Download BioPharm exam Nov 2016 answers

FACULTY OF HEALTH AND MEDICAL SCIENCES
DEPT. OF DRUG DESIGN AND PHARMACOLOGY
UNIVERISTY OF COPENHAGEN
Jagtvej 160
2100 Copenhagen Ø
WRITTEN EXAMINATION IN
In vitro techniques in biochemistry and pharmacology
(SFKKB9091U)
Nov. 9, 2016
(You
have 3THESE
hours)
PLEASE FIRST READ
ALL
INSTRUCTIONS!
Exam Instructions
This exam consists of 20 questions.
All answers must be in English.
No aids are permitted during this exam, except for the software installed on your examination computer.
Read through all the questions first, before you start to answer them.
First answer the questions with which you are the most comfortable.
Your answers should be short, concise and legible.
For the multiple choice questions you should circle your answer.
There is only one correct answer for each multiple choice question.
All questions have equal value.
You have two hours.
Good luck.
Practical Considerations
You may not leave the examination hall during the last 30 minutes of the exam so as to not your disturb
fellow students.
Mobile phones must be shut off and stored away during the exam.
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1) You make a Schild plot for a competitive antagonist and calculate the KB of the antagonist.
About what pharmacological property of the antagonist does KB give information?
Its binding affinity. Its equilibrium dissociation constant. KB = 1/affinity.
2) In the preparation of mouse cortical neuronal cultures (exercise A) you should avoid
excessive bubbles in the cell suspensions. Why?
Bubbles will kill cells and lower your yield of viable neurons (cells).
3) In scintillation counting of tritium (3H) water acts as a chemical quenching agent. Explain
the mechanism behind this phenomenon.
Water can absorb the energy of the emitted beta particle (electron) produced by a decayed
tritium atom but water does not re-emit this energy as a photon. i.e. water doesn’t fluoresce.
So the total amount of photons that the scintillation counter detects in the presence of water is
lower than it would be in the absence of water.
4) Why is it necessary to include FBS (fetal bovine serum) in the neuronal growth medium
(exercise A)?
FBS contains growth factors required for the neurons to survive in culture.
5) You measure the IC50 of an uncompetitive antagonist (a channel blocker) at AMPA
receptors using a TEVC assay in X. lævis frog oocytes. You then conduct a radioligand
binding assay at these same AMPA receptors where you make an L-glutamate competition
curve in the absence, and then in the presence, of the IC50 concentration of the channel
blocker. Explain what you would anticipate to happen to the L-glutamate competition
curve in the presence of the channel blocker, compared to the control curve in the absence
of the channel blocker, and why?
Nothing. The uncompetitive antagonist does not affect the binding at the orthosteric
binding site where L-glu binds. It binds in the channel pore to block the channel.
6) In exercise D you used an HEK293 EPAC149 and 2-AR-expressing cell line to measure
intracellular cAMP changes. Geneticin and zeocin are drugs used in the growth medium of
this cell line. Why do we need them in the growth medium?
They are antibiotics needed to give selective pressure on the HEK293 cells to continue to
produce the EPAC149 biosensor and the 2-AR. In the absence of these antibiotics the cells in
culture will eventually stop producing EPAC149 and 2-AR.
7) How does propofol affect GABAA receptors?
a) it competitively antagonizes GABAA receptors
b) it channel blocks GABAA receptors
c) it potentiates the response of GABA
d) it inhibits the response of GABAA receptors
e) it does not affect GABAA receptors
8) In the Xenopus oocyte electrophysiology (TEVC) experiments (exercised F), two glass
electrodes are inserted into the oocyte. In the voltage-clamp mode one electrode
measures the current. What parameter does the other electrode measure?
the membrane potential
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9) An irreversible, competitive antagonist will:
a) reduce the measured receptor density
b) increase the measured agonist affinity
c) reduce the agonist concentration
d) reduce the radioligand concentration
e) reduce total protein concentration
10) Cytochrome P450 enzymes are:
a) lipoproteins
b) ATP dependent proteins
c) glycoproteins
d) sulfoproteins
e) NADPH oxidizing proteins
11) What do you expect would be the physiological outcome of giving an experimental animal
(e.g. a rat) an injection of a potent CNS permeable GABA transporter (GAT) inhibitor?
Sedation. Since a GAT inhibitor would increase synaptic GABA levels and thereby
increase GABAA receptor activity, and GABAA receptors are inhibitory.
12) Define what specific activity (SA) is for a 3H-labelled compound.
radioactivity per mass of compound
units: Bq/mmol or Ci/mmol or dpm/mmol
13) A rat has experimentally induced epilepsy. You then give this animal an injection of a
compound that is a potent CNS permeable positive allosteric modulator (a potentiator) of
AMPA receptors. What would you predict to be the physiological outcome in the rat and
why?
Increased # seizures and/or increased seizure strength since potentiation of AMPA-R activity
should lead to increased excitatory neuronal activity.
14) Explain the mechanism of how the EPAC149 biosensor allows us to measure cAMP levels
in the 2-AR EPAC149 HEK293 cell line.
When cAMP binds to the EPAC149 biosensor protein it causes a conformational change of
the protein so that the FRET acceptor and donor protein domains move away from each
other. Since this acceptor-donor distance is crucial for FRET, the FRET signal will
decrease when these domains move apart. Thus as the intracellular [cAMP] increases
the FRET signal will decrease accordingly, in a concentration-dependent manner.
15) In your GABA transporter experiments (exercise B) you made some uptake
measurements at 0C (on ice). Why?
This gives a measure of the GABA transporter independent uptake and membrane binding of
3H-GABA since the transporters won’t operate at 0C.
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16) Mitochondria create a membrane potential by generating a proton gradient across the
inner membrane. The proton gradient is produced by respiratory complexes I, III and IV;
thus, any drug inhibiting the electron transport chain will affect the membrane potential.
Which of the following substrates is best suited for probing drugs inhibiting complex II?
a) malate
b) succinate
c) pyruvate
d) citrate
e) acetate
17) A newly developed 2-AR competitive antagonist gives a linear Schild plot with a slope of
1.00 when it is tested in rat blood vessels. However, when you subsequently test this
compound in the presence of IBMX in HEK293 EPAC149 2-AR cell line (exercise D)
you get a surprising result. The compound produces a small increase in intracellular
cAMP. Explain how this can happen.
The new compound is actually a very, very weak partial agonist. In the 2-AR over-expressing
HEK293 EPAC149 2-AR cell line, when IBMX blocks cAMP degradation, there is a
sufficiently large receptor density that such a weak partial agonist can produce enough
activated 2-AR to stimulate some adenylyl cyclase molecules to produce a small amount
of cAMP.
18) Which statement about the dose-ratio is correct?
a) The dose-ratio depends on the concentration of agonist.
b) The dose-ratio does not depend on the concentration of the antagonist.
c) The dose-ratio depends on the size of the response produced by agonist.
d) The dose-ratio describes the extent of the right shift of the dose-effect curve.
e) The dose-ratio applies for both competitive and non-competitive antagonism.
19) In 2D gel electrophoresis, proteins are separated in two dimensions. These separations
are based upon which two physical properties of proteins?
Mr (or mol size or mol weight)
pI or isoelectric point
20)
At what concentration of a radioligand do we achieve 50% receptor occupancy?
Kd = equilibrium dissociation constant of the radioligand.
a) 0.5x Kd
b) Kd
c) 2x Kd
d) 10x Kd
e) 100x Kd
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