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Triamcinolone reduces IGF-I expression in diaphragm (PMID: 9872851).
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Six or 24 hours of 10-1000 nM Dexa determined increased release of radioactive
 tyrosine from rat L8 fully differentiated myotubes.
 In these cells, Dexa upregulated the expression of m-calpain and cathepsins
 D and B
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key "hong1995effects"

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.
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According to PMID: 9083257, L6 myotubes do not change protein degradation
 rate when treated with Dexa.
 Dexa treatment of L6 
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myotubes
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 upregulates p85 regulatory of PI3 kinase, but decreases PI3 kinase catalytic
 activity bound to IRS-1, probably because p85 alpha confines p110 to the
 cytosol (PMID: 9054447).
 In L6 cells, Dexa downregulates total and phosphorylated IRS-1, and leaves
 S6 kinase activity unchanged (PMID: 9468291).
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C2C12 express MyoD in response to Dexa (PMID: 8744946).
 C2C12 cells do not express MyoD in response to Dexa (PMID: 9187537).
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C2C12 express IGFBP-2 in response to Dexa or to IGF-I (PMID: 8691100).
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C2C12 increase protein synthesis in response to IGF-I when they divide,
 but do not respond any longer when 
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fusing
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 (PMID: 9042337).
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C2C12 fusion is no affected by Dexa (PMID: 9011578).
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C2C12 differentiation is stimulated by Dexa (PMID: 9674941).
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Dexa reduces C2C12 calcium influx by non-transcriptional means (PMID: 9756393).
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Dexa reduces protein synthesis rate in cultured bovine myotubes (PMID: 9781494).
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In a model where C2C12 cells are treated with Dexa, they proliferate less
 (rather unusually), downregulate protein synthesis, and upregulate protein
 degradation (all by tracer).
 (PMID: 8791198).
 T has no significant effect on any of these.
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Possibly useful:
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PMID 61389 in 1976 clams that athletes taking some AAS get higher cortisol.
 
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PMID 3110538 claims exercise increases cortisol.
 PMID 8371654 adds that detraining lowers cortisol.
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PMID 9153447: oxandrolone is as good as prednisone in Duchenne muscular
 dystrophy.
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In chronic heart failure, PMID 9316530 says cortisol / DHEA ratio has a
 negative correlation with BMI.
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Minetto (PMID 20139231) says muscle force is increased with Dexa in healthy
 subjects, and electromyography finds less fatigability.
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PMID 22606232: ketoconazole inhibits endogenous cortisol, but has no effect
 on muscle protein synthesis or degradation
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PMID 22936724: dexamethasone stimulates myogenesis in C2C12 and human
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22936724 is a trial of testosterone and GC; GC reduce endogenous T; GC +T
 is better anabolic than T alone for anabolism
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PMC1474207 says Dexa increase Na, K ATPase activity in some muscle
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PMID 621843: The abuse of doximetasone, a synthetic GC, is also associated
 with hirsutism.
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PMC4039250 says that fiber type distribution does not change with age
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SEE MUSCLE TYPE (a mixed muscle; see PMID: 8847313, PMID: 10090572, PMID:
 22013216)
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You swore to re-read Troncose, Chu201, Baehr.
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Not read:
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contrasts "two-way anova"
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The Ras-Raf-MEK-ERK pathway affects fiber type composition without significant
 effects on muscle fiber size, whereas activation of the PI3K/AKT pathway
 induces muscle hypertrophy by regulation of GSK and mTOR kinases.
 (PMID: 21747283)
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Sandri et al.
 demonstrated that IGF-1 acts through AKT and FoxO1 to suppress atrogin-1
 transcription and that the mTOR/S6K, GSK and NFκB pathways are not important
 in regulating atrogin-1 expression.
 (PMID: 21747283)
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the ability of IGF-1 to prevent ang II-induced wasting was mediated by an
 AKT- and a FoxO-1-dependent signaling pathway that resulted in inhibition
 of atrogin-1 but not of MuRF-1 expression (PMID: 21747283)
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Glucocorticoids have been shown to be required for stimulation of the ubiquitin-
proteasome pathway in diabetes, fasting and metabolic acidosis, and we have
 shown that ang II infusion increases glucocorticoid levels and that RU486,
 a glucocorticoid receptor antagonist, significantly blocked the ang II-induced
 weight loss.(PMID: 21747283)
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However muscle also has a characteristic pattern of amino acid metabolism:
 the branched-chain amino acids leucine, isoleucine and valine are rapidly
 degraded as are other nonessential amino acids, including alanine, glutamate
 and aspartate.
 However unlike other tissues, muscle does not degrade the carbon skeletons
 of several other amino acids such as phenylalanine and tyrosine.
 Leucine is an important energy source for muscle tissue particularly during
 fasting, and under these conditions leucine inhibits oxidation of glucose.
 In muscle the breakdown of branched-chain amino acids is accelerated after
 injury or during fasting, and yields amino groups, which are used for synthesis
 of alanine (from pyruvate) and glutamine (from glutamate).
 The de novo synthesis of alanine and glutamine is coupled to degradation
 of the branched-chain amino acids, and alanine and glutamine are normally
 exported from muscle in much larger amounts than predicted simply from
 their occurrence in muscle protein.
 Alanine is avidly taken up by the liver and used for gluconeogenesis, but
 glutamine is an important energy source for cells such as leucocytes and
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fibroblasts and is also metabolised extensively by both the gut and kidney.
 Glutamate is central to all transamination reactions in muscle, and is
 one of the most abundant amino acids in proteins and in the free amino
 acid pool in skeletal muscle, but is present at a low concentration in
 plasma.
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In one study of eight underweight patients with emphysema and muscle wasting,
 reduced plasma concentrations of glutamine, glutamate and alanine were
 reported [32].
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In humans, the techniques commonly used to estimate total protein turnover,
 or synthesis or degradation rates, include 3-methylhistidine (3-MH) excretion,
 amino acid balance studies and tracer studies employing infusion of stable
 isotopes of amino acids (13C, 2H or 15N).
 3-MH, acquires its methyl group after translation, is not re-utilised for
 protein synthesis or metabolised after release during proteolysis, and
 is excreted in the urine.
 As w90% of 3-MH residues are in
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actin and myosin in skeletal muscle, timed urinary excretion can be used
 to estimate whole body skeletal muscle proteolysis.
 3-MH can also be measured in blood draining from a limb to give an indication
 of proteolysis in the limb muscle.
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In COPD, muscle wasting doesn’t change 3-MH → not caused by increased proteolysi
s.
 Same in hypoxia: proteolysis cannot increase because it actually requires
 energy.
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Muscle wasting causes: lack of IGF / insuline / T; hypoxemia / acidosis;
 inflammatory citokines (?); steroid treatment (gluco?).
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(DOI: 10.1183/09031936.03.00004608)
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eIF4G (Fig.
 12) is a large scaffolding protein, responsible for the assembly of the
 cap-binding
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complex and recruitment of the 43S IC to the 5k-cap of mRNA.
 It has binding sites for eIF4E,
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eIF4A (one in yeast, two in human), PABP, mRNA and other factors.
 Human eIF4G also binds
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to eIF3 whereas yeast eIF4G has not been found to bind eIF3, but was reported
 to bind eIF1 and
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eIF5.
 (Marintchev)
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Several studies have examined the effect of myostatin excess or deficiency
 on expression of
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atrogin-1 and MuRF1.
 Both of these E3 ubiquitin ligases were thought to be critical determinants
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of muscle protein degradation, but there is more evidence that atrogin-1
 interferes with protein
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synthesis via degradation of the translation factor eIF3f (3, 22).
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2010: The insulin-like growth factor-1 can block the transcriptional upregulatio
n of MuRF1 and MAFbx via the phosphatidylinositol-3 kinase/Akt/Foxo pathway.
 MuRF1's substrates include several components of the sarcomeric thick filament,
 including myosin heavy chain.
 Thus, by blocking MuRF1, insulin-like growth factor-1 prevents the breakdown
 of the thick filament, particularly myosin heavy chain, which is asymmetrically
 lost in settings of cortisol-linked skeletal muscle atrophy.
 Insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt signaling
 also dominantly inhibits the effects of myostatin, which is a member of
 the transforming growth factor-[beta] family of proteins.
 Deletion or inhibition of myostatin causes a significant increase in skeletal
 muscle size.
 Recently, myostatin has been shown to act both by inhibiting gene activation
 associated with differentiation, even when applied to postdifferentiated
 myotubes, and by blocking the phosphatidylinositol-3 kinase/Akt pathway.
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While inactivation of myostatin leads to muscle growth in vivo, excess levels
 of myostatin induces cachectic-like muscle wasting.
 Molecular analyses reveal that excess levels of myostatin induce Atrogin-1
 expression by reducing Akt phosphorylation and thereby increasing FoxO1
 activity.
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myostatin administration may induce the expression of Foxo1 and the subsequent
 up-regulation of the atrophy-inducing genes MuRF1 and MAFbx.
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Approximately 50% of cancer patients exhibit muscle
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wasting [73] that is primarily derived from protein
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hypercatabolism with little alteration in protein synthesis
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[6].
 In a cancer cachexia model where rats were injected
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with Yoshida AH-130 ascites heptoma cells, skeletal
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muscle mass and IGF-1 mRNAwere reduced while MAFbx
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and MuRF1 mRNA levels were elevated [18].
 Treatment
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with a combination of pentoxifylline (TNFα inhibitor) and
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formoterol (β2-adrenergic agonist) protected against muscle
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wasting as well as reducing MuRF1, but not MAFbx,
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mRNA levels.
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FoxO1 does not directly increase MAFbx or MuRF1
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levels but instead blocks the IGF-1 inhibition of their upregulation
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[105].
 FoxO3a binds directly to the MAFbx promoter.
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Ectopic overexpression of the eIF3a, eIF3b, eIF3c, eIF3h and eIF3i subunits
 leads to transformation of cultured NIH-3T3 cells.
 This probably occurs through the stimulation of global protein synthesis,
 as well as increased translation efficiency of specific mRNAs that are
 normally poorly translated, including cyclin D1 (Ccnd1), Myc, ornithine
 decarboxylase 1 (Odc1) and fibroblast growth factor 2 (Fgf2)
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The eIF4E–4E-BP complex might remain bound to the m7GpppN cap (as shown),
 possibly aiding the inhibition of cap-dependent mRNA translation owing
 to blockade of the mRNA 5′ end.
 Increased levels of hypophosphorylated 4E-BPs, in conjunction with elevated
 levels of eIF4G, can then function as a switch, as observed in certain
 locally advanced cancers, impairing the initiation of translation on purely
 cap-dependent mRNAs but enhancing translation of dual mechanism mRNAs that
 also contain an internal ribosome entry site (IRES) to which eIF4G may
 bind directly.
 These mRNAs include vascular endothelial growth factor A (VEGFA), fibroblast
 growth factor 2 (FGF2), BCL2 and hypoxia-inducible factor 1α (HIF1A).
 This switch is activated by hypoxia and other stresses to preserve tumour
 cell viability and promote tumour angiogenesis.
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Ectopic expression of eIF2Bε in the TA muscle rescued the sepsis-induced
 deficit in GEF activity and muscle protein synthesis.
 (doi: 10.​1152/​ajpendo.​00151.​2010)
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Muscle protein synthesis decreased 26% after exercise and was associated
 with a fourfold increase in the amount of eIF4E present in the inactive
 eIF4E ⋅ 4E-BP1 complex and a concomitant 71% decrease in the association
 of eIF4E with eIF4G.
 Refeeding the complete meal, but not the carbohydrate meal, increased muscle
 protein synthesis equal to controls, despite similar plasma concentrations
 of insulin.
 Additionally, eIF4E ⋅ 4E-BP1 association was inversely related and eIF4E
 ⋅ eIF4G association was positively correlated to muscle protein synthesis.
 ( http://ajpcell.physiology.org/content/274/2/C406.full )
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IGF-I/IGFBP-3 ameliorates alterations in protein synthesis, eIF4E availability,
 and myostatin in alcohol-fed rats
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Alcohol decreased protein synthesis in WT mice, a change associated with
 less 4EBP1 phosphorylation, eIF4E-eIF4G binding, and raptor-4EBP1 binding,
 but greater mTOR-raptor complex formation.
 However, the content of eIF2α was not altered in muscle from alcohol-fed
 rats (Table 2).
 Furthermore, administration of IGF-I/IGFBP-3 did not alter the content
 eIF2α in either control or alcohol-fed rats.
 eIF-2α also undergoes reversible phosphorylation, and the extent of eIF2α
 phosphorylation is inversely proportional to changes in protein synthesis
 (33).
 However, there was also no change in the phosphorylation status of eIF2α
 in response to either alcohol feeding or the binary complex.
 These data suggest that effects of the binary complex on protein synthesis
 are independent of changes in eIF2α protein and phosphorylation state.
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Simvastatin represses protein synthesis in the muscle-derived C2C12 cell
 line with a concomitant reduction in eukaryotic initiation factor 2B
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Women have less muscle than men but lose it more slowly during aging.
 To discover potential underlying mechanism(s) for this we evaluated the
 muscle protein synthesis process in postabsorptive conditions and during
 feeding in twenty-nine 65–80 year old men (n = 13) and women (n = 16).
 We discovered that the basal concentration of phosphorylated eEF2Thr56
 was ~40% less (P<0.05) and the basal rate of MPS was ~30% greater (P = 0.02)
 in women than in men; the basal concentrations of muscle phosphorylated
 AktThr308, p70s6kThr389, eIF4ESer209, and eIF4E-BP1Thr37/46 were not different
 between the sexes.
 Feeding increased (P<0.05) AktThr308 and p70s6kThr389 phosphorylation to
 the same extent in men and women but increased (P<0.05) the phosphorylation
 of eIF4ESer209 and eIF4E-BP1Thr37/46 in men only.
 Accordingly, feeding increased MPS in men (P<0.01) but not in women.
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eIF3i has been reported as TRIP-1, a phosphorylation substrate of the transforma
tion growth factor-β type II (TGF-βII) receptor, that acts as a negative
 modulator of TGF-β pathway [131] L.
 Choy and R.
 Derynck, The type II transforming growth factor (TGF)-beta receptor-interacting
 protein TRIP-1 acts as a modulator of the TGF-beta response,
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Guinea pig maseter and temporal muscle are rescued by testosterone after
 castration (Kochiakian, 1948).
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Semimembranous muscle: “adult rabbit fast-twitch semimembranosus accessorius
 (SMa; approximately 100% type II fibres) and the slow-twitch semimembranosus
 proprius (SMp; 100% type I fibre) “
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1.
 Brenet F, Socci ND, Sonenberg N, Holland EC.
 Akt phosphorylation of La regulates specific mRNA translation in glial
 progenitors.
 Oncogene.
 2009;28(1):128-139.
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Perfomed immunoblot for La on various fractions.
 Used A254 and Northern for 28S, 18S to confirm fractions.
 No pooling.
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Polyribosome profiling in glial progenitors
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N-tva/Z and N-tva/RA cells were incubated for 10 min with 0.1 mg/ml of cyclohexim
ide at 37°C and lysed in 2 ml of lysis buffer containing 125 mM NaCl, 100
 mM sucrose, 50 mM HEPES, 2 mM potassium acetate, and 40 U/ml of an RNase-inhibi
tor, RNasin (Promega).
 The homogenate was first centrifuged for 5 min at 4000 × g, and the supernatant
 was centrifuged again for 10 min at 14,000 × g.
 The resulting postmitochondrial supernatanst was then lysed in a buffer
 containing 50 mM Tris/HCl, pH 7.5, 1% NP40, 50 mM NaCl, 4 mM MgCl2, 45 µg/ml
 cycloheximide.
 The lysate was layered on top of 1.5 ml of 15% sucrose and centrifuged for
 10 min at 400,000 × g.
 The resulting pellet was resuspended in 500 µl of a buffer containing 20
 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 100 mM NH4Cl, 2 mM KAc, 225 µg/ml cycloheximid
e.
 The resulting sample was carefully layered onto a 15 to 40% linear sucrose
 gradient made in 20 mM Tris/HCl, pH 9.0, 80 mM NaCl, 3 mM MgCl2, and 0.02%
 b-mercaptoethanol.
 Gradients were centrifuged for 90 min at 41,000 rpm and fractions were
 collected at a rate of 1 ml per min.
 RNAs were extracted from 500µl of each fraction with Trizol (Invitrogen)
 according to the manufacturer instructions and the ribosomal subunits,
 monosomes, and polysomes were detected at a wavelength of 254 nm using
 a spectrophotometer.
 Proteins from the others 500µl of each fraction were TCA-precipitated overnight
 and analyzed by 10% SDS-PAGE and western blot.
 For EDTA treatment, EDTA was added to a final concentration of 30 mM before
 loading on the gradient.
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2.
 Genolet R, Araud T, Maillard L, Jaquier-Gubler P, Curran J.
 An approach to analyse the specific impact of rapamycin on mRNA-ribosome
 association.
 BMC Med Genomics.
 2008;1:33.
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Lung fibroblasts.
 Compares light and heavy polysomes (2-4 ribosomes vs.
 5+ ribosomes) in microarray.
 Used A254 to confirm fractions.
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MRC-5 cells (Coriell Cell Repository) were cultured in Minimal Essential
 Medium (Gibco) supplemented with 1 mM sodium pyruvate (Sigma), 0.1 mM non-essent
ial amino acids, 10% foetal calf serum (Brunschwig), 1% penicillin/streptomycin,
 in a humidified atmosphere containing 5% CO2.
 For polysome analysis, cells in the growing phase (60% confluence) were
 hypertonically shocked by shifting to medium containing 300 mM NaCl for
 50 min.
 They were then placed in normal isotonic medium for 30 min.
 When rapamycin was used, 100 nM rapamycin (LC laboratories) or 0.01% DMSO
 (the negative control) was added during the hypertonic shock, 20 min before
 the transfer back to isotonic conditions.
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After treatment, cells were scraped into the culture medium and pelleted
 for 4 min at 100 g.
 The pellets, consisting of 5 × 106 cells, were lysed for 15 min on ice
 in 400 μL of 100 mM KCl, 50 mM Tris-Cl pH 7.4, 1.5 mM MgCl2, 1 mM DTT, 1
 mg/mL heparin, 1.5 % NP40, 100 μM cycloheximide, 1% aprotinin, 1 mM AEBSF
 and 100 U/mL of RNasin.
 Nuclei were pelleted by centrifugation in a microfuge, 10 min at 12000
 rpm.
 The supernatant was loaded onto a 20–60% sucrose gradient (in 100 mM KCL,
 5 mM MgCl2, 20 mM HEPES pH 7.4 and 2 mM DTT).
 Extracts were fractionated for 3 h 30 min at 35,000 rpm at 4°C in a Beckman
 SW41 rotor, and the gradients were recovered in 3 fractions [monosome,
 light polysome (2 to 5 ribosomes) and heavy polysome (> 5 ribosomes)] using
 a Brandel gradient fractionator equipped with an ISCO UA-6 flow cell set
 to 254 nm.
 RNA was isolated from the light and heavy polysome fractions by adding
 an equal volume of TriZol (Invitrogen).
 Samples were mixed and incubate for 15 min.
 on ice, then 0.3 volumes of chloroform was added.
 After centrifugation, the upper phase was collected and the RNA precipitated
 with 0.7 volumes of isopropanol.
 The pellet of RNA was re-suspended in water.
 Prior to microarray analysis the pooled RNA fractions were further purified
 using the Qiagen RNeasy kit.
 The total yield of RNA in each pooled fraction was ~2 μg.
 RNA quality was checked on an Agilent 2100 bioanalyser.
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3.
 Lü X, de la Peña L, Barker C, Camphausen K, Tofilon PJ.
 Radiation-Induced Changes in Gene Expression Involve Recruitment of Existing
 Messenger RNAs to and away from Polysomes.
 Cancer Research.
 2006;66(2):1052 -1061.
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4.
 Kumaraswamy S, Chinnaiyan P, Shankavaram UT, et al.
 Radiation-induced gene translation profiles reveal tumor type and cancer-specif
ic components.
 Cancer Res.
 2008;68(10):3819-3826.
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Brain tumor cell lines.
 Microarray between exprimental condtions using solely polyribosomal mRNA
 (with pooling).
 Westernes and Northerns between fractions (no pooling).
 Used A254 and Northern for 28S, 18S to confirm fractions.
 One 150 mm2 plate per condition, apparently.
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Cells were grown to ∼80% confluence in 150-mm2 tissue culture dishes and
 incubated in 100 μg of cycloheximide/mL for 15 minutes before collection.
 Cytoplasmic RNA was obtained by lysing cells in 1 mL of polysome buffer
 [10 mmol/L Tris-HCl (pH 8), 140 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.5% NP40,
 10 mmol/L DTT, 100 μg/mL cycloheximide, 500 μg/mL heparin, 1 mmol/L phenylmetha
nesulfonyl fluoride, and 500 units/mL RNasin (Promega, Madison, WI)].
 After 10 minutes on ice, lysates were centrifuged (10,000 × g for 10 minutes),
 and the resulting cytosolic supernatant was layered onto a 10% to 50% sucrose
 gradient.
 Gradients were then centrifuged at 35,000 × g for 3 hours at 4°C and 1-mL
 fractions collected using an ISCO Density Gradient Fractionation System
 (ISCO, Lincoln, NE) with continuous monitoring based on A254.
 The RNA in each fraction was extracted using TRIZOL and used for Northern
 analysis, or fractions 5 to 11 (corresponding to polysome-bound RNA) were
 pooled and subjected to microarray analysis as described above.
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5.
 Cridge AG, Castelli LM, Smirnova JB, et al.
 Identifying eIF4E-binding protein translationally-controlled transcripts
 reveals links to mRNAs bound by specific PUF proteins.
 Nucleic Acids Res.
 2010;38(22):8039-8050.
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6.
 Smirnova JB, Selley JN, Sanchez-Cabo F, et al.
 Global Gene Expression Profiling Reveals Widespread yet Distinctive Translation
al Responses to Different Eukaryotic Translation Initiation Factor 2B-Targeting
 Stress Pathways.
 Mol.
 Cell.
 Biol.
 2005;25(21):9340-9349.
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Yeast.
 Compares monosomal-to-polysomal mRNA ratios between experimental conditions
 by microarray.
 Pooling for monosomal includes 40S.
 Pooling for polysomal excludes oligopolysomes (not 2, nor 3 ribosomes /
 mRNA).
 Microarray confirmed by qPCR, and functinal confirmation by protein level
 (densitommetric immunoblot).
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Cell extracts were prepared essentially as described previously (3).
 Briefly, after incubation with cycloheximide (100 µg/ml at 4°C), cells
 were pelleted and washed twice in 50 ml of ice-cold lysis buffer (3).
 The cell pellets were resuspended in 500 µl of lysis buffer and rapidly
 frozen in liquid nitrogen.
 Lysates were prepared by grinding the cell pellets under liquid nitrogen.
 The ground yeast was thawed on ice and cleared by successive centrifugation
 steps (5,000 x g for 5 min at 4°C in a clinical centrifuge, and then 16,000
 x g for 30 min at 4°C in an Eppendorf microcentrifuge).
 Sixty A260 units were layered onto 35-ml 15 to 50% sucrose gradients.
 The gradients were sedimented via centrifugation at 16,900 rpm for 13 h
 in a Beckman ultracentrifuge.
 The gradients were collected as described previously (3) and fractionated
 into 15 2.2-ml aliquots.
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7.
 Hittel D, Storey KB.
 The translation state of differentially expressed mRNAs in the hibernating
 13-lined ground squirrel (Spermophilus tridecemlineatus).
 Archives of Biochemistry and Biophysics.
 2002;401(2):244-254.
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10 fractions without pooling, analyzed by Northern for 18S and some genes
 of interest.
 No A254.
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Samples (500 mg) of frozen kidney or BAT were pulverized under liquid N2
 by mortar and pestle and then homogenized in 5 ml of polysome lysis buffer
 [25 mM Tris–HCl, pH 7.6, 25 mM NaCl, 10 mM MgCl2, 250 mM sucrose, 100 μg/ml
 cyclohexamide, 5 U/ml RNAse inhibitor (Promega)] using 10 strokes of a
 Douce homogenizer.
 The homogenate was centrifuged at 16,000g at 4 °C for 15 min.
 The supernatant was removed and was mixed with 0.1 vol of detergent (5%
 w/v sodium deoxycholate, 5% v/v Triton X-100).
 Aliquots (1 ml) of the supernatant were layered on a 5-ml continuous sucrose
 gradient, 0.5 to 1.5 M, prepared in 300 mM NaCl, 10 mM Tris–HCl, pH 7.6, 10
 mM MgCl2, 100 μg/ml cyclohexamide, and 5 U/ml RNase inhibitor.
 The gradients were centrifuged at 4 °C for 2 h at 40,000 rpm in a SW41
 rotor.
 Gradients were drained into 10 fractions of 500 μl each and immediately
 frozen at −70 °C.
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8.
 Kolle G, Ho M, Zhou Q, et al.
 Identification of Human Embryonic Stem Cell Surface Markers by Combined
 Membrane‐Polysome Translation State Array Analysis and Immunotranscriptional
 Profiling.
 STEM CELLS.
 2009;27(10):2446-2456.
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The atrophic effect of testosterone (T) deprivation and the opposite, rescuing,
 effect of T administration on muscle mass are induced by a multitude of
 cellular mediators, in accordance with T’s pleiotropic nature.
 Earlier findings show that at least part of its effect is at the level
 of protein translation1.
 We hypothesize that T regulates protein translation, in both non-specific
 (cap-dependent initiation, elongation factors, ribosomal recycling) and
 specific manners (internal ribosome entry site).
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The effects of T are typically observed only at organism level, at which
 a distinction between these concurrent regulatory pathways is difficult
 to make.
 In order to determine changes in the non-specific regulation of translation,
 we will determine changes of pattern in the proteins that associate with
 polysomes in vivo, as an effect of testosterone deprivation and/or testosterone
 treatment.
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A required initial step is the isolation of translating mRNAs (polysomes)
 and of ribosome-free mRNAs.
 We will obtain specimens of levator ani, a slow-twitching muscle, from
 male mice that underwent castration, with or without T treatment.
 The castration effects will be observed at 14 days after surgery or sham
 intervention, while testosterone’s effect will be observed after 7 days
 of treatment.
 The muscle polysomes will be isolated from levator ani, obtained by necropsy
 from 3 animals for each experimental condition.
 The resulting sample size is comparable to that used in similar earlier
 experiments, 50 mg2.
 Cells will be lysed in the presence of 0.1 g/L cycloheximide (blocks peptide
 elongation, locking the translating ribosomes in place), 1 g/L heparin
 or other RNAse inhibitor (Protector), 0.5 mM DTT (maintains cytosol-like
 reductive environment), 5 mM Mg2+ (stabilizes mRNA structures), in addition
 to regular lysis buffer, and lysed by the standard procedure (incubation
 with Triton).
 The lysate will be precleared by gentle centrifugation, as 12000g, recommended
 by Arava3, might lead to microsomal loss, favoring mRNA for soluble proteins.
 The sucrose gradient will be obtained as described3, comprising the interval
 10-50% sucrose.
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For further analysis, we will pool the fractions as follows: the first fractions
, lighter than 40S subunits, will constitute the ribosome-free mRNAs pool;
 the latter group of peaks, after 80S, will include translated mRNAs pool.
 The distinction between monoribosomal mRNA’s and polyribosomal mRNA’s is
 of reduced theoretical importance, and will be technically challenging,
 according to Arava3.
 Also, the 43S sub-peak, consisting of mRNA’s that are stalled at initiation,
 will be difficult to purify, and relatively difficult to use in a comparison,
 because the counterpart, the translating mRNA pool, will also contain similarly
 stalled ribosomes.
 A comparison between 43S (or 48S) initiating complexes and translating
 ribosomes would be possible by using larger specimens, by using a less
 steep gradient, e.g.
 10-30%, or by using an apparatus similar to that presented by Esposito
 et al.4
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At this time, we should be able to confirm earlier observations, by determining
 differences in the ratio translated-to-untranslated mRNA, which can be
 quantified by qPCR in the two pools with olygo-T primers.
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To determine non-specific regulatory mechanisms acting on protein translation
 as an effect of T treatment, we will compare the proteins in the translating
 mRNA’s fraction between treatments.
 We will approach the problem in two ways, without and with a priori knowledge.
 In the former approach, we will perform mass spectroscopy studies on the
 proteins of the translating ribosome pool, in which case we expect to find
 quantitative and/or qualitative (PTM) differences in the presence and absence
 of T.
 In the latter approach, we will quantify by immunoblot and densitometry
 the following proteins:
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● total rpS6 (required for ribosome count), and phosphorylated S235.S236-rpS6
 (might or might not correlate with S6 kinases activation)
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● total eIF2α and phosphorylated S51-eIF2α
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● eIF3f (core component of eIF3, regulated by atrogin)
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● eIF3j (not a core component, regulatory, stabilizes eIF3 on 40S)
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● total eIF4B and phosphorylated S422-eIF4B (controlled by S6K)
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● total eIF4E and phosphorylated S209-eIF4E (controlled by Mnk)
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● eIF4G
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● Mnk1 (kinase for eIF4E, also binding partner for it and eIF4G)
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● PABP
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● Hsc70
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● and any T-modulated protein found by microarray.
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To determine effect of T on translation of specific mRNAs, the same two
 approaches can be taken.
 The approach without a priori knowledge will consist in the microarray
 analysis comparing translating-to-untranslated ratios of mRNA between presence
 and absence of T.
 The informed approach will involve the same type of comparisons by qPCR
 or densitometric Northern blot for the mRNAs that were found to be regulated
 by T in the microarray and/or any protein which is known to be regulated
 by T (e.g., atrogin).
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Bibliography:
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1.
 Boissonneault, G., Gagnon, J., Ho-Kim, M.A., Rogers, P.A.
 & Tremblay, R.R.
 Depressed translational activity in the androgen sensitive levator ani
 muscle of the rat.
 J.
 Steroid Biochem 32, 507-513 (1989).
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2.
 Wernerman, J., von der Decken, A.
 & Vinnars, E.
 Polyribosome concentration in human skeletal muscle after starvation and
 parenteral or enteral refeeding.
 Metab.
 Clin.
 Exp 35, 447-451 (1986).
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3.
 Arava, Y.
 Isolation of Polysomal RNA for Microarray Analysis.
 Functional Genomics 224, 79-88 (2003).
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4.
 Esposito, A.M.
 et al.
 Eukaryotic Polyribosome Profile Analysis.
 JoVE (2010).doi:10.3791/1948
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Other ideas:
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* Best way to show eIF4E is under the control of 4E-BP (or not) is by pulling
 it down from cell lysate using methyl-7-GTP.
 The pulled down protein will be bound to 4EBP or eIF4G, visible by immunoblot.
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* Repressed mRNA’s might be under the control of miRNA.
 Database search?
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Dexamethasone on C2C12: 53 articles
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PMID 22613845: 100 nM Dex for up to 1.5 during differentiation - no outcome
 of interest, but alters MyoD level.
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PMID 22750276: 100 nM Dex for 2 d post-differentiation doubles MAFbx mRNA
 in C2C12, with seemingly additive effect of 10 mg/L IGF-I (serum replaced
 by BSA)
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PMID 21764995: 1 uM for 2 d post diff (although the 20% protein loss is
 seemingly in the first 24h).
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PMID 21745490: 10 uM with no effect on adipoR and data not shown.
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PMID 21389279: 1 uM for 6h after 6 days of differentiation reduce phosphorylated
 Foxo and activate a Foxo reporter.
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PMID 19631210: 10 uM for 1d post-differentiation eliminate myogenin protein.
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Dexamethasone on mouse muscle:
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http://www.ncbi.nlm.nih.gov/pubmed/22354783
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