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192 Cards in this Set

  • Front
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Name the two way of investigating signalling pathways?

Top down - start with the message and 'dig' down the signal cascade until you reach the bottom


bottom up - effect and 'dig' up to message

List three approaches to investigating cell signalling

work with purified proteins and try to reconstruct system in vitro


use isolated cells from tissues/organs/cell culture


use whole animals


use humans

list the advantages of using purified proteins

Advantages


- ease of manipulation


- clean data, easy to control conditions and incorporate controls


- reproducible


- molecular data points

list the disadvanatges of using purified proteins

non-physiological


artificial, inappropriate conditions


subject to artefacts


need to know components to mix together


making proteins can be difficult


promiscuity

What are the type of cell cultures

1. primary cell cultures- derived form animal/human tissues/organs/samples


2. cell lines - cells originally derived form the same source as above, however then immortalised in some way

what are the two ways immortalisation can occur

through modification or as a result of from where the sample was derived

Advantages of using cell cultures

more physiologically accurate


cells relatively easy to grow, manipulate and maintain


data can be 'clean' as conditions are easy to control


get a lot of data from one cell prep

disadvantages of using cell cultures

can be easily infected


need to be fed regularly


still not truly physiological


method is expensive


cell preps may not be pure if from tissues/organs


cells can change during extended periods of culture

advantages of either whole animal or animal organ/tissues studies

whole animal studies are truly physiological


relatively cheap and easy


model of specific disease states/conditions available

disadvantages of whole animal or organ/tissue studies

data can be noisy


ethical reasons


few experiments per animal


not exactly the same as humans


primary cultured cells ca rapidly change


cell preps may not be pure

list some animal models

diabetes type 1- streptozotocin - injection into rats destroys pancreatic cells


diabetes type II - ob/ob mouse - mouse fails to make leptine (regulates appetite) thus is fat


diabetes type II - sucker rat - defective hypothalamic receptor for leptin - does not stop eating

Advantages of using humans for studies

truly physiological


great feedback


model of specific diseases available

disadvantages of using human studies

need informed consent


concerns over data protection


noisy data


ethical issues


few experiments per human


primary cultured cell can rapidly change


shortage of material


multiple cell types in cell prep

list three methods of cell signalling studies and the problem

use inhibitor/antagonists to 'block' receptors/signalling cascades


use of radio-labels


use of report genes/KO


use of FRET




problem - cells don't like having their signalling pathways messed with! compensation etc

List some substances that effect cAMP

IBMX, caffeine, theophylline - block the action of phosphodiesterases converting cAMP-AMP


Forskolin - activates adenyl cyclase giving max stimulation of system


cholera and pertussis toxins - activate/deactivate key proteins in the cAMP generation

What effect does lithium have on Inositol signalling

blocks a number of enzymes incl. myo-inositol-1-phosphotase


stops recycling on inositol to PIP2


cycle stops so IP pool can be measured



How do you track cAMP and IP3

cAMP- measure in a sample by using a binding assay, cAMP competes for a known amount of radio labelled cAMP


IP3 - pre load the cells with tritiated 3H inositol, block recycling with Li, purify IP products (which are radio labelled) measure levels



How would you prepared to cells in a cAMP experiment

- measure cAMP levels in cell prep


-pre-treat cells with a PDE inhibitor e.g. IBMX


- expose cells to different conditions

what conditions would you expose cells to in a cAMP study

1. nothing - control, basal levels expression


2. agonist - drug being investigated


3. antagonist - block the drug, test for action of cells


4. agonist and antagonist - block drug, explore


5. Forskolin - max activation - positive control, determine action of cAMP

what would be the results of the example study on cAMP

1. low levels if cyclic AMP


2. high levels of cAMP


3. half levels of CAMP


4. still some cAMP - dampening down production - drive reaction to inactive form, small amount of CAMP already in active form


5. high levels cAMP - positive control

what is FRET

FRET is a molecule that we use to detect cAMP levels using fluorescence resonance energy transfer microscopy



How does FRET allow us to detect cAMP levels

cAMP causes a conformational change in the molecule and a change in fluorescent wavelength


when CAMP binds YFP and CFP move apart, YFP doesn't absorb fluorescence instead it gets given off by CFP at 480nm


at resting YFP produces a strong signal at 540nm because of FRET


YFP no longer gets FRET from CFP after cAMP binds



How do you prepare cells for a Inositol Phosphate study?

measure IP3 levels in a cell prep


pre-load cells with 3H-inositol


pre-treat cells with Lithium


expose them to certain conditions

What do you expose cells to in a inositol phosphate study

1. nothing - control


2. agonist - drug being investigated


3. antagonist - block the drug


4. antagonist and agonist - block drug, determine levels of 3H inositol phosphate



How do you prepare cells for phosphorylation studies

pre-load cells with 'hot' phosphate 32P, so the ATP pool is hot


- 32P gets transferred to protein being phosphorylated


add radioactive phosphate to gama position on ATP


add activators of Kinases and inhibitors

what are some inhibitors of Kinases available for studies

straurosporine - inhibit a range


wortmannin PI-3 kinases


Rapamycin p70s6 kinases


PD98059 MAP kinase pathway





What makes a kinases inhibitor more specific

if they block the substrate binding region and not the ATP binding region

list some activators of kinases

phorhol esters - tumour promotors PKC


8- BrcAMP (3'5' cycling monophosphate)



what is the problem with phosphorylation studies

as fast as phosphate is added, it can be removed using a phosphatase



list some phosphatase inhibitors available

okadaic acid - inhibits protein serine/threonine phosphatase


vanadate - inhibits protein tryosine phophastase





what would be the typical results of a phosphorylation study that has


1. control, 2.agonist 3. Ag + antag 4. phorbol


5 - 8. - same again, but in presence of staurosporine

1. bit of phosphorylation


2. shift in gel - a band


3. less band


4. strong band due to activation of PKC


5-8. nothing in staurosporine presence





How can you purify ad identify the results phosphor-protein after a phosphorylation study

phosphorylation sites can be identified by phosphor amino acid analysis and phosphopeptide mapping


mass spec used as neutral ion and peptide sequencing


phosphor specific antibodies available for proteins shifted on gels



list some genetic approaches to invetisgating signalling pathways

antisense technology - antisense nucleic acid sequence which binds to target sense RNA strand


dominant negative protein - introduce a mutated protein that still interacts with pathway but is not functional


constitutively active proteins- add a protein that is permanently active


modified protein - change function/location/role



What can go wrong with cell signalling at the receptor levels and name some example of resulting conditions

1. over production of messenger ligand - hyperarathyroidism


2. under production of inactive messenger - nephrogenic diabetes insipidious


3. failure of receptor to recognise ligands - nephrogenic diabetes insipidious, AIDS


4. failure of receptor t activate G-protein - diabetes insipidious AIDS


5. failure of receptor to deactivate or always active - precocious puberty





what are the domains/components of a 7TM receptor

TM domains - forming core and communication of signals outside and inside cell


N-terminal, exoloops and core interacts with ligands


cytoloops 2, 3 and the C-terminal interact with G-proteins downstream

what is going wrong in the signalling pathway in diabetes insipidious

body fails to recover water from urine as filtered by kidneys


- cycling AMP levels are not activated due to receptor failure thus proteins kinase ! is not activated thus not phosphorylating it downstream target



how does the retina work

foto of light hit the retinal


this is absorbed


5 Amstrong movement


conformational change of receptor


phosphodiesterase turned on


cycling GMP broken down to GMP


closing of calcium channels

how many chromophores are there for three colours

one

where are the genes for green and red colours of the eye

x chromosome


recombination can result in altered gene levels

list some G-protein and their link to disease

GA12 defect - platelet dysfunction


Gat (transducin) - night blindness


Gas - albright syndrome, cushing syndrome


GB3 - hypertension


Gai2 - adrenal coritcal tumour


Gas- cholera toxin


Gai- whooping cough





How does cholera cause disease

cholera is a gram-negative rod shaped bacteria that colonises intestinal epithelial cells and causes a large influx of fluid


it has a single catalytic a subunit and and a pentameter of B subunits


a subunit is taken into the cell and cleaved


NAD+ dependent ADP robosylate GSa a subunit just before GTP binding site = activation


activated causes activation of adenyl cyclase


increase in cAMP causes mis-regulation of ion channels and an efflux of chloride ions





list some results of an increase in adenyl cyclase and thus cAMP levels

increase in PKA activity


adipose tissue - incr in triglyceride hydrolysis


cardiac muscle - epinephrine incr in contraction rate


kidney - vasopressin reabsorption of water


bone cells - reabsorption of Ca2+ from bone


liver/muscle - incr glucose production



what are some functions of adipocytes - a newly discovered dynamic endocrine organ

lipid and lipoprotein metabolism food intake and SNS activation, immune system and acute phase reactants, glucose metabolism/energy homeostasis, extracellular metric metabolism, vasculature and angiogenesis

list some causes of insulin resistance

mutations in signalling components


changes in level/activity of signalling components


alterations in complementary/antagonist pathways


altered metabolic preferences

list some symptoms of trisomy 21

down syndrome


upper respiratory infections


cardiac abnormalities


GI/feeding problems


speech/language difficulty


visions/hearing problems


behaviour problems



list some symptoms of trisomy 18

Edwards syndrome


poor life expectancy - 50% die within 1st week


kidney malformations


structural heart defects


intestines protruding from body


mental retardation



list some symptoms of trisomy 13

patau syndrome


very small, poorly developed eyes


weak muscle tone


extra fingers or toes, hands clenched


cleft lip/palete


absent eyebrows

how can we therapeutically target cancer

epidural growth fact and vascular eptihelial growth fact signalling inhibitors - getting past check point


cycling depends kinases inhibitors - throughout


cycle


telomerase inhibitors - cell can become immortal


PARP inhibitors - involved in DNA damage repair

How does slippage work

cells can escape mitotic arrest by slippage, by destroying their cyclin B due to incomplete inhibition of APC


- falls below threshold required to maintain CdK1 activity so cell exits mitosis without dividing and returns to G1 phase of cell cycle in tetraploid state


death signal slowly accumulates, if breaches threshold before slippage then cell will undergo death





how is apoptosis stimulated

stimuli results in pro-apoptotic proteins such as BAD and BIM are activated


these block natural anti-apoptotic BCI2 family proteins


pore forming proteins are thus made and from pore in mitochondria


cytochrome c is released and triggers activations of caspase cascade to chew up DNA

what do mitotic drugs do to the cell

take it into cell arrest due to chronic activation of spindle check point

list some compounds designed to target various aspects of mitosis

spindle microtubules


mitotic cyclin-dependent kinases


non-CDK kinases


motor proteins


multiple complexes such as SAC

list some evaluated and used mitotic drugs

CDK1, AuRKA, MTAs, PLK1, AURKB

list some potential emerging mitotic drugs

PLK4, NEKs, MASTL, HASPIN, BUB1, BUBR1, Kinesins, Separase

list some microtubule targeting agents

paclitaxel - breast cancer, ovarian


docetaxel - breast cancer, prostate


estramustine - prostate

list some anti-cyclin dependent kinase agents

flavopiridol


dinaciclib


both interfere with mitosis and transcription

list some aurora kinase inhibitors

danusertib


alisertib

list some polo kinase inhibitors

BI2536


Volasertib

give two emerging anti-mitotic drug targets

PLK4 inhibitors - PLK4 is a conserved key regulator of centriole duplication


MPS1 inhibitors- dual specific kinase crucial for recruitment of SAC proteins to unattached kinetochores, MCC formation and APC/C inhibition and chromosome alignment and error correction

what is hapsin

kinase that phosphorylates histones during pro metaphase


phophorylation promotes centric recruitment and activation of aurora B


depleting of haspin by RNAi, or microinjection of H3T3 antibodies = alignment defects and mitosis failure



what oncogene addiction and examples of therapies

when a tumour develops, oncogene pathway activated and the cell depends solely on that pathway


this can be targeted as other cells do not depend on the same pathway thus would survive


- RAS, Aurora kinases, ABL, CML, VEGF

what are the pitfalls of chemotherapy

cytotoxity


drug resistance


intrinsic (heterogenous cell population within the tumour)


acquired

how can cells become acquired - more prevelant

redundant mechnisms/pathways


transported expression - drug exported from cell


resistance to drug - induced apoptosis


detoxifying mechanism


alteration of drug targets


compartmentalisation


alteration of cell cycle checkpoint



why did mTOR only looked promising

plays a part in protein synthesis leading to cyclin D1


ACT pathway could compensate when mTOR inhibited



how can DDR inhibitors be used therapeutically

DDR coordinates repair of DNA


dysregulation can lead to genomic instability that promotes cancer - thus down regulation of DDR pathway can render tumours sensitive


loss of pathway leads to compensatory pathways targeting these may render endogenous DNA damage cytotoxic by synthetic lethality


tumour specific target





How can PARP inhibitions treat cancer

PARP promotes the repair of DNA damage


is it is inhibited then repair is insufficient and DNA damage persist leading to cell death


cells have functional HRR will survive

what is oesteoarthiritis

slow but progressive loss of ECM and Chrondrogenic phenotype in articular cartilage due to mechanical degradation without obvious cause


sever pain, limitation in joint movement


common in elderly

how have mice been study in relation to OA

DMM mouse - destabilisation of the medial meniscus


- cut medial meniscus brings bones together more than usually, develop OA really quickly


thus tests have been done to figure out the key break down events - whether its ADAMTS (aggrecan) of MMP (collagen)


DMM in normal versus ADAMTS5 null mouse - reduced disease in ADAMTS5


DMM in normal versus MMP-13 null mouse - virtually no diease


OA more dependent on collagen cleavage than aggrecan


mice with mutation, enzymes can't break down aggrecan or collagen

what are the best candidate genes to date for OA

GDF5


RUNX2


PTHLH


SMAD3

what are the current treatments for OA

surgery


Non- steroidal anti-inflammatories


identify key proteinases and target


genetic screening may allow to see susceptibility

what is RA

rheumatoid arthritis


common in young patients


progressive loss of ECM and chrondrogenic phenotype due to immune cell mediated damage - linkage to HLA-DR4 presenting peptides on lass II molecules


severe pain, joint limitation



what is the rheumatoid factor and what does it do

autoantibody IgM binds IgG

what are citrullinated peptides and how do they link to RA

a peptide that antibodies have been shown to react with


arginine post translation modification - citrulline


60% patients have auto-antibodies that specifically recognise citrullinated versions of self proteins


RA is likely to progress worse with them than if you dont have them





what factors rasie your chances of getting RA

smoking and drinking to much coffee



what lowers your chances of getting RA

mediterranean diet, anti-oxidants, alcohol and 0oestrogens

RA is characterised by presence of specific B and CD4Thelper cells, how do they mediate auto-immunity

CD4Th activate macrophages, resulting in production of pro-imflammatory cytokines (TNF-a) and sustained inflammation


cytokines induce production of MMP and RANK ligand by fibroblasts


MMPs attack tissues, activation of bone-destroying osteoclasts


= joint destruction



what are the current therapies for RA

non-steroidal inflammatory drugs


identification of TNF-a as potential target - introduction o Infliximab, sometimes doesn't work, blocks action of TNF-a


mono-clonal antibody - Rituximab specific for CD2) - kills B cells


better genetic screening

Outline the stages of Mitosis and how long they last

G1 phase - duration 10 hours. metabolic changes prepare the cell for division. The restriction point has to be met - committed to cell division


S phase - duration 6 hours - DNA synthesis, replicates genetic material


G2 phase- duration 3-4 hours. Metabolic changes assemble the cytoplasmic materials necessary for cytokenesis and mitosis


M phase- 2 hours. Nuclear division followed by ell division

Outline M phase stages

Prophase - chromosomes replicate, control spindles, replicate and move to end of cells


prometaphase- nucleus dissolves, mitotic spindle attach to kinetochores


metaphase - chromosomes align at centre of cell


anaphase - mitotic spindle pulls sister chromatids apart


telophase mitotic spindle disappears, nuclear envelope reforms and cell division begins


cytokinesis

outline the structure of chromosomes

centromere is constricted region of chromosome containing specific DNA sequence, bound to two discs of protein called kinetochores


kinetochores are the points of attachments for microtubules

which three ways are CDKs regulated

1. cdk levels remain the sam and in lareg excess, levels of cyclin partner directly regulate cdk activity


2. specific cdk inhibitor proteins


3. phosphorylation and de-phosphorylation

when does cdk activity turn off

after anaphase until half way through G1

which cyclins bind to which CDKs

cyclin D - CDK4/6


Cyclin E-CDK2


Cyclin A- CDK2


Cyclin B- CDK1

what did sea urchin eggs result in the discovery of in regards to cell cycle

first demonstartion of periodic protein degradation

what did yeast result in the discovery of in regards to cell cycle

kinase partner of cyclin


nearly all cycle regulatory genes


how telomeres protect chromosomes



What did Xenopus eggs result in the discovery of

discvory of MPF

How is a very simple linear stimulus response relationship formed when cyclin B and CDK1 bind

cellular concentrations of CDK1 is much higher than the very low dissociation constant of cyclinB-CdK1 interaction


cyclin B binds to CdK1 with very high affinity


all cyclin B binds when levels increase

which CdKs are responsible for which phases of cell cycle

CDk4 & 6 - G1


CdK 2 - G1/S, possibly M


CdK1 - M phase

What does full cdK activity require

cyclin must first be bound to it CdK partner - then this complex is phosphorylated by CdK activating Kinase at T160 of activation loop

How do Wee1, Myt1 and Cdc25 regulate Cdk-cyclin activity

Myt1 and Wee1 phosphorylate thus deactivating the complex


CdC25 enzymes de-phosphorylate the complex activating it

where do Wee1 and Myt1 phosphorylate the complex

Tyr15 and Try14 (myt)

what is the role of Cks1 proteins

an adaptor to target CdKs to phosphoproteins and also mediate APC interaction with Cyclin B and A


CKS1 enhances multiple phosphorylation of some M-CdK substrates by increasing the affinity of an already partially phosphorylated substrate

what are two examples of CkS1 proteins in humans

CksHs1 and CksHs2

what are CdK inhibitor proteins

suppressors of CdK activity in G1


responsible for maintaining G1 arrest during adverse conditions or in the presence of DNA damage


loss of function is associated with cancer



How does positive feedback effect Cdk activation

wee1 inhibits Cdk1-cyclinB activity


Cdc25 activates Cdk1-cyclinB activity


Cdc25 is activated by a trigger, the activation of Cdk1-cyclinB results in the activation of Cdc25 and the inhibition of Wee1

How is Ubiquitin-mediate protein destruction achieved



mediated by the ubiquitin-proteasome pathway


Ubiquitin activating enzyme (E1) is bound to ubiquitin protein, Atp-AMP


E1 looses this protein to E2 (ubiquitin conjugating enzyme)


E2 then looses this to the target protein, coupled by E3 (target specific ubiquitin ligase)


many ubiqutin proteins are added - small chain

What is SCF and what is its structure

an E3 ubiquitin protein ligase - control G1/S transition


3 core subunits - cullin, Skp1 and Rbx1


also an F-box protein


target protein


ring finger bound to ubiquitin


and E2 - ubiquitin conjugating enzyme



what is a ring finger

small zinc binding domain


in SCF it is Rbx1


in APC it is Apc11

What is the APC

Anaphase promoting complex


control metaphase-anaphase transition


ring subunit is Apc11 cullin is Apc


targets cyclin A and B plus securin


requires activators - Cdc20 or Cdh1

How is the Cdk oscillator assembled

cdk activates APC causing cyclin destruction


Cdk inactivation results in APC inactivation allowing cyclin to increase to start the next cycle


adding CCh1-mediated APC activity prolong Cdk inactivation


cdh1-mediated activity increases as Cdk1 goes down


an inhibitor of Cdh1-APC breaks the loop and allows cyclin to rise again

what is the rate of DNA synthesis

50-100 nucleotide/second

how is the 5' 3' synthesis over come

by primers (on Okazaki framents) - new synthesis is primed by short polynucleotide primers by an enzyme called primase


- unwound single stranded DNA coated with RPA proteins


- length of strand synthesised by the DNA polymerase is greatly enhanced by a sliding clamp

what is the role of a sliding clamp

proteins that forms a closed rign around DNA to hold the polymerase on as it makes the new DNA

How is the pre-replication complex assembled

Origin replication complex binds to chromatin (ORC1-6)


followed by the recruitment of Cdc6 and Cdt1


clamping of the MCM2-7 around the DNA

what stages is licensing activity limited to

activity if restricted to a short time at the end of mitosis/G1 and inhibited once S phase has begun

How does the cell limit the pre-replication complex formation to g1 only

telomerase synthesises DNA at chromosome end


- adds several repeat DNA regions on the lagging strand template to serve as priming regions so that the lagging strand can be completed


- lagging strand can't complete as there is no place for primer biding at chromosome end - would result in ever shortening chromosomes at each round of division

how is the nucleosome assembled

1. acetylated H3-H4 tetramer complexes assembly factor CAF-1


2. this is recruited to the replication fork and incorporated into the DNA


H2A and H2B dimers then loaded onto the H3-H4 tetramer via another assembly factor NAP-1 to form the octamer

what are the basic units of chromatin

4 core histones H2A, B, 3,4


2 copies of each


147bp of DNA wrapped around each octamer to make nucleosomes


protruding N terminal tails

How do tthe levels of cyclin B and Cdk1 change in M phase

Cyclin B1 levels are high, then drop during pro-metaphase


Cyclin B1-Cdk1 activity is high it metaphase


as B1 enters nucleus at the end of prophase - triggers degradation of nuclear envelope


Cyclin A levels decrease during prophase



What are the levels of activity of key regulators during the entry to mitosis

Wee1 and Myt1 are active at G2 - deactivated by activation of Cdc25B, A and C


Cyclin B-Cdk1 and Cdc25A/C are inactive in G2, activated in prophase


Cdc25B is on way to activation in G2 - fully activated in prophase as deactivated by end of pro-metaphase



What is nuclear accumulation promoted by

phosphorylation of cyclin B1 and Cdc25C


de-phosphorylation of Ser216 and Cdc25C

what are the main steps in centrosome cycle

1. pro-centriole formation, elongation and positioning of right angles


2. centrosome disjunction


3. control separation and spindle formation


4. cytokinesis

what are polo-like kinases in charge of

regulate spindle assembly and mitotic exit


activated in early mitosis


regulate centrosome separation and cleavage furrow formation and ingression

What do Aurora A and B regulate

A - functions at centrosomes to maintain spindle activity


B - part of the chromosomal passenger complex, along with INCEP, surviven and Borealin



what is the CPC in charge of

ais in chromosome condensation


to promote correct attachment of microtubules to kinetochores


on chromosome arms early in mitosis but then moves to centromeres and kinetochores later in mitosis



Is the spindle checkpoint active or inactive in


- amphitelic configuration


- monotelic configuration


- syntelic configuration


- merotelic configuration

-inactive


-active


-active


-inactive

outline the activity of aurora B in mitosis

- migrates to centromere during metaphase


counter acted by pp1 activity


activity jumps off centromeres and sits on spindle


chromatids pulled apart


kinetochores fall out of aurora zone, pp1 takes over and kinetochores come apart



How does the spindle checkpoint work

mediated by mitotic checkpoint complex


Cdc20 activator protein brings MCC to APC complex


when the check point is on, Cdc20 is sequestered, thus substrates arent recruited and cyclin and securin aren't destroyed


Cdc-APC dissociated and now APC is active


Cyclin B and securin are destroyed


inactivation of Cdk1 activity, activation of separase


Anaphase onset as cyclin B1 and securin are destroyed

How does APC recognise substrates

cyclins are recognised by APC-Cdc20 via N-terminal destruction motifs or 'boxes'


arg and leu are ritical points of destruction box in cyclin A, B1 and B2

When does Cyclin A and B destruction occur

Cyclin A - almost immediately after NEB


Cyclin B and securin - almost immediately after alignment of last pair of sister chromatids on the metaphase plate

What is the prophase pathway

destruction of cohesion ring without APC activity


mediated by phosphorylation by cohesion


cohesion falls off arms of chromosomes



why is cohesion not broken down sooner than it is

Sgo1-PP2A protects cohesion during pro-metaphase, counter acts phosphorylation

what are the main differences and similarities between Meiosis and Mitosis

meiosis & mitosis- DNA synthesis occurs in S phase of Interphase


Sister chromatids line up at metaphase plate - Meiosis (metaphase II), Mitosis (metaphase)




Meiosis only - Cross over, homologous chromosomes line up at metaphase plate during metaphase I, synapsis of homologous chromosomes during prophase I




One round of DNA synthesis and two rounds of revision for Meiosis, one reductional and one equational (always equational in mitosis)

What is the synaptonemal complex

complex that holds corssing over chromosomes together


species that holds them together - lateral elements

what are the stages of assembly of the synaptonemal complex

leptotene - homologs pair


sygotene - homologs paired, assemble complex


pachytene - fully-formed complex


diplotene - dissemble, separate chromosomes





when do female eggs stop in the cell cycle

stop in prophase 1 of meiosis 1



what happens instead of the prophase pathway in Meiosis

two stage digestion of cohesion by separase


- first in meiosis I, cleavage by separase on the arms


- second in meiosis II, cohesion was protected on centromeres by shugoshins, second round of cleavage by separase

how does the cohesion complex differ between mitosis and meiosis

mitosis - SmC/3 Scc 1/3


meiosis - Smc1 B, Rec8, STAG3

which subunit is the one cleaved of cohesion

mitosis - scc1


meiosis - rec8

How is cohesion protected on the centromeres in anaphase I

by the action of PP2A


-Sgo2 recruits PP2A to de-phosphorylate Rec8 so it is not able to be cleaved by separase


- inhibitor of PP2A in meiosis II





what is depletion

reduced recruitment of protection, disruption of chromosome structure, impaired bioorientation

what is mono/bi-orientation

mono-orientation - meiosis, sister chromatids attached to same pole, different homologue pairs attached to different spindle poles


bi-orientation - sister chromatids attached to different spindle poles

outline which four main stages cohesion break down/conservation happens, and what happens to chromosomes

metaphase I - homologue pairs cross over and held by cohesion


Anaphase I - MEI-S332/Sgo1 ohesion digestion on arms, homologues separate


Metaphase II - kinetochores become bi-orientated, cleavage of cohesion rings


Anaphase II - separation of sister chromatids alike to mitosis

what is the result of nondisjunction at meiosis I

homologues fail to be separated, pulled to one side of cell


two extra chromosomes in following meiosis II


results -


n+1, n+1, n-1, n-1

what is the result of nondisjunction at meiosis II

n+1, n+-1 and n x2

what are the differences between oocyte and somatic cells

oocytes are much larger, 100um diameter, global spindle assembly with multiple microtubule organisation centres, contain cyclic B1 in excess




somatic cells are much smaller 10-20um diameter, simple spindle assembly

cyclin B can be destroyed whilst the spindle check point is on in female eggs, why

cyclin B is in excess, metapahse occurs later when spindle checkpoint is met, protects Cdk1 activity and gives excess time for cells to organise everything

How do eggs stay in arrest

EMI2 block APC activity, maintaining low activity at metaphase II, cyclin can't be destroyed

How is arrest broken at fertilisation

via calcium mediated pathway (oscillations)

How does calcium oscillation work

cyclin B is conserved due to EMI2 blocking APC


sperm has PLZ(zeta) (phospholipase C zeta), factor that induces calcium to rise through production of IP3, Ca2+ is released


Ca2+ oscilations activate CaMKII


this primiing kinase phosphorylates EMI2, allows polo kinases to bind and further phosphorylate EMI2 resulting in its degradation


becomes and SDF kinase target


block on APC release

in order for a cell to communicate successfully the signals must be:

small outside cell - large inside


organised


targeting must be accurate and timely


signals must be turned off


systems must be reset

give an example of a GPCR that has fast activation and slow deactivation

Rhodopsin

give an example of a GPCR that has slow activation and fast de-activation

B2 adrenergic

who first identified insulin in 1921 and on what animal were the tests done

Banting and Best on their dog

where is insulin released from

pancreatic B cells

what two main responses happen after a carbohydrate-containing meal

stimulates insulin secretion


inhibits glucagon secretion

what is Glucokinase known as

the glucose sensor


has a low affinity for glucose

how does glucose sensing by glucokinase result in exocytosis of insulin

glucose is phosphorylated to glucose-6-phosphate and metabolised by glycolysis & mitochondrial oxidation to generate ATP/ADP


increase in ATP/ADP closes the K+-ATP channels = depolarisation of the plasma membrane from -60mV to -30mV


fall in membrane potential = L type calcium channels opening allowing entry of calcium and consequent exocytosis of insulin



Outline the structure of the insulin receptor

consists of four subunits - 2 alpha and 2 beta


the two alpha subunits are joined by disulphide bonds, the binding of insulin to one inhibits the other binding site


the beta subunits span the membrane whilst the alpha is extracelular

how doe the insulin receptor relay its signal

acts as a tyrosine kinase


- auto phosphorylation on tyrosines

which tyrosine residue is required for substrate binding

960

which tyrosine residues phosphorylation leads to kinase activity

1146, 1150, 1151

what is the IRS and what does it do

Insulin Receptor Substrate


phosphorylated by receptor


acts as docking station for proteins that contain SH2 domains


pulls other proteins together



what is an SH2 domain, what does it do

Src homology 2 domain


binds phosphotyrosine residues surrounded by unique sequences

what is an SH3 domain and what does it do

Src homology 3 domain


binds specifically to proline rich regions

what are the roles of PTB and Ph domains

PTB- phosphotyrosine binding domain - locates to activated receptor


Ph- pleckstrin homology domain - sticks to PM

what does the MAPK pathway consist of

Grb2 SH2/SH3 domain containing protein which interacts with SOS


SOS acts as a GDP/GTP exchange factor activating RAS


RAS activates RAF which activates MAPK-MAPKK


this drives differentiation, survival and growth

highlight what happens in the CAP/Cbl/Tc10 pathway

Insulin receptor in caveolae/lipid raft with Cbl and Tc10


Cbl phosphorylated recruits CrKII-C3G complex to the Caveolae/lipid raft


C3G serves as guanine nucleotide exchange factor for TC10 - activating it


Active TC10 causes translocation of GLUT4 vesicles

What is the NCK

Non-catalytic region of tyrosine kinase - causes cytoskeletal reorganisation



What are some outcomes of the insulin receptor

- gene transcription


- stimulation of receptor tyrosine kinase


- protein synthesis


- glucose transport


- glycogen synthase


- cell devision


- enzyme activity

outline the life of glucose

Glycogen - glucose - pyruvate -fat, protein, TCA cycle

what does the removal of lactate from muscle to live reduce

metabolic burden on muscle

outline the Free Fatty Acid pathway

hormone (epinephrine) activates 7TM receptor, resulting in GTP binding to Adenyl cyclase catalysis ATP to cAMP, this activate a protein Kinase


this phosphorylates Triacyglycerol lipase which turns Triacyglycerol to Diacyglycerol


addition of this to other lipases results in free fatty acids and glycerol

outline the Glucose/fatty acid cycle

in diabetes/starvation


- increase in fatty acid levels


- increase in beta oxidation - increase in NADH


- pyruvate dehydrogenase inhibited by acetyl-CoA hence no glucose to pyruvate


- increase fatty acids to muscle


- increase citrate, leaves mitochondria and inhibits phosphofructolinase hence reducing glucose utilisation

if insulin fails it....

stops the liver producing glucose and stimulates muscles and adipocytes to take up glucose

How is equilibrium reach with glucose levels

insulin shuts down glucose production from the liver and stimulates uptake to tissues




glucagon increases the breakdown of glycogen to give glucose, stimulates release of FFA from adipocytes

when is glucagon needed

where glucose is needed - level falls after food

highlight the PI3K arm

IRS - binds to PIK3, this adds a phosphate group to PIP2 changing it to PIP3 so it remains membrane bound, activates PDK1/2, recruits PKB (Akt) and phosphorylates


PKB is the central protein for 4 pathways

which pathways is PKB the central protein for

mTor, GSK3, PDE3B Fatty acid/Glycerol, Glut 4 translocation

what are the two subunits of PI3K

P85 which contains 2x SH2 domains and 1 SH3


P110 - catalytic subunit

which position does PI3K add a phosphate to on PDK1/2

3rd position of the inositol ring

What is a PH domain

100 aa highly variable primary acid sequence


binds calcium and range of lipids


provides a way for a protein to be activated and associated with the cytoplasmic face of PM

Outline the mTor pathway

mamalian target of rapamycin


- TSC2/1 when non-phosphorylated (active) inter- acts with Rheb this breaks down GTP faster thus inactivating Rheb and shutting down the pathway




- is inactivated and no longer speeds up hydrolysis thus Rheb is active longer and GTP bound, so pathway is active



outline the glycogen synthase pathway

increased glucose uptake


GSK3 inactivated by PKB so cannot phosphorylate GS and cannot inactivate it


inactive glycogen synthase = glycogen synthesis


insulin activates protein phosphatase-1 PP1 which de-phosphorylates GS

what does GSK3 act on other than GS

phosphorylates eIF2B


de-phosphorylated eIF2B activates protein synthesis

what is the result of PKB activation of glycogen synthesis and protein synthesis

they both increase



the effect of insulin on GS

- inhibits the actions of GSK3 - less GS phosphorylated


- activates PP1 which de-phosphorylates GS pathway


- less phosphorylated GS, more active GS, more glycogen made

when is GS inactive

when it is phosphorylated!

what does a kinase do

adds a phosphate group in presence of ATP

what does a phosphatase do

removes a phosphate group

what is the role of HSL

Hormone sensitive lipase - key lipase found in adipose tissue, converts triacyglycerols to glycerol and fatty acids

what is the effect of insulin of Fatty acid/glycerol control

inhibits lipolysis by action on HSL and perilipin


lowers FFA levels


lowers cAMP levels by activating phosphodiesterase 3B (PDE3B) thus reducing PKA activity and reducing levels of phosphorylated HSL

how does insulin control the ffa/glycerol pathway

stimulates uptake of fatty acids by activation of an extracellular lipoprotein lipase


inhibits the breakdown and release of fatty acids by deactivating HSL


lipolysis inhibited by perilipin on the surface of the fat droplet

name some Type I diabetes treatments

insulin injections


insulin pumps/patches/sprays/gels


islet transplants

what could be problematic to increasing levels of insulin to overcome resistance

may cause complications in other signalling pathways

what are some future treatments for Type I diabetes

improved islet transplants


new B cells from embryonic stem cells and/or regeneration

what are some type II diabetes treatments

change in lifetsyle/diet


drug intervention

what are the three categories of drug intervention for Type II diabetes

Sensitisers - make system more responsive


secretagogues - make more insulin


insulin



give two examples of sensitisers for type II diabetes

metformin


rosiglitazone

give an example of secretagogues for type II diabetes

sulphonylureas

what can be a problem associated with secretagogues

can accelerate the failure of the pancreas

what is a stage 3 drug that excretes excess glucose, how it works and a side effect

Dapagliflozin


selective, competitive inhibition of sodium-glucose co-transporter 2 in kidney


causes weight loss