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Protein Synthesis (protein + synthesis)
Kinds of Protein Synthesis Terms modified by Protein Synthesis Selected AbstractsMicroarray Analysis of Ethanol-Treated Cortical Neurons Reveals Disruption of Genes Related to the Ubiquitin-Proteasome Pathway and Protein SynthesisALCOHOLISM, Issue 12 2004Ramana Gutala Background: Chronic ethanol abuse results in deleterious behavioral responses such as tolerance, dependence, reinforcement, sensitization, and craving. The objective of this research was to identify transcripts that are differentially regulated in ethanol-treated cortical neurons compared with controls by using a pathway-focused complementary DNA microarray. Methods: Cortical neurons were isolated from postconception day 14 C57BL/6 mouse fetuses and cultured according to a standard protocol. The cortical neuronal cells were treated with 100 mM ethanol for five consecutive days with a change of media every day. A homeostatic pathway-focused microarray consisting of 638 sequence-verified genes was used to measure transcripts differentially regulated in four ethanol-treated cortical neuron samples and four control samples. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis was used to verify the mRNA expression levels of genes of interest detected from the microarray experiments. Results: We identified 56 down-regulated and 10 up-regulated genes in ethanol-treated cortical neurons relative to untreated controls at a 5% false-discovery rate. The expression of many genes involved in ubiquitin-proteasome and protein synthesis was decreased by ethanol, including ubiquitin B, ubiquitin-like 3, ubiquitin-conjugating enzyme E3A, 20S proteasome ,- and ,-subunits, and members of the ribosomal proteins. Furthermore, the mRNA expression of heat shock proteins, myristoylated alanine-rich protein kinase C substrate, phosphatase and tensin homolog deleted on chromosome 10, and FK506 binding protein rapamycin-associated protein (FKBP) (mTOR) was also decreased in ethanol-treated cortical neurons. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis of genes involved in the ubiquitin-proteasome cascade revealed a down-regulation of these genes, thereby corroborating our microarray results. Conclusions: Our results indicate that chronic ethanol treatment of cortical neurons resulted in decreased mRNA expression of genes involving the ubiquitin-proteasome pathway and ribosomal proteins together with mTOR expression leading to disruption of protein degradation mechanism and impairment of protein synthesis machinery. [source] Restoration of Protein Synthesis in Heart and Skeletal Muscle After Withdrawal of AlcoholALCOHOLISM, Issue 4 2004Thomas C. Vary Abstract: Background: The rate of protein synthesis is diminished after chronic alcohol consumption through changes in both mRNA translation initiation and elongation. It remains unknown how long adverse effects of alcohol on protein synthesis persist after withdrawal from ethanol. Methods: We examined the effect of removal of alcohol from the diet of rats for 72 hr after chronic alcohol exposure (16 weeks) on rates of protein synthesis and potential mechanisms for controlling mRNA translation in heart, skeletal muscle, and liver. Rates of protein synthesis were measured after intravenous infusion of [3H]-l-phenylalanine. The formation of active eukaryotic initiation factor (eIF)4E·eIF4G complex, the cellular content of eukaryotic elongation factor (eEF)1A and eEF2, and the phosphorylation state of eEF2 and S6K1 were measured in each tissue. Results: Withdrawal of alcohol from the diet restored protein synthesis in heart and skeletal muscle to values obtained in pair-fed control rats not exposed to alcohol. However, the organ weight and protein content per muscle was not affected by withdrawal of alcohol from the diet. In both heart and skeletal muscle, the restoration of protein synthesis correlated with reversal of defects in the formation of active eIF4E·eIF4G complex and eEF1A content. Myocardial eEF2 content was also restored to control values after withdrawal of alcohol from the diet. In the gastrocnemius, there was a decrease in the cellular content of eEF2. The lower eIF2 content may have been counterbalanced by an increased activity of eEF2 through a reduction in the phosphorylation state of eEF2 allowing protein synthesis to proceed unimpeded. Conclusions: These studies indicate that changes in protein metabolism observed during chronic alcohol intake are reversible and do not, at this stage, represent an irreversible change in cardiac or skeletal muscle. [source] Prolonging Cell-Free Protein Synthesis by Selective Reagent AdditionsBIOTECHNOLOGY PROGRESS, Issue 3 2000Dong-Myung Kim Factors causing the early cessation of protein synthesis have been studied in a cell-free system from Escherichia coli. We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ATP regeneration, and several amino acids are rapidly degraded during the cell-free protein synthesis reaction. The degradation of such compounds takes place even in the absence of protein synthesis. This degradation severely reduces the capacity for protein synthesis. The lost potency was completely recovered when the reaction mixture was supplied with additional PEP and amino acids. Of the 20 amino acids, only arginine, cysteine, and tryptophan were required to restore system activity. Through repeated additions of PEP, arginine, cysteine,and tryptophan, the duration of protein synthesis was greatly extended. In this fed-batch reaction, after a 2 h incubation, the level of cell-free synthesized chloramphenicol acetyl transferase (CAT) reached 350 ,g/mL, which is 3.5 times the yield of the batch reaction. Addition of fresh magnesium further extended the protein synthesis. As a result, through coordinated additions of PEP, arginine, cysteine, tryptophan, and magnesium, the final concentration of cell-free synthesized CAT increased more than 4-fold compared to a batch reaction. SDS-PAGE analysis of such a fed-batch reaction produced an obvious band of CAT upon Coomassie Blue staining. [source] Protein Synthesis Assisted by Native Chemical Ligation at LeucineCHEMBIOCHEM, Issue 9 2010Ziv Harpaz Triggering leucine: A new ligation strategy of using ,-mercaptoleucine coupled with desulfurization at leucine sites was developed, and its applicability in protein synthesis is presented. The efficiency of our Leu-NCL was examined in several model peptides and utilized for the first total synthesis of HIV-1 Tat protein. [source] Chemical Protein Synthesis by Kinetically Controlled Ligation of Peptide O-EstersCHEMBIOCHEM, Issue 4 2010Ji-Shen Zheng Designer peptides: A large reactivity difference was observed between two peptide O-esters that can undergo peptide ligation through an in situ O-to-S acyl shift. This observation allowed for the design of "one-pot" N-to-C sequential peptide fragment condensation through kinetically controlled ligation with more readily accessible peptide O-esters. [source] Cell-free Protein Synthesis through Solubilisate Exchange in Water/Oil Emulsion CompartmentsCHEMBIOCHEM, Issue 8 2004Adriana V. Pietrini Dr. Abstract This work is aimed at finding conditions under which synthetic compartments used as cell models can fuse with each other and allow reagents contained in the different compartments to react. This goal seems to be best achieved by the use of water in oil emulsions (w/o) with dimensions in the range of 30,60 ,m. In particular, cell-free EGFP (enhanced green fluorescent protein) synthesis takes place in Tween 80/Span 80 w/o emulsions, and the extent of the reaction can be monitored directly by fluorescence. The medium is mineral oil, containing 0.5,% v/v aqueous solution. Different premixing configurations of the components (plasmid, amino acids, E. Coli extract) are used and compared. The in vitro synthesis of EGFP in emulsion droplets proceeds for 1 h, and the yield is 7.5 ng,,L,1protein. EGFP synthesis in aqueous solution takes place for at least 5 h. The yield is 10.5 ng,,L,1protein after 1 h and 15.8 ng,,L,1protein after 5 h.The results with the w/o emulsions show that solubilisate exchange takes place among the different water droplets, but it is not possible to demonstrate clearly that a true fusion takes place. [source] A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometryFEBS JOURNAL, Issue 17 2009Elizabeth Diago-Navarro Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5, of A in the core sequence 5,-UA(A/C)-3,. A model of the Kid toxin interacting with the uncleavable mimetic 5,-AdUACA-3, is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5,-AUACA-3,, and its uncleavable mimetic 5,-AdUACA-3,, used to establish the model and structure of the Kid,RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5,-UUACU-3, efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5,-UUACU-3, substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid,RNA complex. [source] Amino Acid Transport Kinetics and Protein Turnover in HemodialysisHEMODIALYSIS INTERNATIONAL, Issue 1 2003Raj Dominic Background: Protein metabolism is abnormal in patients with end-stage renal disease. However, the etiology of abnormal protein turnover is unclear. Also the role of hemodialysis on protein turnover remains controversial. Abnormal protein metabolism could be due to malnutrition or due to abnormal amino acid transport kinetics Hypothesis: 1) Amino acid transport is abnormal in uremia, 2) Hemodialysis increases fractional protein synthesis rate and c) Net protein accretion is negative during hemodialysis because of increased catabolism. Aim: 1) To study the impact of uremia and hemodialysis on intracellular amino acid transport kinetics and 2) Quantify the fractional protein synthesis rate and degradation in a uremic state and during hemodialysis Methods: Protein turnover and amino acid transport kinetics using stable isotopes of phenylalanine in 2 patients and 2 controls. The patients were placed on a standard diet (1.2 gm/Kg protein and 35 Kcal/Kg) for 2 weeks prior to the study. Acidosis as corrected by NaHCO3 supplementation. Amino acid transport and protein turnover were estimated by compartmental model and precursor product approach respectively. Results: Mean protein intake and HCO3 were 1.4 ± 1 gm/day and 26.8 ± 4.1 meq/L respectively. Inward transport (11.2 ± 2.6 vs. 9.8 ± 2.1 nmol/min,1/100 ml leg,1) and outward transport (10.2 ± 1.2 vs.11.0 ± 1.6 l nmol/min,1/100 ml leg,1) were not different before and during HD. Inward and outward transport in controls were 12.6 ± 3.7 and 16.2 ± 3.5 nmol/min,1/100 ml leg,1 respectively. Protein synthesis was higher than catabolism in the pre-dialysis phase (156.8 ± 66.1 vs. 144.3 ± 53.7 nmol/min/ml leg-1, p = NS), but catabolism was higher than synthesis during HD (172.3 ± 20.5 vs. 186.8 ± 25.8 nmol/min/ml leg-1, p = NS). Protein synthesis and catabolism in controls were 110.8 ± 13.5 and 127.4 ± 12.7 nmol/min/ml leg-1. Conclusion: 1. Inward and outward transport of amino acids are not altered by renal failure or hemodialysis. 2. Protein turnover is increased during hemodialysis, with net balance favoring catabolism [source] A picture says more than a thousand words: Structural insights into hepatitis C virus translation initiation,HEPATOLOGY, Issue 6 2006Pantxika Bellecave Ph.D. Protein synthesis in mammalian cells requires initiation factor eIF3, a ,750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5,-cap or an internal ribosome entry site (IRES). Cryoelectron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5,-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly. [source] Fibrodysplasia Ossificans Progressiva (FOP), a Disorder of Ectopic Osteogenesis, Misregulates Cell Surface Expression and Trafficking of BMPRIA,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2005Lourdes Serrano de la Peña Abstract FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease. Introduction: Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes. Materials and Methods: Cell surface proteins were quantified by fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by [35S]methionine labeling and pulse-chase assays. mRNA was detected by RT-PCR. Results: FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand-stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen-activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells. Conclusions: These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis. [source] Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activityMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Céline Vigneron Abstract Roscovitine, a specific inhibitor of MPF kinase activity, has been shown to block efficiently and reversibly the meiotic resumption of oocytes from different species, including cattle. In view to verify that oocytes maintain germinal vesicle like molecular activities under roscovitine treatment, we compared in the present study the M-phase Promoting Factor (MPF) and Mitogen Activated Protein (MAP) kinase activities; protein synthesis and phosphorylation patterns in oocytes and cumulus cells; and CDK1 and Cyclin B messengers storage under control culture and under roscovitine inhibition. We observed that roscovitine induced a full and reversible inhibition of MPF kinase activity and of the activating phosphorylation of both ERK1/2 MAPK. During in vivo maturation, there was a highly significant increase in the relative mRNA level of both cyclin B1 and CDK1 whereas during in vitro culture, the relative amount of CDK1 messenger was reduced. These messengers may be used as markers for the optimization of in vitro maturation treatment. Roscovitine reversibly prevented this drop in relative quantities of CDK1 messenger. Oocytes cultured in the presence of roscovitine maintained a GV like profile of protein synthesis except that two proteins of 48 and 64 kDa specific of matured oocytes also appeared under roscovitine treatment. However, roscovitine did not prevent most of the modifications of protein phosphorylation pattern observed during maturation. In conclusion, results of this study revealed that the use of roscovitine did not prevent all the events related to maturation of bovine oocytes. Mol. Reprod. Dev. 69: 457,465, 2004. © 2004 Wiley-Liss, Inc. [source] Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of menTHE JOURNAL OF PHYSIOLOGY, Issue 1 2005Adam J. Rose Protein synthesis in skeletal muscle is known to decrease during contractions but the underlying regulatory mechanisms are unknown. Here, the effect of exercise on skeletal muscle eukaryotic elongation factor 2 (eEF2) phosphorylation, a key component in protein translation machinery, was examined. Eight healthy men exercised on a cycle ergometer at a workload eliciting ,67% peak pulmonary oxygen consumption with skeletal muscle biopsies taken from the vastus lateralis muscle at rest as well as after 1, 10, 30, 60 and 90 min of exercise. In response to exercise, there was a rapid (i.e. < 1 min) 5- to 7-fold increase in eEF2 phosphorylation at Thr56 that was sustained for 90 min of continuous exercise. The in vitro activity of skeletal muscle eEF2 kinase was not altered by exercise indicating that the increased activity of eEF2 kinase to eEF2 is not mediated by covalent mechanisms. In support of this, the increase in AMPK activity was temporally unrelated to eEF2 phosphorylation. However, skeletal muscle eEF2 kinase was potently activated by Ca2+,calmodulin in vitro, suggesting that the higher eEF2 phosphorylation in working skeletal muscle is mediated by allosteric activation of eEF2 kinase by Ca2+ signalling via calmodulin. Given that eEF2 phosphorylation inhibits eEF2 activity and mRNA translation, these findings suggest that the inhibition of protein synthesis in contracting skeletal muscle is due to the Ca2+ -induced stimulation of eEF2 kinase. [source] Protein and oil concentration of soybean seed cultured in vitro using nutrient solutions of differing glutamine concentrationANNALS OF APPLIED BIOLOGY, Issue 2 2004ANTONIO E PIPOLO Summary Oil and protein are the most valuable components of soybean seed. Evidence indicates that growth and composition of soybean seed are controlled by supplies of carbon and nitrogen provided by the maternal plant to the seed, but it is difficult experimentally to control and quantify the precise amount of carbon and nitrogen provided to the seed by the whole plant. To examine whether oil and protein concentrations are affected by the supply of nitrogen to the seed, immature soybean seeds (Glycine max cv. Williams 82) were grown in vitro in nutrient solutions containing 20, 40, 60 or 80 mM of glutamine. The seeds were incubated in Erlenmeyer flasks for 8 days at 25°C. The rate of dry matter accumulation changed from 7.2 to 8.3 mg seed,1 day,1 as the glutamine concentration increased from 20 to 80 mM but the differences were not significant (P 0.05). Seed protein concentration increased as glutamine concentration increased from 294 mg g,1 at 20 mM glutamine to as high as 445 mg g,1 at 80 mM glutamine. Typical in vivo protein concentration of mature soybean seeds is about 400 mg g,1. Oil and protein concentrations were negatively correlated (r2= 0.44), which indicates that oil and protein synthesis are interrelated. Protein synthesis was favoured over oil synthesis when nitrogen became more abundant. The seeds used in this study clearly demonstrated a capacity to respond to nitrogen availability with changes in seed protein concentration. [source] Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1CELLULAR MICROBIOLOGY, Issue 4 2008Naoko Morinaga Summary Subtilase cytotoxin (SubAB) is a AB5 type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2, (eIF2,). The phosphorylation of PERK and eIF2, was maximal at 30,60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation. [source] GATA-3 transduces survival signals in osteoblasts through upregulation of bcl-xL gene expression,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2010Ruei-Ming Chen Abstract GATA-3, a transcription factor, participates in regulating cell development, proliferation, and death. This study was aimed at evaluating the roles of GATA-3 in protecting osteoblasts against oxidative stress,induced apoptotic insults and their possible mechanisms. Pretreatment with nitric oxide (NO) for 24 hours protected osteoblasts, prepared from neonatal rat calvaria, against oxidative stress,induced apoptotic insults. Such protection involved enhancement of Bcl-XL messenger (m)RNA and protein syntheses and the translocation of this antiapoptotic protein from the cytoplasm to mitochondria. GATA-3 was detected in rat osteoblasts, and GATA-3-specific DNA-binding elements exist in the promoter region of the bcl-xL gene. NO preconditioning attenuated oxidative stress,caused suppression of GATA-3 mRNA and protein synthesis and the translocation of this transcription factor from the cytoplasm to nuclei. Application of GATA-3 small interfering (si)RNA into osteoblasts decreased the levels of this transcription factor and simultaneously inhibited Bcl-XL mRNA synthesis. Pretreatment with NO lowered the oxidative stress,caused alteration in the binding of GATA-3 to its specific DNA motifs. Oxidative stress,inhibited Runx2 mRNA expression, but NO preconditioning decreased such inhibition. NO pretreatment time-dependently enhanced the association of GATA-3 with Runx2. Knocking down the translation of GATA-3 using RNA interference significantly decreased the protection of NO preconditioning against oxidative stress,induced alterations of cell morphologies, DNA fragmentation, and cell apoptosis. In comparison, overexpression of GATA-3 could promote NO preconditioning,involved Bcl-XL expression and cell survival. Therefore, this study shows that GATA-3 plays critical roles in mediating survival signals in osteoblasts, possibly through upregulating bcl-xL gene expression. © 2010 American Society for Bone and Mineral Research. [source] Resistance exercise increases leg muscle protein synthesis and mTOR signalling independent of sexACTA PHYSIOLOGICA, Issue 1 2010H. C. Dreyer Abstract Aim:, Sex differences are evident in human skeletal muscle as the cross-sectional area of individual muscle fibres is greater in men than in women. We have recently shown that resistance exercise stimulates mammalian target of rapamycin (mTOR) signalling and muscle protein synthesis in humans during early post-exercise recovery. Therefore, the aim of this study was to determine if sex influences the muscle protein synthesis response during recovery from resistance exercise. Methods:, Seventeen subjects, nine male and eight female, were studied in the fasted state before, during and for 2 h following a bout of high-intensity leg resistance exercise. Mixed muscle protein fractional synthetic rate was measured using stable isotope techniques and mTOR signalling was assessed by immunoblotting from repeated vastus lateralis muscle biopsy samples. Results:, Post-exercise muscle protein synthesis increased by 52% in the men and by 47% in the women (P < 0.05) and was not different between groups (P > 0.05). Akt phosphorylation increased in both groups at 1 h post-exercise (P < 0.05) and returned to baseline during 2 h post-exercise with no differences between groups (P > 0.05). Phosphorylation of mTOR and its downstream effector S6K1 increased significantly and similarly between groups during post-exercise recovery (P < 0.05). eEF2 phosphorylation decreased at 1- and 2 h post-exercise (P < 0.05) to a similar extent in both groups. Conclusion:, The contraction-induced increase in early post-exercise mTOR signalling and muscle protein synthesis is independent of sex and appears to not play a role in the sexual dimorphism of leg skeletal muscle in young men and women. [source] Forearm and leg amino acid metabolism in the basal state and during combined insulin and amino acid stimulation after a 3-day fastACTA PHYSIOLOGICA, Issue 3 2009J. Gjedsted Abstract Aim:, Fasting is characterized by a progressive loss of protein, but data on protein kinetics are unclear and few have studied the effects of re-feeding. The present study was designed to test the hypothesis that a combined infusion of insulin and amino acids after fasting would induce compensatory increases in protein synthesis and reductions in protein breakdown at the whole body level and in muscle. Methods:, We included 10 healthy male volunteers and studied them twice: (1) in the post-absorptive state and (2) after 72 h of fasting. Amino acid kinetics was measured using labelled phenylalanine and tyrosine, whole body energy expenditure was assessed and urea nitrogen synthesis rates were calculated. Results:, After fasting we observed an increase in arterial blood concentration of branched chain amino acids and a decrease in gluconeogenic amino acids (P < 0.05). Isotopically determined whole body, forearm and leg phenylalanine fluxes were unaltered apart from a 30% decrease in phenylalanine-to-tyrosine conversion (2.0 vs. 1.4 ,mol kg,1 h,1, P < 0.01). During infusion of insulin and amino acids, amino acid concentrations increased. Conclusion:, Our data indicate that after a 72-h fast basal and insulin/amino acid-stimulated regional phenylalanine fluxes in leg and forearm muscle are unaltered. During fasting concentrations of gluconeogenic amino acids decrease and hepatic and/or renal phenylalanine-to-tyrosine conversion decreases. Thus, as opposed to glucose and lipid metabolism, fasting does not induce insulin resistance as regards amino acid metabolism. [source] AMP-activated protein kinase: a core signalling pathway in the heartACTA PHYSIOLOGICA, Issue 1 2009A. S. Kim Abstract Over the past decade, AMP-activated protein kinase (AMPK) has emerged as an important intracellular signalling pathway in the heart. Activated AMPK stimulates the production of ATP by regulating key steps in both glucose and fatty acid metabolism. It has an inhibitory effect on cardiac protein synthesis. AMPK also interacts with additional intracellular signalling pathways in a coordinated network that modulates essential cellular processes in the heart. Evidence is accumulating that AMPK may protect the heart from ischaemic injury and limit the development of cardiac myocyte hypertrophy to various stimuli. Heart AMPK is activated by hormones, cytokines and oral hypoglycaemic drugs that are used in the treatment of type 2 diabetes. The tumour suppressor LKB1 is the major regulator of AMPK activity, but additional upstream kinases and protein phosphatases also contribute. Mutations in the regulatory ,2 subunit of AMPK lead to an inherited syndrome of hypertrophic cardiomyopathy and ventricular pre-excitation, which appears to be due to intracellular glycogen accumulation. Future research promises to elucidate the molecular mechanisms responsible for AMPK activation, novel downstream AMPK targets, and the therapeutic potential of targeting AMPK for the prevention and treatment of myocardial ischaemia or cardiac hypertrophy. [source] AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?ACTA PHYSIOLOGICA, Issue 1 2009T. E. Jensen Abstract In skeletal muscle, the contraction-activated heterotrimeric 5,-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved in mitochondrial biogenesis and other aspects of promoting an oxidative muscle phenotype. Here, the current knowledge on the expression of AMPK subunits in human quadriceps muscle and evidence from rodent studies suggesting distinct AMPK subunit expression pattern in different muscle types is reviewed. Then, the intensity and time dependence of AMPK activation in human quadriceps and rodent muscle are evaluated. Subsequently, a major part of this review critically examines the evidence supporting a necessary and/or sufficient role of AMPK in a broad spectrum of skeletal muscle contraction-relevant processes. These include glucose uptake, glycogen synthesis, post-exercise insulin sensitivity, fatty acid (FA) uptake, intramuscular triacylglyceride hydrolysis, FA oxidation, suppression of protein synthesis, proteolysis, autophagy and transcriptional regulation of genes relevant to promoting an oxidative phenotype. [source] Cellular oxygen sensing, signalling and how to survive translational arrest in hypoxiaACTA PHYSIOLOGICA, Issue 2 2009M. Fähling Abstract Hypoxia is a consequence of inadequate oxygen availability. At the cellular level, lowered oxygen concentration activates signal cascades including numerous receptors, ion channels, second messengers, as well as several protein kinases and phosphatases. This, in turn, activates trans -factors like transcription factors, RNA-binding proteins and miRNAs, mediating an alteration in gene expression control. Each cell type has its unique constellation of oxygen sensors, couplers and effectors that determine the activation and predominance of several independent hypoxia-sensitive pathways. Hence, altered gene expression patterns in hypoxia result from a complex regulatory network with multiple divergences and convergences. Although hundreds of genes are activated by transcriptional control in hypoxia, metabolic rate depression, as a consequence of reduced ATP level, causes inhibition of mRNA translation. In a multi-phase response to hypoxia, global protein synthesis is suppressed, mainly by phosphorylation of eIF2-alpha by PERK and inhibition of mTOR, causing suppression of 5,-cap-dependent mRNA translation. Growing evidence suggests that mRNAs undergo sorting at stress granules, which determines the fate of mRNA as to whether being translated, stored, or degraded. Data indicate that translation is suppressed only at ,free' polysomes, but is active at subsets of membrane-bound ribosomes. The recruitment of specific mRNAs into subcellular compartments seems to be crucial for local mRNA translation in prolonged hypoxia. Furthermore, ribosomes themselves may play a significant role in targeting mRNAs for translation. This review summarizes the multiple facets of the cellular adaptation to hypoxia observed in mammals. [source] Human soleus muscle protein synthesis following resistance exerciseACTA PHYSIOLOGICA, Issue 2 2004T. A. Trappe Abstract Aim:, It is generally believed the calf muscles in humans are relatively unresponsive to resistance training when compared with other muscles of the body. The purpose of this investigation was to determine the muscle protein synthesis response of the soleus muscle following a standard high intensity bout of resistance exercise. Methods:, Eight recreationally active males (27 ± 4 years) completed three unilateral calf muscle exercises: standing calf press/heel raise, bent-knee calf press/heel raise, and seated calf press/heel raise. Each exercise consisted of four sets of 15 repetitions (,15 repetition maximum, RM, or ,70% 1RM). Fractional rate of muscle protein synthesis (FSR) was determined with a primed constant infusion of [2H5]phenylalanine coupled with muscle biopsies immediately and 3 h following the exercise in both the exercise and non-exercise (resting control) leg. Results:, FSR was elevated (P < 0.05) in the exercise (0.069 ± 0.010) vs. the control (0.051 ± 0.012) leg. Muscle glycogen concentration was lower (P < 0.05) in the exercise compared with the control leg (Decrease from control; immediate post-exercise: 54 ± 5; 3 h post-exercise: 36 ±4 mmol kg,1 wet wt.). This relatively high amount of glycogen use is comparable with previous studies of resistance exercise of the thigh (i.e. vastus lateralis; ,41,49 mmol kg,1 wet wt.). However, the exercise-induced increase in FSR that has been consistently reported for the vastus lateralis (,0.045,0.060% h,1) is on average ,200% higher than reported here for the soleus (0.019 ± 0.003% h,1). Conclusions:, These results suggest the relatively poor response of soleus muscle protein synthesis to an acute bout of resistance exercise may be the basis for the relative inability of the calf muscles to respond to resistance training programs. [source] Plasmodium falciparum myosins: Transcription and translation during asexual parasite development,CYTOSKELETON, Issue 4 2005Jacqueline Chaparro-Olaya Abstract Six myosins genes are now annotated in the Plasmodium falciparum Genome Project. Malaria myosins have been named alphabetically; accordingly, we refer to the two latest additions as Pfmyo-E and Pfmyo-F. Both new myosins contain regions characteristic of the functional motor domain of "true" myosins and, unusually for P. falciparum myosins, Pfmyo-F encodes two consensus IQ light chain-binding motifs. Phylogenetic analysis of the 17 currently known apicomplexan myosins together with one representative of each myosin class clusters all but one of the apicomplexan sequences together in Class XIV. This refines the earlier definition of the Class XIV Subclasses XIVa and XIVb. RT-PCR on blood stage parasite mRNA amplifies a specific product for all six myosins and each shows developmentally regulated transcription. Thus: Pfmyo-A and Pfmyo-B genes are transcribed throughout development; Pfmyo-C is predominant in trophozoites; Pfmyo-D occurs in trophozoites and schizonts; Pfmyo-E though barely present in earlier stages is abundant in schizonts; Pfmyo-F increases steadily throughout development and maturation. It is known that Pfmyo-A and Pfmyo-B are synthesised during late schizogony and we now show that Pfmyo-D expression is also temporally regulated to late trophozoites and schizonts where it distributes close to segregating nuclei. Thus, in asexual stages myosin synthesis does not always parallel transcript accumulation, showing that translation is also regulated. The implication is that the mRNAs are either subjected to turnover, synthesised and degraded, or that they are sequestered in an inactivate form until required for protein synthesis. Cell Motil. Cytoskeleton 60:200,213, 2005. © 2005 Wiley-Liss, Inc. [source] Mitogen-activated protein kinase signal transduction in skeletal muscle: effects of exercise and muscle contractionACTA PHYSIOLOGICA, Issue 3 2001U. Widegren Exercise has numerous growth and metabolic effects in skeletal muscle, including changes in glycogen metabolism, glucose and amino acid uptake, protein synthesis and gene transcription. However, the mechanism(s) by which exercise regulates intracellular signal transduction to the transcriptional machinery in the nucleus, thus modulating gene expression, is largely unknown. This review will provide insight on potential intracellular signalling mechanisms by which muscle contraction/exercise leads to changes in gene expression. Mitogen-activated protein kinase (MAPK) cascades are associated with increased transcriptional activity. The MAPK family members can be separated into distinct parallel pathways including the extracellular signal-regulated kinase (ERK) 1/2, the stress-activated protein kinase cascades (SAPK1/JNK and SAPK2/p38) and the extracellular signal-regulated kinase 5 (ERK5). Acute exercise elicits signal transduction via MAPK cascades in direct response to muscle contraction. Thus, MAPK pathways appear to be potential physiological mechanisms involved in the exercise-induced regulation of gene expression in skeletal muscle. [source] Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteinsCYTOSKELETON, Issue 3 2001Nathalie F. Worth Abstract Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (,-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, ,-NM actin was localised to the cell periphery and basal cortex. The dense body protein ,-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2,3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (,-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. Cell Motil. Cytoskeleton 49:130,145, 2001. © 2001 Wiley-Liss, Inc. [source] Novel functions of ribosomal protein S6 in growth and differentiation of Dictyostelium cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2009Kazutaka Ishii We have previously shown that in Dictyostelium cells a 32 kDa protein is rapidly and completely dephosphorylated in response to starvation that is essential for the initiation of differentiation (Akiyama & Maeda 1992). In the present work, this phosphoprotein was identified as a homologue (Dd-RPS6) of ribosomal protein S6 (RPS6) that is an essential member for protein synthesis. As expected, Dd-RPS6 seems to be absolutely required for cell survival, because we failed to obtain antisense-RNA mediated cells as well as Dd-rps6 -null cells by homologous recombination in spite of many trials. In many kinds of cell lines, RPS6 is known to be located in the nucleus and cytosol, but Dd-RPS6 is predominantly located in the cell cortex with cytoskeletons, and in the contractile ring of just-dividing cells. In this connection, the overexpression of Dd-RPS6 greatly impairs cytokinesis during axenic shake-cultures in growth medium, resulting in the formation of multinucleate cells. Much severe impairment of cytokinesis was observed when Dd-RPS6-overexpressing cells (Dd-RPS6OE cells) were incubated on a living Escherichia coli lawn. The initiation of differentiation triggered by starvation was also delayed in Dd-RPS6OE cells. In addition, Dd-RPS6OE cells exhibit defective differentiation into prespore cells and spores during late development. Thus, it is likely that the proper expression of Dd-RPS6 may be of importance for the normal progression of late differentiation as well as for the initiation of differentiation. [source] pMesogenin1 and 2 function directly downstream of Xtbx6 in Xenopus somitogenesis and myogenesisDEVELOPMENTAL DYNAMICS, Issue 12 2008Shunsuke Tazumi Abstract T-box transcription factor tbx6 and basic-helix-loop-helix transcription factor pMesogenin1 are reported to be involved in paraxial mesodermal differentiation. To clarify the relationship between these genes in Xenopus laevis, we isolated pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2 appeared to be transcriptional activators and were induced by a hormone-inducible version of Xtbx6 without secondary protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential T-box binding sites with which Xtbx6 protein was shown to interact, and a reporter gene construct containing these sites was activated by Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6 and pMesogenin1 and 2 knockdowns caused similar phenotypic abnormalities including somite malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis including that of body wall muscle in Xenopus laevis. Developmental Dynamics 237:3749,3761, 2008. © 2008 Wiley-Liss, Inc. [source] Effects of locomotor stimulation and protein synthesis inhibition on circadian rhythms in size changes of L1 and L2 interneurons in the fly's visual systemDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2007Elzbieta Kula Abstract Axons of monopolar cell interneurons L1 and L2 in the first optic lobe (lamina) of the fly Musca domestica undergo cyclical changes in diameter. These axons swell during the day and shrink during the night. In addition, the axons' size depends on light conditions since they are largest in continuous light (LL), somewhat smaller under day/night (LD) conditions, and smallest under constant darkness (DD). In this study we found that sizes of both cells can further increase in free flying flies under LD conditions, while the visual stimulation alone does not have significant effect on the cross-sectional area of L1 and L2 axons. The stimulation of free flying had no effect on L1 and L2 sizes if it was performed at the beginning of subjective day in LL or DD. Our results indicate that a maximal increase in size of L1 and L2 is observed when stimulation of free flying is synchronized with a fly' daily peak of activity. We also found that protein synthesis is needed to increase size of monopolar cell axons during the day when they normally swell. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in , -cellsDIABETES OBESITY & METABOLISM, Issue 2010R. J. Kaufman Diabetes is an epidemic of worldwide proportions caused by , -cell failure. Nutrient fluctuations and insulin resistance drive , -cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the , -subunit (eIF2,) to attenuate protein synthesis, thereby reducing the biosynthetic burden. , -Cells uniquely require PERK-mediated phosphorylation of eIF2, to preserve cell function. Unabated protein synthesis in , -cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of , -cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases , -cell mass and prevents , -cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes. [source] Autoradiographic observations on developing and growing claws of reptilesACTA ZOOLOGICA, Issue 2 2010Lorenzo Alibardi Abstract Alibardi, L. 2010. Autoradiographic observations on developing and growing claws of reptiles. ,Acta Zoologica (Stockholm) 91: 233,241 The present qualitative autoradiographic analysis aims to present the main features of morphogenesis and growth of claws in reptiles. Lizard embryos treated with tritiated thymidine reveal that epidermal cell proliferation in terminal digits is prevalent in the dorsal side and gives origin to the curved unguis of the claw. Less proliferation occurs in the ventral side of the digit tip where the concave sub-unguis is derived. Adult claws of a turtle show that thymidine-labelled cells are present along most of the epidermis of the claw, especially at the claw tip. Also, injection of tritiated histidine and proline, indicating active protein synthesis, confirm autoradiographic labelling along most of the epidermis of claws, in particular at the apical tip. The present study indicates that proximal matrix regions, as have been described in mammalian nails, are absent in reptiles. This pattern of claw growth probably derives from that of terminal digital scales. In fact reptilian (and avian) claws are formed from a modification of scales, a different condition from that present in mammals. [source] Ambient ultraviolet-B radiation reduces hatchling size in the common frog Rana temporariaECOGRAPHY, Issue 5 2000Maarit Pahkala Effects of ambient UV-B radiation and pH on hatchability and early development of Rana temporaria embryos were studied in field experiments conducted at two sites in Sweden. In neither of the populations did we find clear evidence for reduced hatchability or increased frequency of developmental anomalies of embryos exposed to ambient UV-B levels. However, in both populations hatchling size was significantly larger UV-B blocked as compared to control treatments, suggesting that ambient UV-B levels had a negative effect on early growth of embryos. This result is consistent with the hypothesis that the cellular UV-B damage repair mechanisms are costly and trades-off against early growth. Alternatively, UV-B induced photoproducts inhibiting DNA-transcription and thereby protein synthesis may directly reduce growth rate. Although low pH (5.0) had negative effects on hatchability and early embryonic growth, there was no evidence for synergistic effects of pH and UV-B on hatchability, frequency of developmental anomalies or early growth. The results suggest that increased levels of UV-B radiation may cause fitness loss in natural populations of the common frog. [source] | |||||||||||||||||||||||||||||||||||||||||||