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113 Cards in this Set
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Sarcoglycan complex
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a-, b-, d-, g-sarcoglycan transmembrane proteins
Tightly associated with sarcospan SGs = single pass glycoproteins NO extracellular binding partners |
(proteins, association, binding partners)
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Genes involved in limb girdle muscular dystrophies
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alpha, delta, beta, gamma sarcoglycan
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MD resulting from loss of a-dystroglycan
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Congenital (CMD)
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MD resulting from loss of dystrophin
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Duchenne/Becker (BMD)
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MD resulting from loss of SGs
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Limb Girdle (LGMD)
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How to test sarcoglycan deficiencies
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Immunostaining and Western blot w/ antibodies against SGs
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alpha-SG deficient human muscle
1) loss of 2) reduction in 3) intracellular labeling? 4) dystrophin preserved? |
1) a-SG
2) d-, b-SG 3) slight intracellular labeling 4) dystrophin well preserved |
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beta-SG deficient human muscle
1) loss of 2) reduction in 3) intracellular labeling? 4) dystrophin preserved? |
1) all SGs are absent
2) b-dystroglycan 3) no labeling 4) dystrophin well preserved |
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gamma-SG deficient human muscle
1) loss of 2) reduction in 3) intracellular labeling? 4) dystrophin preserved? |
1-2) varies between patients
- some have dramatic reduction in expression - others close to normal 3) no IC labeling 4) dystrophin well preserved |
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Animal model for SG deficiency?
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Sgca-deficient mice
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How to evaluate sarcolemma permeability in Sgca-deficient mice
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1) Inject hetero and homozygous null mice with EBD
2) dye will accumulate in muscle of Sgca-null mutants |
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muscle-specific pyruvate kinase activity in 7-10-wk-old mice (+/+, null, mdx, heterozygous)
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high levels of PK released from -/- and mdx muscle fiber compared to +/- and +/+
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What do you expect to see when performing SDS-PAGE followed by an immunoblot using antibodies against various DGC components on Sgca-deficient mouse muscle?
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The -/- mutant will almost completely lack a, b, g, and d-SG, as well as a-DG
b-DG is a little lower and DYS is conserved compared to the +/+ |
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What additional information do you gain from performing an immunofluorescence analysis (Ab's against same DGC components as in Western blot) of sarcolemma proteins in Sgca-deficient muscle
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All SGs are still drastically reduced. However, dystrophin staining is reduced in the sarcolemma of skeletal muscle in Sgca-null mice
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CMS
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Congenital Myasthenic Syndrome - genetic flaws at NMJ; like myasthenia gravis, except CMS is hereditary
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CMS by AChR defects
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1) those causing reduced AChR expression
2) those altering its kinetic properties |
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Kinetic defects in AChR causing CMS
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1) slow-channel: increased syn response to ACh
2) fast-channel: decreased syn response to ACh |
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Cause, effects of slow-channel CMS
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cause: prolonged opening of AChR
effects: myopathy manifested by degeneration of the junctional folds, loss of AChR from the folds |
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Slow-channel CMS: therapy
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quinidine and fluoxetine (open-channel blockers of AChR that shorten the duration of channel opening events in a concentration dependent manner)
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Concerns with fluoxetine
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Increased suicidal behavior in depressed adolescents and children
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Fast-channel CMS: symptoms
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Reduction in AChR expression
Fast decay of endplate current Varied clinical severity |
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Fast-channel CMS: treatment
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combination of 3,4-diaminopyridine and cholinesterase inhibitors (prevent degradation of ACh)
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What to look for to determine CMS diagnosis
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1) titration of anti-ACh Ab's in serum
2) electromyography (test for muscle contraction) 3) electrophys of NMJ (test repetitive motor response, increments; search for NM block) 4) muscle biopsy (eliminate diagnosis of myopathy) |
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Two elements of a decisive CMS diagnosis
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1) Absence of AChE at NMJ (signifies AChE deficiency = slow-channel CMS)
2) reduction in AChR numbers |
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Myasthenia gravis: what and how
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auto-immune disorder resulting from failure of neuromuscular transmission
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Myasthenia gravis: mechanism
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Auto-Ab's (against nicotinic AChR or MuSK) bind to proteins involved in NMJ signaling
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Myasthenia gravis: characteristics
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- presence of Ab's at NMJ
- Ig injections from MG patients cause symptoms - anti-AChR Abs from animals cause symptoms - immunization of animals with AChR reproduces the disease - removal of Abs decrease severity of symptoms |
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MG: progression of symptoms
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- begin at extrinsic ocular muscles (EOMs)
- progress to other bulbar and limb muscles (generalized MG) |
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NMJs of EOM: characteristics
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- less prominent synaptic folds
- fewer post-syn AChRs and Na+ channels - reduced safety factor |
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NMJs of skeletal muscle: characteristics
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- greater quantal content
- greater post-syn folding - higher postsynaptic sensitivity to ACh - increased Na+ current in the region |
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NMJs of EOM: susceptibility to MG
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high neuronal firing frequency = more fatigue
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NMJs of skeletal muscle: susceptibility to MG
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properties may make fast-twitch skeletal muscle fibers less susceptible to myasthenic failure than slow-twitch fibers.
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Myasthenia gravis - Mechanisms of anti-AChR Ab's in disrupting NM transmission
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1) binding and activation of complement at the NMJ
2) accelerated degradation of AChR molecules crosslinked by Ab (antigenic modulation) 3) functional AChR block |
1) complement
2) degradation 3) block |
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Myasthenia gravis - Mechanisms of anti-AChR Ab's in disrupting NM transmission:
Binding and activation of complement |
- formation of membrane attack complex (MAC)
- localized destruction of post-syn membrane - simplified morphology of post-syn NMJ membrane (lacks normal folds) |
effect on folding, surface morphology
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Myasthenia gravis - Mechanisms of anti-AChR Ab's in disrupting NM transmission:
Degradation of crosslinked AChRs |
- Ab's crosslink AChRs
- cause endocytosis and degradation of linked AChRs (called antigenic modulation) - leads to reduced # of AChRs on membrane |
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antigenic modulation
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The endocytosis and resulting degradation of crosslinked AChR's in the post-synaptic membrane of the NMJ.
Caused by binding of auto-Ab to AChRs on the post-syn membrane. Prevents muscle activation |
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IgG from MG patients: effect on AChR in vivo/vitro
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causes antigenic modulation
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Myasthenia gravis - Mechanisms of anti-AChR Ab's in disrupting NM transmission:
Functional block |
Auto-Ab's bind to AChRs and prevent function by preventing ACh binding
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Why do MG patients with AIDS see relief of their myasthenic symptoms?
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AIDS patients have reduced numbers of the AChR-specific CD4+ T cells required for the development of MG symptoms
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Blocking muscle activity: effect on viability of motor neurons
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prevents developmental death
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Direct stimulation of muscle: effect on viability of motor neurons
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enhances death of motor neurons
- activity in target could inhibit production of trophic factors |
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Neurotrophic factor hypothesis: findings leading to
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blocking activity = prevents death
stimulation of muscle = enhances death |
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Neurotrophic factor hypothesis
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1) neurons extend axons to vicinity of target cells
2) target cells secrete neurotrophic factors, which bind to cell surface receptors 3) neurons that do not receive enough neurotrophic factor die by apoptosis |
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NGF
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- best characterized neurotrophin, stimulates growth, differentiation, survival and maintenance of neurons during development and after injury
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NGF: produced by
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accessory cells (astrocytes, oligodendrocytes)
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NGF: precursor form
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proNGF
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generating NGF from proNGF
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post-translational processing (cleavage of proNGF by plasmin and MMP7 = generation and secretion of beta-NGF)
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neutrophins
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NGF, brain-derived neurotrophic factor (BDNF), neutrophin 3 (NT3), NT4/5
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neurotrophin receptors
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1) tropomyosin-related kinase (Trk) family (A, B, and C)
2) p75 neurotrophin receptor (p75NTR) - member of tumor necrosis factor receptor superfamily |
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NT binding of Trk
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by extracellular domains (Ig-1 is extracellular)
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mutation in Ig-1 domain of trkA can abolish NGF binding
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Effect of deleting Ig-1 and/or 2
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increases spontaneous receptor dimerization and activation
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the Ig domains normally inhibit dimerization in absence of a ligand
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p75 NTR: what does it do?
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enhances specificity of trkA and trkB for primary ligands (NGF and BDBF)
- increases rate of NGF association w/ TrkA |
involves trk and NGF
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Functions of Trk receptors
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1) proliferation and survival
2) axonal/dendritic growth and remodeling 3) modifications of synaptic functions 4) cytoskeletal remodeling 5) membrane trafficking |
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Factors in Trk receptor activation
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- requires neutrophins
- presence of p75 NTR, which regulates Trk receptor activation by neutrophins |
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Signal transduction pathway triggered by NGF
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1) NGF binds
2) dimerization of trkA 3) biochemical cascade - phosphorylation of Tyr's in cyto domain - potentiation of TK activity - further P-ylation creates docking sites for adaptor proteins |
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PLC-gamma-1 signaling: chain of events
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1) phosphorylation of Y785 on TrkA
2) Y785 binds PLC-gamma1 and activates it 3) PLC-gamma1 hydrolyzes PIP2 --> IP3 and DAG 4a) IP3 --> release of Ca2+ 4b) DAG -->stimulates DAG-regulated PKC isoforms pathways activated by PLC-gamma1 extend to the nucleus |
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PKCdelta in PC12 cells: what is it required for?
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NGF-promoted neurite outgrowth and activation of Erk1 and Erk2
- acts between Raf and MEK |
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What is required for the neutrophin-promoted differentiation of neurons and PC12 cells?
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activation of Ras-MAPK/Erk signaling cascade
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activation of a signaling cascade
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PI3 kinase: relevance to cell survival
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PI3 kinase activates the PK Akt --> phosphorylates proteins important to cell survival
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Akt action
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phosphorylates "Bad", which allows 14-3-3 proteins to bind to Bad, preventing Bad from promoting apoptosis
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Akt and IkB relationship
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phosphorylation of IkB by Akt promotes IkB degradation = liberation of active NFkB (promotes neuronal survival)
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NFkB and IkB relationship
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IkB inhibits NFkB
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NFkB action
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activates gene transcriptions that promote neuronal survival
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effect of adding NGF to PC12 cells
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PC12 cells terated with NGF turn into neurons
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how to eliminate the effects of NGF in mice
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1) add function blocking anti-NGF Ab's
2) null mutation in NGF gene or trkA gene |
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effect of excess NGF during development
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prevents naturally occurring death of neurons
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Apoptosis
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cell death resulting from activation of a genetically determined suicide program
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Features of an apoptotic cell (3)
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1) condensation of cyto and nuclear contents
2) blebbing of plasma membrane 3) fragmentation of nuclei and breakdown into membrane-bound apoptotic bodies |
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Evidence that lack of neurotrophic factors leads to apoptosis
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1) neurons maintained in presence of NGF
2) NGF removal leads to cell death 3) inhibition of protein/RNA synth at time of NGF removal = neuron survival (suggests NGF suppresses an endogenous death program) |
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Genes controlling apoptotic program
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ced-3 and ced-4
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gene negatively regulating apoptotic program
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ced-9 (prevents activation of 3, 4)
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cell death pathway in vertebrates
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Apaf-1 + ATP activates caspases, which cause cell death
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inhibition of cell death in vertebrates
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Bcl-2
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mechanism of caspase activation
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Apaf-1 + ATP + pro-caspase --> cleaved caspase --> cell death
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effect of NGF on cell death pathway in vertebrates
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NGF activates Bcl-2, which inhibits Apaf-1
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intrinsic apoptotic pathway
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activation of cytochrome C --> activ. of caspases
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extrinsic apoptotic pathway
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activ of death receptors --> activ of caspases
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three apoptosis pathways
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1) mitochondria-mediated cell death pathway
2) death receptor pathway 3) ER-stress induced pathway |
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mitochondria-mediated cell death pathway
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- activation of Bax and Bid
- release of cyto. c - cyto c + caspase 9 + Apaf-1 --> apoptosome (leads to caspase activation and cell death) |
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Bcl-2 family: antiapoptotic proteins
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Bcl-2, Bcl-xL, Bcl-w
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Bcl-2 family: proapoptotic proteins
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BAX, BOD, BOK, BAD
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regulatory genes controlling Bcl-2 protein levels
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p53 (upregulation during apoptosis)
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effect of blockage of p53
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Pcl-2 upregulation --> blocks Bax --> survival
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what promotes cytochrome C release?
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high Bax/Bcl-2 ratio
- Bax is proapoptotic - Bcl-2 anti |
relationship of Bax and Bcl-2
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pathway most associated with neuronal apoptosis
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mitochondrial pathway (intrinsic)
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inhibitor of caspase-9, 3
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IAP (inhibitor of apoptosis protein)
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Sarcoglycan complex
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a-, b-, d-, g-sarcoglycan transmembrane proteins
Tightly associated with sarcospan SGs = single pass glycoproteins NO extracellular binding partners |
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Genes involved in limb girdle muscular dystrophies
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alpha, delta, beta, gamma sarcoglycan
|
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MD resulting from loss of a-dystroglycan
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Congenital (CMD)
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MD resulting from loss of dystrophin
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Duchenne/Becker (BMD)
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MD resulting from loss of SGs
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Limb Girdle (LGMD)
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Smac/Diablo effect on mitochondrial pathway
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neutralizes inhibitory effects of IAPs = promotion of caspase activation
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ER pathway induced by
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misfolded/aggregated proteins, other stresses in ER
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results of ER stress
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1) Ca2+ release
2) activation of mitoch. pathway via caspase-12 3) activation of independent ER-assoc caspases |
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ER stress procaspase, effect
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procaspase-12 --> cleaved to form caspase-12 --> activates caspase-9 and 3
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caspase target factors
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DFF and PARP, result in DNA fragmentation and cellular degradation
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cytochalasin
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causes local depolymerization of actin, resulting in "turning" of axon away from cytochalasin
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taxol
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stabilizes microtubules, causing axon to turn towards taxol
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nocodozole
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destabilizes microtubules on one side, resulting in turning of axon away from nocodozole
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molecular guidance cues: extracellular matrix adhesion
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growth cone extends on an extracellular matrix component
laminins in basal laminae interact w/ integrins on growth cones |
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molecular guidance cues: cell surface adhesion
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cell aggregation assay --> see effect of cadherins (adhesive molecule on cells)
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molecular guidance cues: adhesion - dystrophin complex
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dystrophin complex responsible for binding of laminin to muscle fiber at the sarcolemma membrane
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Nissl staining: what it does
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for staining cell bodies (NOT axon tracts)
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Cell adhesion molecule L1: what it stains
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axon tracts
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chemoattractant molecules
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NGF
netrin (in axons expressing Dcc) |
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effect of mutations in netrin and DCC
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disruption of guidance (reduced crossing of floor plate)
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DCC in colorectal cancer
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deleted
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netrin induction of attractive axonal growth
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netrin binds to DCC and activates a signaling cascade that triggers cytoskeletal remodeling, axon formation, and local protein synthesis in the axon
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Src, it's inhibitor, effect
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Src tyrosine kinase - required for netrin-induced axon outgrowth
PP2 inhibits Src, preventing outgrowth |
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chemorepulsion: molecules and mechanism
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Sema 3A (semaphorin)
causes cascade that disrupts cytoskeleton dynamics |
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role of cAMP in axon guidance
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cAMP modulates axon's response to netrin (higher cAMP = greater response to netrin)
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Slit and receptor
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Slit: repellant ligand secreted by floor plate
Robo is the Slit receptor |
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Slit: effects on pre- and post-floor-plate-crossing axons
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Slit repels post-crossing axons
does not affect pre-crossing |
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