Protein Synthesis Factors Research Articles

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers.

ABSTRACTFriedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers.

Modulation of Protein Synthesis by Polyamines in Mammalian Cells.

Polyamines exist mainly as RNA-polyamine complexes in cells. Thus, we looked for proteins whose synthesis is enhanced by polyamines at the level of translation in mammalian cells. Here, we describe how synthesis of Cct2 (T-complex protein 1, β-subunit, a chaperonin assisting in the folding actin, tubulin, and several other proteins) and eEF1A (one of the elongation factors of protein synthesis) is stimulated by polyamines at the level of translation. Polyamines stimulated Cct2 synthesis through the stimulation of ribosome shunting during 5'-processive scanning of 40S ribosomal subunits from the m7G-cap to the initiation codon AUG, and eEF1A synthesis through the structural change of the unusual position of a complementary sequence to 18S rRNA in eEF1A mRNA.

Skeletal muscle morphology and regulatory signalling in endurance-trained and sedentary individuals: The influence of ageing

Muscle mass in humans is inversely associated with circulating levels of inflammatory cytokines, but the interaction between ageing and training on muscle composition and the intra-muscular signalling behind inflammation and contractile protein synthesis and degradation is unknown. We studied 15 healthy life-long endurance runners, 12 age-matched untrained controls, 10 young trained and 12 young untrained individuals. Thigh muscle composition was investigated by magnetic resonance imaging (MRI), where non-contractile intramuscular tissue (NCIT) area (fat and connective tissue) was found to be greater in older but lower in trained individuals. Subcutaneous adipose tissue was also lower in trained individuals but was not affected by age. In vastus lateralis biopsies, no influence of age or training was found on levels of endomysial collagen, determined by Sirius Red and Collagen III staining, whereas perimysial organisation tended to be more complex in older individuals. No clear difference with training was seen on intramuscular inflammatory signalling, whereas lower protein levels of NFkB subunits p105, p50 and p65 were observed with ageing. Gene expression of IL6 and TNFα was not different between groups, while IL1-receptor and TNFα-receptor1 levels were lower with age. Myostatin mRNA was lower in older and trained groups, while expression of MuRF1 was lower in trained individuals and FoxO3 expression was greater in aged groups. The association of increased muscle NCIT with age-associated muscle loss in humans is not accompanied by any major alterations in intramuscular signalling for inflammation, but rather by direct regulatory factors for protein synthesis and proteolysis in skeletal muscle.

Involvement of nucleoside diphosphate kinase b and elongation factor 2 in Leishmania braziliensis antimony resistance phenotype.

BackgroundNucleoside diphosphate kinase b (NDKb) is responsible for nucleoside triphosphates synthesis and it has key role in the purine metabolism in trypanosomatid protozoans. Elongation factor 2 (EF2) is an important factor for protein synthesis. Recently, our phosphoproteomic analysis demonstrated that NDKb and EF2 proteins were phosphorylated and dephosphorylated in antimony (SbIII)-resistant L. braziliensis line compared to its SbIII-susceptible pair, respectively.MethodsIn this study, the overexpression of NDKb and EF2 genes in L. braziliensis and L. infantum was performed to investigate the contribution of these proteins in the SbIII-resistance phenotype. Furthermore, we examined the role of lamivudine on SbIII susceptibility in clones that overexpress the NDKb gene, and the effect of EF2 kinase (EF2K) inhibitor on the growth of EF2-overexpressing parasites.ResultsWestern blot analysis demonstrated that NDKb and EF2 proteins are more and less expressed, respectively, in SbIII-resistant line of L. braziliensis than its wild-type (WTS) counterpart, corroborating our previous phosphoproteomic data. NDKb or EF2-overexpressing L. braziliensis lines were 1.6 to 2.1-fold more resistant to SbIII than the untransfected WTS line. In contrast, no difference in SbIII susceptibility was observed in L. infantum parasites overexpressing NDKb or EF2. Susceptibility assays showed that NDKb-overexpressing L. braziliensis lines presented elevated resistance to lamivudine, an antiviral agent, but it did not alter the leishmanicidal activity in association with SbIII. EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of SbIII, suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy.ConclusionOur findings represent the first study of NDKb and EF2 genes overexpression that demonstrates an increase of SbIII resistance in L. braziliensis which can contribute to develop new strategies for leishmaniasis treatment.

Isolation of bacterial compartments to track movement of protein synthesis factors

Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARSs also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities. Cell fractionation included isolation of the cytoplasm, periplasm, membrane, outer membrane vesicles, and extracellular media. Specific endogenous proteins and RNAs were probed respectively within each fraction via Western blots using antibodies and by Northern blots with primers to unique regions of the nucleic acid.

Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in Caenorhabditis elegans

Upon pathogen infection, microbial killing pathways and cellular stress pathways are rapidly activated by the host innate immune system. These pathways must be tightly regulated because insufficient or excessive immune responses have deleterious consequences. Increasing evidence indicates that the nervous system regulates the immune system to confer coordinated protection to the host. However, the precise mechanisms of neural-immune communication remain unclear. Previously we have demonstrated that OCTR-1, a neuronal G protein-coupled receptor, functions in the sensory neurons ASH and ASI to suppress innate immune responses in non-neural tissues against Pseudomonas aeruginosa in Caenorhabditis elegans. In the current study, by using a mass spectrometry-based quantitative proteomics approach, we discovered that OCTR-1 regulates innate immunity by suppressing translation and the unfolded protein response (UPR) pathways at the protein level. Functional assays revealed that OCTR-1 inhibits specific protein synthesis factors such as ribosomal protein RPS-1 and translation initiation factor EIF-3.J to reduce infection-triggered protein synthesis and UPR. Translational inhibition by chemicals abolishes the OCTR-1-controlled innate immune responses, indicating that activation of the OCTR-1 pathway is dependent on translation upregulation such as that induced by pathogen infection. Because OCTR-1 downregulates protein translation activities, the OCTR-1 pathway could function to suppress excessive responses to infection or to restore protein homeostasis after infection.

In vitro fermentation of conventional diets commonly fed to dairy cows in Central Myanmar

This study consisted of two experiments conducted to evaluate the effective net gas, fermentation kinetics (experiment 1), methane gas concentration, partitioning factor (PF) for microbial protein synthesis and in vitro dry matter digestibility (IVDMD) (experiment 2) of conventional diets commonly fed to dairy cows in Central Myanmar. The conventional diets from four areas [diet from Sin Tel area (Diet-ST), diet from Myay Ngu area (Diet-MN), diet from Ta Pel area (Diet-TP) and diet from Amarapura area (Diet-AM)] were used as experimental diets in this study. In most of conventional diets, rice straw, sorghum stover and natural grass were used as roughage source and cotton seed cake and broken rice were used as concentrate. However in some diets, sesame residue and butter bean residue were used as roughage source instead of sorghum stover and natural grass. The roughage to concentrate ratio and crude protein (CP) content of conventional diets ranged from 53:47 to 72:28 and 11.46 to 17.96%, respectively. In experiment 1, the effective net gas volume of Diet-TP was lower (p<0.05) than Diet-ST and Diet-AM and generally, the fermentation kinetics (a, b, c and a+b) of Diet-TP were also lower than those of other diets. In the experiment 2, the lower value (p<0.05) of short chain fatty acid (SCFA) was found in Diet-MN and Diet-TP while the higher values (p<0.05) of metabolizable energy (ME) and organic matter digestibility (OMD) were observed Diet-AM and Diet-TP. Although methane gas concentration of Diet-TP was higher (p<0.05) than those of other diets, the greater values of IVDMD and PF were observed in Diet-TP. According to these findings, it was perceived that all conventional diets have different nutritional qualities which are useful for production and health of dairy cows; however the Diet-TP possessed the highest nutritional qualities among the conventional diets.

Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.

MKNK2 is a valid target of miR-125b in breast cancer

MAP kinase interacting serine/threonine kinase 2 (MKNK2) belongs to the protein kinase superfamily which phosphorylates and activates eukaryotic initiation factor 4E (elF4E), a rate limiting factor in protein synthesis that enhances the translation of some proteins involved in cell cycle, apoptosis and angiogenesis regulation. In this study, we found that the expression levels of MKNK2 are inversely correlated with the expression levels of miR-125b in human breast cancer development. We showed that miR-125b inhibited tumor cell growth and invasion by regulating MKNK2 at posttranscriptional level. Thus, both miR-125b and MKNK2 could be therapeutically targeted in breast cancer.

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD.

Amino acid utilization of lactating dairy cows when diets are changed from an alfalfa-based diet to cereal straw-based diets

This experiment assessed the utilization of amino acids (AA) by the mammary gland of cows when diets are changed from an alfalfa hay-based diet (AH) to cereal straw-based diets using corn stover (CS) or rice straw (RS). Thirty multiparous Holstein dairy cows were selected and randomly assigned to 1 of 3 treatments (n=10). After 13 weeks’ feeding, arterial and venous plasma were collected every 6h over 2days and AA concentrations measured. The cows fed the CS or RS diet had lower milk and protein yield despite similar dry matter intake. The digestive AA flow was predicted to be lower in the cows fed CS or RS than in AH-fed cows. The arterial concentration of methionine and valine was lower in the cows fed RS than in cows fed AH. The mammary uptake of most essential AA, especially branched-chain AA, methionine, and arginine, was greater in the AH-fed cows than in cows fed CS or RS. The ratio of mammary uptake to milk output of methionine was lower in the RS-fed cows than that in the cows fed AH, with a value below 1 for both. In summary, the insufficient supply of free AA from arterial plasma presented to the mammary gland and lower mammary plasma flow restrict the mammary AA uptake for milk protein synthesis when energy is limited. On the other hand, the utilization of leucine and methionine may be a limiting factor for milk protein synthesis and lactation performance when corn stover or rice straw is fed to dairy cows.

Banana MaEF1A facilitates plant growth and development

Plant translation elongation factor 1 alpha (EF1A) is both a protein synthesis factor and an important component of plant signal transduction, immune responses, protein trafficking, and apoptosis. However, its role in plant growth and development remains unclear. Herein, a full-length EF1A gene was isolated from banana (Musa acuminata L.) fruit and termed MaEF1A. We found that MaEF1A shared a high sequence identify with respective genes in other plants and the deduced amino acid sequence contained conserved regions of GTP-EFTU, GTP-EFTU-02, and GTP-EFTU-03, as well as two tRNA binding domains and six GTP-binding sites which represent functional domains for protein biosynthesis. MaEF1A protein is mainly localized to the nucleus. MaEF1A was constitutively expressed in different banana organs including developing fruits, and the highest expression was detected in ovary 4 stage. Arabidopsis thaliana L. (ecotype Columbia) was transformed with MaEF1A and four transgenic lines were obtained. Three transgenic lines were selected for further phenotypic analyses. Our findings indicate that overexpressed MaEF1A could greatly enhance plant height, root length, and both rhachis and silique length by promoting cell expansion and elongation. These experiments suggest an important role for MaEF1A in plant growth and development.

Enhanced translation initiation factor 4G levels correlate with production levels of monoclonal antibodies in recombinant CHO cell lines.

Using cells to manufacture protein-based therapeutics or biopharmaceuticals is a rapidly expanding industrial activity. Chinese hamster ovary (CHO) cells are the most frequently used mammalian host-expression system for the manufacture of biopharmaceuticals. Over the past ∼30years academic and industrial researchers have studied cell expression characteristics with aims to improve product yield, quality, scalability and reproducibility. Although many steps in the gene expression and secretion pathways have been optimized, little attention has been paid to optimizing protein synthesis factors and regulators during this process. A new study in Biochemical Journal by Mead et al., provides a first systematic study of several protein synthesis factors and finds that the expression level of eIF4G1 correlates with the level of recombinant protein expressed in cultures. Optimizing levels and activities of protein synthesis factors may help to enhance recombinant protein expression of biopharmaceuticals.

Profiling of Up-Regulated Genes Response to Acute Hypo-Osmotic Stress in Hepatopancreas and Gill of the Pacific White Shrimps (Litopenaeus vannamei)

<p class="1Body">Suppression subtractive hybridization (SSH) was applied to screen responsively up-regulation genes in hepatopancreas and gill of Litopenaeus vannamei induced by acute hypo-osmotic stress. In the hepatopancreas, 196 clones were randomly selected and sequenced. 131 non-redundant transcripts, corresponding to 41 genes, were found with elevated expressions. They were functionally clustered into eight biological processes which were protein synthesis and processing, carbohydrate metabolism and energy production, transport, cell growth, apoptosis, cytoskeletal component, cell defense and homeostasis, signal transduction, accounting for 26.2%, 14.3%, 16.7%, 4.7%, 4.7%, 2.3%, 11.9% and 7.1%, respectively. When confirmed by real time qRT-PCR, the gene expression levels of MnSOD, glutathione and glutamine synthetase showed significant increases (2.64-folds, 3.44-folds and 2.16-folds, respectively) in hepatopancreas (<em>P</em><0.05). Totally 81 expressed sequence tags (ESTs) were obtained by random sequencing from the gill SSH cDNA library, and 52 unigenes, including 15 contigs and 37 singlets, after CP3 online matching were gained by clustering and assembling. The sequence alignment results revealed that 30 of them had significant homology to previously identified Genbank database sequences, while 22 unigenes did not match any sequence in the datebase and presumably represented unidentified cDNAs. Functionally, the unigenes were classified into six clusters, including ion channels and transport proteins (1); protein synthesis, translation and transcription factor (2); stress resistance and antioxidant factors (3); energy metabolism (4); signal receptor and transduction (5); cell fibrin and cytoskeletal proteins (6) with proportion of 23.3%, 20.0%, 20.0%, 16.7%, 10.0% and 10.0% respectively. Real time qRT-PCR confirmation revealed significant elevation of Arginine kinase, Carbonic anhydrase and NaK-ATPase-α-subunit expressions (3.73-folds, 2.55-folds and 5.83-folds, respectively) in the gill after acute low-osmotic stress (P < 0.05). These results provided insight into critical physiology processes and pathways constituting the hypo-osmotic stress adaption program in hepatopancreas and gill of <em>L. vannamei</em>.</p>

Induction of eEF2-specific antitumor CTL responses in vivo by vaccination with eEF2-derived 9mer-peptides.

Eukaryotic elongation factor 2 (eEF2) is an essential factor for protein synthesis. Previous studies have shown that the eEF2 gene was overexpressed and plays an oncogenic role in various types of cancers and that eEF2 gene product elicited both humoral immune responses to produce eEF2-specific IgG autoantibody in cancer-bearing individuals and cellular immune responses to induce eEF2 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro. The purpose of the present study was to induce eEF2-specific, antitumor CTL responses in vivo by vaccination with MHC class I-binding eEF2-derived peptide. First, two mouse MHC class I-restricted eEF2‑derived, 9-mer peptides, EF17 (17-25 aa, ANIRNMSVI) and EF180 (180-188 aa, RIVENVNVI) were identified as eEF2-specific CTL peptides, and mice were vaccinated intradermally eight times with either EF17 or EF180 peptide emulsified with Montanide ISA51 adjuvant. Cytotoxicity assay showed that eEF2-specific CTLs were induced in both EF17‑and EF180‑vaccinated mice, and histological study showed no detectable damage in the organs of these mice. Next, to examine in vivo antitumor effects of eEF2 peptide vaccination in a therapeutic model, mice were vaccinated four times with one each of the two eEF2 peptides at weekly intervals after implantation of eEF2-expressing leukemia cells. The vaccination with eEF2 peptides induced eEF2-specific CTLs and suppressed tumor growth, and disease-free survival was significantly longer in EF180-vaccinated mice compared to control mice. The survival was associated with the robustness of eEF2-specific CTL induction. These results indicate that vaccination with MHC class I-binding eEF2 peptide induced eEF2-targeting, antitumor CTL responses in vivo without damage to normal organs, which provided us a rationale for eEF2 peptide-based cancer immunotherapy.

The IGF-1/Akt/S6 Signaling Pathway is Age-Dependently Downregulated in the Chicken Breast Muscle.

The skeletal muscle mass is known to be controlled by the balance between protein synthesis and degradation. The fractional rate of protein synthesis has been reported to decrease age-dependently from 1 to 4 weeks of age in the chicken breast muscle (pectoralis major muscle). On the other hand, age-dependent change of the fractional protein degradation rate was reported to be less in the skeletal muscle of chickens. These findings suggest that protein synthesis is age-dependently downregulated in chicken muscle. We herein investigated the age-dependent changes in protein synthesis or proteolysis-related factors in the breast muscle of 7, 14, 28, and 49-day old broiler chickens. IGF-1 mRNA level, phosphorylation rate of Akt, and phospho-S6 content were coordinately decreased in an age-dependent manner, suggesting that IGF-1-stimulated protein synthesis is downregulated with age in chicken breast muscle. In contrast, atrogin-1, one of the proteolysis-related factors, gradually increased with age at mRNA levels. However, plasma Nτ-methylhistidine concentration, an indicator of skeletal muscle proteolysis, did not coordinately change with atrogin-1 mRNA levels. Taken together, our results suggest that the IGF-1/Akt/S6 signaling pathway is age-dependently downregulated in the chicken breast muscle.

Collagen 3

Obesity is a newly classified metabolic disease that can impair anabolic signaling during skeletal muscle regeneration. Optimal skeletal muscle regeneration includes inflammation and ECM remodeling. Disruption to any of these processes can negatively impact skeletal muscle regeneration. Previous literature has reported dysregulated signaling in the inflammatory response as well as a reduction in protein synthesis and myogenic regulatory factors in obese animal models. As a result, there is blunted skeletal muscle growth exhibited in these individuals. The extracellular matrix serves as a scaffold in skeletal muscle that is also used as a reservoir for proteins and growth factors that promote regeneration. PURPOSE: To determine how diet-induced obesity (DIO) alters ECM remodeling during skeletal muscle regeneration. METHODS: Forty-eight male C57BL/6J mice were randomly assigned to two groups; lean diet (10% fat) and high fat diet (HFD) (60% fat). Within those two groups, mice were randomly administered PBS (uninjured) group or bupivacaine (injured) group. Three or 28 days post-injection, the TAs were extracted and PCR reaction was done to quantify ECM-related gene expression. RESULTS: Collagen-I gene expression was 12-fold and 17-fold greater in the lean and HFD groups, respectively 3 days post-bupivacaine injection (p < 0.05). There was a main effect of injury to decrease collagen-I gene expression 28 days post-bupivacaine-injection (p < 0.05). Collagen-III gene expression was 3-fold greater in the lean group and 43-fold greater in the HFD group (p < 0.05) 3 day post-bupivacaine injection. There were no differences in collagen-III gene expression 28 days post-bupivacaine injection. There was a 55% reduction in collagen-III/I in the lean injured group, but a 4.5-fold increase in the HFD injured group (p < 0.05) 3 days post-bupivacaine injection. Collagen-III/I was 8-fold greater in the lean 28 days post-bupivacaine injection (p < 0.05), but there was no significant differences in obese uninjured and injured. CONCLUSIONS: Obesity reversed collagen III/I acutely during regeneration and was blunted 4 weeks following muscle damage. These findings suggest that obesity may negatively impact ECM composition thus reducing the capacity to regenerate skeletal muscle following injury.

Evolution Experiment With Translation-Coupled RNA Replication System

The ability to evolve is a key characteristic that distinguishes living things from non-living chemical compounds. The construction of an evolvable cell-like system from non-living molecules has been a major challenge. We encapsulated an artificial RNA genome and the factors for protein synthesis into water droplets in oil or lipid vesicles to develop an evolvable artificial cell model. In the micro-compartments, the artificial genomic RNA replicates through the translation of its encoding RNA replicase gene. Using the translation-coupled RNA replication system, we performed a long-term (600-generation) evolution experiment, in which mutations were spontaneously introduced by the translated replicase into its genetic information. At the beginning, the amplified RNA genomes were in the double stranded form, a dead-end product for the translation while a small parasitic RNA appeared by a deletion mutation on the RNA genome and dominated the population by stealing the replicase translated from the RNA genome. But highly replicable mutant RNA genomes gradually evolved to eliminate the two short circuits by reinforcing the interaction with the translated replicase. At the end, two-order acceleration in replication rate was observed whereas the population declined when the same reaction was conducted in a bulk solution. The results indicated that the micro-compartmentalization was essential for the assembly of the bio-polymers to evolve.

Paradoxical sleep: A vigilance state to gate long-term brain plasticity?

Memory consolidation is the process for long-term storage of information and protection against interferences. It has been proposed that long-term potentiation (LTP), the long-lasting enhancementof synaptic transmission, is a cellular model for memory consolidation. Since consolidation of several forms of memory is facilitated by paradoxical sleep (PS) we ask whether PS modulates the cellular and molecular pathways underlying LTP. The long-lasting form of LTP (L-LTP) is dependent on the activation of transcription factors, enzymatic cascades and the secreted neurotrophin BDNF. By using PS deprivation, immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR), we showed that an increase in PS amount (produced by rebound in PS deprived rats) is able to up-regulate the expression level of transcription factors Zif268 and c-Fos as well as Arc and BDNF in the CA1 and CA3 areas of the hippocampus. Several studies involved these factors in dendritic protein synthesis and in long-term structural changes of synapses underlying L-LTP. The present study together with the work of others (Ribeiro et al., 2002) suggest that by this mechanism, a post-learning increase in PS quantity (post-learning PS window) could convert a transient form of LTP to L-LTP.

NT-28 * NEUROPROTECTIVE EFFECTS OF BACOPA MONNIERI AND ROSMARINUS OFFICINALIS SUPERCRITICAL CO2 EXTRACTS

Ethnobotanical evidence suggests that herbs such as brahmi (Bacopa monnieri) and rosemary (Rosmarinus officinalis) may possess anti-oxidant and neuroprotective properties. We compared the anti-oxidant and neuroprotective effects of supercritical extract of Bacopa monnieri (BM) and rosemary anti-oxidant (RA) extract obtained from Rosmarinus officinalis as well as their combination to examine the effects on human glial (U-87 MG) and embryonic mouse hypothalamus (EMH) cells. BM, RA and their combination (1:1) are not cytotoxic in both glial and EMH cell lines up to 200 µg/ml concentration. BM + RA combination has better anti-oxidant potential and anti-lipid peroxidation activity than either agent alone. Although BM + RA showed almost similar inhibition of phospho tau expression as BM or RA alone, the combination has better inhibitory effect on Amyloid Precursor Protein (APP) synthesis and higher brain-derived neurotrophic factor (BDNF) production in hypothalamus cells than single agents. These results suggest that BM + RA is more neuroprotective than BM or RA individually.