Papers

Question 1

Multidrug Resistance in Cancer not done (Before Midterm)

Answer 1

• P-glycoprotein efflux pump is a pump found on the plasma membrane is an energy-dependent efflux pump that can pump out anti-cancer drugs, as well as other things that are damaging to the cell
• P-glycoprotein is found in sensitive and drug resistant cells, but excessive P-glycoprotein is found in drug resistant cells, and the more P-glycoprotein that was present, the more resistant the cells were to the drugs
• This excess P-glycoprotein is due to a single gene mutation
• Discovered that it was an energy dependent pump by treating the cells with cyanide to inhibit cell production and observed that the drug stayed in the cell, but as soon as cyanide was removed, the drug was pumped back out
• Northern blotting was used to show that the mRNA that was believed to code for the P-glycoprotein was more prevalent in cells that were drug resistant
• Southern blotting showed that there was an increase in the P-glycoprotein gene (up to 60 copies) in the resistant cells
• Hydropathy plots and sequencing showed that there are 12 transmembrane regions in the P-glycoprotein, which is characteristic of channel forming proteins, indicating P-glycoprotein resembles a membrane transport protein
• P-glycoprotein also has Intracellular ATP binding regions

Question 2

Dynamin Paper (Before Midterm)

Answer 2

• Main Point: Invaginated coated pits in the plasma membrane become constricted by the assembly of dynamin into rings around their necks, and a concerted conformational change then closes the rings and pinches off the budding coated vesicles
• In endocytosis, clathrin surrounds the vesicle, and dynamic facilitates the budding
• Dynamin is a GTPase
• Dynamin consists of four 100K polypeptides, and its C-terminus proline-rich domain (PRD) is the site of interaction with a lot of GTPase activators
• Dynamin’s PRD is required for dynamin stacking during assembly (oligomerization), but once formed, the PRD is not required to keep the oligomer together
• The presence of GTP isn’t required for ring stacking; GTP doesn’t ned to be hydrolyzed for stacking to occur; GTP hydrolysis is necessary for the the conformation change that constricts the ring (pinchase activity)
• The fact that GTP isn’t needed for dynamin ring stacking supports the fact that ring stacking occurs spontaneously
• Subtilisin is a serine protease that cleaves off the PRD
• When in high salt concentrations, dynamin forms tetramers, but doesn’t stack
• In low salt concentrations, dynamin oligomerizes into rings
• Dynamin levels don’t seem to affect dynamin’s ability to oligomerize
• A Sedimentation spin down assay was used to tell if the dynamin had oligomerized or not in different situations

Question 3

CCHFV Paper (Before Midterm)

Answer 3

• CCHFV uses clathrin-dependent endocytosis to enter cells
• This was essentially figured out by knocking out the gene for clathrin using siRNA and observing significantly lower amount of CCHFV and viral proteins in the cells
• The other type of endocytosis is caveolae-mediated endocytosis
• CCHFV entry also depends on pH and cholesterol, amongst other things
• Since clathrin-mediated endocytosis is controlled by cell surface receptors binding to their ligands in the clathrin coated pit, CPZ and sucrose, which are antagonists of said receptors, were applied to the cells to reduce the number of coated pits, which reduced endocytosis; reduction in CCHFV in the cell was observed
• Clathrin-mediated endocytosis feeds vesicles to the endosome; the pH change in the endosome may assist in the delivery of the CCHFV, so altering this pH environment may prevent the delivery of the fever virus; drugs that prevented the acidification of the endosome, like Bafilomycin or NH4Cl might affect virus delivery; saw a significant reduction in viral entry —-> pH of endosome is important
• Lipid rafts are important for calveolin endocytosis, so altering the cholesterol content of he membrane could affect the receptors, and the cell’s ability to endocytose those receptors’ ligands; applied MBetaCD which removed some cholesterol from the plasma membrane; wanted to do this to make sure that CCHFV did NOT utilize calveolin endocytosis????
• Cholesterol levels were found to be important in CCHFV viral entry

Question 4

Smell of Fear Paper (After Midterm; will be worth 8 points on final)

Answer 4

• Wanted to know how prey detect predators
  Def:
  Kairomones: cues transmitted between species that selectively advantage the prey and disadvantage the predator
  VNO: a specialized chemosensory epithelium in terrestrial vertebrates that contains sensory neurons that are genetically determined to mediate innate behavior and are distinctive from canonical olfactory neurons and serve the function of detecting pheromones
  TMT: a prominent pungent compound isolated from fox feces that causes aversion in rodents and is detected through unidentified neurons in the main olfactory epithelium (MOE)
  Major urinary proteins (Mups): excreted in urine and by mammary, salivary, and lachrymal glands
  c-Fos: a transcription factor that is rapidly unregulated when neurons are exposed to new or intense stimuli
• In the first experiment, mice that had never been exposed to predators before were exposed to rat, cat, and snake odors in a behavioral assay in which they were placed in a cage with the scent on one side of the cage. The mice didn’t spend much time in that area, and were shown to demonstrate stress behaviors, such as avoidance, risk assessment, and increase in stress hormones (ACTH)
• When put in the cage with a non-predator scent (rabbit), they did not show such behaviors
• The VNO is known for detecting pheromones in mice, so wanted to know if the TrpC2, the primary primary sensory transduction channel of VNO sensory neurons (VNs) were important in encoding predator scents, so mice had the gene for this knocked out
• These mice lacking TrpC2 did NOT show stress/risk avoidance behaviors, not even when placed in a cage with a live anesthetized rat!
• The rat experiment demonstrated that the display of avoidance is pretty dependent on the sensory cues detected by the VNO, and that other sensory cues (like seeing the rat) aren’t enough
  -TrpC2 is only necessary for avoidance to certain scents (kairomones) and not necessary for all avoidance behaviors; TrpC2 mice still avoided other scents such as NPHT odors
• The next experiment was done to see if any cellular changes go with the behavioral changes that are initiated in the VNO in response to fear-inducing predator scents; calcium imaging was done, as well as c-Fos staining.
• The mice were able to move freely around
• When wt mice were exposed to predator odors, there was a striking amount of c-Fos expression in the peripheral accessory olfactory bulb (pAOB), in which VNO axons directly projected to
• This activity was absent in TrpC2 lacking mice
  -Experiment 3 was to find the specific protein being detected in the fear response to cat scent (set up with assay like in the second big experiment)
  -Feld4, a cat allergen an Mup protein, was purified from cat saliva and then expressed in a recombinant fusion protein with MBP to see if it was detectable as a kairomone and if it was found to initiate similar avoidance behavior in mice
• The protein was found to initiate fear response in mice, indicating that the detection of cat Feld4 through the VNO sensory system induces defensive behavior