what form is Vitamin A found in Rhodopsin? Bound to rhodopsin at the amino acid lysine #296 Present as an aldehyde is cis form 11-cis-retinal Why is cis form of Vitamin A essential in Rhodopsin? It is the most energetically favorable configuration for its confinement among the helices of the protein What is the linkage between vitamin A and Rhodopsin? Why is the linkage unstable? Aldehyde links with the protein by means of a protonated Schiff base Schiff base is less stable because then vitamin A can be more easily detached upon light stimulation What are the different intermediates of Rhodopsin formed upon light stimulation? Bathorhodopsin, lumirhodopsin, metarhodopsin I and II What are the differences between holoprotein and apoprotein? Holoprotein: conjugated protein; occurs when retinal is attached to lysine #296; 11-cis-retinal plus opsin; HIGHER molecular weight Apoprotein: opsin is an apoprotein; portion of rhodopsin with vitamin A detached; LOWER molecular weight What is bleaching of rhodopsin? Conversion of rhodopsin to all-trans retinal and opsin upon stimulation by light What are the three absorption peaks of Rhodopsin observed upon light stimulation and to what do they correspond? Alpha peak (515 nm): absorption of 11-cis-retinal bound to opsin (holoproteins) Beta peak (375 nm): absorption of all-trans retinal not bound to opsin (apoproteins) Gamma peak (280 nm): absorption peak common to both opsin and rhodopsin (both) How do the cone photopigments differ in the range of sensitivity? Blue photopigments: 440-450 nm Green photopigments: 535-555 nm Red photopigments: 570-590 nm What are the two factors affecting the sensitivity of cone photopigments to specific wavelengths? Varied amino acid environments around the 11-cis retinal Its protonated Schiff base linkage How is the sensitivity of red photopigment for red light increased? To increase the sensitivity of red photopigment, substitute serine for alanine at position 180 in the sequence of red pigment What is spectral shifting? Process which involves shifting the sensitivity by about 5 nm How is color blindness caused? How many amino acid variations are responsible for the disorder? Male individuals inherit variations of red and/or green pigment genes arrayed on the X chromosome Variations are only in 15 different amino acids What are glycoproteins and give their general functions? Glycoproteins: proteins to which short chains of sugars (or oligosaccharides) are bound Functions include structure, orientation, immunological recognition, and biological lubrication What are mucins and what are they comprised of? Mucins: heterogeneous molecules that contain a larger amount of carbohydrates than serum and plasma membrane glycoproteins They also contain short-chain sugars How do ocular mucins differ from other glycoproteins? They have a molecular weight of greater than 2.5x10^5 Daltons Composed of 55% carbohydrates Primarily secreted by conjunctival goblet cells What are the functions of ocular mucins? Maintain stability of tear film Biological lubricant at the epithelial surface Viscoelastic buffer against mechanical shock Support tear film stability How do mucins increase tear film stability? By increasing tear film viscosity By trapping lipids within their structure so that they can be reused after blinking What are the pathological conditions that affect the mucins? Vitamin A deficiency Conjunctival ulcerations Stevens-Johnson syndrome Alkali burns What is the influence of loss of mucin production on tear film stability? Destroyed goblet cells, leading to loss of mucin production Leads to a rapid break up of the tear film despite adequate volume of the aqueous layers of tears What is collagen? Give its general functions. Collagen: extracellular insoluble protein that constitutes 80-90% of the bulk of the eye Functions include: Form and maintain tissue structure Act as scaffolds upon which basement membrane is constructed Act as anchoring devices (hold cells onto noncellular areas) Makes up semi-liquid gel of the vitreous humor What is the basic structure of collagen comprised of? Basic structure of collage: some triple helices (different from alpha-helix form) Minor helix: a helical structure for each chain Major helix: a helical structure formulated by the three chains together Three polypeptide chains wound around each other like a piece of rope Classify different types of collagen with an example for each class? 19 different types of collagen, 12 types found in the eye Two main classifications of collagen: Fibrous Non-fibrous FACIT (fiber associated collagens with interrupted triple interrupted helices) Type IX Sheet-forming collagens Type IV and Type VIII Miscellaneous collagens Type XV What is the function of different collagens? Briefly outline the mechanism involved in cellular production of collagen Single collagen chains: synthesized in the usual manner as all proteins on ribosomes Collagen peptide synthesis: reading specific code (sequence) from mRNA at ribosomes located along the cell’s rough ER Polypeptides of collagen: posttranslational modifications during and after synthesis During synthesis: some of the Lys and Pro amino acids have hydroxy groups added to them After synthesis: three chains associate together along common hydrophobic areas How is tropocollagen assembled into collagen fibers? Hydrolysis of the N- and C- extension peptides completes the synthesis of the unit tropocollagen Go through a process of lateral association lengthwise in close vicinity to the cell’s surface Association made up of hydrophobic interactions and formation of crosslinks of lysine and hydroxylysine Crosslinking adds considerable strength to the growing fibers What is FACIT and give its role? Collagen type IX Fiber associate collagens with interrupted triple helices Participate in the formation of fibrils of other collagens by interacting with their fibrils Do not themselves form their own fibril structures Bend away from the associated fiber Associate with other tissue matrix components such as proteoglycans (protein-core with a side chain of GAGs) Name some sheet forming collagens. How do they form mesh-like structures? Collagen type IV and type VIII Have numerous non-collagenous domains These domains facilitate bending of the molecule and form spider structures or meshes upon binding together What are anchoring fibril type collagens? Give their structural features and function. Type VII Form anchors that connect cells to tissues Are dimers: connect cells and tissues at their globular end domains Dimer joined at the end terminal NC domain, forming feet at either end Multiple dimers join to form large 3-pronged feet Large feet get enmeshed in the anchoring plaques of extracellular tissues and hemi-desmosomes How are collagen fibers arranged in cornea? Collagen fibers form sheets, termed lamellae Long axes of fibers are parallel within the sheet They are at different angles from their adjacent lamellae This arrangement gives considerable strength to the corneal tissues How does collagen fiber diameter vary between corneal stroma and sclera? Stromal collagen fibers have a uniform diameter (30 nm) Scleral collagen fibers have diameters that vary between 40 and 140 nm What are the different types of collagen fibers present in corneal stroma? Majority (70%0 is Type I Other include Type V (15%) and Type VI (15%) What is the role of type V collagen in corneal stroma? Prevents light scatter in the corneal stroma Controls the collagen fibril diameter How are collagen fibrils arranged in vitreous and where do they attach? Arranged in roughly parallel orientation across the entire volume of secondary vitreous Some random fibrils are seen joining the fibers at a few locations But fibers establish FIRM attachments near the ora serrata and the macula What are the different types of collagen fibers found in vitreous and their role? Vtrosin: Type II; main vitreal collagen Type IX: associates with both type II collagen and surrounding proteoglycans in the vitreous Proteoglycans help to form aqueous gel of the vitreous Hybrid collagens V-XI: limit the diameter of the type II fibers How does collagen IV support the basement membrane? Forms a flexible web-like structure similar to an open mesh Interacts with other Type IV collagens by associating at non-collagenous peptide extensions What are the common regions in the eye containing type IV collagen? Bowman’s membrane Lens capsule Blood vessels What is the type of structure formed by collagen VIII in Descemet’s membrane? What are its structural features and function? Type VIII: forms geometric patterns such as that found in boxspring Hexagon and tetragon (basic form for Descemet’s membranes) Increases strength What are the two common types of anchoring fibrils? Give their location. Type VII: between basal epithelial cells of cornea and outermost lamellae of the corneal stroma Type I: anterior stromal lamella, randomly oriented How do anchoring fibrils establish connections between epithelial cells and stroma? Hemidesmosomes of the epithelial basal cells to anchoring plaques on the stromal type I collagen fibers Other anchoring fibrils extend form one anchoring plaque to the next What is the relevance of diabetes to anchoring fibrils? Results in loose adhesion of epithelium to the underlying stroma Interferes in the synthesis of anchoring fibrils Carbohydrates homework questions part I What are carbohydrates composed of and their common role? Carbon and water, with the general formula Cn(H2O)n Used for cellular foods and support tissue mi=orphology What are the three forms of carbohydrate structure and which one is most commonly followed? Fischer, Haworth, Conformational Haworth most commonly followed How do carbohydrates undergo isomerization? Give an example By the action of cellular enzymes C-1 can form an oxygen bridge by itself with C-5 to form a six-membered, closed ring known as a pyran What is the difference between a pyran and a furan ring? How are they formed? C-1 can form an oxygen bridge by itself with C-5 to form a six-membered, closed ring known as a pyran ring C-2 can form an oxygen bridge with C-5 to form a five-membered closed ring known as a furan ring What is anomeric carbon? Why is it reactive? Anomeric carbon- C-1 in a six-membered closed ring Donates electrons Reactive property is useful for determination of glucose concentration in blood and urine How is glucose concentration determined in urine and blood? The aldehyde of C-1 reacts with o-toluidine to form a colored covalent complex, which gives a reliable estimate of glucose concentration Give examples of five and six member ring monosaccharides. What is their role? 5-member ring monosaccharide: fructose 6-member ring monosaccharides: glucose and galactose Nutritional value for cells Serve as metabolic building blocks for more complex carbohydrates What are the components of maltose, sucrose and lactose? What kinds of linkages exist between them? Maltose: 2 glucose units held together by an oxygen bridge Alpha (1-4) linkage Sucrose: 1 glucose and 1 fructose joined by an oxygen bridge Alpha (1-2) linkage Lactose: galactose and glucose joined by an oxygen bridge Beta (1-4) linkage What is the difference between an oligosaccharide and a polysaccharide? Oligosaccharide: carbohydrates with more than two units Polysaccharide: carbohydrate with many sugar units Why does glycogen structure permit its easy storage? What cells in the eye store glycogen? Very compact molecule due to extensive branching which is desirable for storage Corneal epithelial and retinal Muller cells maintain glycogen stores in the eye Why are corneal endothelial cells and photoreceptors unable to store glycogen? They utilize glucose at a higher rate, so they cannot store glycogen What is the difference between anabolic and catabolic reaction? What is their role? Catabolic: involving energy production; extract energy from glucose Anabolic: involving energy consumption; reactions that synthesize cellular components and maintain its functions Together they allow the cell to carry out its anabolic functions, such as making proteins, building cell walls, etc. How does ATP link anabolic and catabolic reactions and bring about muscle contraction? ATP transfers its energy to the muscle protein myosin to initiate muscle contraction in a series of two anabolic reactions. What are high energy compounds? What are the components of ATP? High energy compounds provide a connection between anabolic and catabolic reactions. ATP: adenine, ribose,and three phosphate groups How do ATP components initiate a catalytic reaction? ATP facilitates release of its potential energy by breaking the outermost phosphate bond This powers up an enzyme for catalytic work OR increases the potential energy of a protein for some biological task What is the composition of Acetyl CoA? Acetyl CoA: two carbon unit (acetate) coupled to a carrier molecule (coenzyme A) What is the problem of ATP usage in diabetes and starvation? In diabetes or in starvation, the body uses proteins and lipids to make ATP (instead of using carbohydrates). This is destructive to tissues when usage becomes excessive Outline the general metabolic pathway involving ATP production from proteins, carbohydrates and fats? Proteins, carbohydrates, lipids→Acetyl CoA→Krebs cycle→Electron transfer Glucose and galactose enter as glucose 6-phosphate Fructose and mannose enter as fructose 6-phosphate Glycolysis: enzymatic conversion of all carbohydrates to pyruvate, a 3 carbon triose Small amount of ATP produced in formation of pyruvate Pyruvate converted to acetyl coA in mitochondria Acetyl coA joins Krebs cycle, where GTP, NADH, and FADH2 are released CO2 byproduct formed Oxidative phosphorylation: production of substantial quantities of ATP How do you differentiate between aerobic and anaerobic reactions? Aerobic reactions require oxygen, anaerobic reactions don’t What is glycolysis? Glycolysis is the process in which glucose is broken down in the cytoplasm of cells to produce energy in the form of ATP. It is the first step in both aerobic and anaerobic respiration. Enzymatic conversion of all carbohydrates to pyruvate, a three carbon triose In what form do carbohydrates enter the EM pathway? In their phosphorylated form to prevent their escape from the cells (negative charges on the phosphate group will not allow the carbohydrates to pass through the hydrophobic interior of the plasma membrane of the cell) What is the end product of the EM pathway? Pyruvate What compound enters the aerobic pathway and where does the process take place? Acetyl coA enters the aerobic pathway Takes place in mitochondria What are the electron bearing compounds generated in Krebs cycle? NADH and FADH2 generated in Krebs cycle What is oxidative phosphorylation? Involves shuttling of electron-bearing compounds along the mitochondrial, inner membrane Involves production of substantial quantities of ATP What are the three routes possible for glucose 6-phosphate after its formation? 1. Converted to glycogen for storage purpose 2. Converted to pentoses (pentose) shunt for other metabolic requirements of the cell 3. Converted to pyruvate for the continuation of glycolysis What are the end products of electron shuttle and Kreb’s cycle? Electron shuttle end products: NAD+ and FADH2 Krebs cycle end products: 3 NADH, 1 FADH2, 1 ATP (or GTP, which can be converted to ATP), 2 CO2 How are carbohydrates retained within cells? Why can’t carbohydrates escape out of the cell? Retained because they are phosphorylated, the negative charge from the phosphate groups prevents them from exiting plasma membrane Identify sequentially the products generated and enzymes involved in the glycolytic pathway? FLOW CHART Why is phosphofructokinase a dominating enzyme? What are the factors that stimulate and inhibit the enzyme? Because it controls the rate of the entire pathway Low levels of ATP stimulate the enzyme, whereas high levels of H+ ions inhibit the enzyme What is the relationship between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate? Dihydroxyacetone phosphate is converted to glyceraldehyde 2-phosphate, which enters the next part of the pathway How many ATP molecules are consumed, generated in the Glycolytic pathway? 2 ATP consumed 4 ATP generated So, net gain is 2 ATPs How is pyruvate converted to lactate and how many molecules of ATP are generated? What is the reaction called and what are its advantages? Anaerobic exit of glycolysis: pyruvate converted to lactate by lactate dehydrogenase Yield: 2 ATPs per glucose molecule Anaerobic glycolysis is simple, can run at a faster rate, and can obtain a high-energy supply in a short period of time What is the influence of using hard contact lenses on corneal epithelium and cornea in general? Major problem→80% of the available glycogen is used in just over 8 hours of lens wear compared to soft lenses Metabolic strain on epithelial cells Significant swelling of epithelial and anterior stromal corneal tissues due to an increase in lactate Osmotic strain and consequent swelling Why are rigid gas permeable contact lenses a better choice than gas impermeable contacts? Eliminates problem of oxygen level falling below 54 mmHg (corneal swelling could be as much as 20% of the tissue volume) Allows passage of oxygen more efficiently than soft lenses What product diffuses into mitochondria for aerobic phase? What are the two main functions of mitochondria? Pyruvate diffuses into mitochondria for aerobic phase Functions of mitochondria: aerobic manufacturing of ATP, enzymatic processing of reactions, both of which require a separate inner compartment What are the properties of the inner membrane of the mitochondria? What does it contain? Impermeable to molecules and ions without a transport mechanism Large surface area due to numerous infoldings called cristae Contains insoluble electron transferring proteins and ATP synthase, an enzyme required for aerobic ATP production What are the reactions happening in the mitochondrial matrix and inner membrane? Matrix: site of conversion of pyruvate to acetyl coA Inner membrane: aerobic ATP production What is the primary purpose of Krebs cycle? To supply electrons for the synthesis of ATP Helps in energy production How many reactions are involved in Krebs cycle and where do they occur? Nine enzyme-catalyzed reactions that occur in the mitochondrial matrix Identify the key reactions involved in Krebs cycle and the products generated in those key reactions. Important energy deriving reactions: 4, 5, 6, 7, 9 In these reactions, electrons are removed with either NADH, FADH2, or GTP (high energy compound similar to ATP) GTP is readily converted to ATP NADH and FADH2 diffuse from matrix to inner mitochondrial membrane They donate their electrons to the electron transferring proteins located there What compounds diffuse from matrix to the inner membrane? Matrix: contains numerous soluble enzymes not found in cellular cytoplasm Inner membrane: contains insoluble electron-transferring proteins and ATP synthase How many protein complexes are involved in electron shuttling? What are the proteins responsible and to what complexes do they shuttle electrons? 4 protein complexes Coenzyme Q: shuttles electrons between protein complex 1 and 3 and between 2 and 3 Cytochrome C: shuttles electrons between protein complex 3 and 4 NADH: carries electrons to protein complex 1 FADH2: carries electrons to protein complex 2 Where are hydrogen ions pumped out and how do they re-enter into the matrix? Hydrogen ions are pumped out from the mitochondrial matrix to the intermembrane space They flow back into the matrix through the pores of a fifth protein complex known as ATP synthase How is ATP generated from ATP synthase? The flow of hydrogen ions back into the mitochondrial matrix provides the energy to phosphorylate ADP to form ATP on ATP synthase. The flow also causes the release of ATP from ATP synthase. How many ATP do NADH and FADH2 contribute? Movement of 2 electrons from NADH to water produces 3 ATPs Movement of 2 electrons from FADH2 to water produces 2 ATPs What is the total number of ATP molecules generated from aerobic glycolysis? 36-28 ATP What are the two shuttles contributing to additional sources of ATP? Where do these shuttles occur and what is their ATP contribution? Glycerol phosphate shuttle: brain and muscle cells; FAD as FADH2 contributes to 2 additional ATPs Malate-aspartate shuttle: liver, kidney, and heart cells; NADH contributes to 1 additional ATP Carbohydrates homework questions part II Identify sequentially the products generated and enzymes involved in glycogen synthesis Glucose 6-phosphate→ isomerized to glucose 1-phosphate→ reacts with UTP to form uridine diphosphoglucose→ bound to glucose as an energetic mechanism→ glucose is added to a growing chain of glycogen by the action of glycogen synthase What is the role of glycogen transferase? Glycogen transferase: periodically removes some of the growing terminal chains and binds them onto C-6 of some of the units This occurs every time the main chain grows by 6-21 units What is the enzyme responsible for glycogen synthesis and breakdown? How do phosphate levels play a role in their reciprocal activation? Glycogen synthase: glycogen synthesis Glycogen phosphorylase: glycogen breakdown Reciprocally activated and deactivated based on phosphate levels What are the main functions of the pentose shunt pathway? a. Generation of pentoses b. Production of fatty acids c. Cell detoxification What is the role of NADPH formed in the first step of the pentose pathway? NADH becomes the second product in the reaction Electrons from NADH are used to reduce the tripeptide glutathione How does the redox system function to remove hydrogen peroxide? Glutathione reduces hydrogen peroxide to water What are the products formed as a consequence of reduction of molecular oxygen? Oxygen can be reduced into pathological, highly reactive forms What is the threat of singlet oxygen? These forms contain an unpaired electron that is unstable and attacks both proteins and lipids Identify sequentially the products generated and enzymes involved in the pentose shunt pathway. IN FLOWCHART How is the open ring structure of ribose 5 phosphate converted to a closed ring structure? Transfer of the carbonyl group from carbon 2 to carbon 1 so that a closed-ring pentose can be formed What cells in the cornea and lens utilize the pentose shunt pathway? What is it utilized for? Keratocytes (in corneal stroma)→ utilize pentose shunt to maintain an adequate supply of pentoses for nucleic acids Corneal endothelial cells→ increased energy demand than other corneal cell types in order to work to maintain clarity Energy demands in lens lower than cornea Highest in lens epithelium, lowest in lens nucleus What is gluconeogenesis and how does it differ from glycolysis? Identify the four unique enzymes utilized in this pathway Process by which glucose is synthesized from non-carbohydrate precursors Proceeds reverse of the E-M pathway Pyruvate carboxylase, carboxykinase, fructose 1,6-bisphosphatase, glucose 6-phosphatase What is the principal site of gluconeogenesis and why is it an important source for the photoreceptors? Liver is the principal site of gluconeogenesis Has importance to the eye since photoreceptors have one of the highest demands for a constant supply of glucose and oxygen What is the Warburg effect and what is its significance? Directs excessive amounts of pyruvate to become metabolized to lactate Happens due to relatively high oxygen partial pressure and heavy demand for cellular energy Compare the utilization of aerobic and anaerobic glycolysis by different corneal cells for generation of ATP Corneal epithelial and stromal cells have a high percentage of pentose shunt pathways due to physiological roles of these cells Keratocytes utilize the pentose shunt to maintain an adequate supply of pentoses for nucleic acids Corneal endothelial cells have an increased demand for both aerobic and anaerobic glycolysis compared to keratocytes and epithelial cells Why is the percentage of aerobic glycolysis high in corneal endothelial cells compared to other corneal cell types? How many molecules of ATP are generated? Corneal endothelial cells have to work to maintain clarity Energy yield of ATP per 100 molecules of glucose is 140 (anaerobic) and 838 (aerobic) for a total of 968 molecules of ATP Why is pentose shunt utilized more by epithelial and stromal cells compared to endothelial cells? Epithelium is comprised of 5-6 layers; cells are in a constant state of division There is also a heavy demand for protein and lipid production to achieve cell division and growth Why is the percentage of aerobic glycolysis in lens less compared to cornea? Lens fiber cells lose subcellular organelles as they mature Why is the energy requirement substantial in the ciliary body? Ciliary body is responsible for generation of IOP and preparation of aqueous humor Why is aerobic glycolysis maximum in the retina compared to other parts of the eye? How many ATP molecules are generated per 100 molecules of glucose? Highest in retina due to energy demands of this tissue 1020 ATP molecules are produced per 100 molecules of glucose Why is ATP production rate higher in retina compared to brain? Because oxygen consumption rate as well as blood flow rate is high compared to brain What is diabetes and what are the primary structures affected by this disorder? A metabolic disorder of cellular carbohydrate uptake Primary effects occur on blood vessels of the brain, eyes, kidneys, and external limbs Identify the two types of diabetes. How do they vary in terms of insulin dependence? Juvenile onset diabetes (type 1); autoimmune destruction of the beta cells of the pancreas that synthesize insulin Mature onset diabetes (type 2); insulin receptors in insufficient amounts or in normal amounts that fail to promote sufficient glucose intake What are the mechanisms and proteins involved in glucose transport? Facilitated diffusion and active transport → both mechanisms use transport proteins located in the plasma membranes of cells What are glucose transport proteins, how many types exist and how do they facilitate glucose uptake? Membrane proteins that move glucose into cells 7 members of glucose transport protein (GLUT) family identified Change shape to move glucose across cell membrane Briefly describe the steps involved in glucose transport mechanism involving insulin receptor Binding of insulin to its receptor→ Conformational shift in the insulin receptor protein→ Tyrosine kinase activity of the receptor→ Phosphorylation cascade of intracellular proteins→ Transport of GLUT-4 protein to the cell surface→ Glucose uptake into the cell What is the primary cause of type I diabetes? What are the other functions of insulin? Autoimmune destruction of beta-cells of the pancreas that synthesize insulin What is the genetic involvement in the cause of diabetes? Variation in HLA (human leukocyte antigen) What are insulin dependent cells? Give some examples. Cells that are starved for nourishment: muscle cells, adipose cells, capillary endothelial cells How do muscle cells and fat cells alter their metabolism in response to diabetes? Muscle cells accelerate the breakdown of amino acids to form acetyl CoA, a precursor for ATP production Fat cells accelerate lipid oxidation to form fatty acids that enter the bloodstream How are ketone bodies formed? Identify the compounds and what are their deleterious effects? Ketone bodies are formed when there is a reduction of lipids stores due to fatty acids being taken up by the liver to produce acetyl CoA (for the purpose of synthesizing ATP) Ketone bodies lower blood pH to dangerously low levels How does glycation reaction take place and what is its adverse influence? Identify the compounds of glycation reaction. Glycation: slow and permanent reaction; when high circulating levels of glucose cause them to bind to proteins both extracellularly and intracellularly Results in the production of complex forms and eventual denaturation of proteins (Maillard reaction) What is the role of insulin receptors in Type 2 Diabetes? What is insulin resistance? Insulin receptors are either insufficient or in normal amounts but fail to promote sufficient glucose uptake What is the role of TNF-α in Type 2 Diabetes? Inhibits insulin receptor autophosphorylation How is sorbitol formed in the lens and what is its adverse influence? Sorbitol is formed from glucose via aldose reductase It is a polyol intermediate that cannot escape from lens Leads to high intracellular osmotic pressure and bursting of lens cells Which leads to cellular debris (manifests as cataract) How is sorbitol converted to fructose and why is this conversion not totally protective for the lens? Polyol dehydrogenase can convert sorbitol to fructose Not totally protective for the lens because the rate of conversion is too slow to prevent osmotic destruction of lens cells Carbohydrates Homework Questions Part III What are the major changes happening to retinal blood vessels due to diabetes? Pericytes are destroyed, lumen of the vessel becomes blocked due to vessel swelling, thickening of basement membrane What is the threat of diabetes to retinal blood vessels and vision? Diabetic retinopathy, hemorrhages, retinal detachment Loss of vision in parts or all of retina How is DAG formed and what is its influence on retinal blood vessels? DAG is formed through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC). Increase in glucose→increased DAG→increased PKC→increased ET-1→blocked and leaky vessels in the retina What is the influence of PKC activation and ET-1 on retinal blood vessels? PKC increases blood vessel permeability and thickens blood vessels with the synthesis of basement membrane; Causes induction of ET-1 synthesis ET-1 increases blood vessel permeability as well as its thickening How are AGE products formed in the retina? What is their adverse influence on retinal blood vessels? Protein-glucose ketimines continue to react over time to form complex permanent advanced glycation end products (AGEs) What are the three possible effects of diabetes on cornea? Binding to receptors on vascular endothelial cells to produce a blockage in the vessel Leakage of the vessel Vessel thickening and cell death by apoptosis How does decreased epithelial adhesion affect corneal epithelium? Impairs the ability of corneal epithelium to repair itself after surgery How does decreased sensitivity affect cornea? Impairs patient’s sensitivity to contact, which leads to bacterial infections Can also cause corneal ulcerations What is the effect of corneal swelling on contact lens wear and why does the swelling occur? Corneal swelling can be caused by increased corneal thickness (mainly in stroma) Can affect the ability to wear contact lenses Could be deu to decreased catalytic activity of sodium-potassium-ATPase What is the influence of glycation of laminin on corneal epithelium and the influence of the polyol pathway on corneal endothelium? Polyol pathway could lead to abnormal endothelial morphology; Swelling and glycation can cause epithelial defects Glycation of laminin (protein that forms ECM) leads to inhibition of growth of corneal epithelial cells across its matrix; This could impair spreading and attachment of epithelial cells during healing What are the three enzymes involved in galactose conversion to glucose Galactokinase, Galactose phosphate uridyl transferase (GALT), UDP-galactose epimerase What is galactosemia and the main enzyme contributing to its cause? Galactosemia is caused by a deficiency in any of the above listed enzymes– accumulation of either galactose or galactose 1-phosphate in tissues Genetic defect on chromosome 9 What is the influence of galactosemia on the lens? 30% of patients develop zonular or nuclear cataracts Galactitol could accumulate in the polyol pathway What are the general functions of oligosaccharides when they are linked to proteins? Increase protein hydrophilic solubility Stabilize protein conformation Proper orientation of a protein in a membrane Recognition markers for cell sorting prior to protein transport How does oligosaccharide linkage stabilize rhodopsin and facilitate phototransduction? Prevents rhodopsin from flip-flopping in the outer segment membrane disc (which seriously impairs the process of visual transduction) What are glycosaminoglycans? What role do they play? Sugar polymers that exist predominantly in the ECM and have structural roles Cusion, lubricate, and attach the matrix to various kinds of cells Identify the four common GAGs found in ocular tissues Hyaluronic acid, chondroitin sulfate, keratan sulfate, and dermatan sulfate How does addition of negative charge to GAGs influence its ability to interact with water? Addition of negative charge attracts counter ions, mainly sodium and water What is the GAG present in vitreous and how does it differ from the ones in corneal stroma? Hyaluronic acid– not linked to a core protein Identify the two GAGs found in cornea, their core proteins and their role Keratan sulfate: lumican Dermatan sulfate: decorin Core proteins of the proteoglycans bind between one and three GAGs Role of GAGS i cornea: function as spacer molecules, important to maintain corneal clarity, and corneal dystrophy What is the cause of Mucopolysaccharidoses and Hurler Syndrome and their adverse influence on the eye? Mucopolysaccharidoses cause: deficiency of enzymes that normally breakdown the GAGs; causes opacification of cornea, retinal degeneration, and optic atrophy Hurler Syndrome cause: deficiency of alpha-iduronidase; results in deposition of GAGs (lysosomes) in all parts of the body–brain, joints, and cornea; Can also cause mental retardation, skeletal deformation, and cardiac deficiency HW LIPID 1 1. What is the chemical composition of lipids and give their solubility characteristics? Insoluble and hydrophobic Lipid esters of fatty acids and glycerol Soluble in nonpolar solvents (benzene, chloroform, hexane) Associate together when present in polar solvents 2. What is the amphipathic property of lipids and where is it useful? Amphipathic property: both hydrophilicity and hydrophobicity Useful in cell membrane 3. Identify the major classes of lipids Classified based on the chemistry and chemical properties of the lipids Fatty acids, triglycerides, phospholipids, isoprenoids, esters, eicosanoids, glycolipids 4. What are fatty acids? Fatty acids: varying lengths of hydrocarbons (hydrophobic portion) with a carboxylic group (hydrophilic portion) at one end of the chain 5. What are the two factors influencing their characteristics? Chain length: longer chains mean more hydrophobic and higher melting points Degree of unsaturation: more double bonds lower its melting point and increases its fluidity 6. What is the relation of chain length to hydrophobicity and melting point of fatty acids? Chain length: longer chains mean more hydrophobic and higher melting points 7. What is the relation of degree of unsaturation of fatty acids to fluidity and melting point? Degree of unsaturation: more double bonds lower its melting point and increases its fluidity 8. Name some fatty acids found in precorneal tear film Myristic acid: saturated fatty acid Palmitoleic acid: has one double bond Arachidonic acid: has four double bonds Cervonic acid: has six double bonds 9. What are the characteristics of cervonic acid and where is it found? Cervonic acid: has six double bonds and is present abundantly in membranes of retinal photoreceptors 10. How does increasing carbon length and increasing double bonds affect the fatty acid? Increasing carbon length: thickens a membrane and raises its melting point Increasing double bonds: membrane becomes fluid or flexible (enables the fatty acids to slide freely by one another) and lowers its melting point 11. What are the breakdown products of triacylglycerols under normal and abnormal conditions? Also called triglycerides Represent a storage form of fatty acids One triglyceride: three fatty acids bonded to a glycerol molecule using ester bonds Breakdown of triacylglycerols to acetyl CoA- can occur without ketone body formation (occurs in diabetes) 12. What are phospholipids chemically? Phospholipids: when a phosphate ester bonds the glycerol to one of four kinds of polar groups Structure similar to triacylglycerols Most important lipid class: required for the formation and maintenance of all forms of cellular membranes 13. What are the four kinds of polar groups possible in phospholipids? Ethanolamine, choline, serine, inositol 14. What is the membrane structure comprised of? How are the polar head groups arranged? Two lipid layers (bilayer): fatty acid portions face each other Polar head groups: face the aqueous portions of each side of a cell (plasma membrane) or cell chamber (subcellular organelle) Fatty acid composition: varies based on cell’s functional needs 15. Why does the rod outer segment membrane require a high degree of fluidity? What is the fatty acid responsible for this property? For visual transduction function Cervonic acid 16. How does cervonic acid composition vary between red blood cell and rod outer segment? Cervonic acid percent in rod outer segment is almost 6x greater than that of RBC plasma membranes 17. What are isoprenoids? What are the structural components of cholesterol? Built from 5 carbon units- isoprene Composed of cholesterol, vitamin A, and coenzyme Q 18. What is the contribution of cholesterol for ocular functions? Cholesterol makes up only 8% of the lipids of the disc membranes 19. What is chalazion? How does meibomian gland lipid composition vary in this disease? Chalazion: granulomatous inflammation of the eyelid margins With chalazion, meibomian gland lipids are rich in cholesterol 20. What is the source of increased cholesterol production in Chalazion? From the membranes of inflammatory cells such as neutrophils, lymphocytes, and WBCs Homework Questions for Lipids Part II 1. What are waxes? Where are they found and give its special characteristic? Waxes: esters of long chain fatty acids and long chain alcohols Major component of lipid layer of precorneal tear film Exist as liquids at the temperature of the tear film at around 35 degrees celcius 2. What are eicosanoids? Give an example Eicosanoids: cyclic lipids derived from eicosanoic acids (20 carbon) such as arachidonic acid Examples: prostaglandins and leukotrienes 3. What are glycolipids? Glycolipids: lipids that contain carbohydrates such as galactose They are important membrane components found in nervous, ocular, and other tissues 4. What is a sphingosine and a ceramide? Sphingosine: long chain amino alcohol; basic structure that binds glycolipids Ceramide: when a second fatty acid is bound as an ester to sphingosine 5. What is sphingomyelin and a cerebroside? Sphingomyelin: phosphocholine is esterified to a ceramide Cerebroside and ganglioside: a glycolipid where phosphocholine is replaced by one or more carbohydrates 6. What are cell membranes and their components? Cell membranes: functional barriers to both cell surfaces and interior compartments of cells Comprised of proteins and carbohydrates Simplest membranes contain only lipids 7. How are phospholipids arranged in a cell membrane? Form lipid bilayers in aqueous environment Polar groups extend on either side towards an aqueous environment either inside or outside of the cell Nonpolar regions from both sides of the membrane associate hydrophobically 8. Where is phosphatidylcholine and phosphatidylethanolamine found? More PC molecules on the outer surface of the lipid bilayer Predominance of PE molecules on the inner surface of the lipid bilayer 9. What are the functions of glycolipids in cell membranes? Part of the overall lipid bilayer structure Contribute short chain carbohydrates (sugar arms) into the outer aqueous volume just outside the cells Form part of the cellular glycocalyx (sugar coat) due to their short sugar arms 10.What are the two kinds of proteins found in membranes and how do they differ? Intrinsic (integral): cross or extend into the lipid bilayer of a membrane Extrinsic (peripheral): only associated with either side of the membrane; can be isolated from membranes based on solubility 11. What are the different functions of intrinsic and extrinsic proteins? Give an example for each class Intrinsic: transport, reception (e.g. insulin), transduction (e.g. light), and attachment (to basement membranes); Rhodopsin Extrinsic: passive roles, such as structural (cytoskeletal maintenance), anchoring (glycocalyx component), transduction/signaling, and local movement (myosin and actin); Transducin (extrinsic protein sequentially included in the light transduction process with rhodopsin) 12.What are the two kinds of carbohydrate components found in cell membranes? What are their roles? Glycolipids and glycoproteins Interact with surrounding aqueous environment, biological bridges, bonding agents, found mainly on the extracellular surface of plasma membranes 13.What is the significance of protein to lipid ratio in a membrane? How does it vary in mitochondrial inner membrane and myelin sheath? Helps to gauge the role of membrane– transport, metabolism, and signal transduction Mitochondrial inner membranes: heavily involved in electron transport and ATP production; ratio is 3:1 Myelin membrane: an insulator; ratio is 1:5 14.What is simple diffusion? Give an example Very small molecules oxygen and nitrogen readily cross the membrane Move from higher concentration (outside cell) to lower concentration (inside cell) Example: Movement of glucose inside the cell using the protein GLUT-1 15.What is facilitated transport? What are its characteristics? Protein-assisted transport Limited by the ability of the protein or enzyme to perform the transport Subject to competition or inhibition 16.How does active transport differ from passive facilitated transport? Occurs from an area of lower concentration to an area of higher concentration Enzyme catalysis is required for the energy to operate this pump 17.Give an example of an enzyme involved in active facilitated transport? How does it accomplish the transport function? Na,K-ATPase is involved in active facilitated transport Transports 3 ions of sodium outside the cell and 2 ions of potassium inwards by hydrolyzing 1 molecule of ATP 18.What is uniport, cotransport, symport and antiport? Give examples Uniport: when only a single substance is transported as with GLUT-1 and sugar uptake Cotransport: when two substances are transported simultaneously Symport: when substances are transported in the same direction (ex. glucose transport in kidney tubules) Antiport: substances are transported in opposite directions (ex. Na,K-ATPase) 19.How is tear film formed? What are the three layers present? Formed when tears are separated across cornea after eyelid blinking Three parts: anterior/superficial lipid layer, central aqueous layer, posterior mucus layer 20.Give the composition of each layer of tear film Lipid layer: large variety of waxes and cholesteryl esters Aqueous layer: dissolved salts and proteins Mucus layer: mucoid proteins 21.What is the function of the lipid layer? What is tear-film breakup time? The lipid layer stabilizes the film (15-40 seconds) during its contact time Following stabilization, the tear film breaks and stimulates the next blink That period of time is referred to as tear-film breakup time 22.Identify the different lipid components in lipid layer of tear film Cholesteryl esters Wax esters Triacylglycerols Cholesterol Fatty acids Unidentified components as well Meibomian gland secretions 23.What are the functions of lipid esters found in tear film? Help the lipids flow from their ducts to the eyelid edges Help the lipids form a film over the aqueous layer and maintain contact with it Help the lipids to adhere to the eyelid skin and act as a barrier to the aqueous layer Help the lipids to form a water-tight seal when the lids are closed 24.What are the possible causes contributing to meibomian gland dysfunction? Excessive production of keratin in the ductal epithelium Decreased steryl esters, increased cholesterol, appearance of ceramides Detachment of epithelial cells from the gland (blocks flow of new lipids to the tear film) 25.What is the advantage of high fluidity of the photoreceptor disc membrane? Allows for rotational and lateral movements of rhodopsin needed for phototransduction 26.What is the role of Vitamin E in the retina? What happens in the absence of Vitamin E? Vitamin E absorbs free radicals In the absence of vitamin E, free radicals attack the double bonds of membrane fatty acids and break them up into fragmentary aldehydes 27.To what class of lipids does Vitamin A belong? What are the common dietary sources of Vitamin A? Vitamin A: hydrophobic fat-soluble vitamin; Belongs to isoprenoid class of lipids Beta-carotene: carrots, sweet potatoes Retinyl ester: animal source 28.What are the chemical forms of Vitamin A and their roles? Retinyl ester: storage Retinol: transport, hormonal Retinal: visual transduction Retinoic acid: synthesis 29.What are chylomicra and what is their role? Chylomicra: lipid sphered, transport complexes Hydrophobic lipids→incorporated inferiorly Hydrophilic lipids→coat the surface of the sphere; are complexed with proteins 30.How is retinol conveyed from the liver to target cells? Chylomicra and vitamin A are conveyed to the liver where re-esterification and storage occur Mobilization of retinal takes places after binding to two proteins: retinol binding protein (inside cell) and prealbumin (in bloodstream) 31.What is the fate of retinol after it enters target cells? Upon reaching the cell, retinol is released and transported via a receptor protein into the cell cytoplasm 32.Outline the pathway of transport of Vitamin A in retina All-trans retinal enters RPE either from circulation or from the photoreceptor outer segment Is esterified and isomerized to enzymatically upon entering RPE 11-cis-retinol is converted to 11-cis retinal prior to transport to the rod photoreceptor 33.What are the three disorders of the eye caused due to Vitamin A deficiency? Nyctalopia: loss of night vision Xerophthalmia: dry eyes due to hardening of corneal conjunctiva and subsequent loss of conjunctival secretions Keratomalacia: degeneration of corneal epithelium and corneal perforation 34.What is Xerophthalmia and its associated changes? Xerophthalmia: dry eyes due to hardening of corneal conjunctiva and subsequent loss of conjunctival secretions 35.What does Keratomalacia involve? Keratomalacia: degeneration of corneal epithelium and corneal perforation 36.What is the cause of adverse effects of Vitamin A? What are some common adverse effects? Adverse effects occur when the daily intake of vitamin A exceeds 10,000 IU Abdominal pain, blurred vision, drowsiness, headache, irritability, nausea, and vomiting Also biochemical effects: increased gluconeogenesis and protein turnover 37.What are gangliosides? What is the sugar unit present? Gangliosides: glycolipids mainly located in the membranes of nervous tissue Has a negatively charged sugar: sialic acid 38.What is Tay-Sachs ganglioside? Where does it commonly accumulate? A partially degraded ganglioside Accumulates in neural, ocular, and other tissues in the course of Tay-Sachs disease 39.What is Tay-Sachs disease and what is its cause? A metabolic storage disease Caused by an enzyme defect in the catabolism of glycolipids (deficiency of hexosaminidase A) Hexosaminidase A catalyzes the breakdown of ganglioside molecules as new molecules are synthesized 40.What is the influence of accumulation of GM2 in the retina? Degeneration of the ganglion cells Characteristic cherry-red spot in the macular region Can lead to blindness at a very early age Failure to develop motor and mental capacities (patients die between 3-6 years of age Homework Questions for Nucleic Acids Part I 1. What are the different components of nucleic acids Phosphate, deoxyribose or ribose, and base 2. How do nucleotides and nucleosides differ from each other? Give examples of each class Nucleotide: base, sugar, phosphate; dATP can be incorporated into a molecule of DNA, cyclic GMP (intracellular hormone) Nucleoside: deoxyribose (in DNA) or ribose (in RNA); prefix deoxy used if the sugar is a deoxypentose 3. Mention some common nucleotides and their biological role Deoxyadenosine 5’-triphosphate (dATP): used a building block in DNA synthesis Adenosine 5’-triphosphate (ATP): energy-carrying molecule Guanosine 3’,5’-cyclic monophosphate (cGMP): second messenger molecule Deoxycytidine 5’-monophosphate (dCMP): enzyme that converts deoxycytidylic acid to deoxyuridylic acid 4. What are the main functions of nucleic acids? What are the different activities coordinated by nucleic acids? Maintain code for the amino acid sequence for all proteins in a cell Synthesize those proteins 5. What are the different activities coordinated by nucleic acids? Growth, division, specialized functions, development and hereditary characteristics 6. How does DNA and RNA differ from each other? DNA: nucleic acid form that preserves the code for making all the proteins needed by a cell; has thymine instead of uracil; has deoxyribose instead of ribose RNA: translates that code into specific proteins; has uracil instead of thymine; has ribose instead of deoxyribose 7. What is the requirement for incorporation of 2’ deoxynucleotides into DNA? 2’-hydroxy group is replaced by hydrogen 8. What direction does the synthesis of DNA strands proceed? 5’ to 3’ 9. How is the double helix structure of DNA organized? Deoxypentose phosphates are hydrophilic Hydrophobic bases held internally in a double helix structure of DNA One chain contains the code for the synthesis of specific proteins: coding strand Other chain contains the template or complement of the code: complementary strand 10.What are the different forms in which the DNA helix can exist? A, B, and Z-form 11. What does the DNA code consist of and what does it code for? Code: sequence of sets of 3 bases in the chain 3 bases in succession code for a single amino acid in a protein 12.How many chromosomes does the human cellular DNA consist of? Human cellular DNA is divided in each cell into 46 chromosomes 13.What is a chromosome and what is it comprised of? Thread-like structure made of DNA and proteins found in the nucleus of a cell; Comprised of DNA and proteins 14.What is a genome and how many base pairs are contained in a genome? Genome: complete set of an organism's genetic material, including all of its genes and non-coding sequences of DNA Number of base pairs:human genome contains about 3 billion base pairs 15.What is a nucleosome and how does it differ from a chromatosome? Nucleosome: consists of 8 histone proteins around which the NDA wraps 1.65 times Chromatosome: consists of a nucleosome plus the H1 histone 16. What is a chromatid and give its composition? Chromatid: one half of a single chromosome at metaphase Compose of super-coiled structures (lengths of chromatin fibers) 17.What is the role of Histone and nonhistone proteins? Histone proteins: assist DNA in compacting itself Nonhistone proteins: bound to the DNA at the strands; Control DNA and RNA processing 18.Why is the replication of DNA a semiconservative process? One original strand of the parent duplex DNA becomes incorporated into each of the two new daughter duplex molecules 19.What is a replication fork and leading strand? Replication fork: location of activate replication Leading strand: new strand synthesized in the conventional direction 5’-3’ 20.What are Okazaki fragments? How are they made? Okazaki fragments: new strand made in discrete, short discontinuous fragments Each short fragment in the lagging strand is successively formed backwards in the same direction as that of the leading strand Fragments are joined by a ligase enzyme to become a continuous trend 21.What are the different enzymes involved in the DNA replication process? Give the role of each enzyme Ligase enzyme: binds the last nucleotide to join the fragment with the new growing strand DNA helicase: breaks the hydrogen bonds between the bases to form single strands RNA primase: bound to DNA helicase; Synthesizes short strands of primer RNA that bind to one of the separated parental strands RNA primer: initiator for lagging strand DNA polymerase III: parental template for the lagging strand loops and passes through this enzyme complex Lower polymerase III: synthesizes new DNA bound to the RNA primer to form an Okazaki fragment on the lagging strand DNA polymerase I and ligase: hydrolyze the RNA portion of the primer; Completes the gaps between the DNA Okazaki fragments to form a continuous strand 22.What are the different types of RNA and their role? Which RNA is the most common occurring and give its percentage of occurance? Heterogenous nuclear RNA: initial coding RNA product made from DNA Messenger RNA: cellular refinement of hnRNA made in the nucleus; Carries the exact code for protein sequencing; Also used in the process of protein synthesis known as translation Transfer RNA: short chain RNA that attaches to specific amino acids; Transports amino acids to ribosomes where proteins are synthesized Ribosomal RNA: represents 75% of all RNA in a cell; Its specific role in protein synthesis is not well understood (may have a catalytic role) 23.What is transcription and which nucleic acid serves as a template? Transcription: synthesis of RNA from DNA DNA serves as the template for new RNA formation in this process 24.What are the three types of RNA polymerases and their role? RNA polymerase I: for rRNA synthesis RNA polymerase II: for hn and ultimately mRNA synthesis RNA polymerase III: for tRNA and the smaller species rRNA 25.What site does the transcription begin? Mention the role of the site Transcription begins at a promoter site on DNA Promoter site: helps in rigidly controlling protein synthesis by limiting the amount of RNA that is available 26.What kind of sequences are present at the initiation site and what kind of proteins bind at this region? Contains specific short sequences of DNA such as TATA and/or CAAT These sequences do not code for any amino acids Regulatory proteins bind to this region 27.What regions do regulatory proteins bind? Regulatory proteins: support or inhibit the initiation of hnRNA synthesis Proteins bind to the DNA at their major grooves at linker DNA sites between nucleosomes 28.What region does RNA polymerase bind to start transcription? Binding of RNA polymerase to the promoter element will begin RNA synthesis 29.What is a genetic code comprised of? What is a stop code? A sequence of three bases will code for a single amino acid in a protein Three stop codes: specify only termination of synthesis 30.What are the different roles of noncoding regions of DNA? Spacer molecules: connect coded regions of gene sequences (introns) Promoters/enhancers: signal and control transcription Centromere: attaches to microtubules during mitosis and meiosis Telomere: records the number of cell divisions as an aging marker while preserving chromosome integrity 31.What are telomeres? What kind of sequences occur in telomeres? Telomere: records the number of cell divisions as an aging marker while preserving chromosome integrity Highly repeated sequences such as (TTAGGG)n occur in mammalian telomeres 32.Differentiate between an intron and an exon Exons: gene sequences that code for either all or part of a protein in both DNa and hnRNA Intron: noncoding sequence separating exons; Act as spacers and are eventually looped out of a coding sequence prior to protein synthesis 33.What is the hnRNA comprised of? hnRNA: synthesized in the nucleus and contains two or more coding genes (exons) separated by one or more non-coding regions (introns) A collection of pre-RNA, nRNA, and ribonucleoproteins involved with pre-RNA Considered equivalent to pre-RNA 34.What does hnRNA acquire at its two ends before mRNA synthesis? What is the role of the cap and tail? Acquires a cap of GTP at its 5’-end; Cap protects RNA from degradation and is involved in initiation of protein synthesis Acquires a tail of 100-200 bases of adenosine phosphate at its 3’-end; Tail may help in preventing degradation of RNA 35.What is looping and how is it accomplished? mRNA formation from hnRNA occurs by looping out the introns and splicing the exons together Looping out of introns is aided by specialized small nRNA and small nuclear proteins 36.What is translation? Translation: formation of a polypeptide is a cooperative process between mRNA, tRNA, ribosomes, and specialized proteins Basically translation is protein formation from mRNA 37.What are the RNA types involved in protein synthesis? mRNA and tRNA 38.What are the different regions in tRNA? What is anticodon site comprised of and its role? Anticodon region: site for binding to a 3-base codon of mRNA Acceptor stem (3’ end) Amino acid attachment site 39.What is the role of N,N’-dimethyl guanine in tRNA? Facilitates twisted conformation of tRNA that helps to carry its amino acid to a codon site on a ribosome 40.How does tRNA attach to Ribosome and what does tRNA fetch from cytoplasm? htRNA first binds to its amino acid tRNA bound to amino acid enters the ribosome to attach to the codon portion of mRNA with its anticodon site 41.What are the two parts of Ribosomes and their role? Smaller subunit (40S): houses the mRNA Larger subunit (60S): houses the incoming tRNAs and their amino acids 42.How do ribosomes synthesize proteins? One or more ribosomes move along the mRNA chain and simultaneously synthesize a polypeptide Each tRNA with its amino acid binds to mRNA in succession Each amino acid coming into the 60S subunit forms a peptide bond with the previous amino acid and the peptide chain lengthens 43.How does the peptide bond formation take place in the ribosome? Peptide bond is formed by the catalytic activity of the 23S rRNA located on the ribosome This activity is referred to as peptidyl transferase activity 44.What is the role of 23S rRNA? Peptide bond is formed by the catalytic activity of the 23S rRNA located on the ribosome This activity is referred to as peptidyl transferase activity Ribosome continues to move along the mRNA chain (peptide bond formation) until all of the code is read 45.What is the turnover rate of proteins from one mRNA? 10 proteins can be formed from one mRNA molecule per minute 46.What are the five stages involved in protein synthesis? What happens during initiation Initiation: ribosome is assembled in response to its binding to mRNA and the first tRNA Elongation 1 Elongation 2 Elongation 3 Termination Homework Questions for Nucleic Acids Part II 1. What is the influence of UV on DNA structure and what kind of photoproduct is formed? UV will induce a bond to form between adjacent pyrimidines in DNA DNA helix is distorted and replication cannot occur beyond the dimer 6-4 photoproduct is formed 2. What is the fate of cells having significant damage due to UV? Cells with lots of DNA damage will either die or mutate into a cancer cell 3. What are the enzymes responsible for the repair mechanism? Give the function of each enzyme. Excinuclease: breaks 5’-3’ bonds approximately 5 to 8 bases on both sides of the pyrimidine dimer DNA polymerase I: acts following removal of damaged sequence; Sequences new bases at the gap using the opposite chian DNA ligase: adds the last base to rejoin the two segments of DNA together again 4. What is the influence of pre-existing disease on excinuclease? In individuals with xeroderma pigmentosum, repair mechanisms are defective A pre-existing genetic defect makes imperfect excinuclease molecules 5. How does the cornea respond to exposure to different levels of UV radiation? Low levels of exposure: an inhibition of cellular mitosis Moderate to high levels: swollen nuclei and cell death Extreme levels: complete sloughing of epithelial cells 6. What is the influence of UV radiation on the lens? What kind of cataracts form upon exposure to UV-B radiation? Produces cataracts UV-B exposure causes formation of cortical and posterior subcapsular cataracts 7. What are alkylating agents? Give some examples and their influence on healthy cells Alkylating agents: chemical substances that can produce DNA damage Form covalent alkyl bridges, used to treat cancer, may also damage non-cancerous cells Examples; chlorambucil, carmustine, busulfan 8. What is the adverse influence of 2-Naphthylamine? Known to cause bladder cancer Exposure much be chronic–a single mutation is insufficiency to cause cancer Cancer develops after many years 9. How does cancer develop? Cells lose control over their ability to decide One or more genes in these cells make excessive amounts of proteins These proteins signal cells to divide continuously Inactivation of genes that suppress division These proteins function by binding DNA promoter regions 10.What are the different stages of cell cycle? What happens during G1 and G0 stages? G1: resting stage; no synthetic processes leading to cell division occur G0: subdivision of G1; cells cannot enter into a synthetic proceeds leading to division (insufficient nourishment to begin DNA synthesis) S G2 M phase 11. What happens during S and G2 phase? S (synthetic): synthesis of new DNA G2: interphase prior to mitosis or cell division; 4 sets of chromosomes present 12.What is the significance of M phase and what are checkpoints? M phase: complex process of cell division occurs Checkpoints (aka restriction points): movement or commitment of a cell into its next phase occurs by passage through checkpoints Determined by a variety of biochemical mechanisms 13.What are the proteins influencing cell cycle and how do they influence cell cycle? Mechanism for passage of the cell from G1 to S phase involves proteins E2F and Rb Cells held at G1 phase when E2F and Rb are bound together Rb protein phosphorylated by cyclin dependent kinase 4 → promotes checkpoint passage 14.What is the role of CDK4 on progression of cell cycle? Any influence on the activity of cyclinD/CDK4 complex will influence the ability of the cell to divide (to enter S phase) 15.What proteins affect CDK4 and give their mechanism of inhibition? p53, p63, p73 Cause the synthesis of mRNA for p21, a protein that inhibits cyclinD/CDK4 16.What is the influence of cancer on p53 and how does it affect cell cycle? Ant defect in p53 may allow the cyclinD/CDK4 enzyme complex to allow uncontrolled cell division This occurs in some cancer cells in which p53 occurs in mutated and useless forms 17.Identify the number of genes and exons for each crystalline type Alpha-crystallin: 2 genes with 3 exons per gene Beta-crystallins: 6 genes with 6 exons per genes Gamma-crystallins: 7 genes with 3 exons per gene This amounts to 63 separate DNA and RNA sequences 18.What are heat shock proteins and their role in protecting the organism? Heat shock proteins: made in response to unusual temperature rise in an organism Protect the organism by solubilizing and refolding any heat-denatured proteins 19.What is the significance of chaperone activity of α-crystallin? Alpha-crystallin chaperone function useful in preserving lens protein shape, structure, and function Useful in maintenance of lens clarity 20.How does crystallin synthesis vary during lens development? Crystallins are not renewed in lens fiber cells Lens fibers lose their ability to synthesize new proteins upon maturation 21.Identify the chromosome location for α, β and γ crystallin genes? Alpha-A crystallin genes: chromosome 21 Alpha-B crystallin genes: chromosome 11 Two of the eight beta-crystallin genes: found on chromosomes 17 and 22 6 gamma-crystallin genes: occur on chromosome 2 22.What sequence is present in the promoter element of mouse αA crystallin? What is its role? TATA sequence: responsible for forming preinitiation protein complex needed for transcription to occur 23.What nuclear binding protein binds to promoter sequence of mouse αA crystallin? What kind of special structures are present in this protein and give its role Alpha-A-CRYBP1: nuclear protein that binds to the promoter sequences 66 to 58 of mouse alpha-A crystallin Contains zinc fingers that help the protein to bind to required DNA sequences at their major grooves 24.What event is essential for the synthesis of αA crystallin? Binding of alpha-A-CRYBP1 25.Identify the structures where different crystallins are expressed. Give the chromosome location of the genes involved Alpha-crystallin is expressed by the Cryaa gene and is lens specific Alpha-crystallin subtypes are synthesized in both lens epithelial and newly formed lens fiber cells 26.How do genes contribute to manifestation of disease? By transmission of a defective gene Over/under transcription of normal gene Genetic machinery will produce either abnormal proteins or normals proteins in abnormal amounts These changes can lead to formation of congenital cataracts 27.What contributes to the development of cataract in Philly mice? Deficiency of beta-crystallin subtype beta-B2 28.What kind of mutation occurs in Cryga1Neu? How does this mutation lead to cataract formation? Cryga1Neu mutation: amino acid exchange of Gly for Asp at position 77 of gamma-A-crystallin 29.What chromosomes and regions contribute to anterior polar and zonular pulverulent cataracts? Anterior polar cataracts: may be caused by a mutation oat 17p12-13 Zonular pulverulent cataracts: genetic defect of gamma-crystallin localized to chromosome 2q33-35; Involves an abnormal 5-base insertion 30.What is the role of the Pax-6 gene? What eye abnormalities result as a consequence of mutation of Pax-6 gene? Pax-6 gene: important gene in eye development Pax-6 protein abnormalities: related to congenital cataractogenesis Aniridia: absence of iris with cataract Microphthalmia: abnormally small eyeballs with cataract 31.What is the influence of the Pax-6 gene on lens development? What happens if Pax-6 protein is defective? Defective PAX-6 proteins exhibit repressed formation of mRNA for alpha-A crystallins 32. Why do viruses depend on hosts? What are the major structural features of viruses and the function of each structure? Depend on host because they are unable to reproduce on their own Major structural features: capsid (outer coat composed of several layers of proteins, containing DNA or RNA) In complex viruses–capsid is covered by a lipid membrane Viral nucleic acids are either single-stranded or double-stranded; Some variations in shape of strands 33.What is the structural difference between a simple and a complex virus? In complex viruses–capsid is covered by a lipid membrane obtained from its host’s lipid membranes 34.What are the two kinds of herpes viruses that contribute to corneal infections? Where are they commonly found? Type I: found in the mouth and upper esophagus Type II: found in genital area 35.Identify the different structural features of herpes virus Double-stranded DNA Icosahedral capsid, surrounded by granular zone or tegument, duplex DNA is packed in the form of donut-shaped twisted coils Tegument is covered by an envelope of membrane Protein spikes project from the membrane 36.What are the two states in which the herpes virus can exist? Active state: infects cells and reproduces itself Latent state; becomes quiescent in its host for prolonged periods of time 37.Identify the sequence of events involved in invasion of cornea by Herpes simplex virus 1. Virus particle or virion attaches itself to the cell membranes of corneal epithelial cells 2. Attached by binding to a proteoglycan associated with the membrane surface 3. Viral envelope fuses to the cell membrane and the capsid with its duplex DNA is taken rapidly into the cell 4. Capsid is transported to the nuclear pores of the cell where viral DNA is released into the nucleus 38.What are the three kinds of proteins made by Herpes virus and their role Alpha-proteins: regulatory role Beta-proteins: viral DNA replication Gamma-proteins: envelope/capsid, structural role 39.What is the role of glycoproteins produced by the Herpes virus? Allows the virus to infect other cells Serves as immunological markers for Herpes virus infected cells 40.What are the three consequences to the host cell as a result of herpes infection? Destruction of host nucleolus (where ribosomes are made) Terminal alteration of host membranes Shutdown of host protein synthetic machinery 41.What is the storage site for latent form of virus in Corneal infection? How does it get reactivated? Storage site: gasserian ganglion (located on the larger root of CNV) Reactivated by stressful stimuli, like trauma, fever, sunburn, menstruation 42.What changes occur during latency of Herpes Virus? Viral DNA becomes circular and produces one mRNA known as LAT RNA LAT gene is important to establish viral latency This mechanism actually protect sensory neurons form destruction by shutting down the virus Migration of the virus back to the cornea in rabbits is reduced in herpesvirus null mutants 43.What is the mechanism involved in the action of acyclovir on infected cells? What is the influence of the agent on normal cells? Acyclovir: Catalyzed by viral thymidine kinase in infected cells to AGT Cellular thymidine kinase will not phosphorylate it Viral DNA polymerase incorporates AGT into newly formed viral DNA The polymerase cannot synthesize any new DNA past acycloguanosine due to lack of 3’-OH group on the molecule Influence on normal cells: infection is halted since uninfected cells do not catalyze AGT and the drug is nontoxic to healthy cells 44.What is the gene identified that can undergo mutation in Retinitis pigmentosa? What is the role of the gene under normal conditions? Human rod photoreceptor cGMP-gated, cation channel, beta-subunit protein Normally responsible for the transduction cascade of photoreceptors Defects in the alpha-subunit gene have already been associated with one form of RP 45.How many exons and promoter elements are present in the rod photoreceptor protein? 5 exons and 1 promoter element 46.What are the common mutations identified that can lead to development of Retinitis pigmentosa? Mutations in the genes that make polypeptides for the channel protein Defects in the alpha-subunit gene have already been associated with one form of RP 47.What gene is dysfunctional in retinoblastoma? What is its normal role? What happens if the gene is absent? Rb1 gene on chromosome 13 may be absent or non-functional: causes cell proliferation or uncontrolled growth Normally, 2 Rb1 genes limit cell proliferation through the synthesis of a protein 48.What role does oncogene MYCN in retinoblastoma? What are the characteristics of MYCN tumors? Comes into play later in the disease and is present in extra copies Characteristics of MYCN tumors: large, invasive, aggressive, usually appear before a child is six months old 49.What components were used in the transfection of Y-79 retinoblastoma cell line and what is their role? Vector of plasmid DNA bound to polyethylenimine This formed a charged complex with an inactivated form of adenovirus Vector contained luciferase gene, a reporter gene to confirm transfection A phosphodiesterase gene promoter was also included 50.What reporter gene was utilized in the system? How does it indicate whether transfection has occurred? Luciferase gene Extracted luciferase enzyme catalyzes the conversion of coelenterazine (complex organic molecule) into oxidized coelenterazine Occurs with the release of 482 nm blue light Homework Questions for Ocular Immunochemistry part I 1. What are immunoglobulins? What is their function? Immunoglobulins: large proteins with at least 4 polypeptide chains in each protein Function: bind to a foreign substance, identify, and initiate an immunological response AKA: antibodies Overall conformation similar to the letters Y or T 2. Identify the various domains and structural features of immunoglobulins Heavier chains have four domains, each connected by random coil sequences Lighter chains have just two domains Each domain is held together by an intrachain disulfide bond Both heavy and light chains are associated by interchain disulfide bonds 3. What is the role of C1, C2, C3 domains and oligosaccharides of immunoglobulins? C1 and C2: bind to the complement C3: bind to receptor proteins on macrophages and monocytes (WBCs) Oligosaccharides: attached to immunoglobulins; help in increasing water solubility, protecting against enzymatic degradation, and facilitating secretion from cells producing them 4. In what regions of the eye is IgA found? Identify the structural components of IgA and their role IgA: tends to form a two molecule unit (dimer) when secreted by cells that make it Joined stem-to-stem by a polypeptide called a J chain, where J strands are for joining S-chain is wrapped around the two immunoglobulin tetramer units Prevents proteolytic degradation of immunoglobulin Necessary as the precorneal tear film and other secretions are rich in proteolytic enzymes 5. Where do IgD and IgE occur? Give their role IgD: plays a significant role in connective tissue defense and is present on the membranes of many B cells; May play a role in lymphocyte differentiation and is not normally found in the anterior ocular fluids IgE: occurs in very small quantities in the precorneal tear film; Causes release of histamine from mast cells after binding to their surface; Associated with hypersensitivity reactions in asthma and hay fever 6. Where is IgG present in the eye? What are the special features of IgG? IgG: found in significant quantities in both precorneal tear film and aqueous humor Able to diffuse through cornea by itself Made in high quantities in response to an infection Forms 2nd line of defense (after IgA) Can bind to surface receptor proteins of B lymphocytes in order to stimulate those cells 2nd most important antibody molecule in ocular tissues 7. Where is IgM found in the eye? Identify its structural features IgM: found in precorneal tear film Composed of five tetrapeptide units (pentamer) as well as a J polypeptide One unit has ten antigen binding sites First immunoglobulin to be synthesized after the immunorecognition Forms large molecular complexes around foreign bacteria 8. Where does antigen-antibody reaction take place? What are its requirements? Take places between the ends of the variable domains of the heavy and light chains Requires correct conformational requirements between an enzyme and a substrate 9. What is an antigen? Where does an antigen bind? Antigen: substance that will produce an antibody to which it will bind Binds to antibodies that are part of the surface of B cells and T cells of the immune system 10.What is an epitope and a hapten? Epitope: antigenic determinant; Part of an antigen that is recognized by the immune system, specifically by antibodies, B cell, or T cells Hapten: substances that become antigenic when combined with a large carrier molecule (ex. organic diazonium compounds) 11. What factors contribute to antigen-antibody reactions and what are the characteristics of the factors? Chemical bonds: hydrogen, electrostatic, and van der Waals Nonpolar bonds: hydrophobic Conformation: lock-and-key 12.What is a radioimmunoassay and what does it measure? Give its application Radioimmunoassay: sensitive assays that detect hormones or other compounds present in low concentrations in tissues Percent of radioactivity in the antibodies are measure and intersected with the standard curve to obtain the contraction of the antigen 13.What are B cells? Where are they formed and transported? B-cells: site of immunoglobulin synthesis Formed in the bone marrow Transported to lymphoid tissues prior to full development as B plasma cells 14.What are the different steps involved in synthesis of immunoglobulins? Synthesis of immunoglobulins by the developing B cells begins in the bone marrow B cells become committed to the synthesis of light chains initially and then heavy chains to produce IgM and then IgD 15.What are the steps involved in the mechanism of synthesis of light chains? -Uncommitted B cells are present in the bone marrow or fetal liver -J genes codes for a joining peptide between V and C -Code for the constant domain is contained in C gene -When the stem cell becomes committed to B cell development, the DNA containing all these codes recombines by excision of part of the DNA -V gene 29 has been directly joined to J gene 2 -J gene 1 is also excised -V genes 1-28 still present but not directly connected in sequence to the J2 gene and are therefore inactive (also true for J3 and J4) In transcription state of forming hnRNA< only V gene 29, J genes 2-4, and C gene are transcribed -When hnRNA is processed to mRNA, all the introns as well as the inactive J3 and J4 exons are spliced out of the gene sequence 16.What are the three different immunoglobulins that occur in the eye and where are they present? IgA: precorneal tear film; primarily secretory type IgM: limited in penetration of the cornea; blood vessels IgG: most tissues Presence of IGs is limited to precorneal tear film, aqueous, ocular blood vessels, cornea, and sclera 17.What prevents IgA from degradation in the eye? Lacrimal gland epithelial cells synthesize the S-chain polypeptide that surrounds the IgA molecules and protects them from degradation 18. Why are immunoglobulins not found in deeper tissues of the eye? Not present in deeper tissues such as lens, vitreous, and non-vascular parts of retina since no antibodies are normally found 19.What are the drawbacks of collecting tears by sponges and Schirmer strips? Schirmer strips: may cause reflex stimulation Sponges: tend to be contaminated with conjunctival secretions 20.What are the three antibodies made in response to Herpes infection? IgM, IgG, IgA 21.Which areas of eye contain Herpes antigens following infection? As the infection progresses, Herpes antigens are not only present in the cornea, conjunctiva, and tear film, but also in the iris and trigeminal ganglion 22.How does IgA and IgG4 help in case of Herpes infection? Secretory IgA will attack the viruses that are extracellular (hinders virus from attaching to uninfected cells) IgG causes phagocytosis of the antigen virus 23.What happens to IgG4 levels in response to Herpes infection? IgG can reach levels up to 1300 micrograms/mL Normal levels are 4 micrograms/mL! 24.What is a complement? Give its two main functions Complement: collected of related proteins like proteolytic enzymes or membrane binding proteins Destruction of foreign organisms by membrane lysis Activation of phagocytosis (cell engulfment) following chemotaxis (cell movement induced by chemicals) 25.What are the main components of complement? Comprised of 11 protein types: C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9 Homework Questions for Ocular Immunochemistry part II 1. What are the sequential events involved in the activation of C1s? Begins with the binding of the C1 complex to the stem portion of either IgG or IgM (which are themselves bound to the membrane antigens) Upon binding to the IG’s, a conformational shift of the C1 complex induces autocatalytic properties in C1s and C1r The proteins cause their one activation C1s molecules ultimately become a proteolytic enzyme for the next substrate proteins in the sequence C4 and C2 2. What are the sequential events involved from C4 to formation of C6-C9 complex? C4 is broken into fragments C4a and C4b C4b binds to the membrane in the vicinity of the antigen-antibody complex, while C4a does not participate in the sequence C2 is also broken down into a C2a and C2b by C1s C2a binds to the C4b molecule and becomes an enzyme (C3 convertase) C3 convertase splits into C3 and C3a and C3b C3b fragment associates with C4b and C2a to form C5 convertase C5 is split into C5a and C5b C5b fragment binds to a nearby region of the antigen membrane and initiates formation of a complex that contains bound C6, C7, C8, and several molecules of C9 3. What events contribute to the lysis of bacterial membranes? C9 proteins must pierce the cytoplasmic membrane of bacteria in order to be effective This complex forms a channel in the antigen membrane through which Na and water molecules quickly pass Formation of the channel and rapid movement of water and ions causes lysis of gram-negative bacterial membranes Lysis occurs due to the osmotic shock produced by the lesion 4. What is the role of lysozyme entering the channel? The channel may also allow the entrance of lysozyme Lysozyme attacks the thinner peptidoglycan wall between the outer lipoprotein cover and the plasma membrane of the bacteria Viruses exposed to the complement are neutralized Complement prevents a virus from attaching to host cells 5. How does a complement prevent the virus fr

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