C2006/F2402 ’09                      Exam #4  -- Questions & Answers

Each questions and each answer are worth 2 pts unless it says otherwise.

For question 1, circle all right answers for each question and explain part ii in a sentence or two.

1. There are 2 receptors for the hormones cortisol and aldosterone. They are called GR and MR.

    GR = glucocorticoid receptor

    MR = mineralocorticoid receptor

    A.    i. These 2 hormones -- aldosterone & cortisol -- are both (peptides) (steroids) (catecholamines) (amino acid derivatives) (related to acetyl choline) (none of these – the two hormones are not of the same chemical class) (beats me).  No explanation required for this one.

            ii. The 2 receptors -- MR & GR – are both (adrenergic) (cholinergic) (coupled to G proteins) (protein kinases or coupled to protein kinases) (activators of adenyl cyclase) (activators of PLC) (proteins that bind to DNA) (proteins that bind to mRNA) (none of these – they don’t act the same way).  

 These hormones are both lipid soluble, so they use intracellular receptors that are transcription factors.

    B.     i. When the GR is activated, which of the following is the most likely result?

(phosphorylation of the GR cytoplasmic domain) (movement of GR from mitochondria to cytoplasm) (movement of GR from cytoplasm to nucleus)* (movement of GR from nucleus to cytoplasm) (generation of a 2^nd messenger)* (recruitment of proteins to the intracellular side of the cell surface).

            ii. When the GR is activated, which of the following should occur? (mobilization of aquaporins) (inhibition of gluconeogenesis in liver) (inhibition of immune responses) (decrease of Na⁺ reabsorption in kidney) (increase in Na⁺ reabsorption in kidney)  (mobilization of GLUT4) (can’t predict).  Gluconeogenesis = conversion of lactate, amino acids etc. to glucose.

*If you thought the 2 hormones used GPCRs, you received 1 pt for 'generation of a 2nd messenger.'

Explanations: i.  'Movement of GR from cytoplasm to nucleus' is the only answer that makes any sense for a steroid receptor, and it is what actually happens to the GR. (Some steroid receptors reside in the nucleus, but the GR is in the cytoplasm until it binds ligand.) Binding of ligand to a steroid receptor causes the receptor to change conformation. If the receptor is in the cytoplasm, the conformational change unmasks its NLS and the receptor moves to the nucleus. Once inside the nucleus, the receptor can bind to DNA and affects transcription.

    ii. GR is the usual receptor for cortisol. (This follows from the name, and knowing that cortisol is a glucocorticoid. You can also figure it out from information in other questions, although that was not intended.) When the GR is activated, it triggers a series of events that are normally controlled by cortisol. Even if the receptor is activated by a different hormone, it will trigger the same events. It's which receptor you activate, not which hormone you use, that determines the result. (See note below.)  'Inhibition of immune responses' is the only event listed that normally happens in response to cortisol, so it is the only event that should occur if the GR is activated. Cortisol affects gluconeogenesis, but the hormone activates it -- it doesn't inhibit it. (Stimulation of gluconeogenesis provides glucose that the liver can release into the blood.) All the other events involve the actions of hormones that use other receptors. (Mobilization of aquaporins is caused by ADH; mobilization of GLUT4 is caused by insulin. Changes in Na+ reabsorption are caused by aldosterone.)

Note: It doesn't matter which hormone activates a sensor or receptor -- the result is always the same. The activated receptor triggers the same sequence of events, regardless of what ligand is bound to it. (This is assuming one binding site. If there are separate binding sites for different ligands, then binding different ligands may produce different changes in conformation and different results.)

    C. i.  ACTH & angiotensin are both (endocrines) (paracrines) (exocrines) (neuroendocrines)  (neurotransmitters) (tropic hormones)* (juxtacrines) (none of these – none of these terms apply to both).

        ii. Both hormones -- ACTH & angiotensin –

(cause synthesis of a another hormone) (act on the same target gland) (cause synthesis of the same hormone(s))* (act on two different glands that are part of the same organ) (cause release of preformed hormones) (have receptors with an extracellular domain) (none of these – none of these terms apply to both).

1 pt for each correct answer to C. Any two of the following supporting statements were accepted as a sufficient explanation (1 pt each):
    a. Both hormones act on the adrenal cortex.

    b. ACTH stimulates synthesis of cortisol, and weakly stimulates synthesis of aldosterone. (The main factor controlling aldosterone synthesis is angiotensin II, not ACTH, so it was okay to omit aldosterone here.)

    c. Angiotensin II stimulates production of aldosterone.

*Pts were neither given nor deducted for circling 'tropic hormones' or 'cause synthesis of the same hormone' for the following reasons:

Both hormones are tropic hormones according to some definitions, but not according to others.  According to one definition, a tropic hormone is one that stimulates synthesis of another hormone. According to a second definition, a tropic hormone is one made by the AP that stimulates another gland to make a hormone. Angiotensin II fits the first definition, but not the second. ACTH fits both.

ACTH and Angiotensin II both stimulate synthesis of aldosterone, but only ACTH stimulates synthesis of cortisol. Therefore both hormones do not stimulate synthesis of exactly the same hormones. Since the 'exactly' was omitted, the question was considered ambiguous and was discounted.

2. Cells of the pituitary have receptors for cortisol. When cortisol binds to its pituitary receptors, the release of preformed ACTH and synthesis of new ACTH are both affected relatively rapidly.

    A. The most reasonable explanation of the change in ACTH synthesis is a direct affect on (replication) (DNA rearrangements) (transcription) (RNA processing) (translation) (membrane fusion) (transport from ER to Golgi).

    B. The most reasonable explanation for the relatively rapid affect on release of preformed ACTH is an immediate change in _________(membrane fusion) ________. Same choices as in A. Explain both parts.

Explanations: A. The receptor for cortisol is a TF, so it would be expected to affect transcription. This is negative feedback, so when cortisol is high, the change in transcription should decrease the production and/or release of ACTH. In other words, when cortisol binds to its receptor, the complex should inhibit transcription of the gene for ACTH, not activate it. If the mRNA for ACTH is short lived, the inhibition of transcription would lead quickly to an inhibition of translation, and a decrease in the synthesis of ACTH. However, the direct effect of cortisol (bound to receptor) would be on transcription, not translation. 

    B. ACTH is a peptide hormone. Preformed hormone is present in secretory vesicles that release their contents by exocytosis in response to the appropriate signal. When cortisol is high, fusion of vesicles with the plasma membrane should be blocked, exocytosis should decrease, and release of ACTH should decrease.

    Given that the cortisol receptor is a TF, a rapid effect on ACTH synthesis is expected, but a rapid effect on ACTH release is not. If synthesis of a new protein were required to block ACTH release, it would take a while to make that protein and the effect would not be rapid. It is not known how this rapid effect is achieved.

For grading, the explanation to A was ignored, and explanation to B was worth 2 pts. Credit was not given for alternative answers to part A, even if the answers matched the answers to problem 1. This was done because misunderstanding the nature of steroid receptors was considered to be a major error.

3. More questions on the pituitary receptors for cortisol. For this question, circle all correct answers and explain your choices very briefly.

    A.    i. The pituitary receptors are made in cells whose cells bodies are in the (adrenal medulla) (hypothalamus) (adrenal cortex) (posterior pituitary) (anterior pituitary) 

            ii. Which of the following hormones are made in cells with cell bodies in the hypothalamus? (CRH) (TSH) (ACTH) (ADH) (aldosterone) (cortisol) (none of these).

Explanations:  i. ACTH is made by the ant. pit, not the post. pit. Receptors for negative feedback by cortisol would therefore be in the cells of the ant. pit, not in the post. pit. The cells of the ant. pit. are entirely contained in that gland, so the receptors in the ant. pit. would be made entirely within the gland.

    ii. Short explanation: CRH is made in the HT and released from the HT. ADH is made in the HT but released in the Post. Pit.

    Long explanation: The cells of the HT release hormones into the portal circulation between the HT and the ant. pit., and these releasing factors (such as CRH) bind to receptors on the surface of the ant. pit. and trigger synthesis and release of the appropriate hormones (such as ACTH). These releasing factors are made and released in the HT. ADH and oxytocin are made in the HT, but released into the general circulation from the post. pit. The cells that make these hormones have cell bodies in the HT but nerve endings in the post pit. The hormones are made in the cell bodies, and then travel down axons from the HT to the post. pit. before they are released into the circulation.

    B.    i. When cortisol binds to its pituitary receptors, the overall synthesis of cortisol in the body should consequently (stay the same) (stay the same at zero) (increase) (decrease) (can’t predict).

            ii. The pituitary cells with cortisol receptors should also have receptors for (ACTH) (CRH) (aldosterone) (ADH)  (none of these).

            iii. A normal person should have approximately constant blood level(s) of (cortisol) (aldosterone) (TH) (ADH) (insulin) (none of these).

 Grading: For parts i & ii, 2 pts each correct answer; for part iii, 1 pt each correct answer. (1 pt deducted for each wrong answer.) Explanation was ignored (no pts), since the only part that was complicated was iii, and most students missed it (see below). 
 

Explanations: i. There is a negative feedback loop controlling the level of cortisol .(The circuit is similar to the one controlling the level of TH; see class handout.) When the cortisol level is too high, the hormone feeds back to the ant. pit. (& HT) and reduces the release and/or production of ACTH. This reduces the stimulation of the adrenal cortex and the production of cortisol. There is no similar feedback loop for controlling the level of aldosterone (or ADH).

    ii. The production of ACTH in the ant. pit. is primarily controlled by the CRH from the HT.  Therefore the ant. pit. must have receptors for CRH. The ant. pit. makes ACTH, but the hormone does not feedback inhibit the gland that makes it. So the ant. pit. does not have receptors for ACTH. The levels of aldosterone & ADH are not regulated by negative feedback at the ant. pit.

    iii. Aldosterone, ADH, and insulin levels are not regulated. These hormone levels are adjusted as needed to maintain homeostatic levels of other regulated variables such as blood sugar levels, blood pressure, etc. However the levels of TH and cortisol are regulated. These two hormones are needed all the time at relatively constant levels to maintain metabolism, and are kept at approximately constant levels by negative feedback to the AP and HT. (See class handout for TH example.)  Under conditions of stress, additional cortisol is produced, and higher, homeostatic levels are maintained during chronic stress. (The set point for cortisol levels is raised during chronic stress.) Since so many students missed this, it was clearly not stressed enough in class, and the points for part iii were kept to a minimum.

4. The two receptors for cortisol and aldosterone have a common evolutionary origin.

            GR = glucocorticoid receptor = binds cortisol much more tightly than aldosterone.

            MR = mineralocorticoid receptor = binds both hormones equally well.

For each part of this question, pick the best answer and explain briefly.

    A.     i. If you have very high levels of cortisol, you can develop high blood pressure. This is probably caused by cortisol binding to (MR) (GR)  (either one) (neither – must happen some other way).

            ii. The raised blood pressure should be caused by direct changes in the body’s handling of (glucose) (water) (Na+) (glucose or water) (glucose or Na+) (Na+ or water) (any of these) (none of these).

Explanations: Cortisol can bind to the MR and act as an agonist of aldosterone. It doesn't matter which hormone binds to the MR -- if either one binds, the result will be a mineralocorticoid or aldosterone effect. (It's which receptor is activated, and not which hormone does it, that matters. See explanation to 1. B. ii.) If there is excess cortisol, it will bind to the MR and trigger reabsorption of Na+ in the distal tubule, just as aldosterone normally does. Your body won't 'know' you have too much cortisol -- it will respond as if there were a high level of aldosterone. The activated MR triggers synthesis of the components needed to reabsorb Na+. If more Na+ is retained in the blood and body fluids (ECF), then more water will be retained osmotically. The transport of water is not affected directly, but water follows the Na+. A greater Na+ gradient across a membrane will drive more water in the direction of the higher Na+.

    B. In normal blood, the concentration of cortisol is much higher than the concentration of aldosterone. Cells that are targets of aldosterone contain enzyme L that converts cortisol to cortisone. Cortisone does not bind to either receptor. 

            i. Enzyme L is required to prevent (cortisol from binding to the GR) (cortisol from binding to the MR) (aldosterone from binding to the GR) (aldosterone from binding to the MR)

(either hormone from binding to the GR) (none of these – the enzyme must do something else).

            ii. Licorice contains an inhibitor of enzyme L. (I am not making this up. However it takes a lot of licorice to have an effect.) Which of the following is most likely to give you high blood pressure?

(eating licorice) (going on spring break) (studying for finals) (eating licorice while studying for finals)  

(eating licorice on spring break) (either eating licorice or studying for finals) (beats me).

 Explanations: i. Enzyme L converts cortisol to a compound that does not bind the MR. If enzyme L is working, activation of the MR will reflect the level of aldosterone even in the presence of a large excess of cortisol. If enzyme L is not working, the MR will be activated if cortisol is high, regardless of the level of aldosterone. Therefore, if enzyme L is inactive, anything that triggers high levels of cortisol will cause Na+ retention and high BP.

    ii. If cortisol is low, the need for enzyme L is relatively low, so effects of licorice will not be very significant. If stress and cortisol are low, it doesn't matter much if enzyme L is working or not. However, stress will raise the level of cortisol, and increase the need for enzyme L to prevent the cortisol from triggering the MR. If enzyme L is inhibited by licorice, during a stressful period, such as during exams, the high cortisol will cause high BP. Stress alone can cause high BP (although it may primarily be through vasoconstriction), but stress plus licorice will be worse. In other words, the licorice effect is dependent on high levels of cortisol.

5. Be sure to read info below for background information on control of acid base balance.

    A.      i. If you retain too much CO₂ in the body, you increase your breathing rate and increase the rate of CO₂ exhalation. A physiologist would describe this as an example of (negative feedback) (positive feedback) (either type of feedback) (anticipation) feed forward) (adjustment of the set point).

            ii. When the respiratory pacemaker neurons fire, you expect the major muscles of breathing to (relax) (contract) (respond either way – some will relax & some contract) (can’t predict). Base your answer on the background information given.

            iii. Suppose the H+ level is low in the arteries. In the pacemaker cells of the brain, which of the following is most likely to increase? (threshold) (slope of pacemaker potential) (time it takes pacemaker potential to reach threshold)  (effect once threshold is reached) (none of these). For i & ii, no explanation required. Explain iii.

Explanations: i. Negative feedback (in physiology) means that the system makes corrections if the regulated variable deviates too much from the set point (the desired value). Neg. fb. means the system acts to negate the difference between the actual value (level of CO₂ and pH) and the set point (desired level of CO₂ and pH) so as to restore the optimal or desired value. It does NOT mean (in physio) that some process must be inhibited by an end product. In this case, you need to increase breathing rate, increase the rate of CO₂ exhalation, and increase the pH. (To increase the pH you need to decrease the level of CO₂ and acid.) This is negative feedback, because you are decreasing the deviation from the set point, and restoring the desired pH. It is not positive feedback, even though you are making controlled processes (breathing, exhalation of CO₂) go faster.

        ii. When neurons fire and stimulate skeletal muscles, the muscles always contract. Relaxation is the result of no stimulation. The bronchioles of the lung are surrounded by smooth muscle, but the muscles that move the diaphragm and control breathing are skeletal.  When the pacemaker cells fire, the motor neurons should fire and stimulate the major muscles.

        iii. If the H+ level is low, that means the pH is too high and there is too little CO₂ in the body. Therefore you need to breathe more slowly to conserve CO₂ (or lose less) and increase the H+ level (decreasing the pH). You want to decrease the rate of firing by the pacemaker cells. Threshold and effect once it is reached are relatively invariant, and what varies is the slope of the pacemaker potential. You want it to be flatter, so the time it takes to reach threshold will be increased, and the time between APs will be longer.

     B. Most of the chemoreceptors involved in this system actually monitor the CO₂ in the blood, not the actual pH.

            i. If the CO₂ in the blood is too low, the rate of H+ secretion in the kidney should (increase) (decrease) (stay the same) (stay the same at zero) (can’t predict).

            ii. If a person suffers kidney failure, she is most likely to have (acidosis) (alkalosis) (either way), AND

            iii. If the kidney failure is not treated, her urine is likely to be (more acidic than usual) (more basic than usual) (can’t predict).  Explain all 3 parts.

 Explanations: i. Low CO₂ means low H+, and so more H+ should be retained in the body, and less lost in the urine. Secretion refers to the transport of substances from the body to the tubule lumen, not the other way around. (Reabsorption is transport from the tubule lumen to the body.) To retain H+ in the body, H+ secretion should be decreased. (The kidney cannot reabsorb protons; see background info.)

            ii. & iii. The kidney normally secretes lots of H+. If it falls down on the job, that H+ will be retained in the body, and acidosis will result. The lack of the usual H+ in the urine will make the urine more basic.  (If the kidney fails, the volume of urine will decrease, but what there is will be basic.)

    C. The kidney gets input from the sympathetic but not the PS branch of the ANS. (It also gets additional local input about blood pH but we are ignoring that to keep the problem manageable.) Consider all the information provided so far about the regulation of the pH of arterial blood. For each question below, circle all correct answers, and then draw the circuit as explanation.

            i. What is/are the effector(s) in this system?

(kidney) (brain) (lungs) (arteries) (pacemaker cells) (motor neurons) (none of these).

            ii. What is/are the regulated variable(s) here? (rate of breathing) (rate of H+ secretion)  

(rate that pacemaker cells fire) ([H+] in blood) ([H+] in urine) (none of these).

            Draw the homeostatic circuit below. Show what happens to maintain homeostasis during acidosis -- when the pH of the arterial blood falls. (Do not include what happens during alkalosis. Stick to acidosis.) There is more than one way to draw the circuit; any clear, correct version is okay. You might want to try it out on scratch paper first.

Explanations:  i. Effectors are organs or tissues that act to increase or decrease the level of the regulated variable. In this case, the brain is the IC that directs the kidneys and lungs to take appropriate action to raise or lower the pH. So the kidneys and lungs are the actual effectors. The pacemaker cells and the motor neurons are part of the signaling system; the arteries contain some of the sensors.

            ii. The H+ level in the blood is regulated homeostatically -- it is raised if it gets too low and lowered if it gets too high. The processes listed can be fast or slow -- whatever it takes to achieve a constant level of H+ in the blood. They are called 'controlled processes.' The level at which they operate is not regulated -- the level is adjusted to maintain a constant blood pH.

            iii. (4 pts for proper circuit): Circuit should be a single loop, not a double one. It should include sensors (multiple) detect level of CO2 (or pH or H+ level). If pH is too low, sensors send signal to the brain (the IC); the brain signals the effectors to release protons (from kidney) and breathe out CO2 (in lung), thus correcting the pH upwards.

    To put in more details (not required), IC signals pacemaker cells to fire more often (increasing slope of pacemaker potential); pacemaker cells synapse on motor neurons (or on interneurons which synapse on motor neurons); motor neurons fire more often, major inspiratory muscles contract more often, and rate of breathing increases.   

6. See below for description of (fictitious) bacteria mentioned here.  

    A. Cytotoxic T cells kill macrophages infected with viruses or B. botheris. Killing requires a juxtacrine interaction. What components are needed on the cells in order for the juxtacrine interaction to occur?

Circle all correct answers for both parts of A. No explanation required for A.

            i. On the surface of the cytotoxic T cells you need (MHC I) (MHC II) (antibody or BCR) (TCR) (CD4) (CD8) (bacterial or viral epitopes).

            ii. On the surface of the infected macrophages you need (MHC I) (MHC II) (antibody or BCR) (TCR) (CD4) (CD8) (bacterial or viral epitopes).

    B. Do you expect cytotoxic T cells to kill macrophages containing C. catchis? (no) (yes) (can’t predict from info given).

    C. The infected macrophages infected with A. annoyis are probably not killed because they are missing:
(CD4) (CD8) (MHC I) (MHC II) (the correct MHC + A. annoyis epitopes) (both types of MHC) (TCR + A. annoyis epitopes) (antibody or BCR + A. annoyis epitopes) (none of these – something else must be responsible). Explain your answers to B & C.

Part A, 1 pt. each correct answer. 2 pts each for B and C. (Explanation = 2 pts.)

Short Explanation (all that was required): For killing or binding by cytotoxic T cells the target cells must have epitope bound to MHC I. For epitopes to get onto MHC I, the protein from which they are derived must be in the cytosol.

Long Explanation: A. For a cytotoxic T to bind to an infected cell, the T cell must have CD8 and TCR on its surface. The infected cell must have viral or bacterial epitopes on its surface -- the epitopes (pieces of protein from the infecting organism) must be attached to MHC I on the cell surface. The variable part of the TCR must match up with a viral or bacterial epitope displayed on the surface of the infected cell. In order for the cytotoxic T cell to stick to and kill the infected cell, the TCR must match the epitope, and the epitope must be bound to MHC I. The TCR/CD8 combo will only stick to MHC I that has bound an epitope. (It won't stick to empty MHC I or MHC II with epitope.)

    T cells have MHC I, and macrophages have MHC II, but these are not involved in the juxtacrine interaction that leads to killing. 

        B & C. Infected cells must display epitopes on MHC I for the infected cells to bind to cytotoxic T's and be killed. C. catchis epitopes should be displayed on the cell surface with MHC II, not MHC I.  (Cells that have engulfed C. catchis should act like regular APC's and present C. catchis epitopes to helper T cells, not cytotoxic Ts. See explanation of 7B below.) Proteins must be in the cytosol to be degraded by proteasomes, and put onto MHC I (after transport into the ER). The proteins of C. catchis  are trapped inside phagosomes, so they won't reach proteasomes or the ER. If C. catchis epitopes are not displayed with MHC I, the infected cells will not stick to cytotoxic T cells and will not be killed.

        C. A. annoyis replicates inside phagosomes. Proteins have to be in the cytosol to end up on MHC I, as explained above. So A. annoyis epitopes will not be displayed with MHC I, and the cells will not be killed. The A. annoyis cells should have MHC I, like all nucleated cells, but the epitopes derived from the bacteria will not be bound to the MHC I. The cells will lack the combination of MHC I and bacterial epitopes.

     D. See information for problem 6, part D, for description of the DNA samples used here. For each part, circle all correct answers, and explain part ii briefly. Assume no somatic mutation.

            i. You compare your blots from the DNA samples A & B. The blots should look different with the probe(s) for (V_H) (C_H) (MHC I) (MHC II) (none of these) (all of these).

            ii. You compare the DNA sequences from samples S & M. Your sequences from the two DNA samples should look the same in the region that codes for (V_H) (C_H)* (MHC I) (MHC II) (none of these) (all of these).

Explanations: i. The only cells that rearrange their DNA are T cells and B cells, which make TCRs and antibodies respectively. Macrophages do not make antibodies or TCRs, and do not rearrange their DNA. All macrophages have the same chromosomal DNA, so the blots from samples A & B will be the same -- none of the DNA regions should be different or give differences on blots no matter what probe you use.

            ii. MHC part: Genes for MHC I & II are the same in all cells. These genes are never rearranged. Only genes for TCRs and antibodies are rearranged. So the sequences for MHC will be the same in both samples.

    C_H part: The two antibodies are of the same class, meaning they use the same DNA sequence to code for the constant region of the heavy chain.* Therefore the C_H region of the DNA should be the same for both samples. Whether the antibody is secreted or membrane bound depends on alternate processing of the RNA transcript, not on changes in the DNA. (Depending on where polyA is added to terminate the transcript, the last exons code for a hydrophobic or hydrophilic COOH end to the protein. This determines whether the COOH end of the protein will be locked into the ER membrane or translocated entirely into the lumen of the ER. This in turn determines whether the protein will end up in the membrane of a vesicle or in its lumen, and whether the protein will be added to the plasma membrane, or secreted outside the cell.) So the

    V_H part: The two antibodies bind to the same protein, but they don't have to bind to the same epitope (section) of the protein. Even if they do, they may have different amino acid sequences. Why? Each B cell rearranges the V-D-J section of the DNA coding for the variable part of its heavy chain independently. Therefore, the chance that the two clones of B cells have the same DNA coding for the V_H region of the protein is remote.

    *There are some possible complications here -- for example, everyone has two copies of the DNA that are not necessarily exactly the same. Therefore it is possible that the two C_H regions are not the same, if for example, one is derived from the maternal DNA and one from the paternal DNA. If you took these complications into account and explained them properly, that was fine. However it is NOT correct to say the two C_H regions must be different because one codes for secreted antibody and one codes for membrane bound antibody. (See explanation above.)

Note: This question is not about the differences in proteins made in each cell type but about the differences in DNA in each cell type. Changes in gene expression (protein made) do not correspond to changes in the DNA of the genes themselves except in very restricted and special circumstances. Rearrangements occur only in B and T cells, and only in the parts of the DNA encoding TCRs and antibodies.

7.     A. Why are catalase⁺ bacteria a problem in patients with CGD? The catalase⁺ bacteria probably

(do not generate peroxide) (do not degrade peroxide) (destroy the peroxide made by the macrophages)* (destroy their own peroxide) (beats me). Explain why people with CGD can destroy catalase negative bacteria, but not catalase positive  bacteria.

 Explanation: Cat+ bacteria must destroy the peroxide that is normally used to kill them. The macrophages in CGD do not have the enzyme complex needed to make peroxide, yet they destroy Cat- bacteria but not Cat+. So where does the peroxide come from? The simplest solution is that all bacteria make their own peroxide, but Cat+ bacteria destroy the peroxide before it can be used to kill them. (Just as peroxisomes in eukaryotic cells use catalase to destroy peroxide before it can damage the cell.) Macrophages engulf the bacteria, and all bacteria generate peroxide inside the phagosome. However the Cat+ bacteria destroy the peroxide while the Cat- can't. If peroxide remains, which occurs in Cat- bacteria only, the macrophages can use the bacteria's own peroxide against them

     *An alternative explanation: the macrophages in CGD make some peroxide, much less than normal, but enough to kill Cat- bacteria. The Cat+ bacteria survive, because they are capable of destroying the low amount of peroxide made by CGD macrophages. Normal macrophages make larger amounts of peroxide, too much to be destroyed by the catalase of Cat+ bacteria. Therefore normal macrophages can kill both Cat+ and Cat- bacteria. (Full credit for this explanation, but not for the corresponding answer.)

         B. Granulomas form because helper T cells (not cytoxic T cells) bind to the infected macrophages.  Why do granulomas form in patients infected with A. annoyis, but not B. botheris? The simplest explanation is that epitopes of A. annoyis protein (match the TCR of T_H but not T_C) (end up on MHC I) (end up on MHC II) (match the Ab or BCR on the surface of T_H) (don’t fit any of these – there must be some other explanation).

Explanation: For granulomas to form, the infected macrophages must evade destruction by cytotoxic T cells, and the infected macrophages must then stick to helper T cells. Cells infected by B. botheris are killed, since the bacteria replicate in the cytosol. (See explanation to problem 6.) Cells infected by A. annoyis are not killed by cytotoxic Ts because the A. annoyis proteins do not reach the cytoplasm and are not displayed on MHC I.  Therefore the macrophages infected by A. annoyis survive to form a granuloma, but macrophages infected by B. botheris do not.

    Why do helper T cells stick to the macrophages infected with A. annoyis? Helper T cells have CD4, not CD8, and their TCR/CD4 complex sticks to epitopes displayed with MHC II, not MHC I. So the cells in the granuloma must have their epitopes presented with MHC II. This is to be expected. Microorganisms that are engulfed by macrophages generally present epitopes on MHC II, not MHC I. The proteins of the microbes are degraded in endosomes, meet MHC II inside the endosomes, and the complex is transported via vesicles to the plasma membrane. 

Information for problem 5

             Acid base balance is controlled by breathing and by secretion of H+ in the kidney. In all cases, CO₂ and acid are considered equivalent because of the reversible carbonic anhydrase reaction: 
            CO₂ + H₂O ↔ H+ + HCO₃-.

That is, losing CO₂ is equivalent to losing H+ and vice versa.

The terms acidosis (low pH) and alkalosis (high pH) refer to the state of the blood, not to the state of the urine.

            Breathing is controlled by a special area in the brain that contains respiratory pacemaker cells. These pacemaker cells control the firing of motor neurons, which control the major muscles responsible for breathing. When the pacemakers fire, the motor neurons fire, the major muscles respond, the lungs expand, and you breathe in. (Breathing out is normally passive, and follows automatically.)

            There are chemoreceptors for H+ in the arteries and the brain ECF. These send afferent information to the brain.

            If improper breathing leads to improper CO₂ levels, the kidney can compensate. The kidney can secrete protons but cannot reabsorb them. A normal kidney secretes large amounts of H+ per day.

 Information for Problem 6.

Many bacteria are engulfed by phagocytic cells, and end up inside phagosomes (phagocytic vesicles). Most engulfed bacteria are then destroyed, but some replicate inside the phagocytic cells.

Bacterium A (A. annoyis) reproduces inside phagosomes

Bacterium B (B. botheris) escapes from the phagosome and reproduces inside the cytosol.

Bacterium C (C. catchis) is engulfed by phagocytic cells, but does not replicate inside.

(I have made up the names, but all 3 cases occur.)

Cytotoxic T cells kill macrophages (phagocytic cells) infected with B. botheris but not A. annoyis.

Information for problem 6, part D. You have 4 DNA samples. A, B, S, & M.

            A & B are two samples of DNA from infected macrophages from the same person. Sample A is from cells infected by A. annoyis and sample B is from cells infected by B. botheris. You isolate eukaryotic chromosomal DNA from each sample of macrophages. You cut up the DNA in your two samples with restriction enzymes, run gels, and blot with probes for the region coding for V_H, C_H, MHC I & MHC II.

V_H = the variable section of the heavy chain;  C_H = the constant section of the heavy chain.

            S & M are two samples of DNA isolated from two different clones of B cells.

            Both clones of B cells are from the same person. Both clones were isolated separately. Both clones were selected by their ability to make antibody to the same protein from B. botheris. Clone M makes membrane bound surface IgG and clone S makes secreted IgG. You isolate DNA from the two clones and sequence the region(s) coding for the MHCs and the heavy chain of immunoglobulin (antibody).

 Information for Problem 7

    Macrophages usually kill engulfed bacteria by generating superoxide radicals inside the phagosomes. These radicals form hydrogen peroxide and other reactive species that destroy the bacteria.  In CGD (chronic granulomatous disease) the macrophages lack the oxidative enzyme complex needed to produce superoxide radicals. Therefore some engulfed bacteria are not killed effectively. As a result, aggregates of infected macrophages + T cells form nodules called granulomas. CGD is an inherited disease caused by a defect in one of the genes encoding the oxidative enzyme complex. All other genes are normal.

    All the bacteria that are resistant to killing in CGD are catalase positive (produce catalase). Bacteria that are catalase negative (don’t produce catalase) are killed in patients with CGD as well as in normal people.

Catalase catalyzes the reaction:

            RH₂ + peroxide (H₂O₂) à R + 2 H₂0.

    People with CGD get granulomas if they are infected with A. annoyis, but not if they are infected with B. botheris. Binding of helper T cells to macrophages stimulates the ability of normal macrophages to kill engulfed bacteria. T_H cell binding does not help macrophages from people with CGD.

Abbreviations and acronyms

Ab = antibody

ACTH = corticotropin = adrenal cortex tropic hormone

ADH = anti diuretic hormone = vasopressin

Ag = antigen

AP = anterior pituitary

B cells & T cells = lymphocytes (you are supposed to know what they do or don’t do)

BCR = B cell receptor = Ab attached to surface of B cell

CD4 & CD8 = cell proteins (you are supposed to know their location and significance)

CGD  = chronic granulomatous disease

CRH = corticotropin releashing hormone

ER = endoplasmic reticulum

GLUT4 = insulin responsive glucose transporter

GR = glucocorticoid receptor

HT = hypothalamus

IgG = gamma globulin (antibody)

MHC = major histocompatibility complex (you are supposed to know the difference between I and II)

MR = mineralocorticoid receptor

PLC = phospholipase C

PP = posterior pituitary

T_C = cytotoxic T cell

TCR = T cell receptor

T_H = helper T cell

TH = thyroid hormone

TSH = thyroid stimulating hormone = thyrotropin

V_H = the variable section of the antibody heavy chain;  C_H = the constant section of the heavy chain.