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Cell Bio And Disease > Studying Signalling Paths > Flashcards

Studying Signalling Paths Flashcards

1
Q

What are the 2 approaches to studying

A

Top down from signal to effect

Bottom up from effect to signal

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2
Q

What are the 4 methods used

A

Purified proteins
Tissue culture
Animal models
Human models

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3
Q

What is using purified proteins Eg to study the interaction between 2 proteins

A

Overexpression in a system like ecoli then purifying invitro

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4
Q

Is purified proteins easy to manipulate / gain molecular analysis

A

Yes easily manipulated eg addition of phos groups or removal to see effect

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5
Q

What does clean data from purified proteins mean

A

Controlled conditions like ph etc

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6
Q

What does clean data allow

A

Repeated multiple times easily

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7
Q

What is the main disad of purified proteins

A

Not physiological, the conditions are artificial and in vitro

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8
Q

What needs to be known before using purified proteins

A

The pathway components

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9
Q

Is it easy to express and purify proteins

A

No there are some issues with extraction eg if protein becomes insoluble hard to remove or proteins can become inactive in extraction

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10
Q

What is promiscuity in protein purification

A

When 2 proteins meet which wouldnt in vivo

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11
Q

How is ste5 an example of how to use purified proteins promiscuity

A

Can be put in with kss1 components and cause the pathway to occur if starvation

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12
Q

What are the 2 types of cell tissue culture

A

Primary - extracts from human or animal tissue

Cell lines - been immortalised to divide constantly either by Chem treatment or taken from tumour

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13
Q

What are the ads of cell culture

A

More physiological from human/animal

Clean data- still controlled conditions

Easily manipulated

Many experiments can be done (multiple data points)

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14
Q

What conditions needs to be kept for tissue culture

A

Co2 and humidity

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15
Q

What are the disads of tissue culture

A

Not fully physiological eg cell lines Chem treated
Easily infected with bacteria
Expensive
Cells can change over time
Cells might not be pure- may be diff types of cell eg muscle and blood cells

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16
Q

What are ads of using animal models

A

Cheap
Disease models already available eg ob ob
Physiological to an extent

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17
Q

Is data clean with animal and human models

A

No, conditions can’t be controlled, other pathways can interfere, compensation occurs (accommodation to conditions)

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18
Q

What are the disads of animal models/ Human

A

Ethics , not easily repeated/can’t do many experiments on one model, if using cell preps may not be pure and can change, differences between animals, shortage of volunteers

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19
Q

What are the ads of using human models

A

Truly physiological
Can get feedback on how they feel
Can select people with disease

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20
Q

What is the rat treated with streptozotocin modelling

A

Streptozotocin destroys B cells which mimics diabetes 1

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21
Q

Which 2 animal models are for diabetes II

A

Ob ob mouse

Zucker rat

22
Q

What is the ob ob mouse

A

Makes no leptin

23
Q

What is the zucker rat

A

Defective hypothalamic receptor for leptin so can’t respond

24
Q

What are radio markers for

A

Detection of phosphorylation

25
Q

What can be used to study effect of one protein has on path

A

Knock out or reporter gene

26
Q

What are problems with manipulation like Trying to increase camp

A

Cell responds eg pde , de phosphorylate

27
Q

Which 3 chemicals block pde to increase camp

A

Ibmx, caffeine, theophylline

28
Q

How does forskolin increase camp

A

Activates adenylyl cyclase

29
Q

What can be added instead of camp to increase its effect

A

Non hydrolysable analogues

30
Q

Which toxin can used to increase camp

A

Cholera or pertussis

31
Q

What is ip3 sm made from

A

Pip2 breakdown by Phospholipase c into ip3 and dag

32
Q

How do cells convert ip3 back

A

Into ip2 or ip4 then back to inositol then into pip2

33
Q

What blocks the ip3 pathway of recycling

A

Lithium

34
Q

How can you track ip3 production and see if the pathway is active

A

Label

35
Q

How are binding assays used to detect camp levels

A

Radio labelled camp are added which compete with body camp for binding

If more radiolabel binds it means less normal camp was in the body for competition

36
Q

Other than binding assays what can measure camp

A

Fret fluorescence

Fluorescence resonance energy transfer

37
Q

Why is fret used and not probes or dye

A

Can’t use probes or dye for camp

38
Q

What happens when camp binds

A

Change of fluorescence/ emission of light

39
Q

What 2 fp used and what wavelength do they emit without camp

A

Yellow fp

Cyan fp

Cyan absorbs and transfers light into the yellow fp and it emits light at 540nm

40
Q

What happens when camp binds in camp binding domain of fret molecules

A

Conformation change

Light can’t transfer from cyan to yellow fp so it is emitted by cyan at 480nm instead of 540 nm

41
Q

How do you track ip3

A

Add tritiated inositol which would radiolabel ip products by making them and then block path by lithium

42
Q

How are proteins radiolabelled when detecting phos using acrylimide and photography

A

Add radio P onto atp and it is y phosphate so it transfers to proteins

43
Q

How are proteins separated on the aceylimide

A

By mw (changes to heavier when phosphorylated)

44
Q

What activates pkc in phos studies

A

Phorbol esters

45
Q

What activates pka in phos studies

A

8 brc amp

46
Q

What is a general kinase inhibitor which blocks atp binding domain = not specific

A

Staurosporine

47
Q

What do specific kinase inhibitors bind

A

Substrate binding region

48
Q

Which kinase inhibitor is for pkc

A

Wortmannin

49
Q

What needs to be added to phos studies

A

Phosphatase inhibitors

50
Q

What phosphatase is blocked by okadaic acid and vanadate

A

Ser/thr phosphatase

Vanadate- tyr

51
Q

What is added to genes in reporter gene to measure exp levels eg in pathway

A

Luciferase or gfp

Cell Bio And Disease (18 decks)

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