Induction Of Protein Synthesis Research Articles

BASF to invest $200 M to expand Kaolin business.

Ochratoxin A (OTA) is known to induce nephro-toxicity via induction of cellular redox homeostasis perturbation, mitochondrial hyperpolarisation and depolarization, protein synthesis inhibition leading to apoptosis. In the present study, protective efficacy of N-Acetyl-L-Tryptophan (NAT) against OTA induced toxicity was evaluated using Human Embryonic Kidney (HEK-293) cells. Cells were treated with NAT (0–200 μg/ml) before OTA treatment (0–20 μg/ml) and protective efficacy of NAT was evaluated using MTT and SRB assay. OTA-induced intracellular ROS generation and its inhibition by NAT (2.5 μg/ml) pre-treatment was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe. Effects of NAT pre-treatment on OTA treated cells were also evaluated in terms of cell cycle perturbations and mitochondrial membrane potential disturbance using flowcytometry. Results of the study demonstrated significant (∼89 % cell growth in comparison to 50% in OTA alone group; P < 0.05) protection by NAT to the HEK-293 cells against OTA mediated cell death in terms of cell viability. Further, significant reduction in ROS levels and mitochondrial membrane potential disturbance was also observed in NAT pre-treated plus OTA cells as compared to only OTA treated cells. Significant (p < 0.05) arrest in G0 and S phase of cell cycle was observed in OTA treated cells that was found to be inhibited by NAT pre-treatment to OTA treated cells. Also, molecular docking analysis demonstrated higher probability of NAT to bind with OTA binding pocket on phenylalanyl t-RNA synthetase, resulting in inhibition of OTA incorporation in the newly synthesized peptides and thus may ameliorate OTA induced protein synthesis inhibition. Conclusively, present study suggested that NAT offers protection against OTA toxicity in HEK-293 cells by counterbalancing oxidative stress, cell cycle regulation, mitochondrial membrane potential stabilization, protein synthesis inhibition and cell death retardation.

Understanding the mechanism of bias signaling of the insulin-like growth factor 1 receptor: Effects of LL37 and HASF

The development of biased agonist drugs is widely recognized to be important for the treatment of many diseases, including cardiovascular disease. While GPCR biased agonism has been heavily characterized there is a distinct lack of information with respect to RTK biased agonism both in the identification of biased agonists as well as their attendant mechanisms. One such RTK, the Insulin-like Growth Factor 1 Receptor (IGF1R) plays an important role in a range of biological and disease processes. The micropeptide LL37 has been described as a biased agonist of the IGF1R. We were interested to further understand the mechanism by which LL37 promotes biased signaling through the IGF1R. We found that LL37 biased agonism is dependent on β-arrestin 2. Moreover, BRET assays indicated that LL37 biased agonism is explained by the inability of LL37 to promote the recruitment of IRS1 to the IGF1R compared to IGF1. LL37 promotes an altered association of IGF1R with GRK6, which could also serve as an explanation for bias. We also demonstrated a functional consequence of this bias by showing that while LL37 can promote cell proliferation, it does not induce protein synthesis, unlike IGF1, which does both. We have recently identified HASF, a natural protein released by mesenchymal stem cells, as a novel ligand of the IGF1R. HASF is a paracrine factor with potent cardioprotective and cardio-regenerative properties which also acts via IGF1R biased signaling, preferentially activated ERK over Akt.

Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3′ end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

Inflammatory signalling regulates eccentric contraction-induced protein synthesis in cachectic skeletal muscle.

BackgroundSkeletal muscle responds to eccentric contractions (ECC) with an anabolic response that involves the induction of protein synthesis through the mechanistic target of rapamycin complex 1. While we have reported that repeated ECC bouts after cachexia initiation attenuated muscle mass loss and inflammatory signalling, cachectic muscle's capacity to induce protein synthesis in response to ECC has not been determined. Therefore, we examined cachectic muscle's ability to induce mechano‐sensitive pathways and protein synthesis in response to an anabolic stimulus involving ECC and determined the role of muscle signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) signalling on ECC‐induced anabolic signalling.MethodsMechano‐sensitive pathways and anabolic signalling were examined immediately post or 3 h after a single ECC bout in cachectic male Apc Min/+ mice (n = 17; 16 ± 1% body weight loss). Muscle STAT3/NFκB regulation of basal and ECC‐induced anabolic signalling was also examined in an additional cohort of Apc Min/+ mice (n = 10; 16 ± 1% body weight loss) that received pyrrolidine dithiocarbamate 24 h prior to a single ECC bout. In all experiments, the left tibialis anterior performed ECC while the right tibialis anterior served as intra‐animal control. Data were analysed by Student's t‐test or two‐way repeated measures analysis of variance with Student‐Newman‐Keuls post‐hoc when appropriate. The accepted level of significance was set at P < 0.05 for all analysis.Results Apc Min/+ mice exhibited a cachectic muscle signature demonstrated by perturbed proteostasis (Ribosomal Protein S6 (RPS6), P70S6K, Atrogin‐1, and Muscle RING‐finger protein‐1 (MuRF1)), metabolic (adenosine monophosphate‐activated protein kinase, Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α), and Cytochrome c oxidase subunit IV (COXIV)), and inflammatory (STAT3, NFκB, extracellular signal‐regulated kinases 1 and 2, and P38) signalling pathway regulation. Nonetheless, mechano‐sensitive signalling pathways (P38, extracellular signal‐regulated kinases 1 and 2, and Protein kinase B (AKT)) were activated immediately post‐ECC irrespective of cachexia. While cachexia did not attenuate ECC‐induced P70S6K activation, the protein synthesis induction remained suppressed compared with healthy controls. However, muscle STAT3/NFκB inhibition increased basal and ECC‐induced protein synthesis in cachectic Apc Min/+ mice.ConclusionsThese studies demonstrate that mechano‐sensitive signalling is maintained in cachectic skeletal muscle, but chronic STAT3/NFκB signalling serves to attenuate basal and ECC‐induced protein synthesis.

Acute myotube protein synthesis regulation by IL-6-related cytokines.

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis.

Rhaponticum carthamoides and Rhodiola rosea plant extracts: the right combination between both to get the optimal effect on muscle de novo protein synthesis and performance gain

We previously reported in C2C12 myotubes that a Rhaponticum carthamoides root extract (RCE) was able to significantly stimulate protein synthesis and induce an activation of the Akt/mTOR signalling pathway (induction of Akt and S6K1 phospohrylations), that a Rhodiola rosea root extract (RRE) had also the capacity to induce protein synthesis and to inhibit myostatin gene expression and that the combination of both exhibited inhibition of atrogin gene expression (unpublished). Therefore, we aimed to investigate the effects of RCE or RRE alone or combinations of both on acute muscle protein synthesis stimulation and on performance gain following a 4-week resistance training program in adult rats. Acutely, rats were trained on a ladder making 3 repetitions of 10 climbs with a load that successively reached 0%, 50% and 75% of their body mass and were fed (oral gavage) just after training with either placebo, whey protein (910 mg/kg bw) as a positive control, RRE (43 mg/kg bw), RCE (43 mg/kg) and different doses of a 50:50 combination of both (87, 43, 22 or 9 mg/kg bw). RCE alone but not RRE was able to significantly stimulate protein synthesis as measured by western blot quantitative analysis of puromycin incorporation in the flexor digitorum profondus (FDP) and in comparison to placebo (200% versus 100% for RCE and placebo group respectively). Any doses of the combination was significantly superior to RRE alone (300% versus 150%) or RCE alone (300% versus 200%) and also significantly superior to whey proteins (300% versus 150%). The same results were obtained in the biceps and deltoid muscles. In the second part of the study rats followed a 4-week progressive resistance training program while consuming RRE alone (22 mg/kg), RCE alone (22 mg/kg) and different ratio and doses of the combination (50:50 ratio: 43, 22 or 9 mg/kg bw; 83 RCE:17 RRE ratio: 26 mg/kg; 76 RCE:24 RRE ratio: 28 mg/kg; 70 RCE:30 RRE ratio: 15 mg/kg. Training sessions (19) consisted in one set of 10 repetitions with 2 minutes rest between trials and 5 minutes between each set, were held five times a week with initial load fixed at 50% of the rat body weight and increased progressively until 200% after 4 weeks. Maximal repetition was determined as the maximum weight carried up the exercise ladder by the rats in one climb and performance gain was calculated as delta power (post versus pre resistance training). As shown in Figure 1, RCE alone but not RRE was able to significantly increase performance gain in comparison to placebo after 4 weeks of resistance training (0.66±0.08 versus 0.39±0.08 W respectively, + 65%, p<0.05). A synergistic effect on performance gain was detected for the 50:50 combination but interestingly, the combination ratio 70:30 warranted the best improvement of performance gain with the lower daily dosage (15 mg/kg bw) in comparison to placebo (0.62±0.08 versus 0.39±0.08 W respectively, + 59%, p<0.05). These results clearly demonstrated synergy of the combination of extracts on protein synthesis stimulation in the FDP, deltoid and biceps muscles following acute resistance exercise and an improvement of performance gain following a 4-week resistance training program. Those effects are currently investigated in recreationally active men and histological analysis of muscle fibers are under investigations in rats to understand what could drive the effect on performance.

Immunotoxin and bcl-2 inhibitor combination therapy targeting chondroitin sulfate proteoglycan 4.

74 Background: Immunotoxins (ITs) are a class of bifunctional chimeric proteins composed of an antibody fragment linked to a toxin. When ITs internalize into target cells, they induce protein synthesis inhibition and apoptosis. While ITs are highly specific and potent, the efficacy of IT-based therapies in some tumor cells is limited by hyperactive anti-apoptotic pathways and inefficient translocation of ITs from the endoplasmic reticulum to the cytosol. Therefore, to improve the efficacy of IT-based therapies, we evaluated a dual-pathway therapy that combines an IT with the ABT-737, ABT-263, or ABT-199 small molecule Bcl-2 inhibitor. Methods: The immunotoxin 9.2.27-PE38KDEL (9.2.27-IT) was generated by fusing a truncated mutant form of Pseudomonas exotoxin A to a single-chain variable fragment antibody. It targets human chondroitin sulfate proteoglycan 4 (CSPG4), an antigen highly expressed in a variety of cancer cells. We screened and identified 3 human glioblastoma xenografts, 3 human melanoma cell lines, and 5 human breast cancer cell lines resistant to the 9.2.27-IT despite their high levels of cell surface expression of CSPG4 (IC50 of IT alone was &gt;100 ng/ml in all cell lines except for one melanoma cell line). In vitro cytotoxicity of the 9.2.27-IT —alone or in combination with the individual Bcl-2 inhibitors ABT-737, ABT-263, or ABT199—was assessed. Concentrations of ABT analogues were chosen so that ABT alone did not induce cytotoxicity. Results: The treatment groups that responded to the combination therapy yielded IC50 values ranging from 0.04 – 9 ng/ml for glioblastoma xenografts, 0.21-15 ng/ml for melanoma cell lines, and 4.5-50 ng/ml for breast cancer cell lines. The most potent combination group showed &gt;1000 fold improvement of IC50 compared to using the immunotoxin alone. ABT-737 produced the strongest synergistic effects among the ABT analogues. Preliminary results from in vivo studies further demonstrated that this approach engendered a synergistic response and delayed tumor growth in immunotoxin-resistant mouse tumor models. Conclusions: This new combinatorial approach will potentially help to overcome immunotoxin resistance in cancer patients and provide better therapeutic outcomes.

FGF21 Is an Exocrine Pancreas Secretagogue

The metabolic stress hormone FGF21 is highly expressed in exocrine pancreas, where its levels are increased by refeeding and chemically induced pancreatitis. However, its function in the exocrine pancreas remains unknown. Here, we show that FGF21 stimulates digestive enzyme secretion from pancreatic acinar cells through an autocrine/paracrine mechanism that requires signaling through a tyrosine kinase receptor complex composed of an FGF receptor and β-Klotho. Mice lacking FGF21 accumulate zymogen granules and are susceptible to pancreatic ER stress, an effect that is reversed by administration of recombinant FGF21. Mice carrying an acinar cell-specific deletion of β-Klotho also accumulate zymogen granules but are refractory to FGF21-stimulated secretion. Like the classical post-prandial secretagogue, cholecystokinin (CCK), FGF21 triggers intracellular calcium release via PLC-IP3R signaling. However, unlike CCK, FGF21 does not induce protein synthesis, thereby preventing protein accumulation. Thus, pancreatic FGF21 is a digestive enzyme secretagogue whose physiologic function is to maintain acinar cell proteostasis.

Regulation of mRNA Translation by MID1: A Common Mechanism of Expanded CAG Repeat RNAs.

Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT) RNA, which is linked to Huntington's disease (HD), at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analyzed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2), Ataxin3 (ATXN3), and Ataxin7 (ATXN7), that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3, and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3, and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs.

Panaxatriol derived from ginseng augments resistance exercised–induced protein synthesis via mTORC1 signaling in rat skeletal muscle

Resistance exercise activates muscle protein synthesis via the mammalian target of rapamycin complex 1 (mTORC1) pathway and subsequent muscle hypertrophy. Upstream components of the mTORC1 pathway are widely known to be involved in Akt and extracellular signal–regulated kinase 1/2 (ERK1/2) signaling. Previous studies have shown that ginseng stimulated Akt and ERK1/2 signaling. Therefore, we hypothesized that panaxatriol (PT) derived from ginseng triggers mTORC1 signaling and muscle protein synthesis by activating both the Akt and ERK1/2 signaling pathways, and that PT additively stimulates muscle protein synthesis when combined with resistance exercise. The study included male Sprague-Dawley rats. The legs of the rats were divided into control, PT-only, exercise-only, and exercise + PT groups. The right legs were subjected to isometric resistance exercise using percutaneous electrical stimulation, whereas the left legs were used as controls. PT (0.2 g/kg) was administered immediately after exercise. The Akt and ERK1/2 phosphorylation levels were significantly higher in the exercise + PT group than in the exercise-only group 0.5 hour after exercise. The phosphorylation of p70S6K was significantly increased at both 0.5 and 3 hours after exercise, and it was higher in the exercise + PT group than in the exercise-only group at both 0.5 and 3 hours after exercise. Muscle protein synthesis was significantly increased 3 hours after exercise, and it was higher in the exercise + PT group than in the exercise-only group 3 hours after exercise. Our results suggest that PT derived from ginseng enhances resistance exercise–induced protein synthesis via mTORC1 signaling in rat skeletal muscle.

Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress.

Severe ethanol stress (>9% ethanol, v/v) as well as glucose deprivation rapidly induces a pronounced repression of overall protein synthesis in budding yeast Saccharomyces cerevisiae. Therefore, transcriptional activation in yeast cells under severe ethanol stress does not always indicate the production of expected protein levels. Messenger RNAs of genes containing heat shock elements can be intensively translated under glucose deprivation, suggesting that some mRNAs are preferentially translated even under severe ethanol stress. In the present study, we tried to identify the mRNA that can be preferentially translated under severe ethanol stress. BTN2 encodes a v-SNARE binding protein, and its null mutant shows hypersensitivity to ethanol. We found that BTN2 mRNA was efficiently translated under severe ethanol stress but not under mild ethanol stress. Moreover, the increased Btn2 protein levels caused by severe ethanol stress were smoothly decreased with the elimination of ethanol stress. These findings suggested that severe ethanol stress extensively induced BTN2 expression. Further, the BTN2 promoter induced protein synthesis of non-native genes such as CUR1, GIC2, and YUR1 in the presence of high ethanol concentrations, indicating that this promoter overcame severe ethanol stress-induced translation repression. Thus, our findings provide an important clue about yeast response to severe ethanol stress and suggest that the BTN2 promoter can be used to improve the efficiency of ethanol production and stress tolerance of yeast cells by modifying gene expression in the presence of high ethanol concentration.

Proteomics study revealed altered proteome of Dichogaster curgensis upon exposure to fly ash

Fly ash is toxic and its escalating use as a soil amendment and disposal by dumping into environment is receiving alarming attention due to its impact on environment. Proteomics technology is being used for environmental studies since proteins respond rapidly when an organism is exposed to a toxicant, and hence soil engineers such as earthworms are used as model organisms to assess the toxic effects of soil toxicants. This study adopted proteomics technology and profiled proteome of earthworm Dichogaster curgensis that was exposed to fly ash, with main aim to elucidate fly ash effects on cellular and metabolic pathways. The functional classification of identified proteins revealed carbohydrate metabolism (14.36%), genetic information processing (15.02%), folding, sorting and degradation (10.83%), replication and repair (3.95%); environmental information processing (2.19%), signal transduction (9.61%), transport and catabolism (17.27%), energy metabolism (6.69%), etc. in the proteome. Proteomics data and functional assays revealed that the exposure of earthworm to fly ash induced protein synthesis, up-regulation of gluconeogenesis, disturbed energy metabolism, oxidative and cellular stress, and mis-folding of proteins. The regulation of ubiquitination, proteasome and modified alkaline comet assay in earthworm coelomocytes suggested DNA-protein cross link affecting chromatin remodeling and protein folding.

Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR.

As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

α-Lipoic Acids Promote the Protein Synthesis of C2C12 Myotubes by the TLR2/PI3K Signaling Pathway.

Skeletal muscle protein turnover is regulated by endocrine hormones, nutrients, and inflammation. α-Lipoic acid (ALA) plays an important role in energy homeostasis. Therefore, the aim of this study was to investigate the effects of ALA on protein synthesis in skeletal muscles and reveal the underlying mechanism. ALA (25 μM) significantly increased the protein synthesis and phosphorylation of Akt, mTOR, and S6 in C2C12 myotubes with attenuated phosphorylation of AMPK, Ikkα/β, and eIF2α. Intraperitoneal injection of 50 mg/kg ALA also produced the same results in mouse gastrocnemius. Both the PI3K (LY294002) and mTOR (rapamycin) inhibitors abolished the effects of ALA on protein synthesis in the C2C12 myotubes. However, AICAR (AMPK agonist) failed to block the activation of mTOR and S6 by ALA. ALA increased TLR2 and MyD88 mRNA expression in the C2C12 myotubes. TLR2 knockdown by siRNA almost eliminated the effects of ALA on protein synthesis and the Akt/mTOR pathway in the C2C12 myotubes. Immunoprecipitation data showed that ALA enhanced the p85 subunit of PI3K binding to MyD88. These findings indicate that ALA induces protein synthesis and the PI3K/Akt signaling pathway by TLR2.

A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

Babesiosis due to Babesia bigemina is a relevant tick-borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA-1) - have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA-1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA-1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA-1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti-AMA-1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA-1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies.

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser51 phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells.

Investigating the Effects of Paraquat on Protein Synthesis in C2C12 Myoblasts

Reactive oxygen species (ROS) related signaling is important for stress adaptation, although prolonged oxidative stress may contribute to the aging phenotype. Specifically, ROS may alter protein turnover in a duration dependent manner with acute ROS exposure increasing protein synthesis for cellular adaptation, while extended exposure may decrease protein synthesis. Paraquat (PQ) is a mitochondrial complex I inhibitor that induces superoxide production. We hypothesized that acute PQ treatment would induce protein synthesis in cells, but prolonged exposure would reduce overall protein synthesis. We treated C2C12 myoblasts with 0mM, 1mM, or 2mM PQ for 4, 8, and 12 h while using 4% deuterium oxide enriched media to measure protein synthesis of mixed (mix), cytosolic (cyto), and mitochondrial (mito) fractions. PQ increased protein content of HO-1, a marker of oxidative stress. 2mM PQ augmented the mito protein fractional synthesis rate (FSR) at 4 h of exposure., but 8 and 12 h, mito FSR was no different from vehicle treated controls. Cyto FSR was lower at 8 and 12 h with 2mM PQ compared to control. Mix FSR was lower in response to 2mM of PQ at 12 h. Oxidative stress induced by PQ temporarily increased mito FSR. Prolonged PQ exposure, though, reduced protein synthesis in the other fractions by 12 h. Further investigation should examine the effects of PQ on protein breakdown to understand the effects of ROS on protein turnover.

Lewis Lung Carcinoma Regulation of Mechanical Stretch Induced Protein Synthesis in Cultured Myotubes

Mechanical stimuli, such as stretch, can activate muscle and myotube protein synthesis through mTORC1 signaling. However, it has not been determined if stretch can regulate protein synthesis in cac...

Eicosapentaenoic acid in cancer improves body composition and modulates metabolism

ObjectivesThe objective of this review article is to present the most recent intervention studies with EPA on nutritional outcomes in cancer patients, e.g. nutritional status, weight & lean body mass. MethodsFor this purpose a PubMed® and MedLine® search of the published literature up to and including January 2014 that contained the keywords: cancer, sarcopenia, EPA, ω-3 fatty acids, weight, intervention trial, muscle mass was conducted. The collected data was summarized and written in text format and in tables that contained: study design, patient' population, sample size, statistical significance and results of the intervention. The paper will cover malignancy, body composition, intervention with EPA, physiological mechanisms of action of EPA, effect of EPA on weight and body composition, future research. ResultsIn cancer patients deterioration of muscle mass can be present regardless of body weight or Body Mass Index (BMI). Thus, sarcopenia in cancer patients with excessive fat mass (FM), entitled sarcopenic obesity, has gained greater relevance in clinical practice; it can negatively influence patients' functional status, tolerance to treatments & disease prognosis. The search for an effective nutritional intervention that improves body composition (preservation of muscle mass and muscle quality) is of utmost importance for clinicians and patients. The improvement of muscle quality is an even more recent area of interest because it has probable implications in patients' prognosis. Eicosapentaenoic acid (EPA) has been identified as a promising nutrient with the wide clinical benefits. Several mechanisms have been proposed to explain EPA potential benefits on body composition: inhibition of catabolic stimuli by modulating pro-inflammatory cytokines production and enhancing insulin sensitivity that induces protein synthesis; also, EPA may attenuate deterioration of nutritional status resulting from antineoplastic therapies by improving calorie and protein intake as well. ConclusionsIndeed, cancer-related sarcopenia/cachexia is a multifactorial syndrome characterized by inflammation, anorexia, weight loss, and muscle/adipose tissue loss mediated by proinflammatory cytokines, e.g. TNF-α and IL-6, resulting in increased chemotherapy toxicity, costs, morbidity and mortality. With this review we found that EPA can reduce inflammation and has the potential to modulate nutritional status/body composition. In view of the modest survival benefits of chemotherapy/radiotherapy in some cancers, important issues for physicians are to optimize well-being, Quality of Life via nutritional status and adequate body composition. Thus, improvement in nutritional status is a central outcome.

Multiple myeloma proteostasis can be targeted via translation initiation factor eIF4E.

Intensive protein synthesis is a unique and differential trait of the multiple myeloma (MM) cells. Previously we showed that tetraspanin overexpression in MM cell lines attenuated mTOR and PI3K cascades, induced protein synthesis, activated unfolded protein response (UPR), and caused autophagic death, all suggesting breach of proteostasis. Here we assessed the role of translation initiation in the tetraspanin‑induced MM cell death with emphasis on eIF4E translation initiation factor. We showed tetraspanins attenuated peIF4E and its targets [c‑Myc, cyclin D1 (cycD1)]; eIF4E attenuation was Akt-dependent. eIF4E inhibition in MM cells [bone marrow (BM), lines] by siRNA and/or the anti‑viral drug and competitive eIF4E inhibitor ribavirin (RBV) deleteriously affected MM cells in a similar manner to the overexpression of tetraspanins. Furthermore, combined application of RBV and velcade had a synergistic anti‑MM effect. Our results demonstrate that breach of proteostasis via eIF4E inhibition is an attractive therapeutic approach that may be relatively easily achieved by employing RBV, making this strategy readily translatable into the clinic.