Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BMS346 > Essay questions > Flashcards

Essay questions Flashcards

1
Q

Experiment to show that Enac has a function in controlling height of ASL

A
  • Add excess fluid
  • Mimic in vivo conditions of breathing- moving back and forwards
  • look at rate of decrease of height due to change in trans epithelial potential due to changes in Cl secretion and Na reabsorption
  • Butamide and Amiloride inhibition over time
  • 0 hr lots of Amiloride inhibition but decreases to 30% after 48hours
  • Ohr less butamide is 55% inhibition at 48hours
    shows more Cl secretion needed to maintain ASL than Na reabsorption but both help maintain the layer
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

RSV experiments

A
  1. Look at the effect of RSV on amiloride sensitive ssc
    - Add amiloride
    - then add either control or RSV for an hour
    - then re add amiloride and see if there is a change in deflection
    RSV= decrease deflection of amiloride as less enac is functioning
  2. Look at which pathways of ENAC RSV interacts with
    - PKC (BIM inhibitor), Glycoprotein (NA inhibitor), Glycolipid (PMCD)
    - If you add inhibitor then there will be no effect of amiloride as the enac is already blocked- PKC AND GLYCOLIPID
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Experiments for Influenza

A
  1. prove that M2 causes long term influenza
    - Overexpressed Enac and it inhibited Enac
    - decrease open probability of Enac- less deflections when M2 is overexpressed
    - caused endocytosis- western blott showed less alpha subunit and Beta subunit present when M2 overexpressed
    - showed there was less enac due to increased endocytosis- used liddles where endocytosis cant occur and showed that M2 couldn’t endocytose when liddles occurs
  2. look at mechanism of how M2 acts on enac
    - immunostaining- RFP-M2 Or RFP alone
    - ROS= green
    Results- in areas overexpressed with M2 (red) there is also green staining so ROS has accumulated
    Part 2- prove ros is involved
    - inhibited ROS with GSK- antioxidant that scavenges ros
    - also inhibited PKC by Inhib and GO6967 which blocks
    - Inhibiting PKC or M2 expressed more Enac
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How is Cl secreted?

A
  1. Experiment to show DF created- look at rectal shark model
    - oubain- Na/K ATPase inhibitor- reduces NKCC and therefore Cl secretion as cant set up the df. Maintains low IC Na
    - Barium- blocks K- hyperpolarises the cell, Vte -1 to 0- DF for Cl secretion
    - Furoesmide- NKCC inhibitor- stops Cl influx into cell for CFTR to secrete
  2. Above electrochemical gradient
    - no active process then ic would be the same as EC
    - we got 17mM but actually 70mM as active process causing accumulation of cl- this is Na/K ATPase
    - above electrochemical gradient means Cl channels just have open for CL secretion
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Experiment to look at mechanism of CFTR

A

Isolated rat CFTR
looked at the water content- which is dependent on Cl secretion by CF- Secrete Cl and water follow
PGE2- Increases cAMP- increases CFTR- increases Cl secretion
Ach- increases IC Ca- activates apical and basolateral Cl

  • Open CFTR= Cl secretion
  • Open K channel= hyperpolarises
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Experiment to look at the activation of CF and Non CF

A

Ussing chamber experiment
Look at effect of cAMP secretion
- indomethacin- inhibits PG- decrease cAMP
- Imbx- inhibits phosphodiesterase- increase cAMP
- CCB- increase Ach- increase cAMP
- forskin- increase AC- increase CAMP

block amiloride to stop contamination

  • stimulate with ccb- increase Cl secretion- increase neg shift Vte
  • CCB +indomethacin- block cl secretion- DF there but channel is closed
  • CCB+ indomethacin+ imbx- bypass and increase Cl secretion

*CF patient no effect= CFTR channel is not functional

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What experiment would you include in VX770 and G551D

A
  1. Drug screening
  2. Rat thyroid- add VX770 +FSK +G551D
  3. Expression system- Vx770 + PKA and ATP
  4. Look at Vx770 in native cell- Human bronchial cell- amiloride sensitive ssc in double mutant when adding 770 and FSK
  5. Effect of 770 on ASL layer- add fluid and VIP (increase CAMP) and see how 770 increases ASL
  6. Look at how 770 effects CBF
  7. clinical trial in humans- G551D and 770- FEV1%- improvement, pulmonary exacerbation and sweat cl
  8. G551D and Vx770 in Human nasal cells, quirt in Cl free solution and isotopreneral to secrete B agonists and increase CAMP- look at Cl secretion
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Experiment to look at F508

A
  1. drug screening- compounds that drive immature to mature
  2. F508 and VX809 ratio of mature to immature
  3. pulse chase experiment HEK and CFTR - amount of mature and immature depends on the length of time cells are exposed to vx809
    4.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How do you screen for G551D and F508

A

G551D- Increase in fluorescence vm assay when increase membrane potential- which means gating worked

  • tested 228,000 chemicals
  • look at nerst= Cl
  • found VX770

f805- immunoblot assay to see which chemical drive CFTR glycosylation and become mature

  • see 2 bands
  • glycosylated is higher MW band
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Look at the effect of VX770

A

Rat thyroid cells
stimulated G551D and control with FSK (increase cAMP)
G551D + FSK- small increase in isc
GSS1D+ FSK + 770- increases currents of cl secretion- 12-15% of wt- only needs to be that to get normal function
Prove it was CFTR that increase Cl secretion by inhibiting

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Expression system 770

A

In vivo
- look at the effect of PKA and ATP looked at deflections

G551D- few deflections even in presence of PKA and ATP
G551D + 770- channels open, potentiator- increase Cl secretion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Effect of 770 on human cells

A 
Human bronchiole epithelial cells 
Block amiloride= contamination 
- severe CF- contain 2 mutations in G551D/F508 
- add FSK to stimulate 
WT- 56uA FSK induced ssc 
G551D/f508 +fsk= 2.9uA
mutant + FSK + 770= 27uA= 48% of wt 
enough to alleviate the symptoms of CF
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Looking at the effect of 770 on ASL

A

Amiloride to block enac
added excess fluid to the layer
Used VIP- increase cAMP

WT- add excess, stimulate with VIP and the layer decreases then plateaus
G551D +VIP- massive decrease in liquid layer
+ 770- increase ASL- in-between wt and mutant

Looked at the effect on CBF
decrease ASL= decrease CBF
- G551D + VIP- decreased CBF- struggle to beat- restored when liquid is added on top
+779- restores normal layer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Clinical trial for 770

A 
83 patients with CF- mutations in atleast 1 CFTR 
random double blind and placebo 
- Looked at FEV1% 
- Pos= improvement 
neg= worsening

Results
- add VX770 to G551D
FEV1% Improves drastically over 2 weeks and whole time whereas placebo worsens
- Pulmmonary excabertion- Ivacaftor- 67% haven’t, placebo- 40% haven’t
- Sweat cl- drops below threshold

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Part 2 of Trial

A

Inserted Cl free solution containing Isoprenterol (increase cAMP) into nasal passage- initiates Cl secretion
CFTR working then there is a negative shift in potential as more Cl secretion occurring
VX770- 75 and 150mg- increased Cl secretion
direct experiment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Looking at the function of F508 and VX809

A

Looked at ratio of mature to immature
no 809- 0.5%
increased by 30% when VX809 was added

Works by inserting in protein of CFTR and correcting folding so it can be transported to the membrane rather than for degradation

17
Q

Pulse chase experiment Hek and CFTR

A

35s methionine and 35s cysteine
mature cftr
35s removed and compounds left for a duration of time
- formed ratio of Mature Glycosylated 35CFTR and immature 35CFTR
- amount of time left determines ratio

Look at western blot
- cells left for 180 min, radiolabelled immature CFTR
wt= mature shifted, decrease immature
F508- no immature and no mature- degraded
F508 and vx809- slightly more mature but not as much as wt

18
Q

Clincal trial f508

A 
19-19 CF patients 
28 days 
different amounts of F508 homologous 
Looked at FEV1%- NO PATTERN 
Highest sweat cl decrease was still above the cf threshold
19
Q

Combination therapy

A

Combining VX770 and 809
809- not sufficient to treat CF alone
1. change in sweat with 250mg there was a large sweat decrease but none below threshold
2. FEV1% increased by 3%- placebo slowly worsened
3. clinical trial
- 1108 patients, lumacaftor and ivacaftor
placebo, 6000mg + 250mg, 400mg +250mg
- Fev1% improved in first 2 weeks then steadied out, big range of results
- Cough or infection catching- decreased from 40% placebo to 20% ivacaftor
- hospitalisation- decreased
- forrest plot- FEV1%- above black line- better, below- worse, neither

20
Q

Gene therapy

A

Thought they would have done this in 1990s but still hasn’t been done due to the renewal of cells
- Example asthma- gene therapy works until cells are replaced and the new ones wont contain the new
PGM169
- FEV1% Dramatically improved
FVC- lung function improved

Doesn’t improve or worsen- stabilises- if get someone with high lung function it could work well

21
Q

Experiments for A typical CF

A
  1. Show GOF epithelial Na channel- look at mutated ENAC and add amiloride and show similar to Mild CFTR
  2. Prove aW34R was a GOF mutation- bigger function of Enac when was out amiloride
  3. See if aw349r mutation has negative feedback- looked at slightly higher amiloride
  4. Look at the effect of cleaving channels on GOF enac
  5. Self inhibition of Na due to increase Na currents- decreases channel Po
  6. MSET technique to measure Po rather than Patch clamp
  7. Effect of BV348M mutation in Atypical enac
  8. Mouse overexpression B subunit effect on survival
22
Q

Validating ENAC GOF

A
  1. looked at CF, Atypical CF and Normal vte
    - add amiloride- See function of Enac
    - atypical carrier 025mv to -15mv
  2. prove aW345r is GOF
    increase amiloride sensitive currents- increase enac function
    - bigger deflection- bigger function of enac when wash out
  3. to see if aW345R is due to negative feedback
    - started with same number of channels
    - did high and low
    - in wt low- wait for a bit before enac pulled out of membrane so slightly higher amiloride,
    -high Na was the same for WT and mutant- ratio drop same, so not a problem in endocytosis
23
Q

Effect of cleaving on open probability

A

Cleave CFTR- increases Po- more enac work
chymotrypsin= cleaves proteins
WT- add chymotrypsin, increase cleaved ENAC, increase Po,
Know its Enac cleaved as when you add amiloride this decreases
Mutant- response to chymotrypsin is lost, gating looks different but increase PO

Looked at pattern of activity- deflections
WT- add chymotrypsin- increased open probability so increased deflections
Mutant- already has lots of deflections so Po high- irrespective of chymotrypsin

24
Q

Na self inhibition

A

Self inhibition occurs- see spike in Na current as there is too much Na influx so Na channels decrease Po
this is when 100mM of Na is added
Amiloride inhibits this
Mutant- unable to self inhibit, lost even when Na is added

25
Q

Mutation in B subunit to increase ENAC GOF

A
  1. B subunit change in open probability
    - wt= increase current when take away amiloride
    - mutant GOF increases by 40-45% from mutation
    higher Po - increase Na inclux
  2. SCNN1B to see effect on survival of mice
    Atypical CF- overexpress B subunit of ENAC, rate limiting step in Enac function
    Add this to channel the PCL layer decreases
  3. survival curve- decrease surivial of mice overtime as cant excrete mucous
  4. lung slice- plaques and plugs- trap gas in
  5. When you inject the mouse with bacteria
    - leave 2 days
    wt= gone
    mutant- still infected as cant get rid of mucous
26
Q

Experiments for K channels

A
  1. Look at effect of Chromanol 293B on KCNQ1- PT-PCR
  2. Ussing chamber K channel- look to see if Q1 is cAMP activated- added IBMX increased cl secretion
    and look if barium selective- K recycles across cell line= control amount of Cl but is is Q1 selective
  3. How 293B and Ba effect CF and Non CF
  4. Apical UTP on CaCC- cl secretion- Ca activated
  5. Basolateral K channel secretion HSK4 using K channel blockers and UTP
  6. Apical K channel- BK, inhibit using praxiline
  7. Effect of Apical K channel-BK on CBF
27
Q

Q1 channels- 293B inhibition and cAMP activated

A
  1. Looked at fresh nasal epithelium, prove that 293B inhibits Q1
    - RT-PCR
    - BP mw 788bp
    - Q1 expressed in both wt and CF- expressed in the upper respiratory tract
  2. Looked at the effect of cAMP on K channel activation
    - Vte against time
    - added IMBX gradually increasing the conc- Vte became more positive as Cl secretion was occurring
    - block K channel= block Cl secretion
  3. Is Q1 barium sensitive
    - inhibited ssc with 10mg of 293B- didn’t go to 0
    - barium takes to 0
    - K recycling controls CL secretion
  4. Looked at the effect of 293B in CF and Non CF
    - under control and IBMX
    effect on K and cl
    Non CF- when you increase cAMP in 293B - increase Cl secretion
    CF- even if you increase cAMP there is still no secretion as CFTR channels not functional
  5. barium sensitive
    - non CF and CF- increase imbx- increase Ca had no effect on Ba sensitivity- cl secretion is driven by Ba sensitive K channel ontop of Q1 in CF- additional Cl channel
28
Q

Ca effect on Apical channels

A

UTP increased with Increased IC CA
activates Ca activated CaCC channel- initates Cl secretion from apical
increase ssc as upregulated in CF patients however this is not sufficient to alleviate the CF symptoms
- increase UTP- increase PGE- increase cAMP
- hyperpolarising effect as it increases cAMP- increases k efflux from Q1- neg membrane potential- increase CL secretion

29
Q

Effect of UTP on basolateral Cl channels

A
  1. All amiloride and no cAMP
    - effect of 239b and clotrimazole on amiloride
    - UTP stimulated increasing IC cCa
    wt- high cl secretion
    293B- no effect, cAMP low so Q1 not open
    clotrimazole- blocked UTP Ca activated K channel- Cl secretion decreased
  2. All amiloride and cAMP
    - effect on CF and nonCF
    - wt- 293B now has an effect as channel activated through cAMP
    - CF- enhanced Cl secretion 293B contributes to DF in Cl from Ca activated channels
30
Q

Apical K channel

A
  1. inhibit paxiline- Bk inhibitor and see the effects on Cl secretion
    control- add atp- increase Cl secretion
    Paxilline- Add ATP- decrease Cl secretion
  2. KD- BK inhibits Cl secretion by CaCC
31
Q

BK on CBF

A

NHBE cells- lefts for 4.5 days- CBF measured- add PBL- CBF measured
-CBF
- T- no addition to ASL layer- paxiline reduce asl
R- restored

BK

  • T - CBF non existent as KD K= not K efflux= no Cl secretion
  • R- restores
32
Q

B subunit experiments

A
  1. Effect of overexpressing Q1 and E1- patch clamp and voltage dependence
  2. KCNE1 KO mouse and renal function- kidney- Q1 thought to be apical
  3. Clearance study to see the effect of KO on amount of GFR, plasma conc and glucose
  4. is Q1 regulating E1?- KO Q1 293B and if E not working then when you block Q1 there should be no change but there is a shift in FE1% NA
  5. Patch clamp- show its not voltage dependent
33
Q

What does the KCN31 and KNCE2 study show

A

Stimulants Ach, histamine, gastrin
Secretion of HCL from stomach digests food in stomach before small intestine
- Apical Cl open and releases Cl bicarbonate- not enough K to support this

  1. Ammonium pulse technique
    - Ammonia in cell, combine with H, alkylation, increase PH then take away ammonia releasing H ions and an acidification
    lack of recovery= KO
    wt- recover from PH increase
    exchange for H channels
  2. Parietal cell KO
    KO= decrease h
    KO mice increase PH- more alkaline struggle to secrete acid and not as much in stomach
34
Q

Ammonium pulse technique on KNCE2 KO in gastric function

A

WT- recover
HETs- secrete some acid
Hom- no acid secretion- E2 not working so cant regulate their Q1

35
Q

Relationship between E1 and Q1

A
  1. They looked at patch clamp technique
    - 2 electrode voltage clamp in xenopus ooctyes expressing cRNA encoding E1 and Q1
    - thought that E1 modifys/interacts with Q1
  2. Voltage dependence- looked at lines between in Q1 and Q1/E1
    - saw that currents activity was greater
    - slower activation
    - still voltage dependence
  3. E1 ko study in renal mouse- looked at kidney function
    - immunostaining showed that apical E1 and nuclei and where they thought Q1 was stained
    - turned out that Q1 is apical and E1 is proximal
36
Q

Clearance studies

A

To look at the concentration of urine and plasma in the KCNE1 ko mouse

  1. Anaesthetise
  2. Cannulate jugular vein
  3. Collect urine
  4. Blood sample
  5. BP- used to ensure the mouse is still out of it
  6. Heated pad to ensure temp maintained
  • KO had no effect on GFR or plasma Na/Cl/fluid
  • plasma glucose conc was different
  • looked at the FE% = rate of filtration/ rate of absorption
    100= all filtered
    WT- all absorbed non secreted
    KO- struggle to absorb Na/Cl/fluid no problem with glucose bc further down proximal tubule it can be absorbed

Results repeatable in the Sheffield lab- struggle with glucose as it plays role in late proximal tubule

Shows the KO E1 has an effect on tubular function in the nephron

37
Q

Is Q1 regulating E1?

A
  1. Used chromanol 293B inhibitor of Q1
    - if E1 regulates Q1 then if you block Q1 in an E1 ko there should be no difference
    -fe% Na shift
    - same for Cl and water
    E1 regulates a chromanol sensitive k channel
  2. Patch clamp technique
    - extracted Q1/E1 k currents- looks voltage dependent
    When actually look at it it’s not voltage dependent
38
Q

Looking at the KCNE1/2 function in gastric function from acid secretion of parietal cells

A

Ammonium pulse technique
- ammonia in joins with H and causes an alkylation
- ammonia moved out of cell and H released causing an acidification
Look at cells ability to recover
- wt= fast recovery
-ko= no recovery, wait for new H to come in for exchange, decrease in PH
Parietal cell Ko- decrease H, increase PH, alkylation, can’t secrete acid so can’t digest food in stomach

  1. Histamine stimulated- superimposed on top of each other in absence of Na
    WT= recover
    HETS= some acid secretion
    HOM= no acid secretion from acid load- no K secretion as E2 isn’t there to regulate Q1

BMS346 (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions