Brent Pickrell Block 1

Block 1 Practice Questions *Lectures 1&2 were introductory. No testable lecture content* Lecture 3 – Epithelium: Kretzer Q: What’s the purpose of fixating tissues? A: In order to stop enzymatic activity of proteins and thus minimize postmortem artifact. The tissue will digest itself (autolysis) if not fixed. Q: What fixatives are common? What are the effects on the tissues? A: Formaldehyde (formalin) and glutaraldehyde are common. They coagulate/precipitate protein and tend to damage enzymes. In general, fixation denatures protein and spreads out their charge groups so that the groups are more available to bind to stains. Q: Describe the concepts of dehydration and infiltration. A: We dehydrate so that we can take water out and get paraffin/epoxy resin into the tissue (water and paraffin aren’t miscible with each other). Tissue is usually dehydrated in organic solvents like acetone, alcohol, and toluene in order to replace polar water with non-polar solvents. These non-polar solvents extract lipid and further denture protein by competing with their hydrophobic internal bonds. We infiltrate with warm paraffin and then cool it to get a hard block that can be sectioned on a microtome to a thickness of about 8 microns. Q: Compare dehydration for LM versus TEM. A: For LM, there’s fast dehydration starting at 70% concentration of solvent. This fast dehydration is good because shrinkage is beneficial for LM. Once infiltrated with paraffin, we make about 8 micron thick sections with metal blades on the microtome. In contrast, TEM needs a slow dehydration starting at 30% solvent. Slow dehydration is necessary because we want minimal shrinkage. The tissue is then infiltrated with plastic and sectioned to .1 microns using a diamond on the microtome that is propelled forward by thermal expansion. Q: What is eosin? What color is it and what cell organelles readily bind it? A: Eosin is a red dye that is negatively charged and forms non-specific salt linkages with positively charged groups in denatured tissue in the paraffin section. There is nothing specific about eosin binding. The redness in the LM is called eosinophilia or acidophilia. Tons of denatured mitochondria have positively charged proteins and thus bind eosin and are termed eosinophilic. Q: What is hematoxylin? What color is it and what cellular structures does it bind? A: Hematoxylin is a blue dye that is a positively charged dye that forms non-specific salt linkages with negatively charged groups in denatured tissue in a paraffin section. There is nothing specific about hematoxylin binding. Blueness in the LM is called basophilia. Tons of RER are basophilic—therefore cells that are massive protein synthesizers are

Brent Pickrell Block 1 basophilic (ie Pancreas—makes insulin and glucagon). Hematoxylin also stains DNA of the cell nucleus. Q: What is toluidine blue used for? A: Commonly, LM is being done with tissue infiltrated with plastic instead of paraffin. There is less mechanical distortion during microtomy and the sections are thinner and the resolution is greater. However, H&E doesn’t penetrate plastic so toluidine blue is used. Q: What’s an example of a very specific stain for LM paraffin sections? A: Periodic acid Schiff (PAS). It is specific for hexose sugars and it specifically stains glycogen, cell coats, basement membranes, and the ground substance in connective tissue. The paraffin section is incubated with periodic acid which oxidizes free hydroxyl groups on two adjacent carbon atoms to aldehydes. The aldehydes then react with the Schiff reagent to produce a red-colored complex (shows as a magenta ring around the base of cells). Q: If you were in the OR, what is a common way you might preserve tissues? A: Frozen section using a cryostat. There’s no fixation, no dehydration. The samples are dipped in liquid mounting media and then flash frozen in liquid nitrogen. The samples are then cut using a cryostat and subsequently stained with H&E. Q: What’s the dilemma regarding fixation? A: The margin of any tissue sample, although fixed most rapidly, suffers mechanical damage, while the center of the sample, although free of mechanical damage, suffers autolytic damaged because it’s fixed last. Lecture 4 – Superficial back/shoulder: Glen Lecture 5 – Amino Acids: Goodman Q: What are the essential amino acids? A: PVT MT HILL (Private Matt Hill is essential); phenylalanine, valine, tryptophan, threonine, methionine, histidine, isoleucine, lysine, leucine Q: What are the nonessential amino acids? A: alanine, aspartic acid, arginine, asparagine, cysteine, glutamine, glutamate, glycine, proline, serine, tyrosine Q: Given that the pI for glycine is 6.1, to which electrode, positive or negative, will glycine move in an electric field at pH 2? (Adopted from Lippincott Text) A: Negative electrode. When pH < pI the charge on glycine is positive. Q: Methionine, tryptophan and proline are examples of what kind of AAs? A: nonpolar Q: Which type of AAs cannot be synthesized by the body? A: essential amino acids

Brent Pickrell Block 1 Q: Which AA is in charge of disulfide bond formation and it frequently in the active sites of many enzymes? A: cysteine Q: Which AAs can be phosphorylated? A: serine, threonine, tyrosine Q: Which are oligosaccharide attachment points? A: serine, threonine Q: Which amino acid changes in sickle cell anemia? What type of disease is this? What type of mutation is sickle cell? What disease are sickle individuals immune to and why? A: polar acidic glutamate ! non-polar valine. Results from a point mutation, but more specifically a missense mutation. This causes “pockets” of hydrophobicity in the beta chains. The Hb is now referred to as HbS instead of HbA. It’s the most common autosomal recessive disease in African-Americans. They are immune to malaria because their RBCs lyse early and therefore don’t allow the malaria-causing parasite to finish its intracellular portion of development. Sickling of the cells frequently block to flow of blood in narrow capillaries and this interruption in the supply of O2 leads to localized anoxia in the tissue causing pain and eventually death of cells in the vicinity of the blockage. Q: What’s the pH range for arterial blood? A: 7.35-7.45 Q: True or False: Drugs get into cells best if they are charged. A: False. It is best if they are uncharged and lipid soluble. Lecture 6 – Cell Membranes 1: Kretzer Q: What is the trilaminar/unit membrane? A: The plasma membrane exhibits a 3 layer structure after fixation in osmium tetroxide—osmium is deposited on the hydrophilic phosphate groups present on each side of the internal bilayer of fatty acids. The 3 layer trilaminar structure is designated the unit membrane. It’s visible on TEM. Q: Are the P face and E face of the plasma membrane the same? A: No, they are very different and can have different compositions/fluidities. For example, the glycocalyx is found exclusively on the extracellular domain. There is thus lipid asymmetry allowing different lipid environments to have their own unique lipid signatures. Q: What factors affect fluidity of the membrane? A: Length of hydrocarbon tails, the degree of unsaturation of the tails, and cholesterol Q: What are the 3 types of lipid found in the membrane? A: Those that are free in the bilayer (spinning along their vertical axis in their respective leaflet), those that are prosthetic groups that have their tails within a protein, and those

Brent Pickrell Block 1 that surround their proteins that comprise lipid rafts/lakes (these are essentially spheres of low fluidity around proteins). Q: Compare/contrast integral and peripheral membrane proteins. A: Integral proteins are incorporated within the lipid bilayer and thus are not salt-extractable. They require detergent (often SDS) to be removed. Integral include transmembrane proteins that span the membrane and therefore have both E & P sides. Interestingly, some integral proteins undergo membrane triggered protein folding where they undergo a conformational change upon encountering a membrane that allows them to insert. Peripheral proteins have loose associations with one of the two membrane surfaces—they are held in placed by charged groups and thus are easily extracted. Those that associate with the P leaflet were soluble in the cytosol and happened to bump into the membrane. Those that reside on the E leaflet were secreted via exocytosis. Lecture 7 – Protein Structure 1: Goodman Q: Name two types of secondary structure. Which one is “like” Velcro? A: Alpha Helices and Beta-pleated sheets. Beta is like Velcro Q: Give some examples of supersecondary structure. A: Beta-barrel, helix-loop-helix (seen in transcription factors).

Q: What 4 molecular interactions stabilize tertiary structure? A: hydrophobic interactions, disulfide bonds, hydrogen bonds, ionic bonds Q: What proteins are involved in protein folding quality control? A: Chaperones; Chaperons are “heat shock proteins” and interact with the polypeptide at various stages during the folding process. They make sure they protein isn’t folding improperly--it’s a quality control system Q: What’s the biochemical basis of Alzeheimer’s disease? A: There is abnormal proteolytic cleavage resulting in the production and aggregation of A-beta fibrils called amyloid plaques. These are neurotoxic. From text: The A-beta amyloid that is deposited in the brain is derived by proteolytic cleavages from the larger amyloid precursor protein—a single transmembrane protein expressed on the cell surface in the brain and other tissues. Q: What’s the pathology behind Mad Cow Disease? A: An abnormal protein (different 3D shape, but same primary structure) causes a normal protein to refold into the infectious type. This change in conformation confers relative resistance to proteolytic degradation. The infectious agent is thus an altered version of a

Brent Pickrell Block 1 normal protein which acts as a template for the conversion of the normal protein to the pathogenic conformation. NOTE: prions are pure protein. This contrasts most other infectious agents that use nucleic acid. Q: Discuss any genetic polymorphisms related to Mad Cow disease. A: Residue 129 can either be methionine or valine. MM and VV are susceptible to the disease, whereas MV is disease resistant. However, if given the option, it’s better to be valine/valine than met/met. Dr. Goodman is a prion protein 129 heterozygote! Lecture 8 – Protein Structure II: Goodman Q: What metal do hemoglobin and myoglobin possess that allows reversible O2 binding? A: Iron Q: Where does myoglobin reside in the body? How many O2 molecules can it bind? A: Cardiac and skeletal muscle. It can only bind 1 O2 molecule because it only contains one heme group. Q: Who has a higher affinity for O2, myoglobin or hemoglobin? Explain. A: Myoglobin has a higher affinity and thus lower Km for O2. Myoglobin is designed to bind oxygen released by Hb. Q: What’s the difference between hemoglobin and myoglobin’s binding curves? A: Hb has a sigmoidal shape indicating cooperative binding. Myoglobin is hyperbolic since it can only reversible bind one O2 molecule.

Q: What is the Bohr Effect? A: It causes a rightward shift in the O2-dissociation curve resulting from low pH, higher pCO2, and higher temperature. Conversely, raising the pH or lowering CO2 results in a greater affinity for O2 and a leftward shift. Q: How is 2,3-BPG production affected at higher altitudes? How about someone with COPD?

Brent Pickrell Block 1 A: Individuals at high altitudes increase 2,3-BPG production to facilitate O2 offloading to the tissues. Its presence causes a rightward shift in the hemoglobin O2 dissociate curve. The same is true for someone with COPD (ie emphysema) or chronic anemia. Q: Explain the interaction between 2,3-BPG and Hb. A: It binds to a pocket formed by the two beta chains of Hb. The pocket contains several positively charged amino acids that form ionic bonds with the negatively charged 2,3-BPG. Q: Beta-Lysine 82 in HbA is important for the binding of 2,3-BPG. In Hb Helsinki, this amino acid has been replaced by methionine. How will this affect Hb Helsinki’s ability to bind O2? (Adopted from Lippincott Text) A: Substitution of lysine by methionine decreases the ability of negatively charged phosphate groups in 2,3-BPG to bind the beta subunits of Hb. Because 2,3-BPG decreases the O2 affinity of Hb, the reduction of 2,3-BPG should result in increased O2 affinity and decreased delivery of O2 to tissues. Increased O2 affinity (decreased delivery) results in a left shift in the O2 dissociation curve. Q: What’s wrong with the blood at the blood bank (transfused blood)? A: 2,3-BPG has been degraded and thus Hb is lousy at O2 offloading. Q: How does CO binding affect the O2 binding curve? How do we treat CO poisoning? A: CO binds with much higher affinity and in doing so stabilizes the R form of Hb. This causes the binding curve to shift left, change from sigmoidal to hyperbolic and ultimately prevents O2 offloading. CO poisoning is treated with 100% oxygen at high pressure

(hyperbaric O2 therapy).

Q: What type of Hb is most prevalent in adults? A: HbA Q: What type of Hb are we interested in for diabetic patients? A: HbA-1c. We measure HbA1c to see how well one’s diabetes is controlled.

Brent Pickrell Block 1 Q: Compare/contrast the hemoglobinopathies Hb S and Hb C. A: HbS is sickle cell anemia where glutamate ! valine in both beta chains. In HbC, glutamate ! lysine in both beta chains. Both have a single amino acid substitution in the 6th position of the beta globin chain. HbC have more mild hemolytic anemia

Q: What is the pathology associated with a patient with Hb SC? A: the patient has hemolytic anemia and sickle cell crisis. Some beta-globin chains have the sickle cell mutation whereas others have the mutation found in Hb C. Q: What is the compensation mechanism for thalassemia? A: Make different isoforms (like HbF or HbA2) to compensate. Q: What type of collagen is found in both cartilage and intervertebrae disks? A: Type II collagen Q: What type of collagen is found in skin? A: Type I collagen Q: What alpha chain repetitive sequences are found in collagen? What co-translational modification take place? A: Glycine – X – Y, where X is frequently proline and Y is frequently hydroxylproline or hydroxylysine. There is hydroxylation of lysine and proline residues. Q: What vitamin is necessary to carry out the hydroxylation of proline and lysine? A deficiency in this vitamin is known as what? How do patients present with this particular illness? A: Vitamin C (ascorbic acid/ascorbate). In the case of ascorbic acid deficiency (thus lack of prolyl and lysyl hydroxylation) interchain H-bond formation is impaired as is formation of a stable triple helix. Also collagen fibrils cannot be cross-linked. The result

Brent Pickrell Block 1 is defective collagen synthesis that causes the disease scurvy. Patients often have bruises on their limbs as a result of subcutaneous extravasation of blood due to capillary fragility. Connective tissues are fragile, teeth fall out of sockets (since they’re held in place by collagen). Lecture 9 – Enzymes: Uncle Reddy Q: What’s an example of an oxido-reductase enzyme? Where does this enzyme function? A: HMG-CoA reductase. It is the rate-limiting step of cholesterol synthesis. It is anchored in the membrane of the endoplasmic reticulum. Q: How do enzymes work? A: They decrease the energy of activation and thus increase the rate. Equilibrium is not changed. Q: Drugs ending in –navir are what type of drugs? A: Protease inhibitors. These were a large breakthrough in HIV treatment. Q: Why can antibodies act as enzymes? A: Because antibodies can bind and stabilize substrate in its transition state. Q: What is the definition of turnover number? What does a high turnover number indicate? A: Amounts of product per unit time. High turnover indicates a very efficient enzyme Q: What are the advantages of a slight fever in fighting infection? A: Enzymatic activity initially increases as temperature increases. Thus, our immune system is more active and allows us to fight disease better. White cells divide and move faster to the site of infection. Q: What does Km signify? Whose Km is lower—hexokinase or glucokinase? What is there relationship to each other called and how is it defined? A: Km signifies the affinity an enzyme has for its substrate. Km does not vary with the concentration of enzyme. By definition, Km=[substrate]@ 1/2Vmax. Hexokinase has a lower Km than glucokinase and thus has a higher affinity for glucose. They catalyze the same reaction and are thus isozymes. Q: Where is glucokinase found? How about hexokinase? A: Glucokinase is found in the liver and pancreas. Hexokinase is found in essentially all cells. Q: When velocity is proportional to substrate concentration, is this an example of 1st, 2nd, or Zero order reaction? I.e when [s]<<<Km A: 1st order reaction. Q: Alcohol intoxication is an example of 1st order or Zero order kinetics? A: Zero order. Metabolizing enzymes are limiting and the substrate (alcohol) is way in excess.

Brent Pickrell Block 1 Q: Draw and label a general Lineweaver-Burk plot.

Q: How will a competitive inhibitor affect the slope of the Lineweaver-Burke plot? A: Since the apparent Km increases, there will be different x-intercepts that indicate the change in Km. However, Vmax will not change (so it intersects the y-axis at the same point). This ultimately results in a steeper line.

Q: How will a noncompetitive inhibitor affect the slope of the Lineweaver-Burke plot? A: Since Vmax is decreased in the presence of a noncompetitive inhibitor, there will be a different y-intercepts and the slope of the line will increase. However, Km remains unchanged so thee will be the same x-intercept. Q: What is creatine (phospo) kinase (CPK)? A: it catalyzes the conversion of creatine to createin kinase, consuming an ATP. The reaction is reversible. Lecture 10 – Epithelium II: Kretzer Q: What are the patched transmembrane proteins associated with tight junctions/zonula occludins? A: occludin, claudin; the degree of tightness of tight junctions depends on the how many occludins/claudins there are. Tightest junctions are in the brain, leaky ones in intestine Q: What is the patched transmembrane protein of desmosomes/macula adherens? A: desmoglein Q: Intermediate filaments for desmosomes attach to what plaque protein?

Brent Pickrell Block 1 A: desmoplakin (which connects to desmoglein) Q: What junctional complexes wrap all the way around cells? A: tight junctions and adherent junctions Q: What is the patched transmembrane protein of adherent junctions? A: cadherin E (for epithelial) Q: What is the transmembrane protein for hemidesmosomes? A: desmopenetrin Q: What cytoskeletal filaments are involved in focal contacts? A: actin Q: What is the transmembrane protein for focal contacts? A: integrin Q: What are gap junctions composed of? A: Connexons, which are made of 6 connexins Q: What are the layers of the basement membrane? A: lamina rara (lucida), lamina densa, lamina reticularis Q: What cytoskeletal filament is involved in adherent junctions? A: actin Q: Name the 5 types of intermediate filaments. A: Keratins (cytokeratins), vimentin, desmin, neurofilaments, GFAP Q: Which intermediate filament is found in contractile cells? A: desmin Q: Which intermediate filament is found in neurons? A: neurofilament Q: What intermediate filament is found on cells that arise from the neural crest? A: vimentin Q: What type intermediate filament would be found in the Langerhans cell? A: vimentin (all blood cells are vimentin positive) Q: Which cytoskeletal protein undergoes dynamic instability? A: microtubules Q: What 2 motor proteins are associated with microtubules? A: kinesins (carry organelles/stuff toward + end) and dynein (opposite) Lecture 11 – Cell Membranes II: Goodman

Brent Pickrell Block 1 Q: What are the normal electrolyte levels in the ECF and ICF? A: Extracellular Space: Na+=145mM, K+=5, Cl=110, Ca2+=1-2 Intracellular space Na+=5-15, K+=140, Cl=5-15, Ca2+=.0001 Q: Which body compartment contains a higher percentage of TBW, ICF or ECF? A: The ICF contains 2/3 of TBW while the ECF contains 1/3 of TBW

Q: What is the only form of transport that is not carrier mediated? A: simple diffusion Q: What types of molecules don’t need transporters to get across the membrane? A: O2, CO2, NO, steroid hormones Q: What is a reflection coefficient? Which molecules have a RC of 1? Or zero? A: Number ranging from 0 to 1 that describes the ease with which a solute crosses the membrane. An RC = 1 means the membrane is impermeable to that solute (ie serum albumin, intracellular proteins, charged ions). RC = 0 means the membrane is freely permeable to that substance (ie water, urea). Q: Explain the parameters that will increase diffusion. Think Fick’s principle. A: surface area, concentration gradient, permeability, decreased membrane thickness. Think of absorption of nutrients in the gut (across microvilli) or gas exchange in the lungs. Q: Explain the characteristics of an integral membrane transporter protein. A: Hydrophobic surface where the protein is in contact with membrane lipid. Hydrophilic interior through which a polar substance docks or travels. E and P domains are in contact with aqueous phase. Q: Which is faster, facilitated diffusion or passive diffusion? Which can be saturated?

Brent Pickrell Block 1 A: Facilitated diffusion is faster than passive diffusion. Facilitated diffusion can be saturated and involves channels (pores) or permeases (carriers). Therefore, there is a degree of specificity associated with facilitated diffusion. Q: Compare/contrast permeases vs channels. A: Permeases (carriers) bind to their substrate and move it across the membrane. They do this by undergoing a conformational change. Channels (pores) form aqueous pores in the membrane (ie aquaporin). Both are passive. Q: True or False. Membrane transport proteins are multi-pass transmembrane proteins? A: True Q: Is active transport saturable? A: Yes. Active transport also requires energy, is specific, and moves molecules against their electro-chemical gradient (in the case of primary active transport). Q: What is the main difference between ATP-binding cassette (ABC) and solute carrier transporters (SLC)? A: ABC uses ATP directly to transport molecules. SLC includes secondary active transport and facilitated diffusion. Q: Name 4 examples of primary active transporters. Which ones are antiports? Which one is the most abundant? A: Na+/K+ATPase (antiport), H+ATPase (uniport), Ca2+ ATPase (uniport), H+/K+ATPase (antiport). Sodium potassium pump is present in the membranes of all cells and is therefore the most abundant (uses ~25% of total ATP in animal cell). Q: What are the affects of Ouabain on Na+/K+ ATPase? A: It blocks the Na+/K+ATPase. It’s a cardiac glycoside. Q: Are Na+ and K+ obligatory ions for the Na+/K+ATPase? A: K+ is obligatory since no other ion can substitute. There are ions needed to bind to the intracellular side for the pump to work, but Rb+ can substitute for Na+. Q: Describes the steps in the mechanism of Na+/K+ ATPase. A: 3 Na+ bind, ATP phosphorylates, conformational change and release of Na+ to ECF, 2 K+ bind, Mg2+ catalyzes dephosphorylation of enzyme, conformational change and release of K+ to ICF. Q: What are the functions of the Na+/K+ ATPase? A: control of osmolarity and cell volume, creates concentration gradients used for secondary active transport, maintains and restores resting potential, generates heat Q: Give some (2) specific examples of antiport secondary active transporters. A: Na+/H+ antiporter, Na+/Ca2+ antiporter Q: What is SGLT? Where is it at in the body? A: Sodium glucose transporter. It’s a symporter in the intestine.

Brent Pickrell Block 1 Q: What are the downstream affects when there’s inhibition of Na+/K+ ATPase? A: All secondary active transport processes are diminished by inhibitors of Na+/K+APase because their energy source, the Na+ gradient, is diminished. Q: What is the stoichiometry of the Na+/Ca2+ antiporter? A: 3 Na+, 1 Ca2+. Helps maintain low intracellular levels of Ca2+ Q: What is the mechanism of digitalis and it’s treatment for heart failure? A: Digitalis inhibits Na+K+ ATPase, thus increasing intracellular Na+ concentration. The increase in intracellular Na+ concentration alters the Na+ gradient and thereby alters the function of Ca2+Na+ exchanger. As a result, Ca2+Na+ exchange decreases so less Ca2+ is pumped out of the cell, thus increasing intracellular Ca2+. Q: What specifically does digitalis block on the ATPase? A: Digitalis blocks dephosphorylation. It binds near the K+ binding site Q: What are the two cardiac glycosides? A: Digitalis and ouabain Q: What’s unique about the sweat of people with CF? A: it’s very salty Q: Why did pediatricians used to kiss babies during checkups? A: to check for salty sweat and possibility of CF. Q: What type of genetic disease is CF? Which race is most likely to get it? A: It’s an autosomal recessive disorder most common in Caucasians. Q: Where is the most common mutation in the CFTR gene? What actually happens at the cellular level to those with CF? Why does CF still persist in large populations? A: Most common mutation is at F-508. The mutation still produces a functional CFTR but there’s defective protein trafficking so it never makes it to the membrane. This defect persists in large populations because there’s a heterozygote advantage—some resistance to diarrheal diseases like cholera Lecture 12 – Axilla & Arm: Glen Lectures 13 & 14 – Skin I & II: Kretzer Q: What type of epithelium is skin A: stratified squamous epithelium Q: What collagen is unique to skin? A: Skin has type VII collagen Q: What junctional complexes does skin NOT have? A: No tight junctions, no gap junctions, no adherent junctions

Brent Pickrell Block 1 Q: What are the 6 functions of skin? No specific details necessary. A: Barrier, sensory organ, thermoregulation, UV protection, immune, sexuality Q: Describe the thermoregulation function of skin. A: The hypodermis contains adipose tissue that serves as insulation. Skin also has eccrine sweat glands in the dermis and there are capillary beds in the dermal papillae that open and close to allow for the conduction of heat. Q: What is the immune function of skin? A: SALT – skin associated lymphoid tissue. Keratinocytes secrete factors that assist in the maturation of T lymphocytes (similar to what happens in the thymus). There are also Langerhans cells that are macrophages/APCs (therefore of monocyte origin, vimentin positive). Q: Describe the barrier function of skin. A: The inert dead cells at the top of the corneum (keratin layer) serve as a barrier to infection. The stratum granulosa secretes vesicles called Odland bodies/keratinsomes/lamellar bodies that are filled with phospholipid that confer the water barrier of skin (replaces the need for tight junctions). Keratohyaline is also a secretory product of the granulosa but it’s NOT packed into vesicles (it has an anti-protease function). The skin also serves as a barrier to friction through the keratin layer and the dermal papillae and epidermal pegs. Q: How does skin function in UV protection? A: melanocytes secrete melanin Q: Describe the sensory function of skin. A: The skin has naked nerve endings in the first 3 layers that sense hot, cold, vibration, and pain. The nerve nets around the hair follicle, Merkel cells (stratum basale), Meissner’s corpuscles, and Pacinian corpuscles sense touch and pressure. Q: Describe the sexuality function of skin. A: Apocrine glands secrete protein pheromones (arm pit, areola, anal rim) and sebaceous glands secrete lipid pheromones (holocrine secretion). Q: What type of connective tissue is found in the hypodermis? A: dense irregular connective tissue (there’s also adipose tissue and Pacinian corpuscles) Q: Name the connective tissue types in the dermis. What glands reside in the dermis? A: lower has dense irregular connective tissue, the upper has loose irregular CT. The eccrine, apocrine, and sebaceous glands all reside within the dermis. Recall that capillary beds and Meissner’s corpuscles reside within the dermal papillae. Q: What defines epithelial tissue? A: it’s a layer of cells that rests upon a basement membrane and is held there by hemidesmosomes and focal contacts. They’re PAS+

Brent Pickrell Block 1 Q: What junctional complexes are found in the skin? A: desmosomes (lots of them), hemidesmosomes, focal contacts Q: What are the layers of the epidermis from basal to apical? A: stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, stratum corneum Q: Which layer is extremely synthetic? What types of products are made here? A: stratum granulosum. Makes keratohyalin, involucrin, odland bodies, and lysosomes. Because they’re so synthetic they’ve given up mitosis Q: What two layers are mitotically active? A: stratum basale and stratum spinosum Q: What layer is not evident histologically? A: stratum lucidum Q: What is the mitotic rate of the stratum basale controlled by? A: thyroid hormones, estrogen, testosterone, unknown signals from granulosum and corneum Q: What layer has its nucleus and organelles dissolve as a result of lysosomal bursting? A: Stratum lucidum. In addition, intracellular protein precipitates resulting from involucrin precipitating around tonofibrils. Q: What are the non-keratinocytes in the epidermis? Which are vimentin positive? A: Merkel cells, Langerhans cells, melanocytes. Langerhans and melanocyte are vimentin positive. Q: What is merocrine secretion? A: fusion of an intracellular vesicle with the plasma membrane; exocytosis into the extracellular space Q: What is apocrine secretion? A: the apical portion of the cell ruptures, emptying cytoplasmic contents + apical portion of the membrane into the extracellular space Q: What is holocrine secretion? A: Entire cell disintegrates (non-apoptotic cell death) and forms the secretory product Q: What type of secretion does the melanocyte perform? A: cytocrine secretion. Melanosome passes from cytoplasm of melanocyte to the recipient keratinocyte. Melanin then aggregates above the nucleus in characteristic melanin cap that protects nuclear DNA from UV radiation Q: What is a stage 1 melanosome? A: has only tyrosinase (no melanin yet)

Brent Pickrell Block 1 Q: Pertaining to melanocyte secretion, how do light and darker skin come about? A: In darker skin, melanocytes extend their cytoplasmic processes to stratum granulosum whereas people with lighter skin only have extensions into the basale and spinosum. Also, lighter skin have only stage II and III melanosomes, whereas dark skin individuals have predominantly stage III and IV. Lastly, in lighter skin the melanosomes fuse with lysosomes and the lysosome degrades the melanin, and in darker skin there’s suppressed lysosomal degradation. Q: Which gland has no lumen? A: sebaceous Q: Which gland has a large lumen? A: apocrine Q: What are characteristics of sebaceous glands? A: no lumen, secrete lipid pheromones by holocrine secretion, stratified squamous duct, associated with every hair follicle + non-hair areas (lips, glans penis, glans clitoris, areola of breasts), active at puberty, no myoepithelial cells surrounding Q: What are characteristics of apocrine glands? A: large lumen, coiled, tubular, proteinaceous pheromones, merocrine secretion. Apocrine is seen in mammary glands, myoepthelial cells surround gland and contract upon sympathetic stimulation, stratified cuboidal duct, found in circumanal region, areola of breasts, axillae Q: What are some characteristics of eccrine glands? A: small lumen, coiled, never associated with hair follicles, active at birth, have myoepithelial cells Q: Which layer of the stratum corneum desquamates? A: stratum disjunctum Q: What’s the pathology associated with Pemphigus? A: Mutation is desmoglein that causes intraepidermal blisters Q: What pathology is associated with a mutation is desmopenetrin, causing the epidermis to lift off the basal membrane causing sub-epidermal blisters? A: bullous pemphigoid Q: What causes psoriasis? A: A mutation in any of the proteins of the mature keratin pattern (cytokeratin, involucrin, or keratohyalin) sends a feedback signal to the stratum basale that tells them to divide faster. Plaques build up because we can’t increase the rate of oxidation and desquamation. Q: What pathology is associated with cancer is the stratum spinosum? A: squamous cell carcinoma

Brent Pickrell Block 1 Q: What is the cancer of the stratum basale? A: basal cell carcinoma Q: What is cancer of the mechanoreceptors in the stratum basale? A: Merkeloma Q: What is the extremely invasive cancer of the skin? A: Melanoma—melanocytes are not of epithelial origin so they don’t have many junctional complexes to hold their cells there Q: Why don’t albinos make melanin? A: There are 3 possibilities. There was no neural crest invasion of melanocytes into the basale. There’s no functioning tyrosine uptake channel so they lack this amino acid. There’s no tyrosinase activity. Q: Merkel cells contain what intermediate filament? A: They are epithelial and therefore cytokertatin + Q: The dermis is of ________ origin. A: mesodermal Q: What two layers of the epidermis comprise the malphigian layer? A: basale and spinosum Q: What’s the pathology of vitiligo? A: characterized by degeneration of melanocytes; depigmentation disorder Q: Where is thick skin found? A: palms and soles Q: What junctional complexes hold the basale to the basement membrane? A: hemidesmosome and focal contacts Q: What is the thickest epidermal layer? A: Stratum spinosum Q: In which layer of the skin keratinocytes produce desmosomes like crazy? What else happens in this layer? A: stratum spinosum. There’s also the production of filaggrin which bundles intermediate filaments together into tonofibrils Q: Where does the water barrier of the skin originate? (what layer) A: stratum granulosum Q: What product of the granulosum is not packaged into vesicles? A: keratohyalin. It aggregates into granules Q: In the stratum lucidum there is lysosomal rupture. What does this cause?

Brent Pickrell Block 1 A: The nucleus and the organelles of the keratinocyte dissolve, intracellular proteins precipitate, involucrin precipitates around the inside of the plasma membrane. All these steps allow the keratinocyte to be transformed into a dead package. Q: What three layers of the epidermis will the keratinocyte nucleus not be present? A: granulosum, lucidum, corneum Q: What motor protein brings melanosomes to the tip of the melanocyte dendrites? A: kinesin is the motor proteins that transports along microtubules in the + direction Q: What motor protein in the keratinocyte brings the melanosome towards the nucleus? A: dynein Lecture 15 – Cell Membranes III: Goodman Q: What’s the Nernst equation used for? A: Used to calculate the equilibrium potential for an ion at a given concentration difference across a membrane (assuming permeability to that ion). The equation converts a concentration difference for an ion into a voltage. Q: The RMP is close to the equilibrium potentials for which two ions? What does this mean in terms of permeability? A: It’s close to the equilibrium potentials of K+ and Cl- because the permeability to these ions at rest is high. Q: At rest, what are the states of the activation and inactivation gates of Na+ channel? A: At rest, the activation gate is closed and the inactivation gate is open. Q: Graph and document the ionic basis of the action potential.

Q: True or False: Myelin allows fewer channels to be required in the axon. A: True. VG sodium channels are positioned in nodes of Ranvier along the axon. Q: What’s the pathophysiology of Multiple sclerosis?

Brent Pickrell Block 1 A: Multiple sclerosis: loss of the myelin sheath around the nerves causes a decrease in membrane resistance, which means that current “leaks out” across the membrane during conduction of local currents. Thus, local current decay more rapidly as they flow down the axon and may be insufficient to generate an AP when they reach the next node of Ranvier. The host’s own immune system destroys its own myelin and it’s thus classified as an autoimmune disease. Q: How are lidocaine and tetrodotoxin similar? A: both block Na+ channels and thus inhibit APs. Q: What voltage-gated channel is responsible for exocytosis of NT? What is the sequence of events? A: Sequence of events: an AP in the presynaptic cell causes VG Ca2+ channels to open and this influx of Ca2+ causes the NT to be released via exocytosis. The NT diffuses across synaptic cleft and binds to receptors (ligand gated) on the postsynaptic membrane and produces a change in membrane potential on the postsynaptic cell. The change can be either excitatory or inhibitory.

Lecture 16 – Enzymes II: Uncle Reddy Q: True or false: good inhibitors bind with high affinity and dissociate slowly? A: True Q: On a hyperbolic curve, what changes when we add a competitive inhibitor? A: The apparent Km changes, but Vmax remains unchanged. Q: How do we treat methanol poisoning? How is enzyme kinetics relevant? A: If someone has methanol poisoning, enthanol is given (which has a lower Km for alcohol dehydrogenase) so as to inhibit the conversion of methanol to formic acid Q: What is lovastatin a competitive inhibitor for? A: It competes with HMG-CoA for the active site of HMG-CoA reductase. The reaction catalyzed by HMG-CoA reductase is the rate-limiting step in cholesterol synthesis. Statins competitively inhibits this enzyme and plasma cholesterol levels go down. Q: What drugs are #1 in lowering plasma lipid levels of cholesterol? A: Statins

Brent Pickrell Block 1 Q: What is changed on a hyperbolic curve when we add a noncompetitive inhibitor? What isn’t changed? A: Vmax changes, but Km is unchaged. Competitive inhibitors bind to the enzyme (not in active site) forming a covalent bond or bind so tight that the enzyme is not available to bind the substrate, so we end up having less enzyme. No matter how much substrate we have, we can’t attain the same Vmax.

Q: What are some examples from class of noncompetitive inhibitors? A: Aspirin, nerve gases (ie sarin—inhibits acetylcholine esterase), proton pump inhibitors (PPIs) Q: What’s the bottom line for noncompetitive inhibition? Hint: what happens to the enzyme and how do we overcome this effect. A: Some enzyme is inactivated by covalent modification. Only way to recover is to make new enzyme. Q: In what 3 ways can allosteric effectors affect the function of enzymes? A: Alter Vmax, alter Km, alter both Vmax and Km Q: What enzyme catalyzes the conversion of L-tryosine to L-Dopa? A: tyrosine hydroxylase Q: What enzyme is the rate-limiting step in catecholamine synthesis? What are the catecholamines? A: tyrosine hydroxylase is the rate-limiting step. Catecholamines are dopamine, norepinephrine, epinephrine Q: List the order of fastest to slowest mechanisms for regulating enzyme activity. A: Substrate/Product >> Covalent >> alter amount of enzyme Q: What is the #1 marker for heart damage? A: Cardiac troponin Q: Liver damage would indicate increased levels in what enzyme(s)? A: ALT/AST (alanine/asparagine aminotransferases)

Brent Pickrell Block 1 Q: What 3 markers would be used to diagnose damage of heart muscle after heart attack? Which is the least specific? A: CK-MB, cardiac troponin, lactate dehydrogenase (LDH). LDH is not very specific. Q: What tissue marker is used to diagnose brain damage? How about skeletal muscle? A: CK-BB in brain. CK-MM in skeletal muscle Q: What is the action of creatine kinase? What’s it also called? A: It transfers high-energy phosphate between ATP and creatine. It’s also called creatine phosphokinase Q: When does CK-MB peak in the plasma after myocardial infarction? What about LDH? A: CK-MB peaks about 24 hours after infarction. LDH activity peaks about 36-40 hours after infarction. Lecture 17 – DNA Replication: Uncle Reddy Q: Other than the nucleus, where else is there DNA in the eukaryotic cell? A: Mitochondria Q: What comprises a nucleotide in DNA? A: nitrogenous base, deoxyribose sugar, and phosphate Q: How many BPs per turn in DNA? A: 10 base pairs Q: How many bonds are there between AT and GC? What would we expect to see, in terms of replication, at the origin of replication and promoter? A: AT has two bonds, GC has 3 bonds. Origins of replication & promoters are AT rich. Need less energy to separate DNA strands and start replication. Q: Who absorbs more light, single stranded or double stranded DNA? A: Single-stranded DNA absorbs more light. Q: Who has a higher Tm, GC or AT rich DNA? A: Higher GC content the higher the Tm Q: What form does DNA predominantly consist? A: B-DNA is most predominant Q: What are the characteristics of A-form DNA? A: Right handed, 11 BP per turn. See in RNA/RNA and RNA/DNA. Q: What form of DNA do we see in active chromatin? A: Z-form DNA. Have 12 BP per turn and is left-handed. Q: What many origins of replication do bacteria have? How many for eukaryotes? A: bacteria have 1 or a few, eukaryotes have multiple origins of replication

Brent Pickrell Block 1 Q: What is the role of topoisomerase I? How about topo II? A: Topo 1 cuts and ligates one strand, thereby relieving strain due to supercoiling. Topo II cuts and ligates both strands and stabilizes circular DNA. Q: What is the functional equivalent enzyme in bacteria of topoisomerase? What anti-bacterial drugs target this enzyme? A: DNA gyrases are functional equivalent in bacteria. Fluoroquinolones (Cipro) inhibit DNA gyrase and kill bacteria. Q: Which strand is made continuously 5’ to 3’? A: leading strand Q: What enzyme synthesizes the RNA primer? A: primase Q: What’s the main DNA polymerase that synthesizes the leading strand? What capabilities does it have? A: DNA pol III – it is highly processive and remains bound to the template the entire time. It also has 3’ ! 5’ exonuclease activity Q: What is the enzymatic action of Dornase? A: digests DNA. It’s a nuclease Q: What types of bonds are there in cAMP and cGMP? A: phosphodiester bonds Q: Which DNA pol removes the primer and fills in the gap? A: DNA pol I. It has 5’!3’ exonuclease activity that allows it to remove the RNA primer. Q: What’s the action of DNA ligase? A: seals nicks in DNA – forms the phosphodiester bonds between two adjacent nucleotides Q: What is the purpose of SSBs? A: Keep the DNA protected and exposed for copying Q: What’s a tumor suppressor protein that stops cell cycle progression? A: P53 Q: What are telomeres? Do they code for anything? A: Telomeres are complexes of noncoding DNA and protein located at the ends of linear chromosomes. Telomeres shorten with each successive cell division; telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. They thus serve as a defense mechanism to prevent cancer.

Brent Pickrell Block 1 Q: What type of enzyme is telomerase? A: It’s an RNA-dependent DNA polymerase (functions as a reverse transcriptase) Q: Is telomerase present in bacteria? A: No, they don’t have telomeres since their DNA is circular. Q: Do bacteria have histones/chromatin? A: No Q: Where is the telomerase enzyme absent? A: in most somatic cells Q: How could anti-virals drugs work to slow replication? Why are they most nucleosides? A: Incorporation of nucleotides lacking 3’ –OH group leads to chain termination. Most are nucleosides because it’s easier to get them into cells whereas nucleotides have charged polar phosphates attached. Q: What two amino acids are found in abundance in histones? Why does this make sense? A: Arginine and lysine, which are basic amino acids and bear at (+) charge at physiological pH. This charge neutralizes the negatively charged DNA. Histones order DNA into structural units called nucleosomes that resemble beads on a string. Q: Upon inspection, we see methylated DNA and histones. Are these genes active or inactive? A: Methylated DNA and methylated histones correspond to INACTIVE genes. Q: Acetylation and phosphorylation of histones is characteristic of active or inactive genes? A: When histones are unmethylated and acetylated/phosphorylated instead, this corresponds to ACTIVE genes. Q: What are two important characteristics of RBCs? A: They do not have DNA or organelles. They get their energy by anaerobic respiration. Q: Transposition is a process where DNA from one portion of the chromosome moves to another chromosomal location. What cell type experiences this from our notes? A: Neuronal cells. Lecture 18 – Brachial Plexus: Glen Lecture 19 – Cell Organelles: Sifters Q: Which type of chromatin is highly condensed and transcriptionally inactive? A: Heterochromatin Q: Which type of chromatin is much less condensed and transcriptionally active?

Brent Pickrell Block 1 A: Euchromatin Q: Where, within the in the nucleus, are new ribosomes made? A: The nucleolus. Contains genes that synthesize rRNAs. Q: What components make up chromatin? A: Complex of DNA, histones, and nonhistone proteins in eukaryotic cells. Q: Which histone molecule is not part of the “octamer”? A: H1 histone – it’s a “linker” Histone. Q: True or false: In the same cell type, the same chromosomes occupy the same relative positions in the nucleus. A: True Q: True or false: there is unidirectional protein transport between the nucleus and cytoplasm A: False, there’s bidirectional protein transport. For example, making ribosomes requires nuclear transport in two directions. Q: Explain why making ribosomes requires nuclear transport in two directions. Where are the ribosomal proteins made? A: Ribosomal proteins are made in the cytoplasm, then they get targeted back into the nucleolus to get assembled into subunits with rRNA. Then there’s export of the newly assembled ribosomal subunits.

Q: In the nucleus what 3 major types of RNA are transcribed? A: rRNA, mRNA, tRNA. Q: What does the large size of the nuclear pore allow in terms of protein transport that isn’t allowed in the ER and mitochondria?

Brent Pickrell Block 1 A: It allows the NPC to transport completely folded proteins as well as large complexes. Remember, to be transported through the NPC, cargo needs a targeting signal (NLS or NES). Q: True or false: experiments show that charged AAs are required on a NLS? A: True. Q: What are the molecules called that function in recognizing nuclear targeting signals? A: Karyopherins (“nuclear carriers”). Import karyopherins are called importins, while export karyopherins are called exportins. For example, in the cytoplasm the importin binds import cargo, carries it through the NPC, and dissociates in the nuclear interior. Then the importin recycles back to cytoplasm for another round of import. Q: Where is Ran/GDP present in high concentrations—nucleus or cytoplasm? A: Cytoplasm Q: Discuss the mechanism of Ran in nuclear import. A: Ran/guanosine diphosphate (GDP) is present in high concentration in the cytoplasm whereas Ran/guanosine triphosphate (GTP) is present in high concentration in the nucleus. Proteins to be imported into the nucleus form complexes with nuclear localization signals (NLSs) importin alpha; and importin beta. Upon import through the nuclear pore complex, Ran/GTP binds to importin beta, thus releasing importin alpha and the imported protein. To complete the cycle, the Ran/GTP/importin beta complex exits the nucleus to enter the cytoplasm via the nuclear pore complex. Here the Ran/GTPase-activating protein (RanGAP) hydrolyzes GTP, forming Ran/GDP, thus releasing importin beta back into the cytoplasm.

Q: How can some HIV viral proteins get into the nucleus? A: Some HIV viral proteins contain NLSs, allowing viral nucleoprotein complexes to be imported into the nucleus. As an aside, the first discovered NLS was that of the SV40 T antigen (a viral protein).

Brent Pickrell Block 1 Lecture 20 – Cell Organelles II: Sifers Q: Where is the default residence of a protein? A: Cytoplasm Q: What is the organelle that proteins go to in order to enter the secretory pathway? A: Rough Endoplasmic Reticulum Q: The outer nuclear membrane is studded with ribosomes on its cytoplasmic surface. The space between the inner and outer nuclear membrane is called _________. Where are these two membranes united? A: Perinuclear cistern. They are united at the nuclear pores. Q: True or false: the outer nuclear membrane is continuous with the rough ER A: True Q: In order to go to the ER what must a nascent peptide possess? A: Signal sequence Q: What is a characteristic of the signal sequence? A: It’s a stretch of ~20 hydrophobic amino acids (this is the number of AAs required for a polypeptide to span a lipid bilayer as a helix). Q: What types of modifications of proteins take place in the ER lumen? What are the underlying purposes of these mods? A: Glycosylation (more specifically, N-linked glycosylation at asparagine), disulfide bond formation via the action of enzyme protein disulfide isomerase. These aid in producing correct protein folding. ANOTHER modification is the gaining of a GPI anchor. Some membrane proteins lose their transmembrane domain and gain a GPI anchor instead. Q: What molecules (proteins) assist in protein folding? What is their specific name if they are found in the cytoplasm? Or ER lumen? A: Chaperones. They are called HSP70 in the cytoplasm. Called BiP in the ER lumen. Q: What function might a GPI anchor assume for a newly synthesized protein? A: It can act as a sorting signal to direct membrane proteins to special regions of the

plasma membrane.

Brent Pickrell Block 1 Q: Where would we expect smooth ER to be abundant? Think detoxification; now think hormone/steroid synthesis. A: SER is abundant in liver cells because it contains some of the membrane-bound enzymes used to degrade certain hormones and to neutralize noxious substances such as alcohol and barbituates. When large amounts of a certain compound (ie phenobarbital) enter the system, the smooth ER in the liver doubles surface area in a couple days and this reflects the rapid synthesis of detoxification enzymes and the need for more membrane in which to place them. Smooth ER also function in lipid biosynthesis and therefore we’d see an abundance in steroid secreting cells like the Leydig of the testis. Q: What’s another well-known function of smooth ER? A: Lipid biosynthesis Q: How do newly synthesized lipids reach other membranes in the cell? A: Can diffuse to RER which is continuous with the smooth ER. Vesicles can bud off and move along cytoskeletal elements by motor proteins to other membranes. Or by phospholipid-transporting proteins.

Lecture 21 – DNA Repair: Uncle Reddy Q: What does methylation of the cytosines of DNA allow one to ascertain? A: It’s used to differentiate parental versus daughter strands in DNA. Q: What type of enzyme is telomerase? A: RNA dependent DNA polymerase (aka Reverse transcriptase) Q: Regarding anti-virals, why do we use nucleosides instead of nucleotides? A: Nucleotides are charged/more polar and therefore do not enter cells as well.

Brent Pickrell Block 1 Q: What would be the effect of methylation DNA in a promoter region? A: Methylate DNA in promoter region inactivates genes (puts them in “storage”) Q: What is the specific effect of UV light on DNA? How is this repaired? A: UV light can fuse two adjacent thymines to each other. These thymine dimers prevent DNA polymerase from replicating the DNA strand beyond the site of dimer formation. UV-specific endonucleases recognize the thymine dimer and cleaves the damaged strand and the gap is later filled. Q: What is the underlying pathophysiology of xeroderma pigmentosum? A: In XP, the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and consequently early and numerous skin cancers. XP can be caused by defects in any of the several genes that codes for the XP protein required for nucleotide excision repair of UV damage to humans. So bottom line: there’s an absence of UV-specific excinuclease. Q: What is the role of p53 within cells? A: It’s a tumor suppressor protein that stops DNA synthesis and segregation of chromosomes as soon as there is a break in the DNA thus leading to cell cycle arrest. Absence of p53 protein or mutation in p53 gene leads to cancer. Q: The deamination of cytosine results in what compound? A: Uracil Q: True or false: deamination and depurination are common damages to DNA. If true, what is the repair mechanism? A: True. First, the base is removed, then the chain is cleaved, and finally the sugar is removed. After that, add a nucleotide by a DNA polymerase. Close the gap with DNA ligase. Q: If a normal base in DNA changes to another normal base, it is difficult for enzymes to repair since they don’t know the strand in which the mutation occurred. So how do they end up distinguishing? A: Methylation. Q: What are two ways in which double-strand breaks are repaired? A: Nonhomologous end-joining (“quick and dirty” mechanism) OR homologous end joining. Homologous end-joining is more accurate and also more difficult. Lecture 22 – Connective Tissue: Kretzer Q: What is a synonymous term for connective tissue? A: stroma Q: What are the functions of connective tissue? A: Epithelial inducer – we can take epithelial cells from the gut and overlay these cells on the skin CT and they become keratinocytes Immune – filled by B and T lymphocytes and APCs Massive vasculature

Brent Pickrell Block 1 Nerves – neuronal impact on fibroblasts modulate their secretion Latent growth factors – these are activated by cleavage (ie glycoproteins activated this way) or by releasing them from scaffolding Q: What are reticulocytes? A: they are fibroblasts that secrete type III collagen Q: What cell is responsible for the secretion of type IV collagen? A: Epithelial cell; type IV acts as a meshwork/sieve Q: Briefly list the 3 types of connective tissue. A: Loose irregular CT, dense irregular CT, dense regular CT Q: Describe the characteristics of loose irregular CT. What is a synonymous term for loose CT? Include location. A: Has small collagen type I bundles going in all direction, highly cellular, seen in the papillary dermis. It’s also called areolar tissue. Q: Describe the characteristics of dense irregular CT. Include location. A: Has few cells, large bundles of type I collagen running in random direction, is adapted to offer resistance and protection. Seen in the reticular dermis of skin, perichondrium, periosteum, epimysium. Q: Describe the characteristics of dense regular CT. Include location. A: Large collagen type I bundles going in one parallel direction, avascular (think tendon—poorly vascularized and therefore poor repair) Q: Name the layers of the basal lamina. A: Lamina lucida, lamina densa, lamina reticularis Q: What are the cells found in connective tissue? Which is the most common? A: fibroblasts, macrophages, chondroblast, chondrocyte, osteoblast, osteocyte. Fibroblast is the most common Q: Endothelial cells of blood vessels are of what cell line and have what int. filament? A: They are from mesenchyme and thus vimentin + Q: What does the fibroblast synthesize? A: collagen, elastin GAGs, proteoglycans, and multiadhesive glycoproteins; they have 2 stages of activity, active and quiescent. Q: In what context are macrophages involved in connective tissue? A: They act as defense elements by phagocytosing bacteria, cell debris and abnormal ECM elements. They are derived from the monocyte which crosses the wall of capillaries to penetrate connective tissue where they mature into macrophages. Macrophages are APCs and thus are important for the up-take, processing, and presentation of antigens for lymphocyte activation.

Brent Pickrell Block 1 Q: What are proteoglycans? How do they compare to glycoproteins? A: They have a protein core that is covalently linked to many GAGs—they can aggregate and bind to hyaluronic acid through a linker protein. The 3D structure of proteoglycans can be pictured as a test tube brush whereas glycoproteins are globular protein molecules to which branched chains of monosaccharides are covalently attached. Q: What type of collagen is unique to cartilage? A: type II Q: What is the function of type VII collagen? A: anchors skin epidermal basal lamina to underlying stroma Q: What type of collagen is present in all basement membranes? A: type IV (encoded by epithelial cell) Q: What components are found in the lamina densa? A: heparin sulfate (GAG), laminin, type IV collagen, fibronectin (from fibroblasts) Q: Which connective tissue fiber contains the two unique AAs desmosine and isodesmosine? A: Elastic fibers Q: What are GAGs? A: linear polysacchardies formed by repeating disaccharide units Lecture 23 – Organelles III: Sifers Q: The efficiency of protein clearance underlies the existence of many loss-of-function and gain-of-toxic-function disorders. Which one is present in cystic fibrosis? What is fundamentally wrong in CF? A: It’s a loss-of-function disorder because a mutation in the CFTR gene results in a transporter not delivered to the membrane. The mutant protein slightly misfolds (most commonly due to a deletion of Phe508 in CFTR), is trapped in the ER, and then degraded. HOWEVER, the mutation does not appear to affect the ability of the CFTR protein to transport ions, indicating that the mutant protein would probably function if delivered to the plasma membrane. Q: What is the most common mutation in CFTR gene? Does this result in a dysfunctional protein? A: Phe508. The protein misfolds slightly, but it does not appear to affect the ability of the CFTR protein to transport ions. Q: What is alpha1-antitrypsin deficiency? What is the clinical manifestation? Are these examples of loss-of-function or gain-of-toxic-function? A: It’s a genetic disease responsible for chronic obstructive lung emphysema and liver cirrhosis—it’s the most common cause of childhood liver transplantation. Both disorders are caused by mutations in the alpha1-antitrypsin protein. Accumulation in hepatocytes of toxic protein leads to cirrhosis (gain-of-toxic-function). Its hindered secretion is also responsible for lung disease (loss-of-function). So it’s both!

Brent Pickrell Block 1 Q: What molecule is used to tag proteins for degradation? A: ubiquitin Q: What role does the attached sugar modification play in alpha1-antitrypsin deficiency? A: Modification of the attached sugars in the ER generates a crucial component of the degradation signal that allows for the precise timing of signal formation. Q: What’s the retention signal for an ER resident protein? Where do the receptors for this signal lie in the cell? A: KDEL (final amino acids) is found in soluble ER resident proteins. The KDEL receptor lies in the early region of Golgi. Q: How does tetanus toxin work at the cellular level? A: Tetanus toxin is a protease that cleaves receptors used for the movement of synaptic vesicles in inhibitory neurons. This blocks the secretion of inhibitory NTs that normally control muscle movement, leading to convulsive contractions (lockjaw). Q: How does botulinum toxin work? A: Botulinum toxin cleaves receptors that cause the toxin-infected neurons to be unable to release acetylcholine at the neuromuscular junction, leading to descending paralysis. Q: What are some characteristics of lysosomes? What is the signal that targets newly synthesized proteins to lysosomes? Where is this signal added? A: Membrane-bound organelles that have acidic interior pH. They have special coating on their interior to prevent the acid hydrolases from digesting their own membrane components. They degrade proteins, lipids, carbs, DNA, RNA, etc. Mannose-6-phosphate (M6P) is the signal that targets newly synthesized proteins to lysosomes. An N-linked sugar is initially added in the ER, and the M6P is added to the N-linked sugar in the Golgi.

Brent Pickrell Block 1 Q: What causes a lysosomal storage disease? A: When a cell lacks one (or more) of the hydrolytic enzymes (ie nucleases, proteases, glycosidases, lipases, etc) causing the lysosomes to accumulate material that is normally destroyed. Most LSDs are linked to a mutation in a specific acid hydrolase which causes the breakdown of a particular macromolecule to be incomplete, leading to the accumulation within the lysosome. Q: What group does Tay-Sachs disease primarily target? What enzyme is absent and thus what accumulates? Which cell type is particularly enriched in the molecule not broken down? A: Especially prevalent among Jews. The disease results from an absence of hexosaminidase A, wich breaks down glycolipids in the lysosomes. Overtime, this causes the progressive destruction of neurons which are particularly enriched in glycolipids. Q: If you were to look at a brain section from a child with Tay Sachs disease, what might you find? A: Cells with large empty vacuoles that had been filled with glycolipid until extracted with alcohol in preparing the tissue. The vacuoles are the swollen lysosomes. Lecture 24 – Cell Cycle: Haudek Q: What 3 phases compose interphase? A: G1, S, G2 Q: Which phase takes the longest, interphase or mitosis? A: Interphase; composes 90+% of cell cycle Q: What’s the longest phase of interphase? A: G1 Q: What are some defining characteristics of G1 phase? A: Longest phase, high bioactive (RNA and protein synthesis—enzymes are needed for DNA synthesis and enzymes needed to induce S-phase). Cells may go from G1 to Go for a variety of reasons. Q: Cells may go from G1 to G0 instead of S because they are either quiescent, senescent, or post-mitotic cells. Compare/contrast these terms. A: Quiescent cells: stop dividing due to lack of nutrients, growth factors or other physical impediments. Focus 100% on their function and have thus given up replication. They can later return to division upon stimulation. Senescent cells: have irreversible DNA damage (usually due to aging). This occurs once a cell has reached its Hayflick limit (~50 cell divisions). Post-mitotic: they are fully mature/differentiated. They cannot re-enter cell cycle (ie neurons). Q: What stage are histones produced? A: S-phase

Brent Pickrell Block 1 Q: What stage do we see the production of microtubules? A: G2 phase (since they will be needed for M phase) Q: What does mitosis start with (what happens most visibly at first)? A: Chromosomes condense so that they will be easier to separate. Q: When are centrosomes/centrioles duplicated? A: S-phase Q: What are the different types of microtubules found during mitosis? A: There’s kinetochore MT (those attached to kinetochore), Polar MT (attached to other MTs), and Aster MT. **Specific steps of mitosis not discussed. Consult slides. Lecture 25 – Adipose Tissue: Kretzer Q: What junctional complexes are found in adipocytes? A: gap junctions and few focal contacts (no talin) Q: What intermediate filament is found in adipocytes? A: vimentin (they’re mesenchyme origin) Q: How do we bring free fatty acids to the adipocyte? A: chylomicrons from gut and/or VLDL from liver Q: What are the functions of adipose tissue? A: thermoregulator, energy reservoir, shock absorber, endocrine organ, secretes cytokines, shape, placement of organs, heat generator Q: Where are places you will not find adipose tissue? A: skin of the eyelid (need uniform tear fill when you blink), ear pinna (for sound localization), scrotum (spermatogenesis), and penis (switch from para to sympathetic) Q: When looking at adipocytes, the nucleus could be from what cell types? A: adipocyte, fibroblast (reticulocyte secreting type III), endothelial cell Q: What are some characteristics of multilocular adipose tissue? A: Fetal/brown fat, has centrally located nucleus, lots of mitochondria, smaller than unilocular, many small triglyceride lipid droplets, has thermogenin (UCP-1). Cells eventually die off via apoptosis or dedifferentiate to lipoblasts, acclimatization to cold Q: What are some characteristics of unilocular adipose tissue? A: Adult/white fat. Nucleus towards the periphery, has thin rim of cytoplasm where the organelles are found, one huge non-membrane bound triglyceride droplet, nucleus cannot be distinguished from endothelial cell or fibroblast. Q: What type of collagen matrix supports adipocytes?

Brent Pickrell Block 1 A: Type III collagen Q: What junctional complexes do adipocytes lack? A: desmosomes, hemidesmosomes, adherent junctions, tight junctions Q: How does UCP-1 work? A: it permits the backflow of protons without passing through ATP-synthase Q: How does insulin affect the activity of liproprotein lipase? A: insulin activates lipoprotein lipase which cleaves VLDL and chylomicrons into FFA and glycerol Q: Where is lipoprotein lipase located? A: capillary cell membrane (although it’s synthesized by the adipocyte) Q: Free fatty acids broken down by lipoprotein lipase must go through what path in order to be stored in adipocytes? A: plasma membrane of the endothelial cell, basal lamina, matrix of type III collagen, basal lamina of adipocyte, adipocyte plasma membrane Q: Norepinephrine from the sympathetic nervous system activates what enzyme within the adipocyte? What’s the effect of this enzyme’s activation? A: Norepinephrine activates hormone-sensitive lipase which breaks down triglycerides at the surface of the stored lipid droplets. The FFAs diffuse across the membranes of the adipocyte and the capillary endothelium and then they bind the carrier protein albumin in the blood. Mechanistically, NE onto the adipocyte increases cAMP levels which then activates hormone sensitive lipase Q: How does insulin affect the action of hormone sensitive lipase? A: Insulin inhibits hormone sensitive lipase thus reducing fatty acid release Q: What is the effect of cytokine secretion by adipocytes? A: they induce an inflammatory response—the worst is visceral fat which has been shown to produce diabetes and hypertension Q: What is the target of leptin and what does it do? A: It is carried to the hypothalamus where it controls hunger and regulates the amount of adipose tissue Q: What is hyperplastic obesity? A: an increase in the number of adipocytes per unit area; most readily occurs when you overfeed newborns in their first few months of life Q: What is hypertrophic obesity? A: where overeating increases the size of lipid droplets within the adipocyte Q: What are the underlying effects of childhood obesity? A: The interconvertibility between unilocular and multilocular disappears

Brent Pickrell Block 1 Q: If we want to see lipid droplets under the microscope what must we do? A: We need to use a frozen section because lipids are extracted in normal fixation/dehydration technique. We then stain with oil red O or sudan black Q: How do lipid droplets appear in TEM? A: lipid droplets appear black as a result of OsO4 reacting with the glycerol backbone Q: What is a lipoma? A: a benign tumor of a unilocular adipocyte Q: What is a malignant adipocyte tumor? A: Liposarcoma; note: extremely rare Q: What is fibrosis? What is an example of fibrosis pertaining to adipocyte tissue? A: Fibrosis is when fibroblasts secrete excessive type I collagen. Cellulite is when there’s increased fibroblast secretion of type I collagen in the extracellular matrix surrounding adipocytes and endothelial cells. Lecture 26 – Transcription: Uncle Reddy Q: What’s the most abundant type of RNA? A: ribosomal RNA (rRNA) Q: Is the promoter transcribed? What is a promoter sequence? A: No, it’s not transcribed. The promoter is a DNA sequence that the RNA polymerase recognizes and tells it where to start. Promoters are always orientation dependent. Q: What kind of structure is recognized for termination of transcription? A: A hairpin structure and poly A:T signal is recognized for termination by release factor associated with the moving RNA polymerase. The newly synthesized RNA folds to form a “hairpin” Q: How does rifampin work? What does it treat? Include something about mitochondria A: Rifampin binds to bacterial RNA polymerase and to mitochondrial RNA polymerase. It is therefore hypothesized that human mitochondria are of bacterial origin. Rifampin-bound RNA pol is inactive. Rifampin is an anti-TB drug. Q: Drugs ending in –navir do what? A: They are protease inhibitors, usually part of HIV treatment Q: What do fluoroquinalones do? A: They are antibiotics that function as Gyrase inhibitors Q: What do statins do? How do they act? A: Statins are HMG-CoA reductase inhibitors. They reduce cholesterol synthesis and thus reduce plasma cholesterol levels. Q: Which substrate do bacteria prefer, glucose or lactose?

Brent Pickrell Block 1 A: Glucose Q: How does the lac operon work? A: Basically, when glucose is present there’s a repressor protein that binds to the operator and prevents the transcription of 3 genes needed for lactose metabolism. However, when glucose is absent and lactose is present, lactose binds to the repressor protein causing it to change shape and thereby fall off the operator. Now RNA pol is allowed to transcribe the genes needed to breakdown lactose. Q: What are enhancers? Where can they be located? What are insulators? A: Enhancers: increase the rate of initiation of transcription by RNA pol II. Can be located upstream, downstream, be close or far away from promoter, and occur on either strand of the DNA (this is because DNA can bend and thus allow enhancers to interact with the transcription-initiation complex). Enhancers are orientation INDEPENDENT. Insulators are sequences on either side of a gene that insulate it from the influence of adjacent genes. Q: Contrast polycistronic vs. monocystronic. A: Polycistronic refers to one mRNA and its ability to make many different proteins from it. We see this occur in bacteria. Eukaryotes have one mRNA for one protein (monocistronic).

Q: How do steroids alter gene expression? Should we stop steroids abruptly? A: Steroids diffuse into cells, bind receptors that then migrate to the nucleus and bind promoters to alter gene expression. Steroids and other drugs that alter gene expression should not be stopped abruptly—dose should be gradually lowered to facilitate return to normal gene expression. Could take several weeks. Q: Do eukaryotes or prokaryotes contain 5’ cap and 3’ poly-A tail? Which processing reaction occurs first? What’s unique about the 5’ cap linkage? A: Eukaryotes contain both, prokaryotes contain neither. Capping and polyadenylation are unique to eukaryotic mRNAs. Capping occurs very early on the 5’ end of all

Brent Pickrell Block 1 mRNAs. The addition of the poly-A tail occurs after transcription. The cap is attached to the 5’ terminal end of the mRNA forming an unusual 5’!5’ linkage Q: What is the function of cap-binding proteins? What about poly-A tail function? A: Cap-binding proteins help with ribosome binding and the poly(A) tail stabilizes mRNA and facilitates exit from the nucleus. Q: What does the process of splicing remove from pre-mRNA? A: It removes introns. About 70% of pre-mRNA is removed during splicing. Splicing is mediated by the spliceosome which consists of several small RNAs bound to protein (snRNPs). Q: What is alternative splicing? A: Alternative splicing: the pre-mRNA molecule from some genes can be spliced in alternative ways in different tissues. This produces multiple variations of the mRNA and therefore its protein product. In general, splicing joins adjacent exons to each other and can exclude one or more exons. Lecture 27 – Cell Organelles IV: Sifers Q: True of false: mitochondria can undergo division AND are of maternal origin. A: True; new mitochondria arise from the division of pre-existing mitochondria. Q: Where are the majority of mitochondrial proteins encoded? A: The majority of mitochondrial proteins are encoded by DNA in the nucleus. Q: What proteins are encoded by the mito genome? What is the shape of the mito genome? A: The mito genome is circular. It codes for some proteins bound it its inner membrane. Of course, most proteins are encoded by nuclear DNA and imported into the mito. Q: What are significant about “contact sites” in mitochondria? A: These are where protein import into the mitochondria occurs. Q: Describe the signal sequence needed for mito important. A: The signal sequence is amphipathic—the signal is folded as an alpha helix with positively charged residues on one face of the helix while the nonpolar residues are clustered on the opposite face. This configuration is recognized by specific receptor proteins on the mitochondrial surface. Q: What do TOM and TIM stand for as it pertains to the mitochondria? A: TOM = translocator of outer membrane. TIM = translocator of inner membrane

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Q: What is TIM22 complex used for specifically? A: TIM22 is specialized for the translocation of multipass membrane proteins. Q: What complex must mitochondrial produced proteins utilize in order to be inserted into the inner mitochondrial membrane? A: OXA complex Q: If you’re a nuclear encoded protein, how many signals must you have to be an integral membrane protein in the inner mitochondrial membrane? A: 2 signals. First signal will target you to the TOM complex on the outer membrane. The second signal will target you to the inner membrane. Q: What are the general features of peroxisomes? Where does it get its proteins? A: Found in most eukaryotic cells, has no DNA, single bilayer membrane, ALL proteins are imported from the cytosol. Functions: oxidation of fatty acids and other lipids, oxidation of purines/AAs/etc, contain large amounts of catalase which converts H2O2!H20 Q: What are two import signals of peroxisomes? Which signal is not cleaved after translocation? A: PTS1 and PTS2. The PTS1 signal is not cleaved after translocation. PTS = peroxisomal transport signal Q: What’s different about the protein importing process of peroxisomes? Think unfolded vs folded import and consider the fact that there are no chaperones inside peroxisome A: There are no chaperones inside peroxisomes, so the proteins are already folded when they get imported. Q: What is the evidence for the ER being the origin of the peroxisome membrane? A: Clustered peroxisomal membrane proteins are present in the RER membrane. Peroxisomal proteins are detected in vesicles that bud from the ER. Q: What is Zellweger’s syndrome and how does it relate to peroxisomes?

Brent Pickrell Block 1 A: There is a general defect in importing proteins into peroxisomes—there seems to be a mutation in the transport complex so nothing gets into them. Cells exhibit “empty” and disfunctional peroxisomes and causes death soon after birth. Q: What two target organelles don’t have their signal removed after the protein arrives? A: Peroxisome (PTS1) and nucleus Lecture 28 - Cartilage: Kretzer Q: What intermediate filament is found in cartilage? A: vimentin Q: Does cartilage have/secrete a basement membrane? A: No Q: What type of collagen do chondroblasts/chondrocytes secrete? A: type II Q: What are the functions of cartilage? A: bear mechanical stress with reversible deformation, support soft tissues, shock absorber, sliding area for joints, facilitating of bone movements, embryonic skeleton Q: What is the major GAG of cartilage? A: chondroitin sulfate Q: What are the 3 types of cartilage? A: hyaline cartilage, elastic cartilage, fibrocartilage Q: What sheath surrounds cartilage? What is the function of this sheath? A: Perichondrium. It’s a sheath of dense irregular connective tissue that surrounds cartilage. It has a vascular supply and allows nutrients to diffuse from it to cartilage. Because cartilage is avascular and must accept nutrients from the perichondrium, cartilage can only get so thick. Q: What 5 things are found in the perichondrium? A: fibroblasts making type I collagen, lymphatics, vasculature, stem cells that give rise to chondroblasts, and nerves Q: What junctional complexes are found in chondroblasts/chondrocytes? A: gap junctions (communication) and focal contacts (to adhere to ECM) Q: What unique protein does the integrin of the focal contacts in chondroblasts/chondrocytes bind to the ECM? A: chondronectin Q: What junctional complexes does cartilage lack? A: it has no desmosomes, hemidesmosomes, adherent junctions or tight junctions Q: What is the metabolic situation of chondrocytes?

Brent Pickrell Block 1 A: because cartilage is devoid of blood capillaries, chondrocytes respire under low oxygen tension. They glucose for energy but only go to lactic acid because it’s an anaerobic environment Q: Does type II collagen assemble into fibers? A: No, only into fibrils. It never bundles! Q: What is the most common type of cartilage? A: Hyaline Q: Where is fibrocartilage found? A: between intervertebral disks and pubic symphysis Q: Where is elastic cartilage found? A: ear pinna Q: What type of cartilage is the annulus fibrosis? A: fibrocartilage. Breaking this structure causes a ruptured disc causing the nucleus pulposus to leak out Q: What type of collagen is found in fibrocartilage? A: Type II and type I Q: What type of collagen is found in hyaline cartilage? A: type II collagen Q: Describe the matrix of cartilage? A: It functions much like a biomedical spring in that it can deform and rebound. The major GAG is chondroitin sulfate and there’s also keratin sulfate and hyaluronic acid. The anchoring ECM protein is chondronectin Q: Which types of cartilage have a perichondrium? A: hyaline and elastic Q: Where is hyaline cartilage found? A: trachea, temporary skeleton in embryo/child, articular surfaces of joints, ventral ends of rib-sternum Q: What type of growth takes place in cartilage? A: appositional in all three cartilage types—there’s also interstitial growth in hyaline cartilage specifically at the epiphyseal plate Q: What’s unique about the composition elastic cartilage? A: has type II collagen and elastic fibers Q: What collection of chondrocytes appear in groups up to 8 that originated from a single chondrocyte? A: isogenous groups

Brent Pickrell Block 1 Q: What type of secretion does collagen undergo? A: constitutive merocrine secretion (constant, no buildup of vesicles inside fibroblast b/c they are continuously being released) Q: What co-translational process happens to collagen initially in the RER? What additional modification happens shortly after in the RER as well? A: hydroxylation of specific prolyl and lysl residues (vitamin C dependent). There’s glycosylation of hydroxylysyl residues shortly after. Q: Where does assembly of procollagen take place? A: in the RER; 3 polypeptides align Q: What modifications of collagen take place extracellularly? A: After exocytosis of procollagen, some procollagen peptidases cleave most of the nonhelical terminal peptides and thereby transform procollagen into insoluble collagen molecules that aggregate into collagen fibers; NOTE: the registration peptides (also called extension domains) were essential for alignment of chains in the RER but they prevent self-assembly so they must be cleaved Lecture 29 – Mitosis & Meiosis: Haudek Q: What do cyclins bind to exert their influence on the cell cycle? What happens to their expression levels throughout the cell cycle? A: They bind to cyclin-dependent kinases (CDKs)—remember that kinases add phosphate groups to proteins. Their expression levels rise and fall depending on phase. The CDK is the catalytic subunit while the cyclin is the regulatory subunit. Q: What’s the order of cyclin expression during the cell cycle? A: D ! E ! A ! B Q: What is the G1 CDK also called? For S phase? For M phase? A: G1 CDK is also called CDK4. S phase is also called CDK2, M phase is CDK1 Q: What cyclin-cdk complex pushes the M/G1 transition? A: Cyclin D/CDK4. It becomes active after cytokinesis. It promotes synthesis of A/E cyclins and DNA replication complexes. Q: What cyclin-cdk complex pushes the G1/S transition? A: Cyclin E/CDK2. Becomes active at the end of G1 phase. Q: What cyclin-cdk complex pushes the S/G2 transition? A: Cyclin A/CDK2. Becomes active in the MIDDLE of S phase. Q: What cyclin-cdk complex pushes the G2/M transition? A: Cyclin B/CDK1. It becomes active at the end of G2 phase. Also called M phase Promoting factor. Activates chromosome condensation and anaphase promoting complex

Brent Pickrell Block 1 Q: What is the anaphase promoting complex (APC) activated by? What does it promote the degradation of? Which cyclin does it promote the synthesis of? A: Activated by cyclin B/CDK1. It mediates the degradation of B cyclins and thus pushes the M/G1 transition. It also promotes the degradation of structural proteins via ubiquitination of securin and activation of separase. It mediates the synthesis of D cyclins Q: What factors characterize the G1 checkpoint? A: it is controlled by p16, p21 and cyclin D. These factors will decide whether to delay division, enter resting phase Go, or let the cell divide. Q: What is the G2 checkpoint controlled by? A: it is controlled by cdc25 phosphatase—if all DNA is correct, cdc25 removes inhibitory units from the MPF complex Q: When DNA damage is detected by ATM/ATR what do they act on specifically? A: They inhibit cdc25 and increase p53 levels Q: What two things activate the Mitotic spindle checkpoint? A: It activated by improperly attached kinetochores to spindle and by uneven tension among spindle fibers Q: What is the uneven distribution of chromosomes called? A: Aneuploidy Q: What are the most common types of cancer for men and women, respectively? A: Men – prostate, Women – breast. Lung is #2 for both sexes. Q: Is meiosis 1 or 2 similar to mitosis? A: Meiosis II Q: Is there a G2 phase in meiosis? A: there’s no G2 phase Q: What are the binding sites between homologous chromosomes called? A: Chiasmata Q: What is the end result of meiosis I? A: There are now 2 HAPLOID cells Q: How do kinetochores function differently during meiosis I? A: Kinetochores fuse and function as one unit during meiosis I. Q: Compare/contrast the events of mitosis and meiosis. A: Meiosis requires two cell divisions. The products of meiosis are haploid whereas the products of mitosis are diploid. The products of meiosis are genetically different, whereas the products of mitosis are genetically identical. Prophase is much longer in meiosis due to genetic recombination. Kinetochore behavior is different in meiosis.

Brent Pickrell Block 1 Q: One primary spermatocyte turns into how many mature sperm cells? A: results in 4 mature sperm cells Q: One primary oocyte turns into how many ovum? A: 1 mature ovum and 3 polar bodies Lecture 30 – Intro to Radiology: Willis Q: Which imaging modality doesn’t use ionizing radiation? A: MRI, ultrasound Q: Which radiology subspecialty does primarily procedures? A: interventional radiology Q: What is ionizing radiation? A: radiation with enough energy to cause ionizing interactions in the medium being irradiated. Particles or electromagnetic waves with enough energy to detach electrons from atoms or molecules. Q: What factors explain the 10% rise in CT use in the past years? A: Malpractice fear, CT ER triage, patient expectation, self-referral Q: Which patient has the greatest lifetime risk of developing cancer secondary to repeated exposure to ionizing radiation? A: The youngest, especially if below the age of 35. Q: What does ALARA stand for? A: As low as reasonably achievable Q: What are some important places to shield from x-ray? A: thyroid, breast, gonads Lecture 31 - Translation: Uncle Reddy Q: What is the start codon AUG code for? A: methionine Q: Where does the correct amino acid bind on the tRNA? How does this process unfold? A: The AA binds at the 3’ end and this specific process is catalyzed by aminoacyl synthetase. Each tRNA has a specific aminoacyl synthetase. When a tRNA has a covalently attached amino acid it is said to be charged.

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Q: What is the anticodon on tRNA for methionine from 5’ to 3’? A: CAU Q: What are the specific subunits of the prokaryotic ribosome? A: 50S and 30S for a total of 70S. Q: What are the specific subunits of the eukaryotic ribosome? A: 60S and 40S for a total of 80S. Q: What are the different sites on the ribosome? A: Ribosome has 3 binding sites for tRNA molecules—A, P, E. The A site binds the incoming aminoacyl-tRNA, P-site is occupied by peptidyl-tRNA (carrying the chain of AAs already synthesized), and E-site is occupied by the empty tRNA. Q: What is the first amino acid in bacterial proteins? How is the related to immune response? A: formyl-methionine. Human WBCs contain receptors that react with peptides containing formyl-met and migrate to the site of infections. Q: What is the site on the ribosome that the 1st tRNA goes to? A: The initiate tRNA enters the P-site (thereby bypassing the A-site) Q: Where does the peptidyltransferase activity of the ribosome come from? A: It is catalyzed by rRNA (referred to as ribozyme). Q: How is the ribosome positioned on mRNA in bacteria? How is this different in eukaryotes? A: The Shine-dalgarno sequence (upstream of AUG codon) is complementary to the nucleotide sequence of the 16S rRNA of the ribosome. This is in contrast to eukaryotes who use their “cap” at their 5’ end for positioning the mRNA on the ribosome—it then moves down the mRNA until it encounters the initiator AUG.

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Q: What are some examples of post-translational modifications of proteins? A: Phosphorylation, glycosylation, hydroxylation, addition of biotin, fatty acid anchors. Q: What are some effects of a single BP changes? Give (3) names of mutations. A: silent, missense, and nonsense mutation. Could result in a disease such as sickle cell anemia where a single base-pair change results in the change of amino acids within the peptide. Q: Define: missense, silent, nonsense mutations A: Missense: the new codon codes for a different amino acid. Silent: the new codon codes for the same amino acid. Nonsense: the new codon codes for a stop codon Q: What are the effects of inserting/deleting 1 or 2 nucleotides? What about 3 nucleotides? What disease do we know that had a 3 BP deletion? A: If one or two nucleotides are either deleted from or added to the coding region of a message sequence, a frame-shift mutation occurs and the reading frame is altered. If three nucleotides are added, a new amino acid is added to the peptide or if three nucleotides are deleted an amino acid is lost. Loss of 3 nucleotides maintains the reading frame, but can result in serious pathology (ie cystic fibrosis and its loss of phenylalanine residue) Q: What is a triplet repeat disease? What is a neurodegenerative example? A: A sequence of three bases that is repeated in tandem can become amplified in number, so that too many copies of the triplet occur. If it happens in the coding region of the gene, the protein will contain many extra copies of one amino acid. An abnormal protein causes disease. For example, amplification of the CAG codon leads to the insertion of many extra glutamine residues in the Huntington protein, causing Huntington disease. Q: What’s a function of 3’ UTR? What is the effect of exchanging 3’ UTR sequences between mRNAs? A: The 3’UTR determines stability (half-life). Exchanging 3’UTR sequences can dramatically alter half-life. Q: What’s a function a 5’ UTR in mRNA? A: It can regulate the mRNA’s translation. The 5’ UTR can bind a repressor protein and prevent translation. Q: What is the principle iron-storage protein? How does aconitase alter its transcription?

Brent Pickrell Block 1 A: Ferritin is the principle iron-storage protein we use to store iron. We need the protein when there’s an excess of iron. Aconitase will dissociate from 5’ UTR upon binding Fe and allow the translation of ferritin mRNA.

Q: What are transferrin receptors? How does aconitase alter its transcription? A: During iron starvation, we need more transferrin receptors so aconitase binds the 3’ UTR resulting in a longer half-life of mRNA. When iron is in excess, Fe binds to aconitase resulting in its dissociation from the 3’ UTR and causes less protein to be made due to a shorter mRNA half-life. Q: What two vitamins are needed to produce nucleotides and thus DNA? A: Folic acid and B12 Q: What is the method of mRNA degradation in eukaryotes? A: mRNA is degraded in an exosome complex, which consists of several ribonucleases. Exosomes remove the poly(A) tail and the 5’ cap structure. mRNA is then degraded by exonucleases from both the 5’ and 3’ ends. Q: What is macrocytic anemia? A: if there’s a deficiency in B12 and folic acid then there’s a slow-down in nucleic acid synthesis. The cell takes a long time to complete DNA synthesis and divide so the cell gets too large. Q: What is microcytic anemia? A: Due to a problem in hemoglobin synthesis because of an iron deficiency. Cells are dividing normally, however there is too little Hb to fill the cells. Lecture 32 – Forearm Flexors: Duncan

Brent Pickrell Block 1 Lecture 33 – Embryology 1: Brandt Q: How does an extruded oocyte make its way into the fallopian tube? A: It’s swept into the fallopian tube via the fimbriae Q: Where does fertilization normally take place? A: In the ampullary region (the widest part) of the fallopian tube. Fertilization is usually 12 to 24 hours after ovulation. Q: When does a secondary oocyte complete its second meiotic division to become a mature ovum? A: at fertilization Q: What are the three steps to actual fertilization? A: Capacitation of sperm (final maturation step of sperm that takes place in female reproductive tract—mediated by secretions from wall of uterus), acrosome reaction (release of acrosomal enzymes allow sperm to penetrate the zona pellucida), and finally fusion of sperm and oocyte membranes. Q: What three barriers must the sperm penetrate in order to fertilize the egg? A: Corona radiata, zona pellucida, oocyte cell membrane Q: Sperm penetration of zona pellucida causes what to occur? A: It causes cortical granules to release their contents which renders the zona pellucida impenetrable to further sperm. Q: What is pelvic inflammatory disease and how does it affect the fallopian tubes? A: It’s an STD (usually gonorrhea or chlamydia) that results in a purulent infection in the fallopian tubes. This can cause narrowing or occlusion of the fallopian tubes which can prevent successful migration of the fertilized egg. Q: When is the 2-cell stage reached after fertilization? A: approximately 30 hours Q: What another name for the ball of 16 cells that’s present 3 days after fertilization? A: Morula Q: Describe blastocyst formation. A: As the morula enters the uterine cavity, fluid enters the zona pellucida and into the intercellular spaces. Confluence of these intracellular spaces creates a cavity called the blastocele.

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Q: What is blastocyst “hatching”? A: The blastocyst is surrounded by the zona pellucida and it must “hatch” from it in order to be able to implant into the endometrium. Q: What are the relevant structures of the blastocyst? A: Inner cells, also called embryoblast. Outer cells, also called trophoblast, form the wall of the blastocyst. Q: What’s special about the inner cell mass of the blastocyst? A: Embryonic stem cells are derived from the inner cell mass. They’re pluripotent. Q: What are some abnormal embedding sites for the blastocyst? A: These are called ectopic pregnancies. Could be in the fallopian tubes (tubal) or near the cervix (placenta previa) or, even more rarely, in the ovary or in the abdomen (pouch of Douglas). Women with pelvic inflammatory disease have a high instance of ectopic pregnancies. These are dangerous for the mother due to the possibility of internal hemorrhage. Lecture 34 – Forearm Extensors: Duncan Lecture 35 – Cell Organelles V: Sifers Q: What are some examples of vesicle trafficking that lead to lysosomal proteolysis? Hint: one example is involved in organelle destruction A: Phagocytosis (cell eating), Pinocytosis (cell drinking), Receptor mediated endocytosis, transcytosis, and autophagy (organelle destruction). Q: How would an IgG-coated bacterium be eaten by a macrophage or neutrophil? A: IgG antibodies on the surface of bacteria are recognized by Fc receptors on the plasma membrane of macrophages and neutrophils. Binding initiates phagocytosis. Q: What cytoplasmic protein is involved in receptor mediated endocytosis? A: Clathrin—when it coats on the cytoplasmic face it induces vesicle formation. The clathrin cycles between the vesicles and the cytoplasmic membrane. The vesicle will fuse with an early and late endosome before eventual lysosomal fusion.

Brent Pickrell Block 1 Q: The uptake of LDL by cells is an example of what type of endocytosis? What could go wrong in this process? A: Receptor mediated endocytosis. LDL particles contain a core of ~1500 cholesterol molecules surrounded by a lipid monolayer. A large protein organizes the particles and mediates the specific binding of LDL to cell surface receptor proteins. Genetic defects in LDL uptake (as a result of mutant LDL receptors) lead to familial hypercholesterolemia as a result of cholesterol accumulating in the blood. Could result in atherosclerosis over time. Q: Define transcytosis. A: the uptake of a substance from one domain (ie apical) and deliver of that substance to another domain (ie basolateral). Remember that apical domain faces the lumen while the basolateral domain faces the endothelial cells lining the bloodstream. Q: What “little cavities” are involved in transcytosis. What are they? A: Caveolae. They are membrane microdomains (lipid rafts) that are rich in GPI-linked membrane proteins. This is an example of clathrin INDEPENDENT endocytosis. Q: In polarized epithelial cells, what junctions are responsible for preventing the diffusion of membrane proteins between apical and basolateral surfaces? A: tight junctions Q: What type of antibody is secreted by the mammary duct? A: IgA Q: What mechanism is utilized for the uptake of maternal antibodies by nursing infants? A: Transcytosis. Secreted IgA enters the mother’s mammary duct and are released into the milk by transcytosis. The infant nurses, and the maternal IgA travels to the infant’s small intestine. The maternal IgAs bind to Fc receptors on the apical surface of the infant’s intestinal epithelial cells. The receptor and maternal IgAs are internalized and then released into the infant’s bloodstream at the basolateral surface (transcytosis). This provides immunological protection until the infant’s immune system becomes functional. Q: What is the purpose of the cell carrying out autophagy? Where does the engulfing membrane originate from? A: used by cells to destroy their own organelles. It’s especially active in hepatocytes during period of amino acid starvation. The engulfing membrane may originate from the ER or golgi. Q: True or false: autophagy acts as a backup system for degradation. A: True. It’s used in the destruction of proteins accumulating in the cytosol that failed to be degraded by proteasomes. Therefore autophagy is a modifier of gain-of-toxic function diseases. Lecture 36 - Bone: Rowley Q: True or false: Bone is an organ system. A: True. It’s constantly being remodeled and must respond to changes in the environment.

Brent Pickrell Block 1 Q: What are characteristics of woven bone? Does it have an ordered structure? A: It’s primary bone and is the first bone to appear in embryonic development and in fracture repair. It’s not ordered and is characterized by random disposition of collagen fibers. It’s rich in osteocytes. It’s temporary and later replaced by secondary bone (usually when the toddler starts walking). Q: Give details on lamellar bone. A: it’s also called secondary bone and is the type of bone usually found in adults. It has an organized pattern (at approximately right angles) where lamellae are organized either parallel to each other or concentrically around a vascular canal. Q: What are other names for trabecular bone? A: Cancellous bone, spongy bone. Trabecular bone is an inner meshwork Q: What is another name for compact bone? A: Cortical bone. Compact bone has a rigid outer shell Q: What is wrong in osteoporosis? A: there’s a decrease in bone mass. Bone does not remodel very well with age. Also some endocrine issues Q: What is still okay in osteoporosis? A: the ratio of mineral to matrix is normal Q: What deficiency is there is osteogenesis imperfecta? A: There’s a collagen type I deficiency Q: Name the four fundamental cells in bone. Give their roles as well. A: Osteoprogenitor (stem), osteoblast (synthetic), osteocyte (maintenance, repair), and osteoclast (dissolve, remodel). Q: What’s the only way bone can grow? A: through appositional growth Q: Where are osteoprogenitor cells located? A: they line the bone surface Q: What are some characteristics of osteoprogenitor cells? A: They’re mesenchymal derived, mitotic (not synthetic), a pre-osteoblast cell type, spindle shaped (thin) and migratory Q: Are osteoblasts mitotic? A: NO!! The suffix –blast is a misnomer. Instead, they are synthetic. Q: Which cells are responsible for synthesizing all the osteoid? Where are they located? What are the shapes of these cells?

Brent Pickrell Block 1 A: Osteoblasts. They are cuboidal in shape and secrete their materials at the base of the cell. They are located at the surface of the bone matrix. Q: What is the effect of PTH on osteoblasts? What other effects does PTH have on these cells? A: PTH inhibits osteoblast. It also inhibits alkaline phosphatases and collagen synthesis. Q: How do osteoblasts communicate with each other? A: Gap junctions Q: What is the primary function of osteocytes? A: maintenance. They are reserve cells found within the calcified bone matrix in lacunae Q: Who are osteocytes derived from? A: derived from osteoblasts that have been engulfed in the matrix Q: When are osteocytes “activated”? A: They’re activated in repair (they can differentiate back into osteoblasts if there’s a fracture). Also, they are mechanicoreceptors for bone remodeling. Q: What is the primary role of osteoclasts? A: they dissolve bone and are thus involved in remodeling Q: What are osteoclasts derived from? A: they are derived from mononuclear phagocytic system (similar to macrophage) Q: Which bone cells are large and multinucleated? A: osteoclasts Q: Where do osteoclasts reside? How do they attach themselves there? A: They reside on bone surfaces, attached via integrins and cadherins to osteopontin. More specifically, they are found in depressions called “Howships’s Lacunae”. Q: How do osteoclasts exert their influence on bone? A: secrete acid collagenases and acid phosphatases and other proteolytic enzymes that decalcify bone and digests osteoid. Q: Osteoclasts are stimulated by PTH indirectly. Explain. A: Osteoclasts don’t have PTH receptors. PTH stimulates osteoblast to secrete RANKL. Osteoclasts DO HAVE RANKL receptors. Osteoclast action results in elevated blood calcium Q: What hormone inhibits osteoclasts? A: They are inhibited directly by calcitonin (from C-cells of thyroid). Q: What is the bone matrix composed of? A: Organic (osteoid) – fibers are collage type 1. “Glue” is GAGs, proteoglycans, glycoproteins (ie osteonectin) and these help anchor the Ca2+ phosphate crystals.

Brent Pickrell Block 1 Inorganic is hydroxylapatite crystals—the “hardener”. For mineralization to occur the combined local concentration of Ca2+ and phosphate must be above a threshold. Calcium-binding glycoproteins and the phosphatases released in matrix vesicles by osteoblasts promote calcification of the matrix by creating a high concentration of Ca2+ and phosphate locally. The high ion concentrations cause crystals of CaPO4 to form on the matrix crystals. Mineralization lags behind the front of osteoid deposition. Q: Describe the pathophysiology of Padget’s disease. A: Elevated osteoclast activity resulting in elevated resorption of bone and compensatory deposition of primary woven bone. Q: Describe the effects of bone cancer (ie osteosarcoma). A: Malignant tumors of osteoblasts, more common in children, cells represent osteoprogenitor and therefore little/weak matrix is made. The skeleton is often the site of metastases from tumors originating from malignancies in other organs like breast, lung, prostate, and thyroid. Q: What is osteomalacia? What’s the disease called in children? A: Characterized by improper mineralization. It’s called rickets in children. There’s insufficient Ca2+ and phosphate resulting in softening of the bone and therefore increases in fracture. Often a result of vitamin D deficiency which is important for the absorption of Ca2+ and phosphate by the small intestine Q: True or false: both cortical and trabecular bone start out as woven and become lamellar in the adult. A: True. Lecture 37: Diagnostic Exam Lecture 38 – Bone II: Rowley Q: Which parts of cortical bone have osteoprogenitor cells? A: Periosteum and endosteum Q: What connects the periosteum to bone? A: Sharpey’s fibers Q: What are canaliculi and what functions do they perform? A: They are channels that connect the lacunae in osteons essentially allowing exchanges between osteocytes and blood capillaries. Cellular processes go through the canaliculi and there’s diffusion of nutrients and O2 within them. Q: In compact bone, where do external circumferential and inner circumferential lamellae reside? A: Inner are located around the marrow cavity and external are located immediately beneath the periosteum. Q: Describe bone remodeling in cortical bone.

Brent Pickrell Block 1 A: Osteoclasts remove old bone in tunnel-like cavities having the approximate diameter of new osteons. Such tunnels are quickly invaded by many osteoprogenitor cells and sprouting loops of blood capillaries. Osteoblasts develop, line the walls of the tunnels, and begin to secrete osteoid in a cyclic manner, forming concentric lamellae of bone with trapped osteocytes. They use the blood vessel as a guide. Q: What are interstitial lamellae? How do they form? A: When new generations of Haversion systems form they form progressively, and the remodeled remains (fragments) left over become interstitial lamellae. Q: What is the newest lamella of each Haversian system? A: The newest is the innermost layer of the Haversion system because successive deposition of lamellae start from the periphery and proceed inward. As osteoclasts drill open the old bone, osteoprogenitor cells in the reversal zone differentiate into osteoblasts that form onion-like layers from the outside in. Q: What cells are found in lacunae? A: osteocytes Q: Compare/contrast periosteum and endosteum A: Periosteum covers the external surface of bone; it’s dense irregular connective tissue. The innermost cellular layer contains stem cells called osteoprogenitor cells that have the potential to differentiate into osteoblasts. Endosteum lines the marrow cavity, lumen of Haversion/Volkmann’s canals, outer layer of trabecular bones and also contains osteoprogenitor cells and osteoblasts. Endosteum is loose irregular connective tissue. The principle functions of periosteum and endosteum are nutrition of osseous tissue and continuous supply of new osteoblasts for repair or growth of bone. Q: Do osteoclasts have one or several nuclei? A: they have several Q: What type of ossification occurs in flat bones? A: intramembranous—evolves from a mesenchymal membrane Q: What type of ossification occurs in long bones? A: endochondral ossification. The matrix of preexisting hyaline cartilage is eroded and replaced by osteoblasts producing osteoid. Q: Discuss intramembraneous ossification. What type of bone does it occur in? A: Occurs in most flat bones like frontal, parietal, occipital, maxilla, and mandible. It is initiated in a membrane of mesenchyme where the cells condense and come together and differentiate to osteoprogenitor cells and then osteoblasts. Osteoblasts produce osteoid matrix and calcification follows, resulting in the encapsulation of some osteoblasts which then become osteocytes. “Islands” of developing bone emerge and are termed primary ossification centers or “bone blastema”. Several centers develop simultaneously and then fuse together. When they fuse together they form a spongy network called “primary spongiosa”. These thicken via appositional growth. So essentially, intramembranous ossification is a process that initiates in a membrane that contains mesenchyme cells.

Brent Pickrell Block 1 Q: Discuss endochondral ossification. Where does it predominantly occur? What are the two basic phases? Where do secondary ossification centers take place? A: Takes place within a piece of hyaline cartilage whose shape resembles a small version of the bone to be formed. It’s the type of ossification responsible for the formation of short and long bones. Initially the first bone tissue appears as a collar surrounding the diaphysis of the cartilage model (called the bone collar)—this collar is produced by osteoblast activity within the surrounding perichondrium. The collar impedes diffusion of oxygen and nutrients into the underlying cartilage, promoting degeneration. The chondrocytes begin to swell up (hypertrophy). These changes compress the matrix into narrower trabeculae and lead to ossification. Death of the chondrocytes results in a porous 3D structure formed by the remnants of the calcified cartilage matrix. There’s now an invasion of the open chamber by osteogrogenitor cells which then become osteoblasts allow new bone (primary) to be laid down on top of the calcified cartilage matrix. In other words, an “osteogenic bud” composed of osteoprogenitor cells and a developing blood vessel drill through the boney collar and invade the open spaces and thus form a “primary ossification center” midshaft. Osteoprogenitor cells are now in place on top of calcified cartilage and they synthesize and lay down new (woven, primary) bone by appositional growth on top of the calcified cartilage. A secondary ossification center happens (same) in the epiphysis regions. Q: What type of cartilage is responsible for the growth in length of the bone and disappears in adults? A: epiphyseal cartilage Q: What mechanism drives the elongation of bones? Discuss the different zones present in the epiphyseal plate. A: Growth in length of a long bone occurs by proliferation of chondrocytes in the epiphyseal plate. At the same time, chondrocytes in the diaphyseal side of the plate hypertrophy, their matrix becomes calcified, and the cells die. Osteoblasts lay down a layer of primary bone on the calcified cartilage matrix. Because the rates of these two opposing events (proliferation and destruction) are about equal, the epiphyseal plate does not change thickness. The different zones are: resting zone, proliferative zone, hypertrophic zone, calcified cartilage zone, and ossification zone. Q: What is taking place in the proliferative zone? A: chondrocytes are dividing rapidly and form columns of stacked cells Q: What is the resting zone? A: consists of hyaline cartilage with typical chondrocytes Q: What type of ossification is used in the formation of long bones? A: both intramembranous (for boney collar formation) and endocondral. Remember that fracture repair also uses both types of ossification. Q: What is the hypertrophic zone? A: contains swollen chondrocytes whose cytoplasm has accumulated glycogen.

Brent Pickrell Block 1 Q: What is the calcified zone? A: Loss of chondrocytes by apoptosis is accompanied by calcification of cartilage matrix by the formation of hydroxyapatite crystals Q: What goes on during fracture repair? A: The periosteum and the endosteum at the site of the fracture respond with intense proliferation producing a fibrocartilage-like tissue that surrounds the fracture. Primary bone is then formed by a combination of endochondral and intramembranous ossification. Bone is unique in that it heals without forming a scar. Q: What is the ossification zone? A: where bone tissue first appears. Capillaries and osteoprogenitor cells originating from the periosteum invade the cavities left by the chondrocytes. The osteoprogenitor cells form osteoblasts which deposit osteoid over the spicules of calcified cartilage matrix forming woven bone. Lecture 39 – Embryology II: Brandt Q: How many layers do the trophoblast and embryoblast differentiate into? A: Each differentiates into two layers. Q: What layers does the trophoblast differentiate to? What are some characteristics of the layers? A: Cytotrophoblast is the inner layer and is characterized by active proliferation (thus cells divide here and migrate to syncytiotrophobast). The syncytiotrophoblast is the outer layer and is the one that erodes into the maternal endometrium and makes hCG. Ultimately, the trophoblast becomes the placenta. Q: What is hCG and what’s its purpose? A: Human chorionic gonadotropin is a glycoprotein that is secreted by the syncytiotrophoblast to maintain the corpus luteum so that it maintains progesterone production. It’s the molecule tested in urine for pregnancy tests. Q: What are some clinical correlations related to the abnormal growth of the trophoblast with little or no embryonic tissue? A: High hCG levels, usually genetically ALL paternal (double Y)—fusion with oocyte without a nucleus and duplication of spermatic chromosomes in order to be diploid. Q: Approximately what day does the trophoblast and embryoblast differentiate? A: Day 8 Q: What are two forms of gestational trophoblastic disease? Which one is malignant? A: Hydatiform mole (benign), choriocarcinoma (malignant). Genetic analysis of hydatidiform moles indicates that although male and female pronuclei may be genetically equivalent, they may be different functionally. This evidence is derived from the fact that while cells of moles are diploid, their entire genome is paternal. Thus, most moles arise from fertilization of an oocyte lacking a nucleus followed by duplication of the male chromosomes to restore the diploid number. These results also suggest that paternal

Brent Pickrell Block 1 genes regulate most of the development of the trophoblast, since in moles, this tissue differentiates even in the absence of a female pronucleus Q: What layers does the embryoblast differentiate into? A: Embryoblast differentiates into two layers which together form the bilaminar germ disc. The hypoblast and the epiblast. Q: Which layer of the embryoblast is next to the amniotic cavity? A: Epiblast Q: What cell shape types do we see in the epiblast and hypoblast? A: Epiblast are columnar, hypoblast are cuboidal Q: What’s occurring around days 11-12? Hint: Think uteroplacental circulation A: The blastocyst is completely embedded in the endometrium. Cells of the syncyntiotrophoblast penetrate into and erode endothelium of maternal capillaries. With continued erosion, maternal blood flows through trophoblastic system establishing the uteroplacental circulation. Q: What is Heuser’s membrane and how is it formed? A: Flattened cells originating from the hypoblast forms the Heuser’s membrane that lines the inner surface of the cytotrophoblast. This forms the lining of the primitive yolk sac. Q: Discuss the production of extraembryonic mesoderm. A: It fills the space between the trophoblast and the primitive (or primary) yolk sac. A reticulum forms in this space and eventually large cavities are produced within the extraembryonic mesoderm. These large cavities come together to form the chorionic cavity.

Q: What are the boundaries of the extraembryonic somatopleuric mesoderm? A: the part that is adjacent to the cytotrophoblast and the amnion. Q: What are the boundaries of the extraembryonic splanchnopleuric mesoderm? A: The part that is adjacent to the yolk sac.

Brent Pickrell Block 1 Q: What is the space that develops between the splanchonopleuric and somatopleuric extraembryonic mesoderm called? A: the chorionic cavity

Q: Briefly describe Gastrulation. A: It begins with the formation of the primitive streak on the surface of the epiblast and is essentially the creation of three layers. Cells from the epiblast migrate towards the primitive streak and when they arrive, the break off, and dive under it, ending up between the epiblast and the hypoblast. Q: What controls the cells from the epiblast migrating towards the primitive streak? A: FGF8 (fibroblast growth factor)—it downregulates E-cadherin Q: What are the specific names of the three layers formed? A: The surface will be ectoderm, the middle layer will become mesoderm, and the hypoblast is replaced by the invaginating cells to become endoderm. The embryo then becomes a trilaminar disc Q: Cells of the epiblast are what potency at the beginning of gastrulation? A: They’re pluripotent Q: What is the source of all 3 layers? A: the epiblast Q: What is the ectoderm and what does it differentiate into? A: Connects to the “outside”. Produces the nervous system (peripheral and central), the epidermis (skin, nails, hair), sensory epithelium of the ear/nose/eye. Also produces subcutaneous glands, mammary glands, pituitary gland, and enamel of the teeth Q: What does the mesoderm differentiate into? A: It’s the “middle”. Supporting tissue (cartilage, bone), muscle, blood and lymph cells, walls of the blood vessels and heart, genitourinary system, cortical portion of the adrenal, the spleen.

Brent Pickrell Block 1 Q: What will the connecting stalk eventually become? A: the umbilical cord Q: What does the endoderm differentiate into? A: It’s the “inside”. Epithelial lining of GI tract/respiratory tract/bladder/urethra, thyroid, parathyroid, liver, pancreas, lining of tympanic cavity and auditory tube Lecture 40 - Joints: Rowley Q: What kind of joints allow for very limited or no movement? A: synarthroses Q: What type of synarthrotic joint units the skull bones in adults? A: synostosis (no movement, connected by bone) Q: What type of joint connects the 1st rib to the sternum? A: Synchondroses (connected by hyaline cartilage) Q: What type of joint forms the pubic symphysis? A: syndesmosis (connected by dense connective tissue) Q: What type of joint allows for slight movement? Give an example. A: amphiarthroses (ie intervertebral disc) Q: What join allows relatively free movement? What’s another name for this joint? A: Diarthroses or synovial joint. For example, knees and elbows Q: What’s the anatomy of the amphiarthrotic intervertebral disc? A: The disc consists of concentric layers of fibrocartilage called the annulus fibrosus. The annular fibrosus surrounds the nucleus pulposus which allows each intervertebral disc to function as a shock absorber within the spinal column. Q: What are some characteristics of diarthroses joints? A: They generally unite long bones and have high mobility (ie elbow and knee joints). It has a connective tissue capsule that encloses a synovial fluid-filled cavity. The joint cavity is lined by a synovial membrane and secretes the lubricant synovial fluid. Synovial fluid is derived from blood plasma but has a high concentration of hyaluronic acid produced by the cells of the synovial membrane Q: What type of cartilage is found at the tips of long bones in diarthrosis joints? How does this factor into transmitting forces? Be specific. A: Articular cartilage. It contains “gothic arches” of collagen fibers that distribute forces first perpendicular and then become parallel to the surface. Proteoglycan aggregates bound to hyaluronic acid and collagen fill the space among the collagen fiber network and bind a large amount of water. The large assembly of GAGs function as a biomechanical spring in articular cartilage. When pressure is applied, water is forced out of the cartilage matrix into the synovial fluid. When water is expelled, electrostatic repulsion of the negatively charged carboxyl and sulfate groups in the GAGs occur, separating the GAGs again and thus creating spaces for the return of water. When

Brent Pickrell Block 1 pressure is relaxed, water is attracted back into the intersticies of the GAG matrix. There’s no pericondrium b/c it wouldn’t allow the water to move in and out.

Q: Describe the synovial membrane. What are the two prominent cell types? A: The membrane has folds that extend into the cavity and secretes the lubricant synovial fluid. The two cell types are macrophage-like and fibroblast-like. The “synoviocytes” in contact with the synovial cavity are phagocyte and remove wear and tear debris from the synovial fluid. The other fibroblastic synoviocytes produce GAGs which they secrete along with plasma from the capillaries into the synovial fluid. There is no basement membrane or other features of epithelium despite their resemblance. Q: What are some joint disorders associated with aging? A: Herniated disc (degeneration of vertebral disc), osteoarthritis, rheumatoid arthritis Q: What is the pathology of osteoarthritis? A: Degeneration of articular cartilage, loss of hydrated GAGS result in inability to resist compression, erosion of bone, typically occurs in hip and finger joints Q: What is the pathology of rheumatoid arthritis? A: It’s chronic inflammatory condition where the damage is immune mediated. There’s activation/infiltration of macrophages and T lymphocytes. The synovial membrane becomes thick and causes pain/swelling. Articular cartilage is eroded and replaces by fibrous repair. Lecture 41 – Review Diagnostic Exam: Uncle Reddy Lecture 42 – Radiology of Upper Extremities: Willis Q: What is characteristic of a posterior shoulder dislocation? Is this common? What would cause such a dislocation? A: Trough sign; much more rare—only 2-3% of shoulder dislocations; caused by electric shock or convulsive seizers

Brent Pickrell Block 1

Q: What 3 things comprise the ulnar collateral ligament complex? A: anterior bundle, posterior bundle, and transverse ligament Q: Valgus strain is a common result from which action? A: repetitive throwing Q: Ulnar collateral ligament reconstruction is known as what type of surgery? A: Tommy John Q: Which elbow ligament is involved in nursemaid’s pathology? A: Annular Q: Medial epicondylitis is also known as what? A: Golfer’s elbow Q: What is tennis elbow also called? A: lateral epicondylitis; results from repetitive varus stress; more common than medial Q: What’s the pathology of mallet finger? A: Results from the distal interphalangeal joint suddenly being forced into extreme flexion when, for example, a finger is jammed into the base pad. These actions avulse the attachment of the tendon to the base of the distal phalanx and result in the person not being able to extend to DIP Lecture 43 – Embryology III: Brandt Q: Cells of the epiblast are what potency? A: pluripotent; remember that what cells they become in the body depends on their location after they migrate in the embryo Q: Gastrulation begins with the formation of the ___________. A: primitive streak Q: True or false: once the primitive streak is formed on the surface of the epiblast there is not a head (cranial) and tail (caudal) end. A: True; the cephalic end of the streak = primitive node Q: How are three layers created during gastrulation? A: Cells from the epiblast migrate towards the primitive streak and when they arrive, they break off, and dive under it, ending up between the epiblast and the hypoblast. The

Brent Pickrell Block 1 invaginated cells become the endoderm and mesoderm while the cells remaining in the epiblast become the ectoderm.

Q: What growth factor is key in gastrulation? What effect does it have on gene expression? A: FGF8 is produced by streak cells and downregulates E-cadherin (binds epiblast cells together—thus the cells will become loose, break off and move) and it regulates Brachyury (T) which controls cell differentiation into mesoderm Q: What cell layer is the source of all three germ layers? A: epiblast Q: What will the ectoderm eventually become? A: The outside of the embryo (ie skin) and the nervous system Q: Describe the migration of mesoderm. A: Cells move into the primitive streak and then dive out to the edges in a predictable pattern depending on where they dove into the streak. For example, the cells that enter at the primitive node head straight up the middle (cranially). Q: Cells that migrate through the midline are also called what? A: prenotochordal cells—they will form the notocordal plate and the notochordal process

Q: Which of the three germ layers will form the spinal cord? A: ectoderm

Brent Pickrell Block 1 Q: Discuss the formation of the definitive notochord. A: The prenotochordal cells initially become enmeshed in the hypoblast/endoderm forming the notochordal plate (like an “in between” stage). As the hypoblast is replaced by endodermal cells (moving in from the primitive streak), the notorchord plate gets pushed back up to form the definitive notochord.

Q: True or false: the notochord becomes the spinal cord A: False; it more or less serves as a marker or “scaffolding” and then disappears; it’s been hypothesized that the nucleus pulposus is of notochordal origin Q: The notochord originates from which of the three germ layers? A: mesoderm Q: Where is there a temporary connection between the ectoderm and endoderm? What happens if there’s a persistence of this connection? A: the notochordal plate; if there’s a persistence of this connection (in other words, the notochordal process didn’t “bounch” back up) we get a neuroenteric canal

Q: How does the neural plate form?

Brent Pickrell Block 1 A: the appearance of the notochord induces thickening of the ectoderm which forms the neural plate. In other words, the notochord tells the overlying cells of the ectoderm to become the neural plate. The induction of the neuroetoderm is called neurulation

Q: Describe the origin of the neural folds and their role in creation of the neural tube. A: The edges of the neural plate elevate and fold inward creating the depression known as the neural groove. The edges of the neural folds move towards the midline to fuse, creating the neural tube. NOTE: the cells at the crest of the neuroectoderm begin to dissociate from their neighbors and migrate throughout the body.

Q: Where does the fusion of the neural folds first take place? A: Folding starts at the 5th somite and then progresses both caudally and cranially.

Brent Pickrell Block 1 Q: After the neural tube has formed, describe the spatial relationship of the ectoderm, neural crest, and neural tube. A: The ectoderm is on top of both the neural crest and neural tube (think of the skin over your spinal cord). The neural crest drapes over the neural tube.

Q: Which neuropore will close first, the cranial or caudal? A: the cranial neuropore will close first at day 25, while the caudal neuropore closes at day 27. Note: the closure of the neural tube is the final step in neurulation. Q: What pathology is associated with the failure to close the caudal neuropore? A: spina bifida and spina bifida occulta Q: What pathology is associated with the failure to close the cranial neuropore? A: encephalocele and anencephaly Q: What is significant about the cells that sit at the crest of the neural fold? A: They are designated neural crest cells and migrate away from the neuroectoderm to different parts of the body. For example, they can migrate to form melanocytes in the skin, sensory ganglion, or the cartilage/bone in the head. Lecture 44 – Stem Cells: Haudek Q: What are some functions of stem cells? A: Develop into an organism/tissue, maintain an organism/tissue (ie blood, skin—thus involved in homeostasis), repair tissue (regeneration). They’re a constant pool for new cells as long as the individual lives. Q: A fertilized egg has what type of stem cells? A: Embryonic stem cells Q: What types of stem cells are contained in the placenta and umbilical cord blood? A: fetal stem cells Q: Bone marrow and tissues (ie heart, liver, blood, fat, etc) have what type of stem cells? A: Adult stem cells/progenitor cells Q: A stem cell is a single cell that has no special function, what has capacities for what?

Brent Pickrell Block 1 A: Self-renewal (can give rise to a cell of the same type), differentiation, and clonality (a single cell gives rise to multiple tissues). Q: What is the order of stem cells in terms of their capacity to differentiate? A: embryonic > fetal > adult Q: Where are true embryonic stem cells found naturally? A: in the blastocyst; requires destruction of the potential embryo; usually obtained from in vitro fertilization Q: How can we get ES cells through somatic cell nuclear transfer (SCNT)? A: Use a somatic cell nucleus, put it in an egg cell. There’s no sperm necessary and requires the destruction of the potential embryo Q: What are some tidbits on SCNT. A: the new individual is a “clone” of the donor individual of the somatic cell (reproductive cloning); ES cells are a pool of “personalized” cells for donor individual, ES cells allow us to study the exact disease/condition of the donor individual (research cloning); so far, only works in animals, not in humans Q: What are fundamental concerns regarding ES cells? A: Research requires the destruction of an embryo Q: How do we achieve induced pluripotent stem cells (iPS)? A: Viral transfection of genes coding for embryonic transcription factors in a somatic cell; there’s no potential embryo, thus no destruction Q: What are specific ES cell (pluripotent) criteria? A: most grow at least 10-12 months in an undifferentiated state, must express certain stem cell markers on surface, must express certain transcription factors, need intact DNA, must be able to differentiate spontaneously, must be able to differentiate directionally, must be able to regenerate all cells of all 3 germ layer lineages, must be able to forma specific tumor (teratoma) after injection into immune-suppressed mice, must yield viable offspring when injected into empty blastocyst and implanted into mouse Q: What are advantages of ES cells? A: can produce any cell type, easy to isolate/maintain/identify, grow in large numbers, grow fast, large source of blastocysts from in vitro fertilization clinics Q: What are some disadvantages of ES cells? A: Form easily teratomas, major ethical concerns, need to differentiate before application Q: True or false: there is no clear border between fetal stem cells and adult stem cells. A: True Q: Are fetal stem cells pluripotent? A: No, they are multipotent. They give rise to numerous (but not all) cell types. They do not form teratomas.

Brent Pickrell Block 1 Q: Where can we find adult stem cells? Name some characteristics. A: They’re found in almost all tissues, but most prominent: bone marrow, neural, cardiac, epithelial, skin, adipose-derived. They are multi-potent. They can differentiate into other cell types of different tissues Q: What stem cells function in bone marrow transplantation? A: Hematopoietic stem cells Q: Where are mesenchymal stem cells found? Also called what? A: bone marrow, bone, muscle, fat, skin, cartilage. They’re also called stromal stem cells Q: What stem cell is most used for tissue engineering? A: Mesenchymal stem cells Q: Can adult stem cells for teratomas? A: No Q: What are some advantages of adult stem cells? A: Do not form teratomas, are already more specialized towards the desired lineage, no major ethical concerns Q: What are some disadvantages of adult stem cells? A: are rare and in small number, not in every tissue, proliferate slow, self renewal is limited Q: What are the current uses of embryonic stem cells? A: only for research and drug testing; no clinical applications so far Q: What are current uses for fetal stem cells? A: Cord blood is being used to treat hematopoietic and genetic disorders like leukemia, diabetes, brain injury Q: What are the current uses for adult stem cells? A: extensive research application, drug and toxicology testing, several clinical applications; they are currently the best candidates for tissue regeneration Lecture 45 – Gene Regulation: Uncle Reddy Q: What is the function of restriction endonucleases in bacteria? A: They serve as a defense mechanism and thus restrict the expression of non-bacterial DNA through cleavage. Bacterial DNA is protected by methylation of bases at the restriction sites. Q: Define and explain the importance of restriction endonucleases. A: Restriction endonucleases (restriction enzymes) cleave double-stranded DNA into smaller, more management fragments. Cleaves DNA at a specific nucleotide sequence.

Brent Pickrell Block 1 They recognize short stretches of DNA (4-8bp) that are palindromes (the nucleotide sequence on the two strands is identical if each is read in the 5’!3’ direction. Restriction enzymes can produce either “sticky ends” (more useful for cloning purposes) where the resulting DNA fragments have single-stranded sequences that are complementary to each other. Others, produce fragments that have blunt ends that are double-stranded and therefore don’t hydrogen bond with each other Q: What are plasmids, why do bacteria have them, and how can we use them for cloning DNA? A: Plasmids may carry genes that convey antibiotic resistance to the host bacterium. Plasmids can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by restriction endonucleases, and up to 10kb of foreign DNA (cut with the same restriction enzyme) inserted. The recombinant plasmid can be introduced into a bacterium, and large numbers of copies of the plasmid produced. The bacteria are grown in the presence of antibiotics thus selecting for cells contain the hybrid plasmids Q: What are the steps in preparing a cDNA expression library? What is an expression library used for? A: mRNA can be used as a template to make a complementary DNA (cDNA) molecule using the enzyme reverse transcriptase. The resulting cDNA is thus a double stranded copy of mRNA. cDNA can be amplified by cloning or by PCR. It can be used as a probe to locate the gene that coded for the original mRNA. Because cDNA has no intervening sequences, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria. These special plasmids contain a bacterial promoter for transcription of the cDNA, and a shine-dalgarno sequence that allows the bacterial ribosome to initiate translation of the resulting mRNA molecule. Q: Explain southern blotting and its applications. A: Southern blotting is a technique that can detect mutations in DNA. First, DNA is extracted from cells and is cleaved into many fragments using a restriction enzyme. The resulting fragments are separated on the basis of size by electrophoresis. Detection of mutations: mutations cause the pattern of bands to differ from those seen with a normal gene. Longer fragments are generated if a restriction site is lost. Alternatively, a point mutation may create a new cleavage site resulting in the production of shorter fragments. A restriction fragment length polymorphism (RFLP) is a genetic variation that can be observed by cleaving the DNA into fragments with a restriction enzyme. The length of the restriction fragments is altered if the genetic variant alters the DNA so as to create or abolish a site of restriction endonuclease cleavage. Q: How does PCR work? What occurs during each cycle of PCR? A: PCR is a test tube method for amplifying a selected DNA sequence; it uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle of amplification doubles the amount of DNA leading to exponential increase in DNA. First, denature the DNA using heat. Second, the strands are cooled and allowed to anneal to the two primers (one for each strand). Third, DNA pol and deoxyribonucleotides triphosphates are added to initiate the synthesis of two new strands complementary to the original. Can also be used to detect low-abundance nucleic acids: for example, viruses that have a long latency period (like HIV) are difficult to detect at early stage of

Brent Pickrell Block 1 infection. PCR offers a rapid and sensitive method for detecting viral DNA sequences when only a small portion of cells are harboring the virus. Q: When a student runs a PCR on a person with cystic fibrosis, what will they most likely observe? A: They will see a three nucleotide deletion. When the DNA is amplified in the mutated region, one gets a DNA fragment that is shorter by three nucleotides. Q: In DNA fingerprinting, where are the most variable portions located? A: The most variable portions are VNTRs (Variable number of tandem repeats). Tandem repeats: short sequences of DNA are scattered locations in the genome, repeated in tandem. It is unique for any given individual and therefore serves as a molecular fingerprint. Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment Q: What types of polymorphisms are there? A: Single nucleotide polymorphisms (SNPs) and VNTRs/STR (Variable number of tandem repeats or short tandem repeats). Q: True or false: STR data is used in forensic science. A: True. About 100 STR loci are routinely analyzed Q: What are possible sources of fetal DNA? A: Cells in amniotic fluid and the chorinic villus Q: Discuss what a scientist might find in RFLP analysis of the Beta-globin gene of two individuals: one normal and one with sickle cell. A: The A to T mutation within codon six of the beta-globin gene eliminates a cleavage site for a particular endonuclease. As evident through Southern blot analysis, individuals with sickle cell thus have longer fragments due to the absence of a cleavage site. A heterozygote would have one long and one normal fragment. Q: How can one identify the differences in RNA expression between normal and cancer cells? A: Through microarray technology. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag. This mixture is then exposed to a gene chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene. The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the same. Q: HIV infection is initially screen with which technique? What is the diagnosis confirmed with? A: It’s initially screen using ELISA (very inexpensive, and also some false-positives). It’s confirmedy by Western blotting. Q: What sample type is analyzed in Southern blotting? A: DNA; it detects DNA changes

Brent Pickrell Block 1 Q: What sample type is analyzed in Northern blotting? A: RNA; detects mRNA amounts and sizes Q: What does Western blot analyze? A: Measures the amount of protein Q: What does ASO (allele specific oligonucleotide) analyze? A: DNA; detects DNA mutations Q: What is humulin? How is it made? Why don’t they use porcine or bovine insulin? A: Humulin is recombinant human insulin. Mammalian proinsulin mRNA is reverse transcribed to Proinsulin cDNA and then integrated into a plasmid. After prolonged use, humans develop antibodies against porcine and bovine insulin. Q: What is leptin? A: It’s a hormone that control fat metabolism/satiety. We saw how leptin-deficient mice do not limit eating. Lecture 46 – Enzyme Regulation: Uncle Reddy Q: What are some mechanisms by which inactive proteins ! active proteins and vice versa? A: By proteolysis, via G proteins (Gs, Gi, Gq), via second messengers (cAMP, cGMP, Ca2+), by phosphorylation Q: How does the precursor angiotensingon get converted to ang II? What enzymes are involved and what is the significance of angII in the body? A: Renin (a protease from the kidney) cleaves angiotensinogen to angI which is then converted to angII via the action of the enzyme ACE (another protease, endothelial cells). Angiotensinogen ! angI ! angII. Angiotensin II is a potent vasoconstrictor and increases blood pressure. Q: When might the above set of reactions take place? Under what conditions? A: The above pathway is called RAAAS – renin, angiotensin, aldosterone, ADH. It takes place during emergency/fight/flight, low blood pressure (hypovolemia), low sodium (hyponatremia), hypoperfusion of kidneys, extended starvation. Q: What’s are the actions of fibrin and plasmin? A: Fibrin promotes clotting, plasmin dissolves clots Q: What is the mechanism of G proteins? A: In the inactive form of a G protein, the alpha-subunit is bound to GDP. Binding of ligand causes a conformational change in the receptor, triggering replacement of the GDP with GTP. The GTP bound form of the alpha-subunit dissociates from the other two subunits and moves to adenylyl cyclase, which becomes activated. The actions of the alpha-ATP complex are short-lived because it has inherent GTPase activity, resulting in rapid hydrolysis of GTP.

Brent Pickrell Block 1 Q: Compare/contrast Gs, Gi, Gq. A: Gs = stimulatory G protein that activates adenylyl cyclase and thus increases cAMP. Gi = inhibitory G protein that inactivates adenylyl cyclase and thus decreases cAMP. Gq=queer/strange and it activates phospholipase C and thus increases IP3 and Ca2+ Q: What three enzymes are regulated by 2nd messengers? A: Protein kinase A (PKA), protein kinase G (PKG), calcium-calmodulin kinase. Q: How does cAMP exert its effects on PKA? A: Cyclic AMP (cAMP) activates protein kinase A by binding to its two regulatory subunits, causing the release of active catalytic subunits. The active subunits catalyze the transfer of phosphate from ATP to specific serine or threonine residues of protein substrates. The phosphorylated proteins may act directly on the cell’s ion channels, or, if enzymes, may become activated or inhibited. Q: What is the effect of closing K+ channels on membrane potential? A: The cell will depolarize and come closer to threshold. Q: Where is the K+/ATP channel? What is it sensitive to? A: It is located in the pancreatic beta cells. The K+ channels in beta cells are sensitive to ATP/ADP ratio where ATP closes K+ channel (depolarizes cell) where ADP opens K+ channel (hyperpolarizes the cell). High levels of glucose lead to increased ATP which in turn binds to the K+/ATP channel resulting in channel closure. The reduction in membrane potential in turn opens VG-Ca2+ channels increases intracellular calcium which triggers exocytosis of insulin. Q: How does glucose get into pancreatic beta cells? Through what channel? A: GLUT 2 Q: What is the effect of Sulfonylureas on the K+/ATP channel? A: It binds K+ channels, inhibits them and thus depolarizes the cell, causing insulin release. Q: What types of drugs are Sulfonylureas? A: They are oral hypoglycemic drugs (anti-diabetic). They end in ***ide (ie glyburide, glipizide). All these drugs bind to ATP/ADP sensitive K+ channels and increase insulin release from beta cells of pancreas. Q: What are isoenzymes? Give two examples. A: Isoenzymes have the same function but different primary amino acid sequences. For example—hexokinase and glucokinase are isoenzymes in that both add phosphates to sugars. The numerous GLUT transports are also examples of isoenzymes. Q: What are SGLT and GLUT? A: They are two types of glucose transporters. One is an example of secondary active transport (SGLT) while the other (GLUT) is an example of facilitated transport and does not require any kind of energy.

Brent Pickrell Block 1 Q: Where are GLUT 2 transporters found in the body? What’s there Km? A: They are found in the liver and also beta cells of the pancreas. GLUT2. high Km of 15-20 mM. Hence, the rate of glucose transport is proportional to blood glucose levels. Pancreatic Beta cells can sense glucose and adjust insulin secretion accordingly. High Km of GLUT2 in liver also assures that glucose rapidly enters liver cells only in times of plenty. When blood glucose level is low, glucose preferentially enters brain, RBCs, and other tissues because their glucose transporters have a lower Km than that of liver. Q: Where do GLUT 1 & 3 appear? What about its Km value? A: In most tissues—namely RBCs, brain, placenta, neurons, testes. GLUT1 & 3. Km ~1 mM, significantly lower than blood glucose conc. of 4-8 mM. Present in nearly all tissues is responsible for basal glucose uptake. Hence, GLUT1 & 3 continually transport glucose at an essentially constant rate. Q: Which GLUT transporters exhibit an intermediate Km value? A: GLUT4, Km of 5mM. Found predominantly in adipose, skeletal muscle, heart. Insulin sensitive. Q: The intestine contains what type of glucose transporter on its apical surface? A: SGLT 1 is located in the intestine and is Na+-dependent. It actually transports both glucose and galactose. Q: SGLT 2 is located where in the body? Does it only transport glucose? A: Apical membrane of renal tubules. It doesn’t transport galactose like SLGT 1. Q: Specific phosphodiesterases will inactivate which important second messengers? A: cAMP and cGMP—they both have phosphodiester bonds. Q: What amino acid is nitric oxide (NO) synthesized from in the body? A: Arginine Q: What is the function of NO? A: NO is the endothelium-derived relaxing factor, which causes vasodilation by relaxing vascular smooth muscle. Function of nitric oxide (NO) include: relaxes smooth muscle, neurotransmitter, prevents platelet aggregation, macrophage function Q: What is the mechanism of NO causing smooth muscle relaxation? A: NO is synthesized in endothelial cells and diffuses to vascular smooth muscle where it activates guanylate (guanylyl) cyclase to form cGMP. The resultant rise in cGMP causes activation of protein kinase G (PKG) which phosphorylates Ca2+ channels thereby causing decreased entry of Ca2+ into smooth muscle cells. This decreases the calcium-calmodulin activation of MLCK, thereby decreasing smooth muscle contraction and favoring relation. Q: How do nitrates decrease blood pressure? A: Nitrates (specifically nitroglycerin) is metabolized to NO which causes relaxation of vascular smooth muscle and therefore lowers BP

Brent Pickrell Block 1 Q: How does albuterol affect smooth muscle? What receptors does it act upon? A: It causes smooth muscle relaxation by increasing cAMP which inhibits MLCK. It acts on beta-2 receptors. Q: What is the effect or norepinephrine acting on smooth muscle through alpha-1 receptors? A: contraction Q: How is erectile dysfunction treated? A: Keep cGMP levels high. We do this by inhibiting phosphodiesterases. PDE5 inhibitors inhibit the degradation of cGMP in erectile tissue, eyes and pulmonary vasculature. Q: What do drugs end with that are PDE5 inhibitors? A: ***afil. For example, Sildenafil (Viagra), tadalafil (Cialis), vardenafil (Levitra). Q: Where do PDE3 inhibitors exert their effects? A: Cardiac muscle, platelets Q: What are some of the side effects of Viagra? Why do these make sense? A: Problems with hearing and vision (PDE5 also affects eyes/ears). Hypotension since there can be cross-reaction in blood vessels resulting in their dilation. Priapism (erection lastly longer than 4 hours). Contraindication in patients on nitrates since nitrates also dilate blood vessels so the two drugs would work synergistically. Lecture 47 – Embryology IV: Brandt Q: What factor does the notochord secrete that is involved in inducing changes in the ectoderm. A: Sonic hedge hog (Shh); as Shh is secreted from the notochord a gradient is formed

Q: True or false: the notochord AND the neural tube are secreting Shh. If true, which specific part of the tube is secreting Shh? A: True. The notochord and floor plate of the neural tube are secreting Shh.

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Q: What is the roof of the neural tube secreting? A: BMP Q: Describe the gradient that the cells of the neural tube experience. A: Cells experience different percentages of BMP and Shh depending on where they are in the neural tube. This gradient influences the cells’ differentiation. Q: Explain why Shh is only expressed on the left side of the embryo. A: Shh is secreted throughout the primitive node initially. Subsequently, the Activin IIa receptor is expressed on the right side of the node which blocks Shh expression. Shh is thus expressed only on the left side of the node where it activates nodal and lefty. Q: What gene is expressed on the right side of the embryo? A: Snail Q: What two genes are activated as a result of Shh on the left side? A: Shh induces nodal, and then nodal induces lefty Q: What specifically does BMP4 do to the mesoderm? A: it ventralizes the mesoderm (moves it toward the front of the embryo) Q: What 3 proteins are secreted to counteract BMP4? Where are they secreted from? A: Chordin, noggin, and follistatin are secreted by the primitive node and block BMP4 which allows the notochord to stay dorsal. Q: What pathology is associated with mirror image of internal organs? A: situs inversus; note: everything is still functional Q: What pathology is associated with a tumor that contains all three germ lines? What is the most common location of this tumor? A: Teratoma; most common location is saccrococcygeal. If it’s malignant it’s called a yolk sac tumor.

Brent Pickrell Block 1 Q: What does the Brachyury (T) gene regulate in the embryo? What do deficiencies in this gene cause? A: It’s involved in the regulation of dorsal mesoderm formation in the mid and caudal embryo. Deficiencies result in caudal regression. Q: What’s the most severe form of caudal regression syndrome? A: Sirenomelia; note: has to do with regulation of dorsal mesoderm Q: What is a more common and less severe form of caudal regression? A: imperforate anus; note: has to do with regulation of dorsal mesoderm Q: The mesoderm that ingresses on the lateral edge of the primitive streak is called ___. A: paraxial mesoderm

Q: What is the paraxial mesoderm responsible for forming? What are these structures? A: Somitomeres; they are rounded structures that are paired and go on to form blocks of mesoderm called somites. Q: What day do somites start appearing on the embryo? Where do they start forming? What is significant about their progressive formation? A: Somites start around day 20 in the cervical region and 3 somite pairs are added per day for the next 10 days. Because somites appear with specified periodicity, the age of the embryo can be determined by counting the somites.

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Q: As a review, what structures is the mesoderm responsible for forming? A: Supporting tissues (bones, cartilage), muscle, blood and lymph cells, walls of the blood vessels and heart, spleen, cortical portion of the adrenal, dermis of skin Q: Around the 4th week, ventral and medial walls of the somite loosen to become a ____. A: Sclerotome

A: What gene regulates somite differentiation into dermis? A: Pax3 Q: What gene regulates somite differentiation into body wall and limb muscles? A: MyoD Q: What gene regulates somite differentiation into back (epaxial) muscles? A: My15

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Q: What does the sclerotome differentiate into? A: Vertebrae and ribs Q: What does the dermomyotome differentiate into? A: muscles/dermis under the epidermis Q: What portion of the dermomyotome becomes the dermis? A: the mid portion

Q: What portion of the dermomyotome becomes the expaxial (back) muscles? A: dorsomedial portion Q: What’s unique about the origin of the bones of the face? A: They are from the neural crest Q: What does the dorsolateral portion of the dermomytome become? A: limb and body wall musculature Lecture 48 – Biotechnology/Human disease: Uncle Reddy Q: What is SCID? A: Severe combined immunodeficiency disease (SCID): immune deficiency as a result of mutations in the gene for adenosine deaminase (ADA) or a gene coding for an interleukin receptor subunit. Patients with both kinds of SCID have been successfully treated by incorporating functional copies of the appropriate gene into their cells (called gene replacement therapy). Q: What does the ADA enzyme do? A: it converts adenosine to inosine

Brent Pickrell Block 1 Q: What does the pathology of ADA deficiency result in? A: High adenosine levels lead to high dATP. dATP inhibits ribonucleotide reductase, a key enzyme in the synthesis of dCTP, dGTP, and dTTP. No building blocks for DNA synthesis causes the rapidly dividing cells of the immune system to be most affected. Q: How do we treat people with ADA deficiency using gene therapy? A: We take some stem cells/T-cells from the patient and infect them with a retrovirus modified to carry the normal gene for adenosine deaminase. The gene becomes integrated into the cell’s chromosomes and is expressed. We then reintroduce the cells to the patient and they regain immune function. NOTE: must be careful on where we insert—if it goes to the wrong place it could cause cancer. Q: What is the significant of gp120? A: It’s a protein on HIV that binds to the CD4 receptor on the surface of T cells. It thus allows HIV to enter our cells. Q: How does HIV infect cells? A: Viral particle (gp120) bids to CD4 on T cell. Viral envelope fuses with cell membrane allowing viral genome to enter the cell. HIV is a retrovirus. Reverse transcriptase copies viral RNA genome into double-stranded DNA which is integrated into the host DNA. The integration sites are random, although some areas are more prone to accept foreign DNA and thus termed “hot spots”. Q: What enzyme integrates viral DNA into the genome? A: Integrase Q: What are LTR (long terminal repeats)? R: LTR are ends of viral DNA used by the virus to integrate into host chromosome. Q: What is our DNA construct for gene therapy? Hint: give order of elements A: LTR – Enhancer – promoter – ADA cDNA – poly(A) – LTR; from class: including an intron is beneficial Q: If given a choice in gene therapy, what kind of promoter/enhancer do you use? A: Choose a promoter that is highly active in blood cells and is a strong promoter + Enhancer (makes lots of ADA mRNA molecules) Q: What is a Philadelphia chromosome? What disease does it cause? A: It forms as a result of abnormal chromosome translocation. This translocation occurs between a piece of chromosome 9 and a piece of chromosome 22. This exchange results in a longer than normal chromosome 9, and a shorter chromosome 22. The DNA in chromosome 9 contains a gene called ABL, while chromosome 22 contains a gene called BCR. The shorter combination of the two chromosomes, with a gene labeled BCR-ABL, forms what is known as the Philadelphia Chromosome. It causes chronic myelogenous leukemia (CML).

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Q: What does the BCR-ABL mutated gene on the Philadelphia chromosome code for? A: Protein tyrosine kinase. This tyrosine kinase is an enzyme which activates other proteins by adding phosphate to them. This unregulated phosphorylation activates enzymes important in cell division and growth. Q: Where is the BCR-ABL fusion kinase present? A: ONLY in cancer cells—it’s completely absent in normal cells. If one can develop a drug that specifically inactivates cancer-specific fusion kinase, you kill the cancer cells with no effect on normal cells.

Q: What does gleevec do?

Brent Pickrell Block 1 A: It blocks BCR-ABL. It’s given as a one-a-day pill. Since blood cells turnover, when all mutant cells with chromosomal translocation are killed, the patient is cancer-free.

Q: What types of cells lose contact inhibition and pile up? A: Cancer cells. A single base change in an oncogene produces protein that is autosomal dominant and causes cells to proliferate abnormally. Normal cells are very respectful of their neighbors (in other words, they exhibit contact inhibition). Q: What is Ras? What does mutant Ras cause? A: Ras is a GTPase that is involved in signal transduction. Activation of Ras causes cell growth. Since Ras communicates signals from outside the cell to the nucleus, mutations in ras genes can permanently activate it and cause inappropriate transmission inside the cell, even in the absence of extracellular signals. Because these signals result in cell growth and division, dysregulated Ras signaling can ultimately lead to oncogenesis and cancer. G-Proteins have intrinsic GTPase activity that allow it to be turned off. Mutant Ras will cause the protein to stay active and promote cell division (this is b/c the intrinsic GTPase activity is lost). Q: What is the optimal substrate for Dicer? What does it degrade? A: Double-stranded RNA; perfect hybrid and no sequence specificity. It will degrade mutant Ras mRNA. Lecture 49 – Embryology V: Brandt Q: What week marks the end of the embryonic period? A: Week 8 (this is also the beginning of the fetal period) Q: Review the structures that arise from the ectoderm. A: Nervous system (peripheral and central), epidermis (skin, hair, nails), sensory epithelium of the ear/nose/eye. Remember that the ectoderm connects us to the outside.

Brent Pickrell Block 1 Q: Understand the significance of the figure below. It’s a cell fate map.

Q: The epidermis arises from which layer? A: ectoderm Q: The dermis arises from which layer? A: paraxial mesoderm Q: Pigment in the skin is derived from what? A: neural crest; remember that the neural crest cells MIGRATE

Q: Cells that migrate through the midline of the mesoderm are initially called what? What will they form? A: They are “prenotochordal cells” and will form the prechordal plate and the notochordal process

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Q: What is the sequence of maturation of the notochordal process? A: Notochordal process ! notochordal plate ! notochord

Q: Appearance of the notochord induces thickening of ectoderm produce what structure? A: Neural plate

Q: The paraxial mesoderm gives rise to what paired structures? A: Somites (which give rise to dematomyotome and sclerotome) Q: What does the lateral portion of the dermatomyotome give rise to? A: body wall muscle

Brent Pickrell Block 1 Q: What does the mid portion of the dermatomyotome give rise to? A: dermis Q: What does the medial portion of the dermatomyotome give rise to? A: epiaxial muscles Q: What does the sclerotome give rise to? A: bones and cartilage Q: The intermediate mesoderm is the origin of what structures? A: urogenital Q: What is the somatic/parietal mesoderm contiguous with? A: The somatic extraembryonic mesoderm covering the amnion

Q: What is the splanchnic/visceral mesoderm contiguous with? A: the splanchnic extraembryonic mesoderm covering the yolk sac

Q: Understand the significance/implications of the following figure.

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Q: Which layer of the lateral plate mesoderm will cover the organs? A: the Visceral layer, along with underlying endoderm will form the wall of the gut Q: Which layer of the lateral plate mesoderm will form the lateral and ventral body wall? A: Parietal/somatic layer

Q: What is the main organ derived from the endoderm? A: the GI tract Lecture 50 – Anatomy of Hand: Duncan Lecture 51 – Cell Signaling: Cooney Q: Discuss a basic cell signaling pathway. A: An extracellular signal binds, binding of the signal molecule changes the conformation of the receptor, the receptor transduces the signal to the interior of the cell, some intracellular signal proteins translate the signal within the cell (can amplify it or take the signal to where it’s meant to elicit a response). Q: True or false: intracellular receptors depend on ligand chemistry. A: True. If you have an intracellular receptor, the signal molecule must be able to get across the plasma membrane. In contrast, membrane receptors have no dependency on ligand chemistry Q: What are some characteristics of receptors?

Brent Pickrell Block 1 A: They’re found in target cells, exhibit high specificity, have high affinity for their ligand, and elicit a specific response. Receptor affinity is inversely proportional to ligand concentration Q: What are 4 signaling modes? A: contact dependent (neighbors), paracrine (neighborhood), synaptic (long distance but fast), endocrine (long distance and slower)

Q: When a cytotoxic T cells binds a viral infected cell’s MHC/antigen complex what type of signaling is this an example of? A: Juxtacrine/contact dependent signaling Q: Define paracrine signaling. Give examples. A: local diffusion of signaling molecules without involvement of the blood stream. Growth of the oocyte is regulated hormonally and locally. Testosterone diffuses into seminiferous tubules in another example. Q: What endocrine gland is associated with Cushing’s disease and Addison’s? A: adrenals Q: What endocrine gland is associated with Grave’s disease? How about jet lag? A: thyroid; pineal Q: Discuss the term morphogen. A: The morphogen provides spatial information by forming a concentration gradient that subdivides a field of cells by inducing or maintaining the expression of different target genes at distinct concentration thresholds. Thus, cells far from the source of the morphogen will receive low levels of morphogen and express only low-threshold target genes. In contrast, cells close to the source of morphogen will receive high levels of morphogen and will express both low- and high-threshold target genes. Distinct cell types emerge as a consequence of the different combination of target gene expression.

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Q: What is an agonist? A: a chemical/ligand that elicits a positive response when binding to a receptor Q: What is an antagonist? A: A chemical/ligand that inhibits a normal positive physiological response of an agonist Lecture 52 – Intro to Medical Genetics: Potocki Q: Define genomic medicine. A: the promise to tailor health care at the individual level by using patients’ genomic information. Allows us to prescribe the best treatment for each patient, avoid adverse drug reactions, and identify and monitor individuals at high risk from disease Q: What’s the most common skeletal dysplasia? Why is it common? What complications are associated with it? A: Achondroplasia. It’s common because the particular gene is hypermutable. Airway obstruction, obesity, spinal stenosis L1-L4, cord compression. They have short stature (specifically shortening of the bones closest to the trunk, ie femur) but they have normal intellect. Q: What gene is associated with achondroplasia? How does the age of the parents play into risk factors? A: mutations in FGFR3 (99% in the same nucleotide resulting in a missense mutation). It’s autosomal dominant, 100% penetrance, and can happen more frequently with advanced paternal age. Q: What does FGFR3 code for? A: transmembrane TK receptor Q: What is the lethal type of skeletal dysplasia called? A: Thanatophoric dysplasia Q: What mutation causes thanatophoric dysplasia? A: a mutation in FGFR3 (like achondroplasia) results in a missense or nonsense mutation. Q: What is the normal function of FGFR3 (fibroblast growth factor receptor 3)?

Brent Pickrell Block 1 A: It’s a transmembrane TK receptor that binds FGRs. The normal function of FGFR3 is to serve as a negative inhibitor of bone growth during ossification (inhibits proliferation of chondrocytes within the growth plate). Q: Mutations in FGFR3 like we see in Thanatophoric dysplasia, hypochondroplasia, and achondroplasia cause what at the molecular level? A: gain of function mutations cause ligand-independent activation of FGFR3 which inappropriately inhibits chondrocyte proliferation within the growth plate. Because all three disorders are caused by different mutations in the same gene these disorders are allelic. Q: What type of disease is cystic fibrosis? A: autosomal recessive Q: A disorder that has multi-system organ involvement is said to have _____ effects. A: pleotropic Q: What are compound heterozygous mutations? A: mutations on each allele, but they’re different mutations Q: What gene mutation causes cystic fibrosis? A: CFTR; there’s >1000 disease associated mutations Q: All mutations are de novo in _________ dysplasia. A: thanatophoric Q: What form of HbS polymerizes to form sickle cells? A: the deOxy form; this will cause vaso-occlusion Q: When do patients normally present with sickle cell anemia? Symptoms? A: Before age 2; anemia, FTT, splenomegaly, dactylitis (puff hands/feet) Q: Cumulative risk of breast cancer depends on first-degree family history. What does this mean? A: If the age of the relative diagnosed with breast cancer is young (20-29) then there is higher cumulative risk for the first-degree family compared to someone’s family that had a relative diagnosed between age 40-49 Lecture 53 – Immune I: Rowely Q: What defines the innate aspect of the immune system? A: It’s the 1st line of defense and includes the complement system, skin, saliva, mucus, epithelium, macrophages, NK cells. It’s fast, nonspecific, and doesn’t produce memory cells. Q: What is primary lymphoid tissue? A: Includes bone marrow and thymus and functions in lymphocyte production and maturation. It’s where the cells are made and programmed to not attack self. “Training Camp”

Brent Pickrell Block 1 Q: What is secondary lymphoid tissue? A: It’s the “battlefield”. Where the immune response takes place and includes lymph nodes, spleen and diffuse lymphoid tissues like the tonsils. Q: What are the two divisions of the adaptive immune system? Which one is mediated by T lymphocytes? A: humoral and cell-mediated; cellular is mediated by T lymphocytes Q: What antibodies do B lymphocytes have on their surface? A: IgM and IgD serve as receptors Q: What chemicals secreted by helper T cells helps mediate clonal expansion of B lymphocytes? A: interleukins (IL-4, IL-5, IL-6) Q: What are some characteristics of antibodies? A: They’re glycoproteins that interact with antigen epitopes. They’re Y-shaped with a heavy and light chain. There’s 5 classes made by B cells, each with a unique function. Q: Which part of the antibody structure determines the class of the antibody? A: The Fc region (the carboxyl half of the heavy chain). Q: Which antibodies are the first ones secreted in an immune response? A: IgM Q: Which antibodies activate the complement system? A: IgM Q: What’s the most abundance class of antibodies and act as a secondary response? A: IgG Q: What does the activation of the complement system cause? A: Activation of proteases, opsonization of pathogen and ultimately recognition and phagocytosis and direct lysis Q: Which antibodies activate macrophages and neutrophils? What else does this class activate? A: IgG; can also activate the complement system Q: What antibodies can pass from the mother to fetus via the placenta and breast milk? A: IgG Q: Which antibodies are in secretions and transported across epithelial layers? A: IgA Q: Which antibodies are primarily in charge of agglutinating antigen? A: IgA

Brent Pickrell Block 1 Q: Which antibodies are bound to protein J? A: IgA Q: Which antibody stimulates release of histamine, heparine, and cytokines? A: IgE; it serves to activate mast cells and basophils Q: Which antibody is active in allergies? A: IgE Q: What is the effect of histamine in the body? A: Causes vascular dilation and permeability Q: Which antibody induces anaphylactic shock? A: IgE Q: What is another co-receptor (along with IgM) that acts a B cell receptor and function in B lymphocyte activation? A: IgD Q: Which antibody exists as a pentamer? A: IgM Q: Which antibodies have their Fc regions bind to mast cells and basophils? A: IgE Q: What MHC complex do CD8+ cells recognize? A: MHC I complexes Q: What MHC complex do CD4+ cells recognize? A: recognize MHC II Q: What’s the response of cytotoxic T cells when they encounter an abnormal cell? A: Induce programmed cell death or lysis Q: What’s the response of helper T cells when they recognize antigen on MHC II? A: secrete cytokines (interleukins, interferons) to stimulate other immune cells Q: What are the roles of NK lymphocytes? A: Kill viral infected cells and tumor cells in a nonspecific (innate) manner; not through MHC class presentation; they release perforins and fragments to kill cells through membrane lysis and apoptosis; they don’t depend on specific antigen recognition Q: What activates a macrophage to phagocytose a particular antigen? A: They are activated by material coated with IgG (major) and IgM (minor) and complement proteins Q: How do macrophages communicate antigen to a helper T cell? A: They eat and digest antigen, sends it up on the MHC II to show Helper T cell

Brent Pickrell Block 1 Q: What activates neutrophils to phagocytose bacteria? A: They are activated when the bacteria is coated with IgG (major) and IgM (minor) Q: Mast cells and basophils have which antibody bound to their surface? A: IgE is bound to their surface Q: What are the antigen presenting cells (APCs) of the immune system? A: macrophages, B lymphocytes, dendritic cells, follicular dendritic cells Q: How do macrophages and B lymphocytes present antigen to elicit an immune response? A: via MHC II complexes to helper T cells Q: All nucleated cells have which MHC complex? A: All have MHC I complex and thus present to cytotoxic CD8 cells. Q: What are dendritic cells in the skin called? A: Langerhans cells Q: Discuss the process of MHC I presentation. A: Proteins in the cell are continuously digested by proteasomes and antigenic fragments are transferred to the RER where they associate with MHC 1 proteins synthesized there. Peptides are complexed with MHC I and shuttled to the cell surface. Any weird proteins made in the cell are detected by CD8 Q: Discuss the process of MHC II presentation. A: There’s synthesis of MHC II and then transfer of MHC II to the golgi. Golgi vesicle fuses with a lysosome containing antigens processed after endocytosis. Antigens form complexes with MHC II and are shuttled to the surface of the cell. Activates helper T cells Lecture 54 – Cell Signaling II: Cooney Q: How does NO signaling result in smooth muscle dilation? What type of signaling is this an example of? A: A nerve terminal releases acetylcholine onto an endothelial cell which activates NO synthase (NOS). NOS converts arginine to NO and NO diffuses across membranes to bind to guanylyl cyclase. Now active, guanylyl cyclase converts GTP to cGMP and results and relaxation of smooth muscle; this is an example of BOTH paracrine and synaptic signaling

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Q: What enzyme degrades cGMP? A: phosphodiesterase Q: How does PDE5 inhibitor work? A: It inhibits the phosphodiesterase and their the ability to degrade cGMP. Q: Why should people who take nitrates for chest pain not take Viagra? A: because it could lead to an unsafe drop in BP; both Viagra and nitrates act synergistically Q: What are cellular responses to steroid signaling? A: They are genetically based resulting in altered gene expression. This is an example of slower response signaling.

Phosphodiesterase

Nitrates

Endothelial Cell

Brent Pickrell Block 1 Q: G protein coupled receptors pass how many times across the membrane? How do G proteins work? A: Seven; ligand binding causes a change in the alpha subunit such that it now has a high affinity for GTP. The activated protein then activates an enzyme Q: What enzyme synthesizes cAMP from ATP? A: adenylyl cyclase Q: How does PDE set a threshold for cAMP/cGMP? A: The only way to get cAMP/cGMP signaling is if there’s enough to overwhelm the cAMP/cGMP PDE. Therefore, PDE sets a threshold Q: How many molecules of cAMP do you need for activation of PKA? A: You need 4 molecules. You need 4 molecules so the cell isn’t subject to random/stochastic events.

Q: What is the transcriptional consequences of activation of PKA? A: Activated PKA enters the nucleus through the nuclear pore, activates CREB through phosphorylation when then binds CREB-binding protein and together will bind to DNA and activate a target gene resulting in gene transcription. Q: How can a GPCR result in increased cytosolic Ca2+ and activation of PKC? A: A signal molecules binds G(q) and activates phospholipase C, which cleaves PI 4,5-Bisphosphate to IP3 and DAG, IP3 binds to IP3-gated Ca2+ release channel of the endoplasmic reticulum and causes the channel to open and increase cytosolic Ca2+.

Brent Pickrell Block 1 Calcium binds and activates PKC

Lecture 55 – Embryology VI: Brandt Q: True or false: Endovascular invasion into the uterine wall by the trophoblast results in the epithelial to endothelial transformation and, in the process, hybrid vessels with cells from both the embryo and mother. A: True Q: What is the limit of viability for the fetus? (what’s the earliest it can be born?) A: 24 weeks (it’ll be about 1 pound) Q: During the fetal period there’s the fusion of two membranes to form the placenta. What are these two membranes and what are their origins? A: Decidua basilis (uterus) and chorion frondosum (fetal)

Q: There’s also the fusion of the two fetal membranes. Name them. A: Chorionic membrane and amniotic membrane

Brent Pickrell Block 1 Q: What is the part of the chorion bearing villi? A: chorion frondosum Q: What is the nonvillous, membrane part of the chorion? A: chorion leave Q: What are the functions of the placenta? Include hormone production. A: exchange of gases (CO2, O2), exchange of nutrients and electrolytes, transmission of maternal antibodies (IgG), hormone production – progesterone, estriol, hCG, somatomamotropin Q: What structure produces progesterone until the placenta forms? A: corpus luteum Q: What is the function of somatomamotropin? A: It’s human placental lactogen. Tells the mother’s body to give nutrients preferentially to the fetus and also starts changes in breast tissue Q: What type of twins arise from two oocytes? A: dizygotic (fraternal) – have separate fetal membranes Q: What type of twins arise from one oocyte? A: Monozygotic (identical) Q: What type of twins may have one or two amniotic cavities? A: monozygotic Q: What type of twins always have a common chorionic cavity? A: monozygotic Q: What is the space called that develops between the splanchnopleuric and somatopleuric extraembryonic mesoderm? A: chorionic cavity Q: What layer is the source of all three germ layers? A: epiblast Q: The appearance of the notorchord causes what? A: Thickening of the ectoderm (neural plate). The induction of the neuroectoderm is called neurulation. Q: Bones and cartilage of the face arise from what? A: neural crest Q: The dorsomedial portion of the paraxial mesoderm will become what? Dorsolateral? A: epaxial (back) muscles; the dorsolateral portion migrates to become the precursor of limb and body wall musculature

Brent Pickrell Block 1 Q: What is the somatic/parietal mesoderm contiguous with? A: the extraembryonic mesoderm covering the amnion Q: What is the splanchnic/visceral mesoderm contiguous with? A: the extraembryonic mesoderm covering the yolk sac

Q: What layer of the lateral plate mesoderm will cover the organs and form the wall of the gut? A: Visceral/splanchnic layer along with underlying endoderm will form the wall of the gut Q: What layer of the lateral plate mesoderm will line the intraembryonic cavity? A: the parietal/somatic layer along with the overlaying ectoderm will line the intraembryonic cavity Lecture 56 – Energy Metabolism: Gilbert Q: How many high-energy bonds are there in ATP? How about AMP? A: 2 high energy phosphate bonds; deltaG = -7.3kcal/mole; AMP doesn’t have any high energy phosphate bonds Q: What vitamin is necessary to make Acetyl CoA? What’s the deficiency? A: Pantatheine—it’s a B vitamin. There is no known deficiency. Q: Where is pyruvate converted to acetyl-CoA? A: In the mitochondrial matrix. Pyruvate is made in the cytoplasm and a specific pyruvate transporter helps it cross the inner mitochondrial membrane. Q: What kind of transporter brings pyruvate into the mitochondria? A: it’s a pyruvate-OH antiporter. Q: What’s a secondary function of the mitochondria? A: It’s a major storage depot for intracellular calcium and it plays a key role in the signaling apoptosis. Q: How is ATP generated in the mitochondria exported to the cytoplasm?

Brent Pickrell Block 1 A: There is a specific ATP-ADP exchanger that ensures that ADP is brought into the mitochondrial matrix and ATP is exported to the cytoplasm. Q: What’s the function of the pyruvate dehydrogenase complex (PDC)? A: it oxidizes pyruvate to acetyl-CoA resulting in the loss of CO2 and a large negative free energy, which makes it irreversible under physiological conditions. Q: What molecules inhibit the actions of PDC? A: Acetyl-CoA and NADH Q: What cofactors are involved in the PDC? A: NAD, TPP, Lipoic Acid, FAD Q: What would a deficiency in niacin or nicotinamide cause? A: Pellagra skin disease, diarrhea, dementia, and ultimately death Q: A deficiency in thiamin pyrophosphate causes what pathology? A: Beriberi, GI symptoms and neurological symptoms Q: Which cofactor is involved in decarboxylating pyruvate? A: TPP (thiamin pyrophosphate) Q: Which cofactor is involved in acetyl group transfer to CoASH and transfer electrons to riboflavin? A: Lipoic acid Q: Which cofactor accepts electrons from lipoic acid? A: FAD Q: A deficiency in riboflavin causes what symptoms? A: fissures in the corners of the mouth, inflammation of the tongue, skin disease, and often severe irritation of the eye Q: What’s the order of cofactors utilized during PDC reactions? A: TPP ! Lipoic Acid ! FAD ! NAD Q: What enzyme in TCA catalyzes the reaction of oxaloacetate with acetyl-CoA? A: citrate synthase Q: What enzyme in TCA catalyzes the reaction of isocitrate to alpha-ketoglutarate? What molecules are given off? A: isocitrate dehydrogenase; NADH and CO2 Q: What enzyme is involved in substrate-level phosphorylation in TCA? What’s another name for this enzyme? It turns ______ into _________. A: succinate thiokinase; Succinyl-CoA synthetase; it turns succinyl CoA into succinate and a GTP

Brent Pickrell Block 1 Q: How many ATP does one acetyl-CoA generate? A: about 12.5 ATP through oxidative phosphorylation, including the NADH from PDC Q: On average, how much ATP do NADH and FADH2 generate respectively? A: 2.5 ATP, 1.5 ATP Q: Carboxylation of pyruvate by pyruvate carboxylase yields what compound? What cofactor is necessary for this reaction to take place? A: it yields oxaloacetate; the reaction requires biotin as a cofactor Q: What pathology is associated with deficiencies in biotin? A: dermatitis and GI symptoms Q: The flow through PDC and Krebs is stimulated by AMP-like signal metabolites. What are some examples of AMP-like metabolites? A: AMP, ADP, NAD, Ca2+, cAMP, CoASH Q: What are some examples of high energy ATP-like signals? A: ATP, NADH, citrate, acetyl-CoA, malonyl-CoA Q: What turns PDC from active to inactive? A: Protein Kinase Q: What turns PDC from inactive to active? What ion stimulates this enzyme? A: Phosphoprotein phosphatase turns PDC from inactive to active and Ca2+ stimulates the phosphatase. Q: What sort of molecules stimulate the kinase that turns PDC inactive? A: ATP, acetyl-CoA, NADH, citrate, malonyl-CoA Lecture 57 – Immune II: Rowely Q: What is the dendritic cell in the skin? A: Langerhans cell Q: What can a lymphoblast differentiate into? In other words, name the 3 lymphocytes A: B, T or natural killer (NK) cells Q: What are the 4 APCs in the thymus? A: Macrophages, B lymphocytes, dendritic cells, follicular dendritic cells in lymph nodes Q: Which lymphocyte is involved in the innate immune response? A: NK cells Q: When a lymphoid nodule becomes activated as a result of the arrival of antigen-carrying APCs and recognition of the antigens by B lymphocytes, these lymphocytes proliferate in the central portion of the nodule. This middle portion stains lighter and is called what? A: Germinal center

Brent Pickrell Block 1 Q: How do macrophages present antigen? Onto which MHC? A: They present via MHC II to helper T cells (CD4) Q: Which cells do follicular dendritic cells present to? A: B cells; they don’t process antigen and don’t use MHC II Q: What is primary lymphoid tissue? A: Bone marrow and thymus Q: What is secondary lymphoid tissue? A: where the response happens; lymph nodes, spleen, diffuse lymphoid tissue Q: The thymic cortex is composed of an extension population of T lymphoblasts (also called thymocytes) and macrophages in a stroma of epithelia reticular cells. What joins the epithelial reticular cells to one another? A: desmosomes (they are epithelial in origin and thus cytokeratin +); as a side note, occluding junctions between flattened epithelial reticular cells at the boundary between the cortex and medulla help to separate the two regions Q: Where is the thymus located? A: in the mediastinum – peak development during youth, begins involution at puberty. Immature T lymphocytes come to the cortex from the marrow Q: Where does the blood-thymic barrier exist? A: in the thymic cortex Q: Briefly discuss the role of the thymus in T cell maturation. A: When T lymphoblasts arrive at the thymus their surfaces do not yet exhibit the TCR (T cell receptor) or the CD4 or CD8 markers. After entering the thymic cortex they populate the cortex extensively. As thymocytes mature and express T cell markers they undergo thymic selection where they pass through a succession of microenvironments created by different mixes of stromal epithelial reticular cells. Q: Where do T lymphocytes proliferate and program within the thymus? A: in the cortex where there’s positive and negative selection Q: How do T lymphocytes migrate from the cortex to the medulla of the thymus? A: the ones programmed not to recognize self but recognize MHC will migrate as mature cells to the medulla Q: What is AIRE and where is it expressed in the thymus? A: In the thymus, it causes transcription of a wide selection of organ-specific genes that create proteins that are usually only expressed in peripheral tissues, creating an "immunological self-shadow" in the thymus. It is important that self-reactive T cells that bind strongly to self-antigen are eliminated in the thymus (via the process of negative selection), otherwise they can later bind to their corresponding self-proteins and create an autoimmune reaction. So the expression of non-local proteins by AIRE reduces the threat

Brent Pickrell Block 1 of the occurrence of autoimmunity later on by allowing for the elimination of auto-reactive T cells that bind antigens not traditionally found in the thymus (from Wikipedia) Q: What are three key cell types in the thymus? A: epithelial reticular cells (held together by desmosomes), maturing T lymphocytes, and macrophages Q: Where are Hassall’s corpuscles located in the thymus? A: Medulla Q: Discuss thymic selection. A: After entering the thymus, T lymphoblasts populate the cortex where they proliferate extensively. As they mature and express T cell markers, the undergo thymic selection as they pass through a succession of microenvironments created by different mixes of stromal epithelial reticular cells. They are presented with antigens bound to MHC I and MHC II proteins on epithelial reticular cells, macrophages, and dendritic cells. Thymocytes whose TCRs cannot bind MHC molecules at all are nonfunctional and have no future as T cells—they are deleted. Similarly, those thymocytes that strongly bind MHCs containing self peptides are also deleted since they could cause an autoimmune response. Movement into the medulla depends on the action of chemokines and on the interaction of thymocytes with the ECM and cytoreticulum Q: Discuss the blood-thymus barrier. What are the 5 layers? A: regulates exchange of substances between the circulatory system and thymus, providing a sequestered environment for immature T cells to develop. The barrier also prevents the immature T cells from contacting foreign antigens. Endothelial cell, endothelial basal lamina, connective tissue, epithelial reticular cell, epithelial reticular cell basal lamina Q: Why would an adult autoimmune disease be corrected by removing the thymus? A: To prevent further T lymphocyte maturation Q: Discus the overall structure of the lymph node. A: it’s a bean shaped structure that filters regional lymph fluid—all tissue derived lymph is filtered before entering circulation. Lymph nodes have outer convex surface where lymphatics enter (afferent) and have an inner concave surface where a single efferent lymphatic vessel exits. It’s divided into a cortex and medulla which have extensive reticular cells (fibroblasts) and extensive reticular fibers (collagen type III) for filtration. Q: Where are lymphoid (B cell) nodules found in the lymph node? A: in the cortex Q: Where are plasma cells and macrophages located at in the lymph node? A: in the medulla Q: How do lymphocytes enter the lymph node? A: through HEVs in the cortex

Brent Pickrell Block 1 Lecture 58 – Cell Signaling III: Cooney Q: What’s the specific action of the G-protein G(q)? What are the downstream affects? A: it activates phospholipase C-beta which then cleaves PIP2 to DAG and IP3. IP3 diffuses in the cytoplasm and binds to IP3-gated Ca2+ channel on the ER causing an increase in Ca2+ into the cytoplasm. Calcium then binds to Protein Kinase C (PKC) and activates it. Q: What are two mechanisms to deactivate IP3? A: IP3 can be desphosphorylatd to IP2 via p10, or IP3 can be phosphorylated to IP4. Q: What are some ways to decrease intracellular Ca2+ by pumping it out of the cell? A: Na+-Ca2+ exchanger on the cell membrane, or the Ca2+ ATPase on the cell membrane. Q: What are some ways to decrease cytosolic Ca2+ intracellularly? A: Ca2+ pump in the ER membrane, Ca2+ binding molecules in the cytoplasm, active Ca2+ import in mitochondria

Q: What’s chemically similar about receptor kinases? A: all have hydroxyl groups to attach phosphates and they are generally single transmembrane proteins Q: What’s another name for a protein phosphorylase? A: a protein kinase Q: For tyrosine kinase receptors are the kinase domains extra- or intracellular? A: kinase domains are intracellular Q: Discuss the mechanism upon ligand-activation of receptor tyrosine kinases. A: it leads to autophosphorylation of the receptor which sets up a very specific “phospho” code that is further translated during the signaling process

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Q: What is an SH2 domain? A: it’s a phosphotyrosine binding domain Q: What does the SH3 domain preferentially bind? A: it binds proline rich motifs Q: How do we synthesize PIP2? What’s significant about PIP2? A: We first phosphorylate PI and then PI(4)P to get PIP2. The breakdown of PIP2 is critical and generates two intracellular intermediates, IP3 and DAG Q: Discuss the relevant points regarding Insulin/Insulin Receptor signaling. A: Insulin binds its receptor. The activated receptor phosphorylates itself on tyrosines and one of the phosphotyrosines then recruits a docking protein called IRS1 (insulin receptor substrate 1). Then the activated receptor phosphorylates IRS1 on tyrosines and one of these phosphotyrosines recruits the adaptor protein Grb2 via an SH2 domain of Grb2. Next, Grb2 uses one of its two SH3 domains to bind to a proline-rich region of the monomeric GTPase activating protein called SOS (a Ras GEF). Grb2 uses its other SH3 domain to bind a proline rich sequence in a scaffold protein

Q: What’s GEF?

Brent Pickrell Block 1 A: Guanine exchange factor; it regulates monomeric GTPase. GEF displaces GDP and replaces it with GTP. When GTP binds the monomeric GTPase is active. GAP induces monomeric GTP to cleave GTP to GDP

Q: What is Ras? A: it is a monomeric GTPase. Ras is often required when RTKs signal to the nucleus to stimulate cell proliferation or differentiation, both of which require changes in gene expression. If one inhibits Ras, the cell proliferation or differentiation responses normally induced by the activated RTKs do not occur. Conversely, many human tumors have hyperactive mutant forms of Ras, which contribute to the uncontrolled proliferation of cancer cells. Ras functions as a molecular switch, cycling between active (when GTP bound) and inactive (when GDP bound). Ras-GEFs and Ras-GAPs regulate Ras activity. Hyperactive mutant forms of Ras are resistant to GAP-mediated GTPase stimulation and are locked permanently in the GTP-bound active state which is why they promote the development of cancer.

Q: MAP kinase kinase kinase (also called Raf) is activated by what? A: it is activated by Ras! Ras recruits Raf to the plasma membrane and helps activate it. Raf then activates the MAP kinase kinase (also called Mek), when then activates the MAP kinase (also called Erk). Erk then phosphorylates a variety of downstream proteins.

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Q: What are 5 ways to down regulate transmembrane signaling? A: receptor sequestration (taken in by endosomes), receptor down regulation via endosome fusing with lysosome, receptor binding inhibitor, inactivation of signaling protein, production of inhibitory protein

Lecture 59 – Energy Metabolism II: Gilbert Q: Which membrane of the mitochondria is the most permeable? A: The outer membrane is permeable to small molecules and small proteins; the inner membrane is impermeable to small and large molecules Q: Which component of the ETC mediates the transfer of electrons from NADH to the membrane-mobile ubiquinone? A: NADH oxidase (also called NADH dehydrogenase) Q: Where does coenzyme Q (ubiquinone) transfer its electrons to? A: to complex III (cytochrome bc1 reductase); remember that CoQ is a mobile carrier that can accept hydrogen atoms from both NADH dehydrogenase and from FADH2 produced on complex II

Brent Pickrell Block 1 Q: Who does Complex III reduce? A: it reduces the mobile carrier Cytochrome C; cytochrome C is associated with the outer face of the inner membrane Q: Which complexes pump protons into the intermembrane space? A: Complexes I, III, IV Q: Who is the reduced cytochrome C oxidized by? A: Complex IV (cytochrome c oxidase). Q: At what complex is oxygen converted to H2O? A: Complex IV (cytochrome c oxidase). It is the only electron carrier in which the heme iron has an available coordination site that can react directly with O2. At this site, the transported electrons, O2, and free protons are brought together and O2 is reduced to water. Q: Which complex has a covalently bound flavin mononucleotide (FMN) that accepts two electrons? A: Complex I (NADH dehydrogenase); it also contains iron-sulfur clusters that transfer the electrons, one at a time to Coenzyme Q Q: Where can CoQ accept electrons from? A: From both FMH2 produced on NADH dehydrogenase or from FADH2 produced on complex II (succinate dehydrogenase). CoQ transfers electrons to Complex III Q: What’s the mechanism behind the electron transfer to Cytochrome c oxidase? A: The electron from reduced cytochrome c is transferred through a copper center of cytochrome oxidase. There ends up being a collection of 4 electrons from oxidation of cytochrome c bound along with O2. 4 protons are pumped in the process Q: What are reactive oxygen species (ROS)? How do they form? A: Electron transfer in the mitochondria occasionally allows oxygen to escape before it is fully reduced to water. Escape as a superoxide anion radical (-O2) can lead to the formation of hydrogen person or the hydroxyl radical which can induce DNA damage. The superoxide, hydrogen peroxide and hydroxyl radical are known as ROS. Enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase are cellular defenses against ROS. Q: What is the action of superoxide dismutase? What condition is it defective in? A: It’s defective in ALS (Lou Gehrig’s disease). It takes two superoxide anion radicals and converts them to oxygen and hydrogen peroxide. Q: By what mechanism is H2O2 broken down and detoxified? A: Glutathione peroxidase catalyzes the reduction of H2O2 to water. GSH (glutathione) is oxidized to form the disulfide (GSSG). GSH + H2O2 ! GSSG + H20; the GSSG is recycle back to GSH using glutathione reductase and completes the cycle of detoxification of the ROS.

Brent Pickrell Block 1 Q: How is there a generation of a proton gradient? A: At each of the 3 steps where there is a large negative deltaG and thus sufficient energy to drive the pumping of protons into the intermembrane space. The proton movements are driven by conformation changes of the proteins of the complexes. The electrochemical potential of the proton creates a proton motive force that drives protons back through the ATP synthase. The proton motive force, like all electrochemical potentials consists of a concentration gradient (pH gradient) and an electrical gradient (positive potential outside). Approximately 3 protons are used to drive ATP synthesis. Q: How do the inhibitors cyanide (CN), carbon monoxide, and azide (N3) specific inhibit the ETC? A: The block the terminal step, the transfer of electrons to oxygen through binding of cytochrome c oxidase Q: How do uncouplers work? A: They collapse the proton gradient and thus allow protons to re-enter the mitochondrial matrix without energy being captured as ATP—the energy is released as heat and the process is called non-shivering thermogenesis. This prevents the synthesis of ATP but will normally increase respiration and electron transport. Q: What are some examples of uncouplers? A: UCP1 (also called thermogenin; principally expressed in brown fat—generates heat) and dinitrophenol (DNP—membrane permeant in its protonated state; it’s a synthetic uncoupler) Lecture 60 & 61: Cytogenetics: Bacino Lecture 62 – Immune III: Rowely Q: What are some general characteristics and functions of lymph nodes? A: It’s an encapsulated bean shaped filter that filters regional lymph fluid. Lymph nodes have an outer convex surface where afferent lymphatics enter, and they have a concave inner surface where a single efferent lymphatic vessel exits. The subcapsular sinus in the outer cortex is where the afferent lymphatics enter. The interior of lymph nodes is divided into a cortex and medulla. Both the cortex and medulla have extensive reticular cells (fibroblasts) and extensive reticular fibers (collage type III) that function in filtration. Lymphoid (B cell) nodules are found in the cortex and function in B cell proliferation. Q: The _________ is where afferent lymphatic vessels drain into. A: Subcapsular Q: Where are lymphoid nodules found within lymph nodes? A: in the cortex; formed mainly by B lymphocytes Q: Where are the HEVs located within the lymph node? What does HEV stand for?

Brent Pickrell Block 1 A: High endothelial venules; they are located in the cortex/paracortex of the lymph node and have “honing” receptors on the endothelial cells that allow B & T lymphocytes to enter the lymph nodes Q: The lymph node is an important site of lymphocyte proliferation (especially of B cells in the germinal centers) as well as transformation of B lymphocytes into plasma cells. Because of this, what might the lymph leaving the node be enriched in? A: antibodies Q: Where do the majority of plasma cells and macrophages reside within the lymph node? A: There are more plasma cells and macrophages in the medulla than the cortex Q: How are the cells that line HEVs different from normal endothelial cells? A: They are cuboidal shaped and “high”. Usually endothelial cells are flat Q: What cell line are reticular cells in the lymph node derived? Compare to thymus. A: Mesenchymal derived; they are modified versions of fibroblasts and thus make fibers; in the thymus, the reticular cells are epithelial and don’t make fibers Q: What’s the primary function of the spleen? A: It’s a blood filter, also secondary lymphoid tissue Q: What are the general functions of the spleen? A: Defense against microorganisms in circulation, site for detection and destruction of old RBCs. It’s the largest single accumulation of lymphoid tissue in the body and the only one involved in filtration of blood—thus it’s important in defense against blood-borne antigens Q: What does the white pulp in the spleen consist of? A: they consist of lymphoid nodules Q: Describe the STRUCTURE of the spleen. A: The central artery is surrounded by a sheath of cells known as PALS (periarterial lymphatic sheath) that is composed of mostly helper T cells. Surrounding the sheath is a developing B cell nodule that contains marginal sinuses that function to stimulate the B cell nodule. The B cell nodule is considered white pulp. The sheath of the central artery eventually ends and the artery branches off into penicillar arterioles. Some of the penicillar arterioles dead-end to macrophages which are waiting for the antibody covered antigens. Other penicillar arterioles connect directly to the splenic vein and form a closed circulation loop. Q: What are the cords of billroth? A: they are found in red pulp between the sinusoids consisting of reticular cells and reticular fibers and a large population of macrophages. Q: What are 3 examples of MALT (mucosa-associated lymphoid tissue)?

Brent Pickrell Block 1 A: Palantine tonsils, Pharyngeal tonsils, and lingual tonsils. Their purpose is to survey substances brought into the mouth and throat. Q: What structure is being described? Primary lymphoid tissue responsible for the selection of properly programmed T lymphotes A: Thymus Q: If IgM levels are high, but IgG is low what can we conclude? A: There was a recent infection; IgM is the first wave Q: What antibody is present in milk to fetus? A: IgA Q: What structure degenerates with age? A: Thymus Q: Briefly describe the lymph node function. A: Is a secondary lymphoid tissue and surveys lymph for antigen. A single efferent lymphatic vessels leaves with 99% of all antigens removed and the lymph is thus returned to blood circulation cleaned. Q: Briefly describe the spleen. A: Is a secondary lymphoid tissue where blood is surveyed, old RBCs are removed, and the blood is cleaned of antigens Q: What antibody is present in fetal circulation? A: IgG Q: Which antibody is a pentamer that activates complement (C3)? A: IgM Q: What type antibodies are expressed on the B cell surface? A: IgD and IgM (monomer when bound to B cell) Q: What is the first antibody expressed during a response? A: IgM Q: What antibody is the major activator of phagocytosis? A: IgG Q: What antibody do mast cells and basophils have on their surface? A: IgE; they are activated to release histamine and heparin Q: What is the major antibody activator of both macrophages and neutrophils? A: IgG Q: What APC presents directly to B cells without an MHC? A: Follicular dendritic cells

Brent Pickrell Block 1 Q: Explain the term “clock face nucleus”. A: It’s the appearance of a plasma B cell that is actively making antibodies Lecture 63 – Cell Signaling Review: Cooney Q: True or false: 50% of all prescribed drugs target GPCRs A: True Q: What are the three transmembrane receptor paradigms? A: G proteins, ion channels, enzyme coupled receptors Q: Retinoic acid acts as a developmental morphogen to regulate mammalian limb development. This is an example of what type of signaling? (from Cooney’s lecture). A: Paracrine signal; recall that a morphogen is a concentration dependent signal Q: Which of these processes is not associated with RTK function? Activation of GEF, Activation of Ser/Thr kinase, Activation of Tyr kinase, Activation of Adenylyl Cyclase, Activation of Ca2+ release (from Cooney’s Lecture) A: Activation of Adenylyl cyclase Q: What type of receptor kinase is the insulin receptor? A: Tyrosine kinase; Tyr kinases are generally transmembrane proteins Q: How does insulin signal? Through a GPCR, Through a Ser/Thr kinase receptor, Through a receptor tyrosine kinase, Through an ion channel coupled receptor. (From Coney’s Lecture). A: Through a receptor tyrosine kinase Q: What type of signal does NO (nitric oxide) act as when it causes relaxation of smooth muscle? A: NO acts as a paracrine signal

Q: Compare/contrast Gs vs Gq. A: Gs activates adenylyl cyclase resulting in the synthesis of cAMP and subsequent activation of PKA. Gq activates phospholipase-C beta resulting in the cleaving of PIP2 yielding IP3 and DAG. IP3 diffuses in the cytoplasm and opens an IP3-gated Ca2+ channel in the ER. DAG remains bound to the membrane and assists in the activation of PKC

Brent Pickrell Block 1 Q: Some pathways give genomic responses while other do not. What does this mean? A: Some signals result in altered gene expression through DNA binding, for example. This can alter protein synthesis. Other signals result in altered protein function (non genomic). Q: Why doesn’t MAP kinase kinase kinase phosphorylate MAP kinase? A: Each kinase has a catalytic site that only phosphorylates residues that are surrounded by specific amino acids Q: What are the functions of PDE (phosphodiesterase)? A: it degrades the phosphodiester bond (generally in cyclic nucleotides) in the second messenger molecules cAMP and cGMP. Therefore, they terminate the signal. They also set the threshold above which signaling must occur to elicit a response. Recall that Sildenafil (Viagra) is an inhibitor of PDE5 and thus enhances the vasodilatory effects of cGMP. Q: True or false: Both GPCR and RTK can activate phospholipase C. A: True Lecture 64 – Energy Metabolism III: Gilbert Q: True or false: in skeletal muscle, the glucose derived from glycogen is not shared with other organs. A: True. Skeletal muscle is a “selfish” organ. Glycogen from muscle cannot be used as a source of glucose for other organs because muscle lacks glucose-6-phosphatase. Q: Energy generation pathways are activated by low energy signals (AMP-like signals). What are some examples of AMP-like signals? A: AMP, NAD, Ca2+, cAMP, F2,6P Q: Energy generation pathways are inhibited by high energy signals (ATP-like signals). What are some examples of ATP-like signals? A: ATP, citrate, NADH; on the contrary, storage pathways are activated by these ATP-like signals and inhibited by AMP-like signals Q: What metabolic pathways are active when the body has energy (high ATP)? A: Glucose from the blood is taken up and glycogen is synthesized. Q: What metabolic pathways are active when energy is needed? A: Glycogenolysis, glycolysis, oxidative phosphorylation, protein catabolism Q: What type of energy generation is carried out in the absence of oxygen? What type of cell does this? A: Red blood cells; there is anaerobic glycolysis (no TCA or Oxidative Phos.) and glycogenolysis resulting in the end product lactate Q: What enzyme effectively traps glucose into the cell, thus committing it to further metabolism? A: hexokinase; it requires ATP

Brent Pickrell Block 1 Q: What’s the most important control point and the rate-limiting/committed step in glycolysis? What molecule does this enzyme produce? A: the irreversible phosphorylation catalyzed by PFK-1; fructose-1,6-bisphospate Q: What types of signals activate PFK-1? A: AMP-like signals and F2,6P; ATP-like signals inhibit the enzyme (ie citrate) Q: What is the most potent activator of PFK-1? A: Fructose 2,6 Bisphophate Q: Which 3 carbon molecule makes its way through glycolysis, glyceraldehyde 3-phosphate or dihydroxyacetone phosphate? A: glyceraldehyde 3-phosphate Q: What is an example of feed forward activation in glycolysis? A: pyruvate kinase is activated by fructose 1,6-bisphophate (produced from PFK-1). This feed-forward regulation has the effect of linking the two kinase activities. Increased PFK activity results in elevated levels of fructose 1,6-bisphosphate, which activates pyruvate kinase. Q: When oxygen is limiting, such as in vigorous exercise, how does the cell get its ATP? A: through anaerobic glycolysis Q: During anaerobic glycolysis, NAD becomes limited. How does the cell recycle NADH back to NAD? A: Lactate dehydrogenase recycles NADH back to NAD to allow continued flux through glycolysis. Q: What enzyme in glycolysis is responsible for producing NADH? A: glyceraldehyde 3-phosphate dehydrogenase Q: How much ATP is generated from anaerobic glycolysis? A: 2 ATP; each glucose is converted to two molecules of lactate. There is no net production or consumption of NADH Q: How much ATP is generated from aerobic glycolysis? A: about 32 ATP Q: What is the Cori cycle? A: Exercising muscles convert glucose to lactate. This lactate is taken up by the liver and reconverted to glucose (via gluconeogenesis, uses ATP), which is released back into the circulation Q: What is the building block of glycogen? Hint: not just glucose A: UDP-glucose which is synthesized from glucose 1-phosphate and UTP Q: What are the effects of ATP and G6P on glycogen phosphorylase?

Brent Pickrell Block 1 A: they inhibit the enzyme Q: What is the effect of G6P on glycogen synthase? A: it activates the enzyme Q: Activation of cAMP dependent protein kinase (PKA) will have what affect on glycogen? A: During a fasting state, glucagon/epinephrine will bind to membrane receptors and lead to an increase in cAMP. This second messenger will activate cAMP-dependent protein kinase A which then phosphorylates (and ACTIVATES) the enzyme “Glycogen phosphorylase kinase”. Active glycogen phosphorylase results in the degradation of glycogen. Note: at the same time, the enzyme glycogen synthase is INACTIVATED by phosphorylation Q: True or false: F26P acts like an AMP-like signal. A: True Q: What are the actions of PFK-2 and FBPase2? A: Increased concentrations of fructose-6-phosphate stimulates PFK2 activity and generates more F26P which stimulates PFK1 and activates glycolysis. FBPase2 dephosphorylates F26P back to fructose-6-phosphate. Both PFK-2 and FBPase2 function together as a bifunctional enzyme. In the liver, phosphorylates inhibits PFK2 and activates FBPase2. Q: Would ATP and citrate activate or inhibit FBPase? A: They would activate Lecture 65 - Muscle: Kretzer Q: In general, if a cell contracts it is desmin positive. What’s an exception to this rule? A: Vascular smooth muscle is vimentin +; also pericytes and myofibroblasts Q: What glands have myoepithelial cells around them? Which one doesn’t? A: Eccrine and apocrine have myoepithelial cells; sebaceous do not (don’t need them) Q: What is the cytoplasm of muscle called? A: sarcoplasm Q: What is the plasma membrane of muscle called? A: sarcolemma Q: What is a muscle cell referred to as? A: myofiber Q: What are 3 non-muscle cells that contract? A: myoepithelial cells, myofibroblasts, pericytes Q: Which muscle type has only one nucleus? A: Smooth

Brent Pickrell Block 1 Q: List the different muscle types in order of decreasing diameter. A: Skeletal (100 microns), Cardiac (25 microns), Smooth (10 microns) Q: Which muscle types are striated? A: Skeletal and cardiac Q: Which muscle cell type has a halo around its nucleus? Why is this? A: Cardiac; because there’s lipid and glycogen surrounding the nucleus Q: What intermediate filament type do pericytes possess? A: vimentin Q: Which muscle type arises from the fusion of myoblasts? A: skeletal Q: Which muscle type can undergo hyperplasia? A: Smooth (ie pregnant uterus) Q: Which muscle types can be considered multinucleated? A: Skeletal and cardiac—cardiac CAN have 2 nuclei Q: After myocardial infarct, what possibilities exist for regeneration? A: If you can suppress fibrosis, there’s a latent stem cell in the bone marrow that can migrate and regenerate the muscle. Q: What’s the paradox of cardiac muscle hypertrophy? A: It can occur in either a well-trained athlete or someone with pathological hypertension Q: Can skeletal and smooth muscle show hypertrophy? A: yes Q: Which muscle type has a peripheral nucleus? A: skeletal Q: Which muscle types have central nuclei? A: cardiac and smooth Q: What cell allows regeneration of skeletal muscle? A: Satellite cell Q: Which muscle type has more precise striations, skeletal or cardiac? A: Skeletal Q: What would indicate pathology/disease in skeletal muscle? A: One central nucleus Q: What non-muscle cell that contracts is involved in wound repair?

Brent Pickrell Block 1 A: myofibroblasts Q: What cells play a role in the pathology of diabetic retinopathy? A: Pericytes die, causing the endothelial cells to be compromised and leak plasma/blood Q: When looking at cross section of skeletal muscle myofiber, a nucleus could be from what? A: Satellite cell, endothelial cell, fibroblast, or the peripheral nuclei of muscle fiber Q: What cells and junctional complexes are pericytes responsible for maintaining? A: endothelial cells and tight junctions Lecture 66 – Thigh: Blutt Lecture 67 – Pattern of Inheritance I: Potocki Q: The mitochondrial genome is not nuclear. How is this significant? A: It’s derived from the oocyte mitochondria and thus exhibits matrilineal inheritance Q: What are the characteristics of an autosomal dominant disease? A: one mutant allele is sufficient to cause the disease, 50% chance of transmission with each pregnancy, there’s vertical transmission, male to male transmission, affected males = affected females; the graph below shows two woman who were non-penetrant

Q: What type of inheritance pattern is shown in achondroplasia? A: autosomal dominant single gene disorder; it’s a new mutation dominant disorder—90% of infants with achondroplasia are born to parents with normal stature; increased new mutation rate associated with advanced paternal age; has 100% penetrance Q: Who is the lethal cousin of achondroplasia? A: Thanatophoric dysplasia (remember they’re allelic) Q: What type of inheritance pattern is shown in thanatophoric dysplasia? A: new mutation autosomal dominant; ALL infants with thanatophoric dysplasia are born to unaffected parents

Brent Pickrell Block 1 Q: True or false: There’s increased instances of Marfan syndrome and Achondroplasia with advanced paternal age. A: True; continuous series of mitotic cell divisions in spermatogonia result in 1in 10 having deleterious mutation Q: What is fitness? A: the probability of transmitting one’s genes to the next generation as compared to the average probability for the population; fact, fitness for TD = 0 Q: What is “brittle bone disease”? What’s its inheritance pattern? What’s the lethal form? A: Osteogenesis imperfecta – inherited disorder of type I collagen; it’s an autosomal dominant disease; type II is lethal and therefore the reproductive fitness is zero Q: What is Huntington’s disease? A: Autosomal dominant disorder that shows age-dependent penetrance; people who carry the gene do not know they are affected when they are having children (mean age of presentation is 35-45 years – age of onset depends on degree of expansion – people with greatest number of repeats have the earliest onset); penetrance is 100% by age 70; it’s a disorder in which all affected have unstable trinucleotide CAG repeat expansion (polyglutamine disorder); characterized by progressive movement disorder, cognitive decline and changes in personality Q: What’s the penetrance for achondroplasia at birth? A: 100% Q: Compare/contrast the terms penetrance versus expressivity. A: Penetrance is whether or not the trait is manifested; expressivity is the degree to which a trait is manifested (contributing factors to variability in expression include modifier genes, environment, stochastic factors) Q: What’s an example of a disorder with variable expressivity? A: Neurofibramatosis type I; it’s autosomal dominant but have extremely variable expressivity; development of patches of brown pigmentation (café-au-lait macules), benign nodules of the iris, axillary or inguinal freckles Q: What’s an example of a disorder with age-dependent penetrance? A: Huntington Disease Lectures 68 & 69 – Pattern of inheritance II/III: Potocki Q: What are the characteristics of autosomal recessive inheritance? A: two mutant genes are required to cause disease, males and females affected equally, child of affected person is (at least) obligate carrier

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Q: What’s the most common autosomal recessive disorder in Caucasians? A: cystic fibrosis Q: What’s the pathology of phenylketonuria (PKU)? A: there’s a buildup of phenylalanine because there’s a shortage of the enzyme (phenylalanine hydroxylase) that converts it to tyrosine; the disease is caused by mutations in the PAH gene resulting in a primary deficiency of phenylalanine hydroxylase; it’s autosomal recessive Q: What is the coefficient of inbreeding, F? A: The probability that a homozygote has received both alleles at a locus from the same ancestral source Q: True or false: For X-linked inheritance, all daughters of affected males are carriers and all sons of affected males are “normal”. 50% of daughters of carrier females are carries and 50% of sons of carrier females are affected. A: true Q: What is lyonization? A: The term used for the phenomenon of X inactivation. One X (either maternal or paternal) is rendered transcriptionally inactive in somatic cells of females. It begins soon after fertilization (complete by 1st week). It’s randomly determined yet irreversible in somatic cells such that the inactive X in a particular cell remains inactive in all descendants of that cell. The inactive X chromosome is heterochromatic (Barr body) and heavily methylated at gene control regions Q: What lysosomal storage disease results from a deficiency in hexosaminidase A? A: Tay-Sachs; it’s a neurodegenerative disease that presents shortly after birth and causes death by 2-4 years old Q: What are characteristics of X-linked dominant disorders? A: All daughters of affected males are affected, no male to male transmission, affected woman have affected and unaffected daughters

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Q: What makes Y linked disorders easy to identify? A: father passes it on to all his sons (ie Hypertrichosis Pinnae auris, Sertoli cell only syndrome) Q: Describe the clinical manifestation of incontinentia pigmenti. A: Progressive skin rash in linear distribution, conical teeth, alopecia Q: What are characteristics of X-linked recessive inheritance? A: Disorder expressed almost exclusively in males. Females may manifest mild or no phenotype. An example is Hemophilia A – Factor VIII deficiency

Q: What are some characteristics of Duchenne Muscular Dystrophy? How does this compare to BMD (Becker’s muscular dystrophy)? A: Mutation in dystrophin gene on X chromosome resulting in proximal muscle weakness, gower sign, pseudophertrophy of calf, elevated CPK, median age of death is 18 years. Becker’s is less severe – dystrophin protein still preserved. Female carriers often have elevated CPK as well Q: What’s the most common form of inherited mental retardation? How is this similar to Huntington’s? A: Fragile X syndrome. Results in larger ears, relatively large chin, longer faces, larger testes. Results from a triplet repeat expansion of FMR1 (5’ UTR), thus it’s a triplet repeat expansion like Huntington’s. More repeats = more severe. Have fragile sites on the X chromsome Q: X-linked recessive phenotypes can happen in females if:

Brent Pickrell Block 1 A: Skewed lyonization due to post-inactivation selection, variability in mosaicism, turner syndrome (45, X), homozygosity for mutation (ie colorblind), X-autosome translocation Q: Describe the significant features of the mitochondrial genome. Talk about inheritance. A: circular genome, encodes 13 proteins (imports the rest from the cytosol), the majority of mitochondrial disorders are not caused by mutations in mitochondrial DNA—they are caused by mutations in nuclear DNA. Follows matrilineal inheritance and thus mom transmits to all kids, but her son doesn’t transmit to any of his kids

Q: What happens when mitochondria fall prey to disease? Who is most affected? A: When mitochondria fail, less energy (ATP) is generated within the cell and death follows. Cells that are damaged the most are: brain, heart, liver, skeletal muscle, kidney, and endocrine system Q: What is heteroplasmy as it pertains to mitochondrial inheritance? Threshold effect? A: pertains to having different mitochondria within the cell. Threshold effect: symptoms depend upon the percentage of mutant mtDNA. Intrafamilial variability.

Q: What is multifactorial/polygenic inheritance? A: disorders or traits that result from complex interactions between many or few genes; there is not a simple mendelian pattern of inheritance; for example: autism, schizophrenia, diabetes type I/II, etc. Q: The risk of NTD (neural tube defects) is inversely correlated with maternal serum __________ levels during pregnancy. A: folic acid; folic acid 400-800 micrograms/day reduces risk of NTD by 75%

Brent Pickrell Block 1 Lecture 70 – Muscle II: Kretzer Q: Which muscle type has intercalated disks? A: cardiac Q: What is the ONLY junctional complex in skeletal muscle? Is there talin? A: focal contacts; there no talin or vinculin—they are replaced by dystrophin Q: What muscle type has a neuromuscular junction? A: skeletal Q: What junctional complexes are present in cardiac muscle? A: gap junctions, adherent junctions, desmosomes, focal contacts Q: If the dystrophin gene is sequenced, then why is Duchenne’s muscular dystrophy still a problem? A: It’s hard to replace in gene therapy because the gene is so large Q: What muscle types are affected in Duchenne’s muscular dystrophy? A: It affects mainly skeletal where there’s lots of focal contacts and dystrophin replaces vinculin and talin in all of them; it somewhat affects cardiac where there aren’t many focal contacts and dystrophin replaces vinculin and talin in about half of them Q: Discuss the relevant connections of filaments in skeletal focal contacts. Contrast this to what is found in epithelial cells. A: (+) end of Actin ! alpha actinin ! dystrophin ! integrin; in epithelial cells the (+) end of Actin ! alpha actinin ! vinculin ! talin ! integrin Q: Why do individuals with Duchenne’s have large calf muscles? A: due to fibrosis (type I collagen) in the endomysium Q: What kind of connective tissue is found in perimysium and epimysium? A: dense irregular CT Q: What connective tissue surrounds the entire muscle? A: epimysium Q: What connective tissue surrounds each fascicle? A: perimysium Q: What connective tissue surrounds each muscle fiber? A: endomysium Q: What is the pathology behind myasthenia gravis? A: it’s an autoimmune disorder characterized by progressive muscular weakness caused by a reduction in the number of functionally active ACh receptors in the sarcolemma of the NMJ. This reduction is caused by circulating antibodies that bind to the receptors

Brent Pickrell Block 1 Q: What muscle type experiences hyperplasia? A: smooth Q: Describe how exercise causes skeletal muscles to hypertrophy. A: it is caused by the formation of new myofibrils and a pronounced growth in the diameter of individual muscle fibers Q: Skeletal muscle fibers are classified into three types. What determines the differentiation of muscle into red, white, or intermediate? A: It’s determined by the nature of its NMJ—it’s frequency of impulses from its motor innervations. If nerves to red and white fibers are exchanged, the fibers change their morphologic and physiologic characteristics to conform to the innervating nerve Q: What muscle type corresponds to red fibers? A: Type I—lots of myoglobin and are mainly involved in aerobic metabolism; for example, postural muscles of the back; for slow, continuous contractions Q: What is skeletal muscle type IIb adapted for? What’s its color? A: It’s white due to having much fewer mitochondria and myoglobin and having abundant glycogen. Depends largely on glycolysis for energy and are adapted for rapid contractions Q: What muscle type corresponds to intermediate oxidative-glycolytic fibers? A: Type IIa; they utilize both oxidative metabolism and anaerobic metabolism and are intermediate between the other fiber types in both color and in energy metabolism; they have many mitochondria/myoglobin but also considerable amounts of glycogen; used in athletics Q: Why is there some cardiac dysfunction in individuals with Duchenne’s? A: Because cardiac muscle does have SOME focal contacts (not many) where some of the vinculin/talin is replaced with dystrophin Q: What are two ways to regenerate skeletal muscle? A: Can re-innervate with motor neuron (frequency determines what kind of fiber it will become) and there’s myoblasts (satellite cells). Q: Why is there not really any basophilia associated with skeletal/cardiac muscle? A: because the proteins they make are soluble in the cytoplasm (not secreted). Q: Is smooth muscle basophilic? Why or why not? A: Yes, they are very synthetic (almost like fibroblasts) and thus have lots of RER Lecture 72 – Muscle III: Kretzer Q: What is the endocrine function of atrial myofibers? What kind of secretion? A: secrete atrial natriuretic hormone (aka atrial natriuretic factor). This is regulated merocrine secretion (secretory vesicles are visible)

Brent Pickrell Block 1 Q: What is the function of atrial natriuretic hormone? A: acts on kidneys to secretion Na+ and H20; reduces BP; it opposes the actions of aldosterone and ADH Q: What are the specialized myocardial ventricular cells that conduct electrical impulses? A: purkinje fibers Q: Mitochondria comprise what percentage of cytoplasmic volume of cardiac muscle? A: 40%, it reflects the need for continuous aerobic metabolism in the heart Q: What junctional complexes are present in the transverse portions of the intercalated disks? A: Adherent junctions and desmosomes (note that desmosomes are usually seen in epithelial, but not here! Desmosomes have desmin, not cytokeratin!!!) Q: What junctional complex is present along the longitudinal portion of intercalated disks? A: gap junctions for electrical conduction Q: What is the appearance of purkinje fibers? A: they oftentimes look like adipocytes because they have lots of glycogen Q: What type of smooth muscle is connected via gap junctions (ie gut)? A: single unit Q: What type of smooth muscle is found in the eye? A: multi-unit (allows for more precise movements). Lectures 73, 74, 75 – Lower extremity anatomy, others had review material or no testable content Lecture 76 – Muscle IV: Kretzer Q: What junctional complexes are “resurrected” in cardiac muscle? A: desmosomes (they’re usually epithelial in origin) Q: What protein is used in a cardiac muscle biopsy to determine the age of a person? A: Lipofuscin Q: What proteins are Z lines made out of? A: alpha-actinin Q: How do actin filaments attach to Z lines? What end of actin attaches? A: They attach via the cap Z protein; it’s the (+) end of actin that embeds Q: What is the length of the actin thin filament? A: 1 micron

Brent Pickrell Block 1 Q: Where are focal contacts found in skeletal muscle? A: The last sarcomere in a polymer; the (+) end of actin ! cap Z ! alpha-actinin ! dystrophin ! integrin Q: Which muscle type has both nebulin and titin? A: skeletal Q: What is the function of nebulin? A: it extends laterally from the Z line in skeletal muscle and is a very good measuring rod Q: What is the function of titin? Where is titin found? A: it is found in skeletal and cardiac muscle; it extends from Z line and is very stretchy (like a giant rubber band); it’s not as good of a measuring rod as nebulin and is the only form of measurement in cardiac muscle (which is why cardiac isn’t as ordered) Q: Which muscle type has neither titin nor nebulin? A: smooth muscle Q: What is the structure of myosin? A: Has 2 heavy chains that function as rigid coiled coils, which give rise to 2 globular heads which each bind 2 different light chains Q: Which band is composed of thin filaments only? A: the I band Q: What protein is associated with the M line? A: myomesin; it functions to keep myosins apart; the M line bisects H zone Q: What is the H zone? What zone resides within it? A: H zone is the area where there’s no thin filaments; the bare zone is within it Q: What is the size of the bare zone? A: .2 microns!!!! Q: True or false: thick and thin filaments may overlap in the A band. A: True Q: What protein anchors the entire myofibrillar array to the cell membrane of skeletal muscle? A: dystrophin Q: Which protein passes through the thick filaments and acts like a spring? A: titin; as the entire length of the sarcomere changes, so does the elastic portion of the titin molecule Q: What protein helps to center the thick filaments in the sarcomere? A: titin

Brent Pickrell Block 1 Q: What protein functions as a molecular “ruler” and is associated with actin? A: nebulin Q: Why are the bands/striations in cardiac muscle not as “exact” as skeletal? A: cardiac has no nebulin, lots of mitochondria, and a central nucleus Q: What is the length of a myosin molecule? A: 1.6 microns Q: Which portion of the myosin molecule is able to bind ATP? A: S1 Lecture 77 – Adult Genetics: Dhar Q: What are the reasons for referral to an adult genetics clinic? A: Diagnosis/initial assessment in a symptomatic patient, further evaluation/management of a known diagnosis, risk assessment in an asymptomatic patient with + family history Q: What is the utility of genetic testing? A: confirm the diagnosis, more focused care, test at-risk family members, assist in reproductive decision-making Q: What is a PMGP? A: Personalized medical genome profile Lecture 78 – Challenges families face: Potocki Q: What are challenges for the pediatric patient/family? A: diagnostic odyssey, management marathon (once the diagnosis is made there can be a long list of recommendations for the parents to follow), undefined itinerary (sometimes the diagnosis is so RARE/NEW to the medical community that there are no clear guidelines to follow and parents have no idea about potential problems), household havoc, transition threshold Q: What are the ACMG requirements for any genetic testing protocol? A: A knowledgeable professional should be involved, consumer should be fully informed, the scientific evidence on which a test is based should be clearly stated, clinical lab must be accredited, privacy concerns must be addressed. Lecture 79 – Spinal Cord: Glen Lecture 80: Q: How are the Z-lines aligned in adjacent myofibrils? A: Because desmin is between them Q: Name all the contractile proteins in the I band of skeletal muscle. A: actin, tropomyosin, troponin T/C/I, nebulin, titin

Brent Pickrell Block 1 Q: Name all the contractile proteins in the I band of cardiac muscle. A: actin, tropomyosin, troponin T/C/I, titin; remember that there’s NO NEBULIN Q: Discuss excitation-contraction coupling in skeletal muscle. A: AP travels down T tubules (continuous with sarcolemma), dihydropyridine receptors on T-tubules (are voltage sensitive) undergo a conformational change and physically pries open ryanodine receptor on SR, the SR releases Ca2+ into cytoplasm where Ca2+ binds troponin C and causes troponin I to move tropomyosin out of the way, there’s cross-bridge cycling, then there’s Ca2+ uptake back into SR by SERCA Q: What is the function of SERCA? Include something about calsequestrin. A: it’s a Ca2+ ATPase in the sarcoplasmic reticulum that pumps Ca2+ from the ICF of the muscle fiber into the interior of the SR. Within the SR, Ca2+ is bound to calsequestrin which keeps Ca2+ concentration low and thus reduces the work of the Ca2+ ATPase pump. Q: True of false: Extracellular Ca2+ is utilized in skeletal muscle contraction. A: False Q: What’s unique about the cardiac AP? A: during the plateau a different isoform of dihydropyridine (L-type channels) allows Ca2+ influx into the cell (this is called trigger Ca2+ and is extracellular); this Ca2+ induces Ca2+ release from the SR through the ryanodine receptor – called Ca2+ induced Ca2+ release (CICR) Q: What factors determine how much Ca2+ is released from the SR during CICR? A: the amount of Ca2+ previously stored and the size of the inward Ca2+ current Q: What are some characteristics of smooth muscle? A: mitotic, fusiform, no striations, no halo, no NMJ, no fibrils, lots of ECM because they’re interconvertible with fibroblasts Q: What contractile proteins are missing in smooth muscle? A: cap Z, tropomyosin, troponin T/C/I, nebulin, titin Q: Since smooth muscle doesn’t have troponin, what Ca2+ binding protein regulates the interaction between actin and myosin? A: calmodulin; Ca2+-calmodulin regulates MLCK which regulates cross-bridges; also, the Ca2+-calmodulin complex leads to the phosphorylation of caldesmon and calponin which normally bind to and inhibit myosin Q: In smooth muscle, what mechanisms contribute to the increase in intracellular Ca2+ levels during EC coupling? Note: THERE’S MULTIPLE SOURCES A: there’s VG Ca2+ channels in the sarcolemma, hormones/NTs can bind to a GPCR that activates phospholipase C where the product IP3 binds to an IP3-gated channel on the SR, there’s also ligand gated channels (also a GPCR), there’s lastly a stretch gated Ca2+ channel in the sarcolemma

Brent Pickrell Block 1 Q: What is the function of MLCK? A: it is activated by Ca2+-calmodulin; it phosphorylates the myosin light chain which allows myosin to bind actin Q: How is myosin arranged differently in smooth muscle? A: It’s not bipolar, instead it’s “side polar” Lecture 81 – Radiology of Lower Extremity: Willis; nothing apparently testable Lecture 82 – Molecular Embryology: Refer to slides Lecture 83 – Genetics Lecture: Potocki Q: What is HHT? A: Hereditary Hemorrhagic Telangiectasia (HHT) is a multisystem vascular dysplasia characterized by the presence of multiple arteriovenous malformations (AVMs) that lack intervening capillaries and result in direct connections between arteries and veins. Small AVMs, called telangiectases, close to the surface of skin and mucous membranes often rupture and bleed. The most common clinical manifestations are spontaneous and recurrent epistaxis and multiple telangiectases, which commonly appear on the lips, face, tongue or hands in adulthood. A minority of individuals with HHT have symptomatic GI bleeding, which most commonly begins after age 50 years. Large AVMs often cause symptoms when they occur in the brain or lung; complications from bleeding or shunting may be sudden and catastrophic. Telangiectases in the nose, along with the nosebleeds they cause, are the most common symptom of HHT. About 95% of people with HHT have recurring nosebleeds by the time they reach middle age. Lecture 84: Nervous system Histo: Refer to slides Lecture 85/86: Genetics small group discussions Lecture 87 – Grand Synthesis: Goodman Q: True or false: The RATIO of an ion inside and outside establishes the membrane potential. A: True (think of the Nernst equation); remember that potassium permeability establishes the resting potential Q: What is the duration of the neuronal AP? A: 1-2 msec

Brent Pickrell Block 1

Q: Describe the characteristics of the cardiac AP. A: The initial depolarization is dependent on Na+ channels; Na+ channels start closing and K+ channels open to start repolarization. Sustained depolarization depends on long-lasting Ca2+ channels (L-Channels). Repolarization occurs when L-channels begin to close and more K+ channels open.

Q: What’s the duration of a cardiac AP? A: about 200msec Q: What’s the result of extracellular Ca2+ coming into the cell? A: it causes Ca2+ induced Ca2+ release from the sarcoplasmic reticulum. About ¼ of Ca2+ comes in from the outside and then induces the release of ¾ of Ca2+ to be released from SR—this augments myocardial contractility

Brent Pickrell Block 1 Q: Are ligand gated ion channels ionotropic or metabotropic? A: They flux ions and are thus ionotropic. Q: Define ionotropic receptors. A: They are ligand-gated ion channels that can be either excitatory or inhibitory. They are fast, produce a GRADED response (local depolarization or hyperpolarization that’s integrated in the soma), and have the structural motif of TM4 (4 transmembrane spanning regions). Q: Give two examples of excitatory neurotransmitters. A: ACh, glutamate Q: Give two examples of inhibitory neurotransmitters. A: GABA, glycine Q: What is a local depolarization by a ligand binding called? A: EPSP (excitatory post synaptic potential) Q: What are general characteristics of metabotropic receptors? A: They are slower, more complex (can involve 2nd messengers, transcriptional regulation), and have a common structural motif of TM7 (7 transmembrane spanning regions). Most commonly they are G-protein receptors Q: All neurotransmitters must be inactivated. What is the major means of inactivation and what is a major exception to this “rule”? A: The major means of inactivation is re-uptake by the pre-synaptic neuron. The exception is acetylcholine which is inactivated by enzymatic degradation (cholinesterase)

Q: Which organs in the body are energy hogs? A: Brain, muscle, liver, kidney. These utilize aerobic metabolism almost exclusively and are thus MOST LIKELY to be affected by a mitochondrial disorder/pathology. Q: Erythrocytes utilize what form of bioenergetics? A: Anaerobic metabolism. They are just bags of hemoglobin, only glycolysis