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192 Cards in this Set
- Front
- Back
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 |
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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 |
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list the advantages of using purified proteins |
Advantages - ease of manipulation - clean data, easy to control conditions and incorporate controls - reproducible - molecular data points |
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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 |
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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 |
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what are the two ways immortalisation can occur |
through modification or as a result of from where the sample was derived |
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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 |
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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 |
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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 |
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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 |
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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 |
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Advantages of using humans for studies |
truly physiological great feedback model of specific diseases available |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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what is FRET |
FRET is a molecule that we use to detect cAMP levels using fluorescence resonance energy transfer microscopy |
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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 |
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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 |
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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 |
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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 |
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what are some inhibitors of Kinases available for studies |
straurosporine - inhibit a range wortmannin PI-3 kinases Rapamycin p70s6 kinases PD98059 MAP kinase pathway |
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What makes a kinases inhibitor more specific |
if they block the substrate binding region and not the ATP binding region |
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list some activators of kinases |
phorhol esters - tumour promotors PKC 8- BrcAMP (3'5' cycling monophosphate) |
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what is the problem with phosphorylation studies |
as fast as phosphate is added, it can be removed using a phosphatase |
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list some phosphatase inhibitors available |
okadaic acid - inhibits protein serine/threonine phosphatase vanadate - inhibits protein tryosine phophastase |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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how many chromophores are there for three colours |
one |
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where are the genes for green and red colours of the eye |
x chromosome recombination can result in altered gene levels |
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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 |
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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 |
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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 |
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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 |
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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 |
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list some symptoms of trisomy 21 |
down syndrome upper respiratory infections cardiac abnormalities GI/feeding problems speech/language difficulty visions/hearing problems behaviour problems |
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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 |
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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 |
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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 |
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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 |
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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 |
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what do mitotic drugs do to the cell |
take it into cell arrest due to chronic activation of spindle check point |
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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 |
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list some evaluated and used mitotic drugs |
CDK1, AuRKA, MTAs, PLK1, AURKB |
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list some potential emerging mitotic drugs |
PLK4, NEKs, MASTL, HASPIN, BUB1, BUBR1, Kinesins, Separase |
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list some microtubule targeting agents |
paclitaxel - breast cancer, ovarian docetaxel - breast cancer, prostate estramustine - prostate |
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list some anti-cyclin dependent kinase agents |
flavopiridol dinaciclib both interfere with mitosis and transcription |
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list some aurora kinase inhibitors |
danusertib alisertib |
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list some polo kinase inhibitors |
BI2536 Volasertib |
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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 |
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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 |
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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 |
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what are the pitfalls of chemotherapy |
cytotoxity drug resistance intrinsic (heterogenous cell population within the tumour) acquired |
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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 |
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why did mTOR only looked promising |
plays a part in protein synthesis leading to cyclin D1 ACT pathway could compensate when mTOR inhibited |
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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 |
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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 |
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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 |
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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 |
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what are the best candidate genes to date for OA |
GDF5 RUNX2 PTHLH SMAD3 |
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what are the current treatments for OA |
surgery Non- steroidal anti-inflammatories identify key proteinases and target genetic screening may allow to see susceptibility |
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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 |
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what is the rheumatoid factor and what does it do |
autoantibody IgM binds IgG |
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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 |
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what factors rasie your chances of getting RA |
smoking and drinking to much coffee |
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what lowers your chances of getting RA |
mediterranean diet, anti-oxidants, alcohol and 0oestrogens |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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when does cdk activity turn off |
after anaphase until half way through G1 |
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which cyclins bind to which CDKs |
cyclin D - CDK4/6 Cyclin E-CDK2 Cyclin A- CDK2 Cyclin B- CDK1 |
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what did sea urchin eggs result in the discovery of in regards to cell cycle |
first demonstartion of periodic protein degradation |
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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 |
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What did Xenopus eggs result in the discovery of |
discvory of MPF |
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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 |
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which CdKs are responsible for which phases of cell cycle |
CDk4 & 6 - G1 CdK 2 - G1/S, possibly M CdK1 - M phase |
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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 |
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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 |
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where do Wee1 and Myt1 phosphorylate the complex |
Tyr15 and Try14 (myt) |
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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 |
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what are two examples of CkS1 proteins in humans |
CksHs1 and CksHs2 |
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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 |
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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 |
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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 |
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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 |
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what is a ring finger |
small zinc binding domain in SCF it is Rbx1 in APC it is Apc11 |
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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 |
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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 |
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what is the rate of DNA synthesis |
50-100 nucleotide/second |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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What is nuclear accumulation promoted by |
phosphorylation of cyclin B1 and Cdc25C de-phosphorylation of Ser216 and Cdc25C |
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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 |
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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 |
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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 |
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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 |
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Is the spindle checkpoint active or inactive in - amphitelic configuration - monotelic configuration - syntelic configuration - merotelic configuration |
-inactive -active -active -inactive |
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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 |
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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 |
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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 |
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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 |
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What is the prophase pathway |
destruction of cohesion ring without APC activity mediated by phosphorylation by cohesion cohesion falls off arms of chromosomes |
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why is cohesion not broken down sooner than it is |
Sgo1-PP2A protects cohesion during pro-metaphase, counter acts phosphorylation |
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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) |
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What is the synaptonemal complex |
complex that holds corssing over chromosomes together species that holds them together - lateral elements |
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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 |
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when do female eggs stop in the cell cycle |
stop in prophase 1 of meiosis 1 |
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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 |
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how does the cohesion complex differ between mitosis and meiosis |
mitosis - SmC/3 Scc 1/3 meiosis - Smc1 B, Rec8, STAG3 |
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which subunit is the one cleaved of cohesion |
mitosis - scc1 meiosis - rec8 |
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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 |
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what is depletion |
reduced recruitment of protection, disruption of chromosome structure, impaired bioorientation |
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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 |
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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 |
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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 |
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what is the result of nondisjunction at meiosis II |
n+1, n+-1 and n x2 |
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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 |
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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 |
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How do eggs stay in arrest |
EMI2 block APC activity, maintaining low activity at metaphase II, cyclin can't be destroyed |
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How is arrest broken at fertilisation |
via calcium mediated pathway (oscillations) |
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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 |
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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 |
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give an example of a GPCR that has fast activation and slow deactivation |
Rhodopsin |
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give an example of a GPCR that has slow activation and fast de-activation |
B2 adrenergic |
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who first identified insulin in 1921 and on what animal were the tests done |
Banting and Best on their dog |
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where is insulin released from |
pancreatic B cells |
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what two main responses happen after a carbohydrate-containing meal |
stimulates insulin secretion inhibits glucagon secretion |
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what is Glucokinase known as |
the glucose sensor has a low affinity for glucose |
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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 |
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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 |
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how doe the insulin receptor relay its signal |
acts as a tyrosine kinase - auto phosphorylation on tyrosines |
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which tyrosine residue is required for substrate binding |
960 |
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which tyrosine residues phosphorylation leads to kinase activity |
1146, 1150, 1151 |
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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 |
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what is an SH2 domain, what does it do |
Src homology 2 domain binds phosphotyrosine residues surrounded by unique sequences |
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what is an SH3 domain and what does it do |
Src homology 3 domain binds specifically to proline rich regions |
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what are the roles of PTB and Ph domains |
PTB- phosphotyrosine binding domain - locates to activated receptor Ph- pleckstrin homology domain - sticks to PM |
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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 |
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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 |
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What is the NCK |
Non-catalytic region of tyrosine kinase - causes cytoskeletal reorganisation |
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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 |
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outline the life of glucose |
Glycogen - glucose - pyruvate -fat, protein, TCA cycle |
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what does the removal of lactate from muscle to live reduce |
metabolic burden on muscle |
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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 |
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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 |
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if insulin fails it.... |
stops the liver producing glucose and stimulates muscles and adipocytes to take up glucose |
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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 |
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when is glucagon needed |
where glucose is needed - level falls after food |
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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 |
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which pathways is PKB the central protein for |
mTor, GSK3, PDE3B Fatty acid/Glycerol, Glut 4 translocation |
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what are the two subunits of PI3K |
P85 which contains 2x SH2 domains and 1 SH3 P110 - catalytic subunit |
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which position does PI3K add a phosphate to on PDK1/2 |
3rd position of the inositol ring |
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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 |
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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 |
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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 |
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what does GSK3 act on other than GS |
phosphorylates eIF2B de-phosphorylated eIF2B activates protein synthesis |
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what is the result of PKB activation of glycogen synthesis and protein synthesis |
they both increase |
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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 |
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when is GS inactive |
when it is phosphorylated! |
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what does a kinase do |
adds a phosphate group in presence of ATP |
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what does a phosphatase do |
removes a phosphate group |
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what is the role of HSL |
Hormone sensitive lipase - key lipase found in adipose tissue, converts triacyglycerols to glycerol and fatty acids |
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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 |
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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 |
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name some Type I diabetes treatments |
insulin injections insulin pumps/patches/sprays/gels islet transplants |
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what could be problematic to increasing levels of insulin to overcome resistance |
may cause complications in other signalling pathways |
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what are some future treatments for Type I diabetes |
improved islet transplants new B cells from embryonic stem cells and/or regeneration |
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what are some type II diabetes treatments |
change in lifetsyle/diet drug intervention |
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what are the three categories of drug intervention for Type II diabetes |
Sensitisers - make system more responsive secretagogues - make more insulin insulin |
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give two examples of sensitisers for type II diabetes |
metformin rosiglitazone |
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give an example of secretagogues for type II diabetes |
sulphonylureas |
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what can be a problem associated with secretagogues |
can accelerate the failure of the pancreas |
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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 |