Studying Signalling Paths

Question 1

What are the 2 approaches to studying

Answer 1

Top down from signal to effect

Bottom up from effect to signal

Question 2

What are the 4 methods used

Answer 2

Purified proteins
Tissue culture
Animal models
Human models

Question 3

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

Answer 3

Overexpression in a system like ecoli then purifying invitro

Question 4

Is purified proteins easy to manipulate / gain molecular analysis

Answer 4

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

Question 5

What does clean data from purified proteins mean

Answer 5

Controlled conditions like ph etc

Question 6

What does clean data allow

Answer 6

Repeated multiple times easily

Question 7

What is the main disad of purified proteins

Answer 7

Not physiological, the conditions are artificial and in vitro

Question 8

What needs to be known before using purified proteins

Answer 8

The pathway components

Question 9

Is it easy to express and purify proteins

Answer 9

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

Question 10

What is promiscuity in protein purification

Answer 10

When 2 proteins meet which wouldnt in vivo

Question 11

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

Answer 11

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

Question 12

What are the 2 types of cell tissue culture

Answer 12

Primary - extracts from human or animal tissue

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

Question 13

What are the ads of cell culture

Answer 13

More physiological from human/animal

Clean data- still controlled conditions

Easily manipulated

Many experiments can be done (multiple data points)

Question 14

What conditions needs to be kept for tissue culture

Answer 14

Co2 and humidity

Question 15

What are the disads of tissue culture

Answer 15

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

Question 16

What are ads of using animal models

Answer 16

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

Question 17

Is data clean with animal and human models

Answer 17

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

Question 18

What are the disads of animal models/ Human

Answer 18

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

Question 19

What are the ads of using human models

Answer 19

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

Question 20

What is the rat treated with streptozotocin modelling

Answer 20

Streptozotocin destroys B cells which mimics diabetes 1

Question 21

Which 2 animal models are for diabetes II

Answer 21

Ob ob mouse

Zucker rat

Question 22

What is the ob ob mouse

Answer 22

Makes no leptin

Question 23

What is the zucker rat

Answer 23

Defective hypothalamic receptor for leptin so can’t respond

Question 24

What are radio markers for

Answer 24

Detection of phosphorylation

Question 25

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

Answer 25

Knock out or reporter gene

Question 26

What are problems with manipulation like Trying to increase camp

Answer 26

Cell responds eg pde , de phosphorylate

Question 27

Which 3 chemicals block pde to increase camp

Answer 27

Ibmx, caffeine, theophylline

Question 28

How does forskolin increase camp

Answer 28

Activates adenylyl cyclase

Question 29

What can be added instead of camp to increase its effect

Answer 29

Non hydrolysable analogues

Question 30

Which toxin can used to increase camp

Answer 30

Cholera or pertussis

Question 31

What is ip3 sm made from

Answer 31

Pip2 breakdown by Phospholipase c into ip3 and dag

Question 32

How do cells convert ip3 back

Answer 32

Into ip2 or ip4 then back to inositol then into pip2

Question 33

What blocks the ip3 pathway of recycling

Answer 33

Lithium

Question 34

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

Answer 34

Label

Question 35

How are binding assays used to detect camp levels

Answer 35

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

Question 36

Other than binding assays what can measure camp

Answer 36

Fret fluorescence Fluorescence resonance energy transfer

Question 37

Why is fret used and not probes or dye

Answer 37

Can’t use probes or dye for camp

Question 38

What happens when camp binds

Answer 38

Change of fluorescence/ emission of light

Question 39

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

Answer 39

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

Question 40

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

Answer 40

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

Question 41

How do you track ip3

Answer 41

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

Question 42

How are proteins radiolabelled when detecting phos using acrylimide and photography

Answer 42

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

Question 43

How are proteins separated on the aceylimide

Answer 43

By mw (changes to heavier when phosphorylated)

Question 44

What activates pkc in phos studies

Answer 44

Phorbol esters

Question 45

What activates pka in phos studies

Answer 45

8 brc amp

Question 46

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

Answer 46

Staurosporine

Question 47

What do specific kinase inhibitors bind

Answer 47

Substrate binding region

Question 48

Which kinase inhibitor is for pkc

Answer 48

Wortmannin

Question 49

What needs to be added to phos studies

Answer 49

Phosphatase inhibitors

Question 50

What phosphatase is blocked by okadaic acid and vanadate

Answer 50

Ser/thr phosphatase Vanadate- tyr

Question 51

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

Answer 51

Luciferase or gfp