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Sample records for stress protein synthesis

  1. Effects of moisture stress on germination and protein synthesis in ...

    African Journals Online (AJOL)

    ... 3, 5 triphenyl tetrazolium chloride (TTC), and their abilities to synthesize protein after stress by incorporating L- 4,5-3H leucine into their root tips. Les graines de dolique non pigmentées, TVX 3236 (crème et brune) et IT81S-818 (blanche), étaient exposées aux conditions d'humidité constantes plus stressantes (-0.1 et ...

  2. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-31

    The research described in this final report focused on the influence of stress agents on protein synthesis in crop plants (primarily soybean). Investigations into the `heat shock` (HS) stress mediated changes in transcriptional and translocational regulation of protein synthesis coupled with studies on anaerobic water deficit and other stress mediated alterations in protein synthesis in plants provided the basis of the research. Understanding of the HS gene expression and function(s) of the HSPs may clarify regulatory mechanisms operative in development. Since the reproductive systems of plants if often very temperature sensitive, it may be that the system could be manipulated to provide greater thermotolerance.

  3. Environmental-stress mediated changes in transcriptional and translational regulation of protein synthesis in crop plants

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.

    1982-04-14

    Research progress is reported for the current year in the following areas: analysis of heat shock-induced mRNAs using cloned cDNA sequences and identification of a heat shock protein(s) with each cloned cDNA sequence; comparative analysis of heat shock protein synthesis in pea, soybean and millet; physiological implications of the heat shock response: protection to very high temperatures by an intermediate heat shock temperature; and comparative analyses of different stresses using heat shock cDNA clones.

  4. Stress protein synthesis and peroxidase activity in a submersed aquatic macrophyte exposed to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Siesko, M.M.; Grossfeld, R.M. [North Carolina State Univ., Raleigh, NC (United States); Fleming, W.J. [National Biological Service, Raleigh, NC (United States). North Carolina Cooperative Fish and Wildlife Research Unit

    1997-08-01

    Sago pondweed (Potamogeton pectinatus L.) was exposed to CdCl{sub 2} to evaluate peroxidase (POD) activity and stress protein (SP) synthesis as potential biomarkers of contaminant stress in an aquatic plant. Peroxidase activity did not increase in sago pondweed incubated for 24 h in a liquid culture medium containing 0.5, 0.75, or 1 mM CdCl{sub 2}. By contrast, at each of these CdCl{sub 2} concentrations, SPs of 162, 142, 1122, 82, and 61 kDa were preferentially synthesized, and synthesis of a 66-kDa protein was reduced relative to controls. Peroxidase activity also did not change in sago pondweed rooted for 21 d in agar containing 1 mM CdCl{sub 2}, despite the lower growth rate, lower protein content, and brown discoloration of the plants. Only when the plants were grown 7 or 21 d on agar containing 10 mM CdCl{sub 2} were the growth retardation and phenotypic deterioration accompanied by significantly increased POD activity. In contrast, plants rooted for 7 d in agar containing 1 mM CdCl{sub 2} were not significantly discolored or retarded in growth, yet they preferentially synthesized SPs of 122, 82, and 50 kDa and synthesized proteins of 59 and 52 kDa at reduced rates relative to controls. Similar changes in protein synthesis were accompanied by signs of depressed growth after 21 d of incubation with 1 mM CdCl{sub 2} and with 7 or 21 d of exposure to 10 mM CdCl{sub 2}. These data indicate that changes in SP synthesis may precede detectable alterations in growth of aquatic plants and, therefore, may be a potentially useful early biomarker of contaminant stress. However, further studies will be required to determine whether the SP response is measurable during exposure to environmentally relevant contaminant levels.

  5. Errors in macromolecular synthesis after stress : a study of the possible protective role of the small heat shock proteins

    NARCIS (Netherlands)

    Marin Vinader, L.

    2006-01-01

    The general goal of this thesis was to gain insight in what small heat shock proteins (sHsps) do with respect to macromolecular synthesis during a stressful situation in the cell. It is known that after a non-lethal heat shock, cells are better protected against a subsequent more severe heat shock,

  6. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.; Nagao, R.T.

    1991-06-01

    The major research activities accomplished during the renewal period focused on defining regulatory mechanisms operative in the heat shock (HS) response and assessing the mechanism of HS-induced thermotolerance in Arabidopsis. HS gene regulation was studied by transcriptional run-off assays, and self regulation of heat shock protein (hsp) levels and synthesis was studied through use of amino acid analogs and protein synthesis inhibitors. Also studied were subcellular localization of hsps during HS and HS recovery, characterization of membrane bound hsps and their effects on membrane proton transport, and the influence of over- or underexpression of HS mRNAs and hsps plant phenotype using transgenic plants. In addition, hsp70 and hsp83 cDNAs/genes are being characterized and their expression patterns will be evaluated. 10 refs. (MHB)

  7. Boron stress activates the general amino acid control mechanism and inhibits protein synthesis.

    Directory of Open Access Journals (Sweden)

    Irem Uluisik

    Full Text Available Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.

  8. Boron stress activates the general amino acid control mechanism and inhibits protein synthesis.

    Science.gov (United States)

    Uluisik, Irem; Kaya, Alaattin; Fomenko, Dmitri E; Karakaya, Huseyin C; Carlson, Bradley A; Gladyshev, Vadim N; Koc, Ahmet

    2011-01-01

    Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.

  9. Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

    Science.gov (United States)

    Uluisik, Irem; Kaya, Alaattin; Fomenko, Dmitri E.; Karakaya, Huseyin C.; Carlson, Bradley A.; Gladyshev, Vadim N.; Koc, Ahmet

    2011-01-01

    Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance. PMID:22114689

  10. An emergency brake for protein synthesis The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

    Czech Academy of Sciences Publication Activity Database

    Hronová, Vladislava; Valášek, Leoš

    2017-01-01

    Roč. 6, APR 25 (2017), s. 1-3, č. článku e27085. ISSN 2050-084X Institutional support: RVO:61388971 Keywords : synthesis of proteins * eukaryotic cells * eIF2 Subject RIV: EE - Microbiology, Virology Impact factor: 7.725, year: 2016

  11. Environmental stress mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Progress report, August 1980-April 1981

    Energy Technology Data Exchange (ETDEWEB)

    Key, J L

    1981-04-01

    The response of soybean seedling tissues to heat stress and to a lesser extent to other stresses including water, anaerobiosis, DNP, salt, and supraoptimal levels of 2,4-D was studied. A detailed analysis of polyribosome/monoribosome transitions was made in the case of heat shock, and salt and water stresses. Poly(A) RNAs isolated from stressed tissues were translated, and cDNA made to poly(A) RNA from heat shock tissue was cloned, and selected clones used to assess changes in the homologous mRNA(s) during stress induction/recovery. cDNAs were also used in hybridization analyses to assess the complexity and abundance distribution of poly(A) RNAs of control and heat shocked tissue. In vivo labeling studies were made to assess the pattern of protein synthesis during the induction of and recovery from heat stress. There was a rapid loss of polyribosomes following transfer of tissue from 28/sup 0/ to 40/sup 0/. During water stress and salt stress, there was a gradual and near complete loss of polyribosomes. Polyribosomes and poly(A) RNA isolated from those polyribosomes isolated from both 28/sup 0/ and 40/sup 0/ tissue were translated in in vitro systems. In both cases a normal spectrum of translation products was obtained at both 28/sup 0/ and 40/sup 0/; in addition, a number of new proteins was observed in the 40/sup 0/ system. Intact seedlings or slices of hypocotyls incubated at 40/sup 0/ demonstrated a new pattern of in vivo labeled proteins. Synthesis in vivo of normal (28/sup 0/) proteins is greatly inhibited at 40/sup 0/ to 42/sup 0/. The normal 28/sup 0/ in vivo pattern of protein synthesis is restored rapidly following transfer of 40/sup 0/ to 42/sup 0/ heat-shocked tissue to 28/sup 0/. (ERB)

  12. PROTEIN SYNTHESIS GAME

    Directory of Open Access Journals (Sweden)

    J.C.Q. Carvalho

    2004-05-01

    Full Text Available The theoretical explanation of biological concepts, associated with the use of teaching games andmodels, intensify the comprehension and increase students interest, stimulating them to participateactively on the teaching-learning process. The sta of dissemination from Centro de BiotecnologiaMolecular Estrutural (CBME, in partnership with the Centro de Divulgac~ao Cientca e Cultural(CDCC, presents, in this work, a new educational resource denoted: Protein Synthesis Game. Theapproach of the game involves the cytological aspects of protein synthesis, directed to high schoolstudents. Students are presented to day-by-day facts related to the function of a given protein in thehuman body. Such task leads players to the goal of solving out a problem through synthesizing aspecied protein. The game comprises: (1 a board illustrated with the transversal section of animalcell, with its main structures and organelles and sequences of hypothetical genes; (2 cards with thedescription of steps and other structures required for protein synthesis in eukaryotic cells; (3 piecesrepresenting nucleotides, polynucleotides, ribosome, amino acids, and polypeptide chains. In order toplay the game, students take cards that sequentially permit them to acquire the necessary pieces forproduction of the protein described in each objective. Players must move the pieces on the board andsimulate the steps of protein synthesis. The dynamic of the game allows students to easily comprehendprocesses of transcription and translation. This game was presented to dierent groups of high schoolteachers and students. Their judgments have been heard and indicated points to be improved, whichhelped us with the game development. Furthermore, the opinions colleted were always favorable forthe application of this game as a teaching resource in classrooms.

  13. In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

    Science.gov (United States)

    Rebnegger, Corinna; Graf, Alexandra B; Valli, Minoska; Steiger, Matthias G; Gasser, Brigitte; Maurer, Michael; Mattanovich, Diethard

    2014-04-01

    Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h(-1) . At very slow growth (μ = 0.015 h(-1) ) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes. © 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made.

  14. Environmental stress mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Kochert, G.; Key, J.L.

    1984-05-01

    This report focuses on the development of thermotolerance in soybeans under varying heat shock (hs) regimes, and specifically addresses the isolation of genomic clones to hsp's 70 and 84 kd, the purification of hsp's for use in antibody preparation and chloroplast protein synthesis during hs. 4 references.

  15. Protein stress and stress proteins: implications in aging and disease

    Indian Academy of Sciences (India)

    2007-04-02

    Apr 2, 2007 ... Environmantal stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so called stress or heat shock proteins. The stress response as well as stress proteins are ubiquitous, highly conserved ...

  16. A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.

    Science.gov (United States)

    Gaide Chevronnay, H P; Janssens, V; Van Der Smissen, P; Liao, X H; Abid, Y; Nevo, N; Antignac, C; Refetoff, S; Cherqui, S; Pierreux, C E; Courtoy, P J

    2015-06-01

    Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered

  17. Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: suppression of stress-activated protein kinase/c-Jun N-terminal kinase.

    Science.gov (United States)

    Yamamoto, Naohiro; Otsuka, Takanobu; Kuroyanagi, Gen; Kondo, Akira; Kainuma, Shingo; Nakakami, Akira; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Tokuda, Haruhiko

    2015-01-01

    Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Beneficial roles of melatonin on redox regulation of photosynthetic electron transport and synthesis of D1 protein in tomato seedlings under salt stress

    Directory of Open Access Journals (Sweden)

    Xiaoting Zhou

    2016-11-01

    Full Text Available Melatonin is important in the protection of plants suffering various forms of abiotic stress. The molecular mechanisms underlying the melatonin-mediated protection of their photosynthetic machinery are not completely resolved. This study investigates the effects of exogenous melatonin applications on salt-induced damage to the light reaction components of the photosynthetic machinery of tomato seedlings. The results show that melatonin pretreatments can help maintain growth and net photosynthetic rate (PN under salt stress conditions. Pretreatment with melatonin increased the effective quantum yield of photosystem II (ΦPSII, the photochemical quenching coefficient (qP and the proportion of PSII centers that are ‘open’ (qL under saline conditions. In this way, damage to the photosynthetic electron transport chain (PET in photosystem II (PSII is mitigated. In addition, melatonin pretreatment facilitated the repair of PSII by maintaining the availability of D1 protein that was otherwise reduced by salinity. The ROS levels and the gene expressions of the chloroplast TRXs and PRXs were also investigated. Salt stress resulted in increased levels of reactive oxygen species (ROS, which were mitigated by melatonin. In tomato leaves under salt stress, the expressions of PRXs and TRXf declined but the expressions of TRXm1/4 and TRXm2 increased. Melatonin pretreatment promoted the expression of TRXf and the abundances of TRXf and TRXm gene products but had no effects on the expressions of PRXs. In summary, melatonin improves the photosynthetic activities of tomato seedlings under salt stress. The mechanism could be that: (1 Melatonin controls ROS levels and prevents damaging elevations of ROS caused by salt stress. (2 Melatonin facilitates the recovery of PET and D1 protein synthesis, thus enhancing the tolerance of photosynthetic activities to salinity. (3 Melatonin induces the expression of TRXf and regulates the abundance of TRXf and TRXm gene

  19. mTOR is essential for the proteotoxic stress response, HSF1 activation and heat shock protein synthesis.

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    Shiuh-Dih Chou

    Full Text Available The target of rapamycin (TOR is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP, including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1, we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

  20. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.

    1983-01-01

    Work during the 2 1/2 years of this contract has focused on: (1) descriptive biology of the heat shock (hs) response of soybean seedlings, (2) cDNA cloning and characterization of several hs specific mRNA sequences, (3) comparative analysis of the hs response in several crop plant systems using in vivo labeling/2D gel analysis of the newly synthesized proteins and soybean hs cDNA clones in northern hybridization analysis of hs mRNAs, (4) comparative analysis between different environmental stresses and hs in soybean using cloned cDNA, (5) an analysis of the physiological significance of the hs response in crop plants - thermal tolerance and selective localization of the hs proteins (hsp's) and (6) the influence of hs on normal (28/sup 0/) RNA metabolism.

  1. Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos.

    Science.gov (United States)

    Kojima, T; Udagawa, K; Onishi, A; Iwahashi, H; Komatsu, Y

    1996-04-01

    The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42 degrees-45.5 degrees C and for 10-180 min) was examined. Synthesis of 70 kDa hsp (hsp70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42 degrees C-10 min (236.6 +/- 71.4; P heat stress compared to control (82.5 +/- 47.3 microns), while heat stress with 43 degrees C for > or = 60 min, 44 degrees-44.5 degrees C for > or = 30 min, or 45 degrees-45.5 degrees C for > or = 10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42 degrees C-180 min, 43 degrees C-10 min, 43 degrees C-30 min, 44 degrees C-10 min, or 45 degrees C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42 degrees C for 180 min, 43 degrees C for 30 min, 44 degrees C for 10 min, and 45 degrees C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute

  2. Solid phase protein chemical synthesis.

    Science.gov (United States)

    Raibaut, Laurent; El Mahdi, Ouafâa; Melnyk, Oleg

    2015-01-01

    The chemical synthesis of peptides or small proteins is often an important step in many research projects and has stimulated the development of numerous chemical methodologies. The aim of this review is to give a substantial overview of the solid phase methods developed for the production or purification of polypeptides. The solid phase peptide synthesis (SPPS) technique has facilitated considerably the access to short peptides (peptides have stimulated the development of solid phase covalent or non-covalent capture purification methods. The power of the native chemical ligation (NCL) reaction for protein synthesis in aqueous solution has also been adapted to the solid phase by the combination of novel linker technologies, cysteine protection strategies and thioester or N,S-acyl shift thioester surrogate chemistries. This review details pioneering studies and the most recent publications related to the solid phase chemical synthesis of large peptides and proteins.

  3. Exposure of gnotobiotic Artemia franciscana larvae to abiotic stress promotes heat shock protein 70 synthesis and enhances resistance to pathogenic Vibrio campbellii

    Science.gov (United States)

    Pineda, Carlos; MacRae, Thomas H.; Sorgeloos, Patrick; Bossier, Peter

    2008-01-01

    Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority. PMID:18347942

  4. Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

    Directory of Open Access Journals (Sweden)

    Richa Tyagi

    2015-02-01

    Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

  5. Insect heat shock proteins during stress and diapause.

    Science.gov (United States)

    King, Allison M; MacRae, Thomas H

    2015-01-07

    Insect heat shock proteins include ATP-independent small heat shock proteins and the larger ATP-dependent proteins, Hsp70, Hsp90, and Hsp60. In concert with cochaperones and accessory proteins, heat shock proteins mediate essential activities such as protein folding, localization, and degradation. Heat shock proteins are synthesized constitutively in insects and induced by stressors such as heat, cold, crowding, and anoxia. Synthesis depends on the physiological state of the insect, but the common function of heat shock proteins, often working in networks, is to maintain cell homeostasis through interaction with substrate proteins. Stress-induced expression of heat shock protein genes occurs in a background of protein synthesis inhibition, but in the course of diapause, a state of dormancy and increased stress tolerance, these genes undergo differential regulation without the general disruption of protein production. During diapause, when ATP concentrations are low, heat shock proteins may sequester rather than fold proteins.

  6. Environmental stress mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Kochert, G.; Key, J.L.

    1985-04-01

    This progress report summarizes research accomplished during 1985. This research has focused on: (1) an analysis of the transport of heat shock proteins into chloroplasts and isolation of cDNA clones to those proteins, (2) development of monoclonal antibodies to 15 to 18 kD heat shock proteins, (3) additional analyses of heat shock protein localization focusing on ''heat shock granules'' and ribosome association of 15 to 18 kD heat shock proteins, (4) isolation of cDNA and genomic clones to heat shock proteins, hsp 70 and hsp 84, (5) Cadmium/arsenite-mediated induction of heat shock mRNAs and proteins and (6) some aspects of the regulation of heat shock mRNA levels.

  7. Termination of protein synthesis.

    Science.gov (United States)

    Tuite, M F; Stansfield, I

    1994-05-01

    One of three mRNA codons--UAA, UAG and UGA--is used to signal to the elongating ribosome that translation should be terminated at this point. Upon the arrival of the stop codon at the ribosomal acceptor(A)-site, a protein release factor (RF) binds to the ribosome resulting in the peptidyl transferase centre of the ribosome switching to a hydrolytic function to remove the completed polypeptide chain from the peptidyl-tRNA bound at the adjacent ribosomal peptidyl(P)-site. In this review recent advances in our understanding of the mechanism of termination in the bacterium Escherichia coli will be summarised, paying particular attention to the roles of 16S ribosomal RNA and the release factors RF-1, RF-2 and RF-3 in stop codon recognition. Our understanding of the translation termination process in eukaryotes is much more rudimentary with the identity of the single eukaryotic release factor (eRF) still remaining elusive. Finally, several examples of how the termination mechanism can be subverted either to expand the genetic code (e.g. selenocysteine insertion at UGA codons) or to regulate the expression of mammalian retroviral or plant viral genomes will be discussed.

  8. Major E. coli heat-stress protein do not translocate: implications for cell survival.

    Science.gov (United States)

    Yatvin, M B; Clark, A W; Siegel, F L

    1987-10-01

    When Escherichia coli are exposed to heat stress, the majority of proteins in the process of synthesis at the time of heat stress are rapidly translocated to the outer membrane of the bacterium. The synthesis of most of these proteins appears to take place on membrane-bound polyribosomes. With the temperature shift, overall protein synthesis is inhibited while the synthesis of a small group of proteins is initiated. These proteins are not translocated, but remain in the cytosolic compartment, and they are identifiable as heat-stress proteins. Both the translocation phenomenon and the retention of heat-stress proteins in the cytosolic compartment in proximity to the nucleoid could counteract the effects of heat stress. The translocated proteins may operate by stabilizing the outer membrane prior to the induction of heat-stress proteins and the latter, which are confined to the cytoplasmic compartment, may serve to protect the integrity of the nucleoid structures.

  9. Protein synthesis regulation by leucine

    Directory of Open Access Journals (Sweden)

    Daiana Vianna

    2010-03-01

    Full Text Available In vivo and in vitro studies have demonstrated that high protein diets affect both protein synthesis and regulation of several cellular processes. The role of amino acids as substrate for protein synthesis has been established in the literature. However, the mechanism by which these amino acids modulate transcription and regulate the mRNA translation via mTOR-dependent signaling pathway has yet to be fully determined. It has been verified that mTOR is a protein responsible for activating a cascade of biochemical intracellular events which result in the activation of the protein translation process. Of the aminoacids, leucine is the most effective in stimulating protein synthesis and reducing proteolysis. Therefore, it promotes a positive nitrogen balance, possibly by favoring the activation of this protein. This amino acid also directly and indirectly stimulates the synthesis and secretion of insulin, enhancing its anabolic cellular effects. Therefore, this review aimed to identify the role of leucine in protein synthesis modulation and to discuss the metabolic aspects related to this aminoacid.Estudos in vivo e in vitro verificaram que dietas hiperprotéicas influenciam a síntese protéica e regulam vários processos celulares. O papel dos aminoácidos como substrato para a síntese de proteínas já está bem evidenciado na literatura, porém as formas como esses aminoácidos modulam a etapa da transcrição e regulam a tradução do RNAm, pela via de sinalização dependente da mTOR, ainda não estão totalmente esclarecidas. Tem-se verificado que a mTOR é uma proteína responsável por ativar uma cascata de eventos bioquímicos intracelulares que culminam na ativação do processo de tradução protéica. Dentre todos os aminoácidos, a leucina é a mais eficaz em estimular a síntese protéica, reduzir a proteólise e, portanto, favorecer o balanço nitrogenado positivo, possivelmente por favorecer a ativação desta proteína. Al

  10. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Progress report, August 1983-August 1985

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.

    1985-10-01

    Research has focused on: (1) physiological significance of the heat shock (HS) response to crop plants in terms of the acquisition of thermotolerance and selective localization of HS proteins; (2) an analysis of the influence of HS on organelle gene expression and localization of HS proteins in organelles; (3) HS gene expression; (4) comparative analysis of the HS response with other stress responses (e.g., arsenite, cadmium, anaerobiosis, and cold); (5) monoclonal antibodies to HS proteins; and (6) isolation and analysis of HS cDNAs and HS genes.

  11. Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

    Science.gov (United States)

    Walsh, Derek; Mathews, Michael B.; Mohr, Ian

    2013-01-01

    Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

  12. A karyopherin acts in localized protein synthesis

    NARCIS (Netherlands)

    Veenhoff, Liesbeth M.; Meinema, Anne C.; Poolman, Bert

    2010-01-01

    Multiple mechanisms are in place to regulate adequate synthesis of proteins, ranging from ways to ensure sequence fidelity, polypeptide folding and protein modification, to control of amounts and subcellular localization of the molecules. Some of these mechanisms act at the level of mRNA export and

  13. Genetic Synthesis of Periodic Protein Materials

    OpenAIRE

    Fournier, M J; Creel, H. S.; Krejchi, M. T.; Mason, T L; Tirrell, D.A.; McGrath, K. P.; Atkins, E. D. T.

    1991-01-01

    Genetic engineering offers a novel approach to the development of advanced polymeric materials, in particular protein-based materials. Biological synthesis provides levels of control of polymer chain architecture that cannot yet be attained by current methods of chemical synthesis. In addition to employing naturally occurring genetic templates artificial genes can be designed to encode completely new materials with customized properties. In the present paper we: 1) review th...

  14. 70 kD stress protein (Hsp70) analysis in living shallow-water benthic foraminifera

    Digital Repository Service at National Institute of Oceanography (India)

    Heinz, P.; Marten, R.A; Linshy, V.N; Haap, T.; Geslin, E.; Kohler, H-R.

    Hsp70 is a phylogenetically highly conserved protein family present in all eukaryotic organisms tested so far. Its synthesis is induced by proteotoxic stress. The detection of Hsp70 in foraminifera is presented here. We introduce a standard...

  15. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  16. Intracellular proteins produced by mammalian cells in response to environmental stress

    Science.gov (United States)

    Goochee, Charles F.; Passini, Cheryl A.

    1988-01-01

    The nature of the response of mammalian cells to environmental stress is examined by reviewing results of studies where cultured mouse L cells and baby hamster kidney cells were exposed to heat shock and the synthesis of heat-shock proteins and stress-response proteins (including HSP70, HSC70, HSP90, ubiquitin, and GRP70) in stressed and unstressed cells was evaluated using 2D-PAGE. The intracellular roles of the individual stress response proteins are discussed together with the regulation of the stress response system.

  17. Protein stress and stress proteins: implications in aging and disease

    Indian Academy of Sciences (India)

    Madhu Sudhan

    2007-04-02

    Apr 2, 2007 ... cells reaching 1–5% of total cellular protein, which shows that a continuous intense demand is present to .... stem (and tumor) cell proliferation and cell survival. Hsp90 ensures, amongst several hundred ... interventions focusing to preserve the protein turnover is an attractive therapy in anti-aging research.

  18. Stress proteins are induced by space environment

    Science.gov (United States)

    Takahashi, Akihisa; Ohnishi, Takeo

    The space environment contains two major biologically significant influences such as space radiations and microgravity. Almost all organisms possess essential recognition and response systems for environmental changes. The famous one of cellular stress responses is the gene induction of heat shock protein (HSP). HSP expression is increased under elevated temperatures, and also increased by other sources of cellular stress, including ionizing radiation, oxidative injury, osmotic stress and the unfolded protein response. HSPs assist in the folding and maintenance of newly translated proteins, the refolding of denatured proteins and the further unfolding of misfolded or destabilized proteins to protect the cell from crisis. Based on our space experiment, we report the results and discussion from the viewpoint of HSP expression after exposure to space environment.

  19. Plant Desiccation and Protein Synthesis 1

    Science.gov (United States)

    Oliver, Melvin J.; Bewley, J. Derek

    1984-01-01

    Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)− RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)+ RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)− RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)+ RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples. Poly(A)− RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)+ RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)+ RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)+ RNA for protein synthesis. The rate of synthesis of poly(A)+ RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)+ RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance. PMID:16663533

  20. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants: Progress report, 1986-1987

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.

    1987-04-01

    Research during the last year has focused on aspects of the phenomenon of thermotolerance, certain aspects of regulatory events associated with temperature shifts, continued analysis of both cDNA and genomic clones to certain families of HS proteins, and construction of various HS promoter/gene combinations for further analysis of the transcriptional aspects of the HS response. 8 refs.

  1. Fanconi anemia proteins and endogenous stresses

    Energy Technology Data Exchange (ETDEWEB)

    Pang Qishen [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Research Foundation, Cincinnati, OH (United States); Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH (United States); Andreassen, Paul R., E-mail: Paul.Andreassen@cchmc.org [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Research Foundation, Cincinnati, OH (United States); Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH (United States)

    2009-07-31

    Each of the thirteen identified Fanconi anemia (FA) genes is required for resistance to DNA interstrand crosslinking agents, such as mitomycin C, cisplatin, and melphalan. While these agents are excellent tools for understanding the function of FA proteins in DNA repair, it is uncertain whether a defect in the removal of DNA interstrand crosslinks (ICLs) is the basis for the pathophysiology of FA. For example, DNA interstrand crosslinking agents induce other types of DNA damage, in addition to ICLs. Further, other DNA-damaging agents, such as ionizing or ultraviolet radiation, activate the FA pathway, leading to monoubiquitination of FANCD2 and FANCI. Also, FA patients display congenital abnormalities, hematologic deficiencies, and a predisposition to cancer in the absence of an environmental source of ICLs that is external to cells. Here we consider potential sources of endogenous DNA damage, or endogenous stresses, to which FA proteins may respond. These include ICLs formed by products of lipid peroxidation, and other forms of oxidative DNA damage. FA proteins may also potentially respond to telomere shortening or replication stress. Defining these endogenous sources of DNA damage or stresses is critical for understanding the pathogenesis of deficiencies for FA proteins. We propose that FA proteins are centrally involved in the response to replication stress, including replication stress arising from oxidative DNA damage.

  2. Cell-free protein synthesis: applications come of age.

    Science.gov (United States)

    Carlson, Erik D; Gan, Rui; Hodgman, C Eric; Jewett, Michael C

    2012-01-01

    Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Oxidative stress causes plasma protein modification.

    Science.gov (United States)

    Tetik, Sermin; Kiliç, Arzu; Aksoy, Halil; Rizaner, Nahit; Ahmad, Sarfraz; Yardimci, Turay

    2015-01-01

    We investigated the effect of oxidative systems on plasma proteins using Chloramine-T, a source of free radicals. Plasma specimens from 10 healthy volunteers were treated with 40 mmol/L Chloramine-T (1:1 v/v). Total protein and plasma carbonyl levels were evaluated spectrophotometrically. Identification of plasma proteins modifications was performed by SDS-PAGE, protein and lipid electrophoresis. Protein fragmentation was evaluated by HPLC. Total protein levels of oxidised plasmas were significantly lower (4.08 ± 0.12 g/dL) than control (7.86 ± 0.03 g/dL) (P < 0.01). Plasma carbonyl levels were higher (1.94 ± 0.38 nmol/mg protein) in oxidised plasma than that of control (0.03 ± 0.01 nmol/mg protein) (P < 0.01). Plasma oxidation had no significant effect on the levels of proteins and lipids. Protein fragmentations were detected in oxidised groups compared to those of the control. We conclude that protein modifications have direct effect on the protein functions, which are related to stress agent, its treatment period(s), and the methodology used for evaluating such experimental results.

  4. Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

    Science.gov (United States)

    Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S.; Smorodinsky, Nechama I.; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

    2011-01-01

    We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection. PMID:21795382

  5. SHORT-TERM MEMORY IS INDEPENDENT OF BRAIN PROTEIN SYNTHESIS

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Hasker P.; Rosenzweig, Mark R.; Jones, Oliver W.

    1980-09-01

    Male Swiss albino CD-1 mice given a single injection of a cerebral protein synthesis inhibitor, anisomycin (ANI) (1 mg/animal), 20 min prior to single trial passive avoidance training demonstrated impaired retention at tests given 3 hr, 6 hr, 1 day, and 7 days after training. Retention was not significantly different from saline controls when tests were given 0.5 or 1.5 hr after training. Prolonging inhibition of brain protein synthesis by giving either 1 or 2 additional injections of ANI 2 or 2 and 4 hr after training did not prolong short-term retention performance. The temporal development of impaired retention in ANI treated mice could not be accounted for by drug dosage, duration of protein synthesis inhibition, or nonspecific sickness at test. In contrast to the suggestion that protein synthesis inhibition prolongs short-term memory (Quinton, 1978), the results of this experiment indicate that short-term memory is not prolonged by antibiotic drugs that inhibit cerebral protein synthesis. All evidence seems consistent with the hypothesis that short-term memory is protein synthesis independent and that the establishment of long-term memory depends upon protein synthesis during or shortly after training. Evidence for a role of protein synthesis in memory maintenance is discussed.

  6. Chromatin proteins: key responders to stress.

    Directory of Open Access Journals (Sweden)

    Karen T Smith

    Full Text Available Environments can be ever-changing and stresses are commonplace. In order for organisms to survive, they need to be able to respond to change and adapt to new conditions. Fortunately, many organisms have systems in place that enable dynamic adaptation to immediate stresses and changes within the environment. Much of this cellular response is coordinated by modulating the structure and accessibility of the genome. In eukaryotic cells, the genome is packaged and rolled up by histone proteins to create a series of DNA/histone core structures known as nucleosomes; these are further condensed into chromatin. The degree and nature of the condensation can in turn determine which genes are transcribed. Histones can be modified chemically by a large number of proteins that are thereby responsible for dynamic changes in gene expression. In this Primer we discuss findings from a study published in this issue of PLoS Biology by Weiner et al. that highlight how chromatin structure and chromatin binding proteins alter transcription in response to environmental changes and stresses. Their study reveals the importance of chromatin in mediating the speed and amplitude of stress responses in cells and suggests that chromatin is a critically important component of the cellular response to stress.

  7. RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

    DEFF Research Database (Denmark)

    Bhowmick, Rahul; Minocherhomji, Sheroy; Hickson, Ian D

    2016-01-01

    Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis...... replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective...

  8. Cell-specific monitoring of protein synthesis in vivo.

    Directory of Open Access Journals (Sweden)

    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  9. Cognitive and emotional information processing: protein synthesis and gene expression.

    Science.gov (United States)

    Sajikumar, Sreedharan; Navakkode, Sheeja; Korz, Volker; Frey, Julietta U

    2007-10-15

    Recent findings suggest that functional plasticity phenomena such as long-term potentiation (LTP) and long-term depression (LTD) - cellular processes underlying memory - are restricted to functional dendritic compartments. It was also shown, however, that a relatively strong activation of a synaptic input can abolish compartment restrictions. Our data support these findings and we present one cellular pathway responsible for uncompartmentalization of the normally localized plasticity processes by the action of rolipram, an inhibitor of type 4 phosphodiesterases. In contrast with compartment-restricted information processing, uncompartmentalization requires transcription. In the search for system relevance of compartmentalization versus uncompartmentalization we describe firstly data which show that more cognitive information processing in rats' behaviour may follow rules of compartmentalization, whereas stressful, more life-threatening, inputs abolish compartment-restricted information processing involving transcription. Our findings allow us to suggest that consolidation of processes which take place during the cognitive event most probably depend on local protein synthesis, whereas stress immediately induces gene expression in addition, resulting in a compartment-unspecific up-regulation of plasticity-related proteins (PRPs), providing the entire neuron with a higher level of 'reactiveness'. These data would provide a specific functional cellular mechanism to respond differentially and effectively to behaviourally weighted inputs.

  10. Protein degradation and protein synthesis in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2014-06-01

    Full Text Available Long-term memory (LTM formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly consolidate and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

  11. Predictors of muscle protein synthesis after severe pediatric burns.

    Science.gov (United States)

    Diaz, Eva C; Herndon, David N; Lee, Jinhyung; Porter, Craig; Cotter, Matthew; Suman, Oscar E; Sidossis, Labros S; Børsheim, Elisabet

    2015-04-01

    Following a major burn, skeletal muscle protein synthesis rate increases but is often insufficient to compensate for massively elevated muscle protein breakdown rates. Given the long-term nature of the pathophysiologic response to burn injury, we hypothesized that muscle protein synthesis rate would be chronically elevated in severely burned children. The objectives of this study were to characterize muscle protein synthesis rate of burned children over a period of 24 months after injury and to identify predictors that influence this response. A total of 87 children with 40% or greater total body surface area (TBSA) burned were included. Patients participated in stable isotope infusion studies at 1, 2, and approximately 4 weeks after burn and at 6, 12, and 24 months after injury to determine skeletal muscle protein fractional synthesis rate. Generalized estimating equations with log link normal distribution were applied to account for clustering of patients and control for patient characteristics. Patients (8 ± 6 years) had large (62, 51-72% TBSA) and deep (47% ± 21% TBSA third degree) burns. Muscle protein fractional synthesis rate was elevated throughout the first 12 months after burn compared with established values from healthy young adults. Muscle protein fractional synthesis rate was lower in boys, in children older than 3 years, and when burns were greater than 80% TBSA. Muscle protein synthesis is elevated for at least 1 year after injury, suggesting that greater muscle protein turnover is a component of the long-term pathophysiologic response to burn trauma. Muscle protein synthesis is highly affected by sex, age, and burn size in severely burned children. These findings may explain the divergence in net protein balance and lean body mass in different populations of burn patients. Prognostic study, level III.

  12. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A

    2015-01-01

    mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

  13. Proteomics-based dissection of biotic stress responsive proteins in ...

    African Journals Online (AJOL)

    related proteins, mapping dynamics of their expression and post translational modifications represent an important approach in the research of plant stresses. Biotic stress is one of the major stresses limiting crop productivity and the geographical ...

  14. The Role of Post-Exercise Nutrient Administration on Muscle Protein Synthesis and Glycogen Synthesis

    Science.gov (United States)

    Poole, Chris; Wilborn, Colin; Taylor, Lem; Kerksick, Chad

    2010-01-01

    Nutrient administration following an exercise bout vastly affects anabolic processes within the human body, irrespective of exercise mode. Of particular importance are protein and carbohydrates whereby these two macronutrients portray distinct functions as anabolic agents. It has been confirmed that protein and/or amino acid ingestion following resistance training is required to reach a positive protein/nitrogen balance, and carbohydrate intake during recovery is the most important consideration to replenish glycogen stores from an exhaustive exercise bout. Several factors play significant roles in determining the effectiveness of protein and carbohydrate supplementation on post-exercise protein and glycogen synthesis. Improper application of these factors can limit the body’s ability to reach an anabolic status. The provided evidence clearly denotes the importance these two macronutrients have in regards to post-exercise nutrition and anabolism. Therefore, the purpose of this review is to discuss the impact of dietary protein and carbohydrate intake during the recovery state on muscle protein synthesis and glycogen synthesis. Key points Post-exercise nutrient intake is essential for promoting protein synthesis and glycogen synthesis. The timing and amount of protein and/or carbohydrate ingested affects the rate and amount of synthesis. The type/form of protein and/or carbohydrate ingested after exercise alters anabolic processes during the recovery period. PMID:24149627

  15. The role of post-exercise nutrient administration on muscle protein synthesis and glycogen synthesis.

    Science.gov (United States)

    Poole, Chris; Wilborn, Colin; Taylor, Lem; Kerksick, Chad

    2010-01-01

    Nutrient administration following an exercise bout vastly affects anabolic processes within the human body, irrespective of exercise mode. Of particular importance are protein and carbohydrates whereby these two macronutrients portray distinct functions as anabolic agents. It has been confirmed that protein and/or amino acid ingestion following resistance training is required to reach a positive protein/nitrogen balance, and carbohydrate intake during recovery is the most important consideration to replenish glycogen stores from an exhaustive exercise bout. Several factors play significant roles in determining the effectiveness of protein and carbohydrate supplementation on post-exercise protein and glycogen synthesis. Improper application of these factors can limit the body's ability to reach an anabolic status. The provided evidence clearly denotes the importance these two macronutrients have in regards to post-exercise nutrition and anabolism. Therefore, the purpose of this review is to discuss the impact of dietary protein and carbohydrate intake during the recovery state on muscle protein synthesis and glycogen synthesis. Key pointsPost-exercise nutrient intake is essential for promoting protein synthesis and glycogen synthesis.The timing and amount of protein and/or carbohydrate ingested affects the rate and amount of synthesis.The type/form of protein and/or carbohydrate ingested after exercise alters anabolic processes during the recovery period.

  16. Neutrophil-activating protein (HP-NAP) versus ferritin (Pfr): comparison of synthesis in Helicobacter pylori.

    Science.gov (United States)

    Dundon, W G; Polenghi, A; Del Guidice, G; Rappuoli, R; Montecucco, C

    2001-05-15

    We recently reported that the neutrophil-activating protein (HP-NAP) of Helicobacter pylori is capable of binding iron in vitro. To more fully understand the relationship between iron and HP-NAP the synthesis of HP-NAP was compared to that of Pfr, another iron-binding protein of H. pylori. Synthesis of HP-NAP and Pfr in growing cultures of H. pylori was analysed under iron depletion and iron, copper, nickel and zinc overload. The synthesis of HP-NAP and Pfr in H. pylori was also analysed under conditions of varying pH and oxidative stress. In addition, recombinant HP-NAP and Pfr were produced in Escherichia coli to assess the contribution of the two proteins to increased survival of E. coli under heavy metal overload. Our data reveal that both HP-NAP and Pfr accumulate in the stationary phase of growth. HP-NAP synthesis is not regulated by iron depletion or overload or by the presence of copper, nickel or zinc in liquid medium and it does not confer resistance to these metals when produced in E. coli. Except for an increase in the synthesis of Pfr at pH 5.7 neither the pH or oxidative stress conditions investigated had an affect on the synthesis of either protein. An increase in Pfr synthesis was observed under iron overload and a decrease was observed under conditions of copper, nickel and zinc overload confirming previous reports. Recombinant Pfr, as well as conferring resistance to iron and copper as previously reported, also conferred resistance to zinc overload when produced in E. coli.

  17. N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

    Science.gov (United States)

    Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

    2016-02-01

    Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

  18. Real-time assay for testing components of protein synthesis

    Science.gov (United States)

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Goldman, Yale E.; Cooperman, Barry S.

    2012-01-01

    We present a flexible, real-time-coupled transcription–translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore. PMID:22422844

  19. Variations of total protein content and protein synthesis during microsporogenesis in Larix europaea L.

    Directory of Open Access Journals (Sweden)

    Wiesława B. Chwirot

    2014-01-01

    Full Text Available The variations of the total protein content and protein synthesis were analyzed in successive stages of microsporogenesis in Larix europaea L. Pure fractions of microsporocytes containing the cells in successive phases of meiosis and fractions of the anther wall cells were used to perform the assays. It was revealed, that the protein content and the intensity of protein synthesis undergo considerable changes in the course of meiosis. It was proved also, by means of electrophoretic separation of protein fractions, that no new proteins are synthesised during differentiation of microsporocytes of larch, and that the microsporogenesis process is related rather to disappearance of some protein fractions.

  20. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials

    NARCIS (Netherlands)

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; Duinkerken, Van Gert; Yu, Peiqiang

    2015-01-01

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more

  1. Escherichia coli MazF leads to the simultaneous selective synthesis of both "death proteins" and "survival proteins".

    Directory of Open Access Journals (Sweden)

    Shahar Amitai

    2009-03-01

    Full Text Available The Escherichia coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful.

  2. Starting the protein synthesis machine: eukaryotic translation initiation.

    Science.gov (United States)

    Preiss, Thomas; W Hentze, Matthias

    2003-12-01

    The final assembly of the protein synthesis machinery occurs during translation initiation. This delicate process involves both ends of eukaryotic messenger RNAs as well as multiple sequential protein-RNA and protein-protein interactions. As is expected from its critical position in the gene expression pathway between the transcriptome and the proteome, translation initiation is a selective and highly regulated process. This synopsis summarises the current status of the field and identifies intriguing open questions. Copyright 2003 Wiley Periodicals, Inc.

  3. Adeno-associated virus rep protein synthesis during productive infection

    Energy Technology Data Exchange (ETDEWEB)

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  4. Oxidative stress, free radicals and protein peroxides.

    Science.gov (United States)

    Gebicki, Janusz M

    2016-04-01

    Primary free radicals generated under oxidative stress in cells and tissues produce a cascade of reactive secondary radicals, which attack biomolecules with efficiency determined by the reaction rate constants and target concentration. Proteins are prominent targets because they constitute the bulk of the organic content of cells and tissues and react readily with many of the secondary radicals. The reactions commonly lead to the formation of carbon-centered radicals, which generally convert in vivo to peroxyl radicals and finally to semistable hydroperoxides. All of these intermediates can initiate biological damage. This article outlines the advantages of the application of ionizing radiations to studies of radicals, with particular reference to the generation of desired radicals, studies of the kinetics of their reactions and correlating the results with events in biological systems. In one such application, formation of protein hydroperoxides in irradiated cells was inhibited by the intracellular ascorbate and glutathione. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Polyamines Function in Stress Tolerance: From Synthesis to Regulation

    Directory of Open Access Journals (Sweden)

    Ji-Hong eLiu

    2015-10-01

    Full Text Available Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine,spermidine and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested.

  6. Predictors of muscle protein synthesis after severe pediatric burns

    Science.gov (United States)

    Objectives: Following a major burn, muscle protein synthesis rate increases but in most patients, this response is not sufficient to compensate the also elevated protein breakdown. Given the long-term nature of the pathophysiologic response to burn injury, we hypothesized that skeletal muscle prot...

  7. Nuclear transport factor directs localization of protein synthesis during mitosis

    NARCIS (Netherlands)

    Bogaart, Geert van den; Meinema, Anne C.; Krasnikov, Viktor; Veenhoff, Liesbeth M.; Poolman, Bert

    Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the

  8. Gastrin induction of protein synthesis in isolated gastric mucosal cells

    Energy Technology Data Exchange (ETDEWEB)

    Majumdar, A.N.; Edgerton, E.

    1987-09-01

    Exposure of isolated rat gastric mucosal cells to 10/sup -10/ and 10/sup -9/ M gastrin for 2 hr significantly stimulated (/sup 3/H) leucine incorporation into protein by 100 and 212%, respectively, when compared with the basal levels. Doses beyond 10/sup -9/ M lowered the maximal stimulatory effect of the hormone. Gastrin (10/sup -9/ M) specifically stimulated the synthesis of five proteins in isolated gastric mucosal cells with apparent molecular weights of 105, 76, 71, 63, and 54 kDa. Actinomycin-D completely abolished the gastrin-mediated stimulation of protein synthesis in isolated gastric mucosal cells.

  9. Glucose Synthesis in a Protein-Based Artificial Photosynthesis System.

    Science.gov (United States)

    Lu, Hao; Yuan, Wenqiao; Zhou, Jack; Chong, Parkson Lee-Gau

    2015-09-01

    The objective of this study was to understand glucose synthesis of a protein-based artificial photosynthesis system affected by operating conditions, including the concentrations of reactants, reaction temperature, and illumination. Results from non-vesicle-based glyceraldehyde-3-phosphate (GAP) and glucose synthesis showed that the initial concentrations of ribulose-1,5-bisphosphate (RuBP) and adenosine triphosphate (ATP), lighting source, and temperature significantly affected glucose synthesis. Higher initial concentrations of RuBP and ATP significantly enhanced GAP synthesis, which was linearly correlated to glucose synthesis, confirming the proper functions of all catalyzing enzymes in the system. White fluorescent light inhibited artificial photosynthesis and reduced glucose synthesis by 79.2 % compared to in the dark. The reaction temperature of 40 °C was optimum, whereas lower or higher temperature reduced glucose synthesis. Glucose synthesis in the vesicle-based artificial photosynthesis system reconstituted with bacteriorhodopsin, F 0 F 1 ATP synthase, and polydimethylsiloxane-methyloxazoline-polydimethylsiloxane triblock copolymer was successfully demonstrated. This system efficiently utilized light-induced ATP to drive glucose synthesis, and 5.2 μg ml(-1) glucose was synthesized in 0.78-ml reaction buffer in 7 h. Light-dependent reactions were found to be the bottleneck of the studied artificial photosynthesis system.

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

    Science.gov (United States)

    Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

    2017-11-01

    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. Copyright © 2017 the

  11. Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Matthew B Hudson

    Full Text Available Mechanical ventilation (MV is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1 determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2 establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

  12. Depression of leukocyte protein synthesis, immune function and growth performance induced by high environmental temperature in broiler chickens

    Science.gov (United States)

    Kamel, Nancy N.; Ahmed, Ayman M. H.; Mehaisen, Gamal M. K.; Mashaly, Magdi M.; Abass, Ahmed O.

    2017-09-01

    In tropical and semitropical regions, raising broiler chickens out of their thermal comfort zone can cause an added economic loss in the poultry industry. The cause for the deleterious effects on immunity and growth performance of broilers under high environmental temperatures is still poorly understood. Therefore, the aim of the current investigation was to evaluate the effect of heat stress on leukocytes protein synthesis and immune function as a possible direct cause of low performance in broiler chickens under such condition. In this study, 300 one-day-old male broiler chicks (Cobb500™) were randomly assigned into 2 groups with 5 replicates of 30 chicks each. From 21 to 42 days of age, one group was exposed to non-stressed condition at 24 °C and 50% relative humidity (control group), while the other group was exposed to heat stress at 35 °C and 50% relative humidity (HS group). At 42 days of age, blood samples were collected from each group to evaluate stress indicators, immune function, and leukocytes protein synthesis. Production performance was also recorded. Noteworthy, protein synthesis in leukocytes was significantly ( P leukocyte protein synthesis through increasing the level of eEF2 Thr56 phosphorylation may play a key role in the observed decrease in immune function and growth performance with the high mortality rate encountered in broiler chickens under heat stress environment.

  13. Rewiring protein synthesis: From natural to synthetic amino acids.

    Science.gov (United States)

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2017-11-01

    The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Regulation of protein synthesis during sea urchin early development

    Energy Technology Data Exchange (ETDEWEB)

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  15. Mildiomycin: a nucleoside antibiotic that inhibits protein synthesis.

    Science.gov (United States)

    Feduchi, E; Cosín, M; Carrasco, L

    1985-03-01

    Mildiomycin, a new nucleoside antibiotic, selectively inhibits protein synthesis in HeLa cells, and is less active in the inhibition of RNA or DNA synthesis. An increased inhibition of translation by mildiomycin is observed in cultured HeLa cells when they are permeabilized by encephalomyocarditis virus. This observation suggests that this antibiotic does not easily pass through the cell membrane, as occurs with other nucleoside and aminoglycoside antibiotics. The inhibition of translation is also observed in cell-free systems, such as endogenous protein synthesis in a rabbit reticulocyte lysate or the synthesis of polyphenylalanine directed by poly (U). Finally the mode of action of mildiomycin was investigated and the results suggest that the compound blocks the peptidyl-transferase center.

  16. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    2016-06-01

    Full Text Available Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise.

  17. Proteomic analysis of Arabidopsis thaliana leaves in response to acute boron deficiency and toxicity reveals effects on photosynthesis, carbohydrate metabolism, and protein synthesis.

    Science.gov (United States)

    Chen, Mei; Mishra, Sasmita; Heckathorn, Scott A; Frantz, Jonathan M; Krause, Charles

    2014-02-15

    Boron (B) stress (deficiency and toxicity) is common in plants, but as the functions of this essential micronutrient are incompletely understood, so too are the effects of B stress. To investigate mechanisms underlying B stress, we examined protein profiles in leaves of Arabidopsis thaliana plants grown under normal B (30 μM), compared to plants transferred for 60 and 84 h (i.e., before and after initial visible symptoms) in deficient (0 μM) or toxic (3 mM) levels of B. B-responsive polypeptides were sequenced by mass spectrometry, following 2D gel electrophoresis, and 1D gels and immunoblotting were used to confirm the B-responsiveness of some of these proteins. Fourteen B-responsive proteins were identified, including: 9 chloroplast proteins, 6 proteins of photosynthetic/carbohydrate metabolism (rubisco activase, OEC23, photosystem I reaction center subunit II-1, ATPase δ-subunit, glycolate oxidase, fructose bisphosphate aldolase), 6 stress proteins, and 3 proteins involved in protein synthesis (note that the 14 proteins may fall into multiple categories). Most (8) of the B-responsive proteins decreased under both B deficiency and toxicity; only 3 increased with B stress. Boron stress decreased, or had no effect on, 3 of 4 oxidative stress proteins examined, and did not affect total protein. Hence, our results indicate relatively early specific effects of B stress on chloroplasts and protein synthesis. Copyright © 2013 Elsevier GmbH. All rights reserved.

  18. DNA Nanoparticles for Improved Protein Synthesis In Vitro.

    Science.gov (United States)

    Galinis, Robertas; Stonyte, Greta; Kiseliovas, Vaidotas; Zilionis, Rapolas; Studer, Sabine; Hilvert, Donald; Janulaitis, Arvydas; Mazutis, Linas

    2016-02-24

    The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 10(4)  clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription-translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Protein intake does not increase vastus lateralis muscle protein synthesis during cycling

    DEFF Research Database (Denmark)

    Hulston, CJ; Wolsk, Emil; Grøndahl, Thomas Sahl

    2011-01-01

    PURPOSE: This study aimed to investigate the effect of protein ingestion on leg protein turnover and vastus lateralis muscle protein synthesis during bicycle exercise and recovery. METHODS: Eight healthy males participated in two experiments in which they ingested either a carbohydrate solution (...

  20. QUANTIFYING ELONGATION RHYTHM DURING FULL-LENGTH PROTEIN SYNTHESIS

    Science.gov (United States)

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E.; Cooperman, Barry S.

    2013-01-01

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. PMID:23822614

  1. Retraction Statement: Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

    Science.gov (United States)

    2017-07-01

    IUBMB Life (2014) 66:793-802. DOI: 10.1002/iub.1328 The above article, published online on November 15, 2014 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal's Editors-in-Chief, Angelo Azzi and William J. Whelan, Corresponding Author Tina Wenz, the University of Cologne, and Wiley Periodicals, Inc. The article has been retracted on request of the University of Cologne that, after an investigation, established that the data reported in it are not reproducible. Note from the Corresponding Author: "The paper reports on the influence of mitochondrial acetylation on protein synthesis. After publication, several irregularities appeared and have been thoroughly investigated by the lab of the Corresponding Author in cooperation with the commission of Research integrity of the University of Cologne. Both came to the conclusion that data used for the publication are erroneous and that the presented data are not reproducible by the lab of the Corresponding Author and other labs. The Corresponding Author takes responsibility and regrets not having detected these issues before publication. The appropriate corrective action is retraction of the paper. The Corresponding Author apologizes to the scientific community." Antonella Di Domenico, Annette Hofer, Federica Tundo, Tina Wenz (2014), Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance, IUBMB Life. 66: 793-802, 2014. DOI: 10.1002/iub.1328 © 2017 IUBMB Life, 69(7):553-553, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

  2. Semi-Synthesis of Labeled Proteins for Spectroscopic Applications

    Directory of Open Access Journals (Sweden)

    Luca Domenico D'Andrea

    2013-01-01

    Full Text Available Since the introduction of SPPS by Merrifield in the 60s, peptide chemists have considered the possibility of preparing large proteins. The introduction of native chemical ligation in the 90s and then of expressed protein ligation have opened the way to the preparation of synthetic proteins without size limitations. This review focuses on semi-synthetic strategies useful to prepare proteins decorated with spectroscopic probes, like fluorescent labels and stable isotopes, and their biophysical applications. We show that expressed protein ligation, combining the advantages of organic chemistry with the easy and size limitless recombinant protein expression, is an excellent strategy for the chemical synthesis of labeled proteins, enabling a single protein to be functionalized at one or even more distinct positions with different probes.

  3. Nutritional interventions to promote post-exercise muscle protein synthesis.

    Science.gov (United States)

    Koopman, René; Saris, Wim H M; Wagenmakers, Anton J M; van Loon, Luc J C

    2007-01-01

    Resistance exercise is a powerful stimulus to augment muscle protein anabolism, as it can improve the balance between muscle protein synthesis and breakdown. However, the intake of food during post-exercise recovery is necessary for hypertrophy to occur. Therefore, athletes need to ingest protein following exercise to attain a positive protein balance and maximise their skeletal muscle adaptive response. The interaction between exercise and nutrition is not only important for athletes, but is also of important clinical relevance in the elderly. Exercise interventions combined with specific nutritional modulation provide an effective strategy to counteract or reduce the loss of skeletal muscle mass with aging.

  4. Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

    Science.gov (United States)

    In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

  5. Intestinal DNA concentration and protein synthesis in response to ...

    African Journals Online (AJOL)

    Performance, protein synthesis and mucosal DNA in small intestine of Leghorn hens may be affected by low quality feedstuff. An experiment was conducted in completely randomized design (CRD) in 2 × 2 factorial arrangement. Main factors included diets containing 20 and 40 % barley and black and blue strains of leghorn ...

  6. Iron chelation excludes protein synthesis inhibition in the ...

    African Journals Online (AJOL)

    We have compared the trypanocidal properties of four antibiotics that show bactericidal activities by destabilizing ribosome-mRNA complex to inhibit protein synthesis. Tetracycline and oxytetracycline that have iron chelating properties extended the lifespan of trypanosome infected rats from 6 and 5 days of control to 15 and ...

  7. Digestion and microbial protein synthesis in sheep as affected by ...

    African Journals Online (AJOL)

    Useni , Alain

    sajas.v42i5.9. Digestion and microbial protein synthesis in sheep as affected by exogenous fibrolytic enzymes. W.F.J. van de Vyver. #. & B.A. Useni. Department of Animal Sciences, Stellenbosch University, Private Bag X1, Matieland 7602, South ...

  8. Total chemical synthesis of proteins without HPLC purification.

    Science.gov (United States)

    Loibl, S F; Harpaz, Z; Zitterbart, R; Seitz, O

    2016-11-01

    The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins

  9. Reactive Protein Synthesis in Pregnant Rats

    African Journals Online (AJOL)

    olayemitoyin

    /kg) groups. At terminal gestation day (GD) ranging from 0-20, the rats were sacrificed, and blood samples and amniotic fluids were collected. Thyroid hormone, C-reactive protein (CRP) and leptin assay was carried using the blood samples.

  10. Phospholemman: a novel cardiac stress protein.

    Science.gov (United States)

    Cheung, Joseph Y; Zhang, Xue-Qian; Song, Jianliang; Gao, Erhe; Rabinowitz, Joseph E; Chan, Tung O; Wang, Jufang

    2010-08-01

    Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. Functionally, when phosphorylated at serine(68), PLM stimulates Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L-type Ca(2+) channel. Therefore, PLM occupies a key modulatory role in intracellular Na(+) and Ca(2+) homeostasis and is intimately involved in regulation of excitation-contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na(+) overload and simultaneously preserves inotropy by inhibiting Na(+)/Ca(2+) exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and potentially L-type Ca(2+) channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189-196.

  11. Habituation to low or high protein intake does not modulate basal or postprandial muscle protein synthesis rates: a randomized trial

    NARCIS (Netherlands)

    Gorissen, S.H.; Horstman, Astrid; Franssen, Rinske; Kouw, I.W.; Wall, B.T.; Burd, N.A.; Groot, de C.P.G.M.; Loon, van L.J.C.

    2017-01-01

    Background: Muscle mass maintenance is largely regulated by basal muscle protein synthesis rates and the ability to increase muscle protein synthesis after protein ingestion. To our knowledge, no previous studies have evaluated the impact of habituation to either low protein intake (LOW PRO) or high

  12. THE ROLE OF POST-EXERCISE NUTRIENT ADMINISTRATION ON MUSCLE PROTEIN SYNTHESIS AND GLYCOGEN SYNTHESIS

    Directory of Open Access Journals (Sweden)

    Chris Poole

    2010-09-01

    Full Text Available Nutrient administration following an exercise bout vastly affects anabolic processes within the human body, irrespective of exercise mode. Of particular importance are protein and carbohydrates whereby these two macronutrients portray distinct functions as anabolic agents. It has been confirmed that protein and/or amino acid ingestion following resistance training is required to reach a positive protein/nitrogen balance, and carbohydrate intake during recovery is the most important consideration to replenish glycogen stores from an exhaustive exercise bout. Several factors play significant roles in determining the effectiveness of protein and carbohydrate supplementation on post-exercise protein and glycogen synthesis. Improper application of these factors can limit the body's ability to reach an anabolic status. The provided evidence clearly denotes the importance these two macronutrients have in regards to post-exercise nutrition and anabolism. Therefore, the purpose of this review is to discuss the impact of dietary protein and carbohydrate intake during the recovery state on muscle protein synthesis and glycogen synthesis

  13. Postligation-desulfurization: a general approach for chemical protein synthesis.

    Science.gov (United States)

    Ma, Jimei; Zeng, Jing; Wan, Qian

    2015-01-01

    Native chemical ligation, involving regioselective and chemoselective coupling of two unprotected peptide segments, enabled the synthesis of polypeptide with more than 200 amino acids. However, cysteine was indispensable in this synthetic technique in its initial format, which limited its further application. Thus, considerable effort has been put into breaking the restriction of cysteine-containing ligation. As a consequence, postligation-desulfurization, concerning thiol-mediated ligation followed by desulfurization, was developed. This review describes the development and recent progress on the chemical synthesis of peptides and proteins encompassing postligation-desulfurization at alanine, valine, lysine, threonine, leucine, proline, arginine, aspartic acid, glutamate, phenylalanine, glutamine, and tryptophan.

  14. Proteomics-based dissection of biotic stress responsive proteins in ...

    African Journals Online (AJOL)

    hope&shola

    2010-10-25

    Oct 25, 2010 ... digestion, MALDI-TOF/MS analysis and database searching of some of the identified proteins indicated that the proteins are ... identified as biotic stress responses proteins directly coupled to disease and pathogen infection on wheat. Nevertheless ... and animals (McMullen et al., 1997). Host resistance is.

  15. Ribosomal history reveals origins of modern protein synthesis.

    Directory of Open Access Journals (Sweden)

    Ajith Harish

    Full Text Available The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17 and the oldest substructure (the ribosomal ratchet in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

  16. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    Science.gov (United States)

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. Copyright © 2016. Published by Elsevier Ltd.

  17. Branched-chain amino acids and muscle protein synthesis in humans: myth or reality?

    Science.gov (United States)

    Wolfe, Robert R

    2017-01-01

    The branched chain amino acids (BCAAs) are leucine, valine and isoleucine. A multi-million dollar industry of nutritional supplements has grown around the concept that dietary supplements of BCAAs alone produce an anabolic response in humans driven by a stimulation of muscle protein synthesis. In this brief review the theoretical and empirical bases for that claim are discussed. Theoretically, the maximal stimulation of muscle protein synthesis in the post-absorptive state in response to BCAAs alone is the difference between muscle protein breakdown and muscle protein synthesis (about 30% greater than synthesis), because the other EAAs required for synthesis of new protein can only be derived from muscle protein breakdown. Realistically, a maximal increase in muscle protein synthesis of 30% is an over-estimate because the obligatory oxidation of EAAs can never be completely suppressed. An extensive search of the literature has revealed no studies in human subjects in which the response of muscle protein synthesis to orally-ingested BCAAs alone was quantified, and only two studies in which the effect of intravenously infused BCAAs alone was assessed. Both of these intravenous infusion studies found that BCAAs decreased muscle protein synthesis as well as protein breakdown, meaning a decrease in muscle protein turnover. The catabolic state in which the rate of muscle protein breakdown exceeded the rate of muscle protein synthesis persisted during BCAA infusion. We conclude that the claim that consumption of dietary BCAAs stimulates muscle protein synthesis or produces an anabolic response in human subjects is unwarranted.

  18. Depression of leukocyte protein synthesis, immune function and growth performance induced by high environmental temperature in broiler chickens.

    Science.gov (United States)

    Kamel, Nancy N; Ahmed, Ayman M H; Mehaisen, Gamal M K; Mashaly, Magdi M; Abass, Ahmed O

    2017-04-28

    In tropical and semitropical regions, raising broiler chickens out of their thermal comfort zone can cause an added economic loss in the poultry industry. The cause for the deleterious effects on immunity and growth performance of broilers under high environmental temperatures is still poorly understood. Therefore, the aim of the current investigation was to evaluate the effect of heat stress on leukocytes protein synthesis and immune function as a possible direct cause of low performance in broiler chickens under such condition. In this study, 300 one-day-old male broiler chicks (Cobb500™) were randomly assigned into 2 groups with 5 replicates of 30 chicks each. From 21 to 42 days of age, one group was exposed to non-stressed condition at 24 °C and 50% relative humidity (control group), while the other group was exposed to heat stress at 35 °C and 50% relative humidity (HS group). At 42 days of age, blood samples were collected from each group to evaluate stress indicators, immune function, and leukocytes protein synthesis. Production performance was also recorded. Noteworthy, protein synthesis in leukocytes was significantly (P broiler performance indicate that HS birds had a significant (P growth performance with the high mortality rate encountered in broiler chickens under heat stress environment.

  19. Cadherin-11 modulates cell morphology and collagen synthesis in periodontal ligament cells under mechanical stress.

    Science.gov (United States)

    Feng, Lishu; Zhang, Yimei; Kou, Xiaoxing; Yang, Ruili; Liu, Dawei; Wang, Xuedong; Song, Yang; Cao, Haifeng; He, Danqing; Gan, Yehua; Zhou, Yanheng

    2017-03-01

    To examine the role of cadherin-11, an integral membrane adhesion molecule, in periodontal ligament cells (PDLCs) under mechanical stimulation. Human PDLCs were cultured and subjected to mechanical stress. Cadherin-11 expression and cell morphology of PDLCs were investigated via immunofluorescence staining. The mRNA and protein expressions of cadherin-11 and type I collagen (Col-I) of PDLCs were evaluated by quantitative real-time polymerase chain reaction and Western blot, respectively. Small interfering RNA was used to knock down cadherin-11 expression in PDLCs. The collagen matrix of PDLCs was examined using toluidine blue staining. Cadherin-11 was expressed in PDLCs. Mechanical stress suppressed cadherin-11 expression in PDLCs with prolonged force treatment time and increased force intensity, accompanied by suppressed β-catenin expression. Simultaneously, mechanical stress altered cell morphology and repressed Col-I expression in a time- and dose-dependent manner in PDLCs. Moreover, knockdown of cadherin-11 with suppressed β-catenin expression resulted in altered PDLC morphology and repressed collagen expression, which were consistent with the changes observed under mechanical stress. Results of this study suggest that cadherin-11 is expressed in PDLCs and modulates PDLC morphology and collagen synthesis in response to mechanical stress, which may play an important role in the homeostasis and remodeling of the PDL under mechanical stimulation.

  20. Using Chemical Synthesis To Study and Apply Protein Glycosylation.

    Science.gov (United States)

    Chaffey, Patrick K; Guan, Xiaoyang; Li, Yaohao; Tan, Zhongping

    2018-01-16

    Protein glycosylation is one of the most common post-translational modifications and can influence many properties of proteins. Abnormal protein glycosylation can lead to protein malfunction and serious disease. While appreciation of glycosylation's importance is growing in the scientific community, especially in recent years, a lack of homogeneous glycoproteins with well-defined glycan structures has made it difficult to understand the correlation between the structure of glycoproteins and their properties at a quantitative level. This has been a significant limitation on rational applications of glycosylation and on optimizing glycoprotein properties. Through the extraordinary efforts of chemists, it is now feasible to use chemical synthesis to produce collections of homogeneous glycoforms with systematic variations in amino acid sequence, glycosidic linkage, anomeric configuration, and glycan structure. Such a technical advance has greatly facilitated the study and application of protein glycosylation. This Perspective highlights some representative work in this research area, with the goal of inspiring and encouraging more scientists to pursue the glycosciences.

  1. Emerging roles of ER stress and unfolded protein response pathways in skeletal muscle health and disease.

    Science.gov (United States)

    Bohnert, Kyle R; McMillan, Joseph D; Kumar, Ashok

    2018-01-01

    Skeletal muscle is the most abundant tissue in the human body and can adapt its mass as a consequence of physical activity, metabolism, growth factors, and disease conditions. Skeletal muscle contains an extensive network of endoplasmic reticulum (ER), called sarcoplasmic reticulum, which plays an important role in the regulation of proteostasis and calcium homeostasis. In many cell types, environmental and genetic factors that disrupt ER function cause an accumulation of misfolded and unfolded proteins in the ER lumen that ultimately leads to ER stress. To alleviate the stress and restore homeostasis, the ER activates a signaling network called the unfolded protein response (UPR). The UPR has three arms, which regulate protein synthesis and expression of many ER chaperone and regulatory proteins. However, the role of individual UPR pathways in skeletal muscle has just begun to be investigated. Recent studies suggest that UPR pathways play pivotal roles in muscle stem cell homeostasis, myogenic differentiation, and regeneration of injured skeletal muscle. Moreover, markers of ER stress and the UPR are activated in skeletal muscle in diverse conditions such as exercise, denervation, starvation, high fat diet, cancer cachexia, and aging. Accumulating evidence also suggests that ER stress may have important roles in the pathogenesis of inflammatory myopathies and genetic muscle disorders. The purpose of this review article is to discuss the role and potential mechanisms by which ER stress and the individual arms of the UPR regulate skeletal muscle formation, plasticity, and function in various physiological and pathophysiological conditions. © 2017 Wiley Periodicals, Inc.

  2. Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein

    DEFF Research Database (Denmark)

    Schreiber, K; Boes, N; Escbach, M

    2006-01-01

    activity was detected in the deeper layers of a P. aeruginosa biofilm using a PPA3309-gfp (green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria....... the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress...... proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression...

  3. Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress

    DEFF Research Database (Denmark)

    Kästle, Marc; Reeg, Sandra; Rogowska-Wrzesinska, Adelina

    2012-01-01

    After oxidative stress proteins which are oxidatively modified are degraded by the 20S proteasome. However, several studies documented an enhanced ubiquitination of yet unknown proteins. Since ubiqutination is a prerequisite for degradation by the 26S proteasome in an ATP-dependent manner...... this raises the question whether these proteins are also oxidized and, if not, what proteins need to be ubiquitinated and degraded after oxidative conditions. By determination of oxidized- and ubiquitinated proteins we demonstrate here that most oxidized proteins are not preferentially ubiquitinated. However......, we were able to confirm an increase of ubiquitinated proteins 16h upon oxidative stress. Therefore, we isolated ubiquitinated proteins from hydrogen peroxide treated cells, as well as from control and lactacystin, an irreversible proteasome inhibitor, treated cells, and identified some...

  4. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    Energy Technology Data Exchange (ETDEWEB)

    Horst, M.N. (Mercer Univ., Macon, GA (USA))

    1990-12-01

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.

  5. ZINC STIMULATION OF RNA AND PROTEIN SYNTHESIS IN RHIZOPUS NIGRICANS.

    Science.gov (United States)

    WEGENER, W S; ROMANO, A H

    1963-12-27

    Addition of 1.7 x 10-(5)M zinc sulfate to cultures of Rhizopus nigricans increases growth and substrate utilization. Analysis of cells during the course of growth, after addition of the metal, showed that there was an immediate increase in RNA, followed by a corresponding increase in protein and cell mass. The DNA content was affected to a lesser extent. It is postulated that Zn(++) stimulates growth through a primary effect on RNA synthesis.

  6. Impact of caffeine and protein on postexercise muscle glycogen synthesis.

    Science.gov (United States)

    Beelen, Milou; Kranenburg, Janneau van; Senden, Joan M; Kuipers, Harm; Loon, Luc J C van

    2012-04-01

    Both protein and caffeine coingestion with CHO have been suggested to represent effective dietary strategies to further accelerate postexercise muscle glycogen synthesis in athletes. This study aimed to assess the effect of protein or caffeine coingestion on postexercise muscle glycogen synthesis rates when optimal amounts of CHO are ingested. Fourteen male cyclists were studied on three different test days. Each test day started with a glycogen-depleting exercise session. This was followed by a 6-h recovery period, during which subjects received 1.2 g·kg⁻¹·h⁻¹ CHO, the same amount of CHO with 0.3 g·kg⁻¹·h⁻¹ of a protein plus leucine mixture (CHO + PRO), or 1.7 mg·kg⁻¹·h⁻¹ caffeine (CHO + CAF). All drinks were enriched with [U-¹³C₆]-labeled glucose to assess potential differences in the appearance rate of ingested glucose from the gut. Muscle biopsies were collected immediately after cessation of exercise and after 6 h of postexercise recovery. The plasma insulin response was higher in CHO + PRO compared with CHO and CHO + CAF (P synthesis rates averaged 31 ± 4, 34 ± 4, and 31 ± 4 mmol·kg⁻¹ dry weight·h⁻¹ in CHO, CHO + PRO, and CHO + CAF, respectively (P = NS). In accordance, histochemical analyses did not show any differences between net changes in Type I and Type II muscle fiber glycogen content between experiments. Coingestion of protein or caffeine does not further accelerate postexercise muscle glycogen synthesis when ample amounts of CHO (1.2 g·kg⁻¹·h⁻¹) are ingested.

  7. Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work.

    Science.gov (United States)

    Kent, Stephen

    2017-09-15

    Total chemical synthesis of proteins has been rendered practical by the chemical ligation principle: chemoselective condensation of unprotected peptide segments equipped with unique, mutually reactive functional groups, enabled by formation of a non-native replacement for the peptide bond. Ligation chemistries are briefly described, including native chemical ligation - thioester-mediated, amide-forming reaction at Xaa-Cys sites - and its extensions. Case studies from the author's own works are used to illustrate the utility and applications of chemical protein synthesis. Selected recent developments in the field are briefly discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Fluorinated proteins: from design and synthesis to structure and stability.

    Science.gov (United States)

    Marsh, E Neil G

    2014-10-21

    Fluorine is all but absent from biology; however, it has proved to be a remarkably useful element with which to modulate the activity of biological molecules and to study their mechanism of action. Our laboratory's interest in incorporating fluorine into proteins was stimulated by the unusual physicochemical properties exhibited by perfluorinated small molecules. These include extreme chemical inertness and thermal stability, properties that have made them valuable as nonstick coatings and fire retardants. Fluorocarbons also exhibit an unusual propensity to phase segregation. This phenomenon, which has been termed the "fluorous effect", has been effectively exploited in organic synthesis to purify compounds from reaction mixtures by extracting fluorocarbon-tagged molecules into fluorocarbon solvents. As biochemists, we were curious to explore whether the unusual physicochemical properties of perfluorocarbons could be engineered into proteins. To do this, we developed a synthesis of a highly fluorinated amino acid, hexafluoroleucine, and designed a model 4-helix bundle protein, α4H, in which the hydrophobic core was packed exclusively with leucine. We then investigated the effects of repacking the hydrophobic core of α4H with various combinations of leucine and hexafluoroleucine. These initial studies demonstrated that fluorination is a general and effective strategy for enhancing the stability of proteins against chemical and thermal denaturation and proteolytic degradation. We had originally envisaged that the "fluorous interactions", postulated from the self-segregating properties of fluorous solvents, might be used to mediate specific protein-protein interactions orthogonal to those of natural proteins. However, various lines of evidence indicate that no special, favorable fluorine-fluorine interactions occur in the core of the fluorinated α4 protein. This makes it unlikely that fluorinated amino acids can be used to direct protein-protein interactions. More

  9. Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis.

    Science.gov (United States)

    Stoianov, Alexandra M; Robson, Debra L; Hetherington, Alexandra M; Sawyez, Cynthia G; Borradaile, Nica M

    2015-01-01

    Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.

  10. Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

    Directory of Open Access Journals (Sweden)

    Matti A Kjellberg

    Full Text Available Members of the glycolipid transfer protein superfamily (GLTP are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.

  11. Twister Protein: a ludic tool involving protein synthesis

    Directory of Open Access Journals (Sweden)

    Aline Weyh

    2015-07-01

    Full Text Available Several studies show that students of various grade levels report the Genetics as an abstract theme and difficult to assimilate by the students, with multiple problems in the teaching-learning process and becoming necessary the development of auxiliary practices. Among the teaching tools, the game is the most currently opted playful activity by stimulating multiple intelligences, allowing greater student-teacher interaction. This work seeks the production of an innovative and dynamic educational game, Twister Protein, as a pedagogical resource for Genetics discipline. The development of the game was based on the use of easily accessible and low cost materials by teachers, allowing the knowledge of transcription, translation and protein folding. The activity was proposed and applied in the classroom with pilot undergraduate students. The fun associated with the knowledge of science not only allowed a better memorization of the content addressed, as aroused the curiosity, theme reflection, character building and collaborative spirits, as well as competitiveness through the interaction between class. This practice proved to be an effective tool in the escape from routine and fault repair of the theoretical process.

  12. An Arabidopsis mitochondrial uncoupling protein confers tolerance to drought and salt stress in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Kevin Begcy

    Full Text Available BACKGROUND: Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1 from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants. CONCLUSIONS/SIGNIFICANCE: Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions.

  13. An Arabidopsis Mitochondrial Uncoupling Protein Confers Tolerance to Drought and Salt Stress in Transgenic Tobacco Plants

    Science.gov (United States)

    Begcy, Kevin; Mariano, Eduardo D.; Mattiello, Lucia; Nunes, Alessandra V.; Mazzafera, Paulo; Maia, Ivan G.; Menossi, Marcelo

    2011-01-01

    Background Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs) that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking. Methodology/Principal Findings Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1) from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT) plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants. Conclusions/Significance Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions. PMID:21912606

  14. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y and glial (CCF-STTG1 lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48 residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

  15. Butyrate increases colonocyte protein synthesis in ulcerative colitis.

    Science.gov (United States)

    Frankel, W; Lew, J; Su, B; Bain, A; Klurfeld, D; Einhorn, E; MacDermott, R P; Rombeau, J

    1994-07-01

    Butyrate promotes epithelial cell healing and improves symptoms when administered rectally in patients with distal ulcerative colitis (UC). It was hypothesized that butyrate may enhance healing in patients with UC by stimulating colonocyte proliferation and/or protein production. Mucosa from the descending colon was obtained from patients with UC (n = 5), Crohn's disease (n = 8), diverticulitis (n = 6), and cancer (normal tissue 10 cm from tumor; n = 10). Epithelial cells were isolated using dispase/collagenase and differential sedimentation and incubated for 4 hr at 37 degrees C with either Na butyrate (10 mM) or NaCl (10 mM). Protein synthesis was assessed by [14C]leucine incorporation and proliferation was determined with [3H]thymidine. Mean viability and purity were >88%. Spontaneous proliferation was significantly increased in UC when compared to diverticulitis and normal controls. Butyrate significantly increased protein synthesis in UC epithelial cells when compared to saline control. The therapeutic effects of butyrate in patients with UC may be due to its use by epithelial cells as a metabolic fuel to increase protein production and promote healing.

  16. ER Dysfunction and Protein Folding Stress in ALS

    Science.gov (United States)

    Matus, Soledad; Valenzuela, Vicente; Medinas, Danilo B.; Hetz, Claudio

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is the most frequent paralytic disease in adults. Most ALS cases are considered sporadic with no clear genetic component. The disruption of protein homeostasis due to chronic stress responses at the endoplasmic reticulum (ER) and the accumulation of abnormal protein inclusions are extensively described in ALS mouse models and patient-derived tissue. Recent studies using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive reaction against ER stress, have demonstrated a complex involvement of the pathway in experimental models of ALS. In addition, quantitative changes in ER stress-responsive chaperones in body fluids have been proposed as possible biomarkers to monitor the disease progression. Here we review most recent advances attributing a causal role of ER stress in ALS. PMID:24324498

  17. Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis

    NARCIS (Netherlands)

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J F; Vollmer, Waldemar; Breukink, Eefjan|info:eu-repo/dai/nl/120305100; Terrak, Mohammed

    2017-01-01

    Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert

  18. Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein

    Science.gov (United States)

    Schmidt, Sarah; Tavernaro, Isabella; Cavelius, Christian; Weber, Eva; Kümper, Alexander; Schmitz, Carmen; Fleddermann, Jana; Kraegeloh, Annette

    2017-09-01

    In this study, a novel approach for preparation of green fluorescent protein (GFP)-doped silica nanoparticles with a narrow size distribution is presented. GFP was chosen as a model protein due to its autofluorescence. Protein-doped nanoparticles have a high application potential in the field of intracellular protein delivery. In addition, fluorescently labelled particles can be used for bioimaging. The size of these protein-doped nanoparticles was adjusted from 15 to 35 nm using a multistep synthesis process, comprising the particle core synthesis followed by shell regrowth steps. GFP was selectively incorporated into the silica matrix of either the core or the shell or both by a one-pot reaction. The obtained nanoparticles were characterised by determination of particle size, hydrodynamic diameter, ζ-potential, fluorescence and quantum yield. The measurements showed that the fluorescence of GFP was maintained during particle synthesis. Cellular uptake experiments demonstrated that the GFP-doped nanoparticles can be used as stable and effective fluorescent probes. The study reveals the potential of the chosen approach for incorporation of functional biological macromolecules into silica nanoparticles, which opens novel application fields like intracellular protein delivery.

  19. Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

    LENUS (Irish Health Repository)

    Samali, Afshin

    2010-01-01

    The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

  20. Crosstalk between proteins expression and lysine acetylation in response to patulin stress in Rhodotorula mucilaginosa.

    Science.gov (United States)

    Zheng, Xiangfeng; Yang, Qiya; Zhao, Lina; Apaliya, Maurice Tibiru; Zhang, Xiaoyun; Zhang, Hongyin

    2017-10-18

    The proteomic and lysine acetylation (Kac) changes, accompanying degradation of patulin in Rhodotorula mucilaginosa were analyzed using tandem mass tagging and N6-acetyllysine affinity enrichment followed by LC-MS/MS. Proteomic results showed that expression level of short-chain reductase protein and glutathione S-transferase involved in detoxification was significantly up-regulated. In addition, the expression levels of zinc-binding oxidoreductase and quinone oxidoreductase that are involved in antioxidant process, ABC transport and MFS transport responsible for chemical transport were activated when treated with patulin. The quantitative real time PCR (qRT-PCR) result also indicated these genes expression levels were increased when treated with patulin. Kac changes accompanying degradation of patulin in R. mucilaginosa were also observed. Totally, 130 Kac sites in 103 proteins were differentially expressed under patulin stress. The differentially up expressed modified proteins were mainly involved in tricarboxylic acid cycle and nuclear acid biosynthesis. The differentially down expressed Kac proteins were mainly classified to ribosome, oxidative phosphorylation, protein synthesis and defense to stress process. Our results suggest that patulin exposure prompt R. mucilaginosa to produce a series of actions to resist or degrade patulin, including Kac. In addition, the Kac information in R. mucilaginosa and Kac in response to patulin stress was firstly revealed.

  1. Protein-Polymer Matrix Mediated Synthesis of Silver Nanoparticles

    Directory of Open Access Journals (Sweden)

    Swati Mishra

    2014-09-01

    Full Text Available Silver nanoparticles were synthesized in the protein-polymer matrices of two different ratios to obtain a stringent control over the morphology. UV-visible spectrophotometry showed a single plasmon resonance peak at 416nm and 418nm respectively, confirming the formation of silver nanoparticles. X-ray diffractometry confirmed that the peaks matched with that of the reference silver. Both confocal microscopy and FEG-SEM confirmed the uniform morphology of the synthesized particles dependent on the template ratio. Doubling the protein-polymer concentration results in greater stability, more nucleation sites and hence restricted growth. Photoluminescence of the sample in the doubled matrix was found to be much greater at any given wavelength, meaning the flexibility and rigidity of interacting molecules affects the luminescence signal. The interaction in turn is dependent on the proximity of the proteins and polymer in the dispersion that forms a template and dictates the synthesis.

  2. RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis

    Directory of Open Access Journals (Sweden)

    Sams Carl E

    2006-09-01

    Full Text Available Abstract Background Mean phosphorous:nitrogen (P:N ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER were analyzed under different conditions of exponential growth. Results It was found that P:N ratios calculated from RNA and protein contents in these particular organisms were in the same range as the mean ratios reported for diverse organisms and had similar positive relationships with growth rate, consistent with the growth-rate hypothesis. The efficiency of protein synthesis in microorganisms is estimated as the number of active ribosomes required for the incorporation of one amino acid into the synthesized protein. This parameter is calculated as the SGR:PER ratio. Experimental and theoretical evidence indicated that the requirement of ribosomes for protein synthesis is proportional to the RNA:protein ratio. The constant of proportionality had the same values for all organisms, and was derived mechanistically from the characteristics of the protein-synthesis machinery of the cell (the number of nucleotides per ribosome, the average masses of nucleotides and amino acids, the fraction of ribosomal RNA in the total RNA, and the fraction of active ribosomes. Impairment of the growth conditions decreased the RNA:protein ratio and increased the overall efficiency of protein synthesis in the microorganisms. Conclusion Our results suggest that the decrease in RNA:protein and estimated P:N ratios with decrease in the growth rate of the microorganism is a consequence of an increased overall efficiency of protein synthesis in the cell resulting from activation of the general stress response and

  3. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling

    OpenAIRE

    Lee, Elaine Choung-Hee; Strange, Kevin

    2012-01-01

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is ...

  4. Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.

    Science.gov (United States)

    Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C

    2016-01-01

    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  5. Noncanonical amino acids in the interrogation of cellular protein synthesis.

    Science.gov (United States)

    Ngo, John T; Tirrell, David A

    2011-09-20

    Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other

  6. Determination of human muscle protein fractional synthesis rate

    DEFF Research Database (Denmark)

    Bornø, Andreas; Hulston, Carl J; van Hall, Gerrit

    2014-01-01

    In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically...... labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC-C-IRMS) has thus far been considered the 'gold' standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography-tandem MS (LC...

  7. Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to the stress-inducible yeast protein STI1

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1992-01-01

    in families of fungal proteins required for mitosis and RNA synthesis. In particular, the protein has 42% amino acid sequence identity to STI1, a stress-inducible mediator of the heat shock response in Saccharomyces cerevisiae. Northern blot analysis indicated that the 3521 mRNA is up-regulated in several...... transformed cells. Immunofluorescence studies using a polyclonal antibody raised against the purified protein revealed that the antigen is present mainly in the nucleus of SV40 transformed MRC-5 fibroblasts, while it localizes to the Golgi apparatus and small vesicles in their normal counterparts...

  8. Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma.

    Science.gov (United States)

    Choi, Jinah; Corder, Nicole L B; Koduru, Bhargav; Wang, Yiyan

    2014-07-01

    Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H2O2 formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor β. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa

    Directory of Open Access Journals (Sweden)

    Jackson MARCONDES

    2011-06-01

    Full Text Available Among various physiological responses to salt stress, the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L. under the treatment of 170 mmol/L NaCl. In order to better understand the role of the SALT protein in the physiological processes involving salinity, it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique. This variety turned out to be an excellent model for that purpose, since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer. This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity. Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition. Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization, its lectin-like activity seems to play an important role in the establishment of stable complexes, either to other proteins or to oligosaccharides that are translocated to the chloroplast.

  10. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    Muscle cell culture (L/sub 6/) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 ..mu..M compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of (/sup 3/H) leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using (/sup 3/H) leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 ..mu..M level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.

  11. Production of functional proteins: balance of shear stress and gravity

    Science.gov (United States)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2011-01-01

    A method for the production of functional proteins including hormones by renal cells in a three dimensional culturing process responsive to shear stress uses a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-.alpha.-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D.sub.3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating an in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  12. [Glycation of mitochondrial proteins, oxidative stress and aging].

    Science.gov (United States)

    Naudí, Alba; Jové, Mariona; Ayala, Victoria; Portero-Otín, Manuel; Pamplona, Reinald

    2010-01-01

    Mitochondrial proteins can be modified by glycation reactions from endogenous dicarbonyl compounds such as physiologically generated methylglyoxal and glyoxal. This modification could cause structural and functional changes in the proteins Consequently, dicarbonyl attack of the mitochondrial proteome may be an event leading to mitochondrial dysfunction and thus, to oxidative stress. These protein chemical modifications can play an important role in the physiological aging process and age-associated diseases, where both mitochondrial defects and increased dicarbonyl concentrations have been found. Future research should address the functional changes in mitochondrial proteins that are the targets for dicarbonyl glycation. 2010 SEGG. Published by Elsevier Espana. All rights reserved.

  13. Oxidative stress and protein carbonylation in adipose tissue - implications for insulin resistance and diabetes mellitus.

    Science.gov (United States)

    Ruskovska, Tatjana; Bernlohr, David A

    2013-10-30

    While historically considered simply as a depot for excess energy, white adipose tissue is a dynamically active endocrine organ capable of responding to a variety of efferent stimuli resulting in the synthesis and secretion of peptides, proteins and metabolites that serve as signal transducers to the peripheral and central circulation. Such regulation controls a variety of physiological processes including energy expenditure, food intake, reproductive capacity and responsiveness to insulin. Indeed, the accumulation of inflammatory cells in white adipose tissue is considered to be causative in the development of insulin resistance and eventually type 2 diabetes mellitus. A large body of evidence suggests that oxidative stress in adipose tissue not only correlates with insulin resistance but is also causative in its development. Moreover, using the available plasma oxidative stress biomarkers, many clinical studies have shown the presence of systemic oxidative stress in obese insulin resistant subjects, and its decrease after the successful treatment of obesity. In this review we emphasize the role of protein carbonylation in dysfunctional obese white adipose tissue and its metabolic implications. We focus on glutathione S-transferase A4 as the key enzyme for trans-4-hydroxy-2-nonenal and trans-4-oxo-2-nonenal removal from the cell, thus preventing protein carbonylation. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Effect of Insulin Infusion on Liver Protein Synthesis during Hemodialysis

    DEFF Research Database (Denmark)

    Reinhard, Mark; Frystyk, Jan; Jespersen, Bente

    2011-01-01

    Background Hemodialysis (HD) is a catabolic procedure that may contribute to the high frequency of protein-energy wasting among patients receiving maintenance HD. The present study investigated the additional effect of glucose and glucose-insulin infusion on liver protein synthesis during HD...... compared with a meal alone. Methods In a randomized cross-over study with three arms, 11 non-diabetic HD patients were assigned to receive a conventional HD session with either: • no treatment (NT) • IV infusion of glucose (G) • IV infusion of glucose-insulin (GI) During infusions blood glucose levels were...... maintained at 8.0-10.0 mmol/L by additional glucose infusion. Glucose and glucose-insulin infusions were commenced 2 h prior to HD and continued throughout the HD session. Fasting blood samples were collected at baseline before infusion and followed by the only meal allowed during the study. Results Blood...

  15. How is the balance between protein synthesis and degradation achieved?

    Directory of Open Access Journals (Sweden)

    Rothman Stephen

    2010-06-01

    Full Text Available Abstract Unlike most substances that cells manufacture, proteins are not produced and broken down by a common series of chemical reactions, but by completely different (independent and disconnected mechanisms that possess no intrinsic means of making the rates of the two processes equal and attaining steady state concentrations. Balance between them is achieved extrinsically and is often imagined today to be the result of the actions of chemical feedback agents. But however instantiated, chemical feedback or any similar mechanism can only rectify induced imbalances in a system previously balanced by other means. Those "other means" necessarily involve reversible mass action or equilibrium-based interactions between native and altered forms of protein molecules somewhere in time and space between their synthesis and degradation.

  16. Effects of oxytocin on adreno-medullary catecholamine synthesis, uptake and storage in rats exposed to chronic isolation stress.

    Science.gov (United States)

    Jovanovic, Predrag; Stefanovic, Bojana; Spasojevic, Natasa; Puskas, Nela; Dronjak, Sladjana

    2016-05-01

    The adreno-medullar system represents one of the main systems involved in the response to stressful events. The neuropeptide oxytocin, is highly sensitive to the social environment, and regulates autonomic function. Adreno-medullary activity is dependent on the synthesis of catecholamine, its reuptake, release, degradation and vesicular transport. A direct influence of oxytocin on catecholamine synthesizing enzyme and transports in animals exposed to chronic social isolation stress has not been studied yet. In the present study, we examined the effect of chronic oxytocin treatment on the level of plasma catecholamine and its content, mRNA and protein levels of tyrosine hydroxylase (TH), noradrenaline transporter (NET) as well as vesicular monoamine transporter 2 (VMAT2) in the adrenal medulla of socially isolated rats. Our results show that, by the end of 12 weeks, social isolation did not produce any significant changes in catecholamine content but increased plasma catecholamine level and synthesis in the adrenal medulla. Oxytocin treatment had no further effect either on catecholamine synthesis or content in socially stressed animals whereas a significant elevation of plasma norepinephrine and epinephrine were reduced. On the other hand, chronic isolation caused a significant increase in VMAT2 and decrease in NET protein levels. Oxytocin treatment brought about an increase in protein levels of NET and its return to the levels of control group. Besides, it further increases VMAT2 protein levels in the adrenal medulla of individually housed rats. The present results show that peripheral oxytocin treatment enhances catecholamine uptake and storage in the adrenal medulla of chronically isolated animals.

  17. Stress-induced cell death is mediated by ceramide synthesis in Neurospora crassa

    DEFF Research Database (Denmark)

    Plesofsky, Nora S; Levery, Steven B; Castle, Sherry A

    2008-01-01

    The combined stresses of moderate heat shock (45 degrees C) and analog-induced glucose deprivation constitute a lethal stress for Neurospora crassa. We found that this cell death requires fatty acid synthesis and the cofactor biotin. In the absence of the cofactor, the stressed cells are particul...... type, and phosphorylated OS-2 increased in wild-type cells in response to heat shock and combined heat and carbon stress.......The combined stresses of moderate heat shock (45 degrees C) and analog-induced glucose deprivation constitute a lethal stress for Neurospora crassa. We found that this cell death requires fatty acid synthesis and the cofactor biotin. In the absence of the cofactor, the stressed cells...... are particularly sensitive to exogenous ceramide, which is lethal at low concentrations. When we extracted endogenous sphingolipids, we found that unique ceramides were induced (i) by the inhibitory glucose analog 2-deoxyglucose and (ii) by combined heat shock and 2-deoxyglucose. We determined that the former...

  18. Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants.

    Science.gov (United States)

    Ludwig, Andrea A; Saitoh, Hiromasa; Felix, Georg; Freymark, Gerald; Miersch, Otto; Wasternack, Claus; Boller, Thomas; Jones, Jonathan D G; Romeis, Tina

    2005-07-26

    Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress.

  19. THERMOTOLERANCE IN MAMMALIAN-CELLS - PROTEIN DENATURATION AND AGGREGATION, AND STRESS PROTEINS

    NARCIS (Netherlands)

    KAMPINGA, HH

    Cells that have been pre-exposed to thermal stress can acquire a transient resistance against the killing effect of a subsequent thermal stress. The cause for this phenomenon, called thermotolerance, seems to be an enhanced resistance of proteins against thermal denaturation and aggregation. This

  20. Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana under Oxidative Stress

    Science.gov (United States)

    Jacques, Silke; Ghesquière, Bart; De Bock, Pieter-Jan; Demol, Hans; Wahni, Khadija; Willems, Patrick; Messens, Joris; Van Breusegem, Frank; Gevaert, Kris

    2015-01-01

    Reactive oxygen species such as hydrogen peroxide can modify proteins via direct oxidation of their sulfur-containing amino acids, cysteine and methionine. Methionine oxidation, studied here, is a reversible posttranslational modification that is emerging as a mechanism by which proteins perceive oxidative stress and function in redox signaling. Identification of proteins with oxidized methionines is the first prerequisite toward understanding the functional effect of methionine oxidation on proteins and the biological processes in which they are involved. Here, we describe a proteome-wide study of in vivo protein-bound methionine oxidation in plants upon oxidative stress using Arabidopsis thaliana catalase 2 knock-out plants as a model system. We identified over 500 sites of oxidation in about 400 proteins and quantified the differences in oxidation between wild-type and catalase 2 knock-out plants. We show that the activity of two plant-specific glutathione S-transferases, GSTF9 and GSTT23, is significantly reduced upon oxidation. And, by sampling over time, we mapped the dynamics of methionine oxidation and gained new insights into this complex and dynamic landscape of a part of the plant proteome that is sculpted by oxidative stress. PMID:25693801

  1. Identification of drought-responsive universal stress proteins in viridiplantae.

    Science.gov (United States)

    Isokpehi, Raphael D; Simmons, Shaneka S; Cohly, Hari H P; Ekunwe, Stephen I N; Begonia, Gregorio B; Ayensu, Wellington K

    2011-02-07

    Genes encoding proteins that contain the universal stress protein (USP) domain are known to provide bacteria, archaea, fungi, protozoa, and plants with the ability to respond to a plethora of environmental stresses. Specifically in plants, drought tolerance is a desirable phenotype. However, limited focused and organized functional genomic datasets exist on drought-responsive plant USP genes to facilitate their characterization. The overall objective of the investigation was to identify diverse plant universal stress proteins and Expressed Sequence Tags (ESTs) responsive to water-deficit stress. We hypothesize that cross-database mining of functional annotations in protein and gene transcript bioinformatics resources would help identify candidate drought-responsive universal stress proteins and transcripts from multiple plant species. Our bioinformatics approach retrieved, mined and integrated comprehensive functional annotation data on 511 protein and 1561 ESTs sequences from 161 viridiplantae taxa. A total of 32 drought-responsive ESTs from 7 plant genera Glycine, Hordeum, Manihot, Medicago, Oryza, Pinus and Triticum were identified. Two Arabidopsis USP genes At3g62550 and At3g53990 that encode ATP-binding motif were up-regulated in a drought microarray dataset. Further, a dataset of 80 simple sequence repeats (SSRs) linked to 20 singletons and 47 transcript assembles was constructed. Integrating the datasets on SSRs and drought-responsive ESTs identified three drought-responsive ESTs from bread wheat (BE604157), soybean (BM887317) and maritime pine (BX682209). The SSR sequence types were CAG, ATA and AT respectively. The datasets from cross-database mining provide organized resources for the characterization of USP genes as useful targets for engineering plant varieties tolerant to unfavorable environmental conditions.

  2. Differential effects of insulin deprivation and systemic insulin treatment on plasma protein synthesis in type 1 diabetic people

    OpenAIRE

    Jaleel, Abdul; Klaus, Katherine A.; Morse, Dawn M.; Karakelides, Helen; Ward, Lawrence E.; Irving, Brian A.; Nair, K. Sreekumaran

    2009-01-01

    It remains to be determined whether systemic insulin replacement normalizes synthesis rates of different plasma proteins and whether there are differential effects on various plasma proteins. We tested a hypothesis that insulin deprivation differentially affects individual plasma protein synthesis and that systemic insulin treatment may not normalize synthesis of all plasma proteins. We measured synthesis rates of 41 plasma proteins in seven each of type 1 diabetic (T1DM) and nondiabetic part...

  3. Exercising before protein intake allows for greater use of dietary protein-derived amino acids for de novo muscle protein synthesis in both young and elderly men

    National Research Council Canada - National Science Library

    Pennings, B; Koopman, R; Beelen, M; Senden, J.M.G; Saris, W.H; van Loon, L.J.C

    2011-01-01

    .... The objective was to compare in vivo dietary protein digestion and absorption kinetics and subsequent postprandial muscle protein synthesis rates at rest and after exercise between young and elderly men...

  4. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    Directory of Open Access Journals (Sweden)

    Seok Hoon eHong

    2014-06-01

    Full Text Available Incorporating non-standard amino acids (NSAAs into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L and long-lasting (>10 h in batch operation CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  5. Activation of the Unfolded Protein Response by Lipid Bilayer Stress.

    Science.gov (United States)

    Halbleib, Kristina; Pesek, Kristina; Covino, Roberto; Hofbauer, Harald F; Wunnicke, Dorith; Hänelt, Inga; Hummer, Gerhard; Ernst, Robert

    2017-08-17

    The unfolded protein response (UPR) is a conserved homeostatic program that is activated by misfolded proteins in the lumen of the endoplasmic reticulum (ER). Recently, it became evident that aberrant lipid compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism, however, remained unclear. We show that the most conserved transducer of ER stress, Ire1, uses an amphipathic helix (AH) to sense membrane aberrancies and control UPR activity. In vivo and in vitro experiments, together with molecular dynamics (MD) simulations, identify the physicochemical properties of the membrane environment that control Ire1 oligomerization. This work establishes the molecular mechanism of UPR activation by lipid bilayer stress. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Involvement of endoplasmic stress protein C/EBP homologous protein in arteriosclerosis acceleration with augmented biological stress responses.

    Science.gov (United States)

    Gao, Junhong; Ishigaki, Yasushi; Yamada, Tetsuya; Kondo, Keiichi; Yamaguchi, Suguru; Imai, Junta; Uno, Kenji; Hasegawa, Yutaka; Sawada, Shojiro; Ishihara, Hisamitsu; Oyadomari, Seiichi; Mori, Masataka; Oka, Yoshitomo; Katagiri, Hideki

    2011-08-16

    The processes of arteriosclerosis, including atherosclerosis and vascular remodeling, are affected by interactions among numerous biological pathways such as responses to inflammation, oxidative stress, and endoplasmic reticulum stress. C/EBP homologous protein (CHOP), which is well known to induce cellular apoptosis in response to severe endoplasmic reticulum stress, is reportedly upregulated in plaque lesions. We examined the effects of CHOP deficiency on 2 types of arteriosclerosis: cuff injury-induced neointimal formation and hypercholesterolemia-induced atherosclerosis. Cuff injury-induced neointimal formation was markedly inhibited in CHOP(-/-) mice with suppressed aortic expression of inflammatory factors and smooth muscle cell proliferation-related proteins. A CHOP deficiency also inhibited aortic plaque formation in hypercholesterolemic apolipoprotein E(-/-) mice with suppressed aortic expression of inflammatory factors and oxidative stress markers. Bone marrow transplantation experiments revealed that recipient CHOP deficiency significantly suppressed both cuff injury-induced neointimal formation and hypercholesterolemia-induced atherosclerotic plaque formation to a greater extent than donor CHOP deficiency, suggesting the importance of CHOP in vascular cells for arteriosclerosis progression. Furthermore, in our in vitro experiments, in not only macrophages but also endothelial and smooth muscle cell lines, endoplasmic reticulum stress inducers upregulated inflammation-, adhesion-, or smooth muscle cell proliferation-related proteins, whereas decreased CHOP expression remarkably suppressed endoplasmic reticulum stress-induced upregulation of these proteins. In addition to the well-known signaling for apoptosis induction, CHOP may play important roles in augmenting potentially pathological biological stress responses. This noncanonical role of CHOP, especially that expressed in vascular cells, may contribute to the progression of vascular remodeling and

  7. Nitrosative stress and nitrated proteins in trichloroethene-mediated autoimmunity.

    Directory of Open Access Journals (Sweden)

    Gangduo Wang

    Full Text Available Exposure to trichloroethene (TCE, a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ∼ 250 mg/kg/day via drinking water. TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-κB p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies.

  8. Assessment of protein synthesis in highly aerobic canine species at the onset and during exercise training.

    Science.gov (United States)

    Miller, Benjamin F; Ehrlicher, Sarah E; Drake, Joshua C; Peelor, Frederick F; Biela, Laurie M; Pratt-Phillips, Shannon; Davis, Michael; Hamilton, Karyn L

    2015-04-01

    Canis lupus familiaris, the domesticated dog, is capable of extreme endurance performance. The ability to perform sustained aerobic exercise is dependent on a well-developed mitochondrial reticulum. In this study we examined the cumulative muscle protein and DNA synthesis in groups of athletic dogs at the onset of an exercise training program and following a strenuous exercise training program. We hypothesized that both at the onset and during an exercise training program there would be greater mitochondrial protein synthesis rates compared with sedentary control with no difference in mixed or cytoplasmic protein synthesis rates. Protein synthetic rates of three protein fractions and DNA synthesis were determined over 1 wk using (2)H2O in competitive Alaskan Huskies and Labrador Retrievers trained for explosive device detection. Both groups of dogs had very high rates of skeletal muscle protein synthesis in the sedentary state [Alaskan Huskies: Mixed = 2.28 ± 0.12, cytoplasmic (Cyto) = 2.91 ± 0.10, and mitochondrial (Mito) = 2.62 ± 0.07; Labrador Retrievers: Mixed = 3.88 ± 0.37, Cyto = 3.85 ± 0.06, and Mito = 2.92 ± 0.20%/day]. Mitochondrial (Mito) protein synthesis rates did not increase at the onset of an exercise training program. Exercise-trained dogs maintained Mito protein synthesis during exercise training when mixed (Mixed) and cytosolic (Cyto) fractions decreased, and this coincided with a decrease in p-RpS6 but also a decrease in p-ACC signaling. Contrary to our hypothesis, canines did not have large increases in mitochondrial protein synthesis at the onset or during an exercise training program. However, dogs have a high rate of protein synthesis compared with humans that perhaps does not necessitate an extra increase in protein synthesis at the onset of aerobic exercise training. Copyright © 2015 the American Physiological Society.

  9. Bacillus subtilis During Feast and Famine: Visualization of the Overall Regulation of Protein Synthesis During Glucose Starvation by Proteome Analysis

    Science.gov (United States)

    Bernhardt, Jörg; Weibezahn, Jimena; Scharf, Christian; Hecker, Michael

    2003-01-01

    Dual channel imaging and warping of two-dimensional (2D) protein gels were used to visualize global changes of the gene expression patterns in growing Bacillus subtilis cells during entry into the stationary phase as triggered by glucose exhaustion. The 2D gels only depict single moments during the cells' growth cycle, but a sequential series of overlays obtained at specific points of the growth curve facilitates visualization of the developmental processes at the proteomics scale. During glucose starvation a substantial reprogramming of the protein synthesis pattern was found, with 150 proteins synthesized de novo and cessation of the synthesis of almost 400 proteins. Proteins induced following glucose starvation belong to two main regulation groups: general stress/starvation responses induced by different stresses or starvation stimuli (ςB-dependent general stress regulon, stringent response, sporulation), and glucose-starvation-specific responses (drop in glycolysis, utilization of alternative carbon sources, gluconeogenesis). Using the dual channel approach, it was not only possible to identify those regulons or stimulons, but also to follow the fate of each single protein by the three-color code: red, newly induced but not yet accumulated; yellow, synthesized and accumulated; and green, still present, but no longer being synthesized. These green proteins, which represent a substantial part of the protein pool in the nongrowing cell, are not accessible by using DNA arrays. The combination of 2D gel electrophoresis and MALDI TOF mass spectrometry with the dual channel imaging technique provides a new and comprehensive view of the physiology of growing or starving bacterial cell populations, here for the case of the glucose-starvation response. [This is presented as a movie of B. subtilis's growth/glucose-starvation response, available at www.genome.org and also at http://microbio1.biologie.uni-greifswald.de/starv/movie.htm.] PMID:12566400

  10. Human serum protein adsorption onto synthesis nano-hydroxyapatite.

    Science.gov (United States)

    Mohsen-Nia, M; Massah Bidgoli, M; Behrashi, M; Mohsen Nia, A

    2012-02-01

    Adsorption of human serum proteins (Albumin and total protein) onto high purity synthesis nano-hydroxyapatite (HA), Ca₁₀(PO₄)₆(OH)₂, has been studied in a wide temperature range by UV-visible spectrophotometer. Adsorption isotherm is basically important to describe how solutes interact with adsorbent, and is critical in optimizing the use of adsorbent. In the present study, the experimental results were fitted to the Langmuir, Freundlich, Temkin and Dubinin-Radushkevich (DR) models to obtain the characteristic parameters of each model and square of the correlation coefficients (R²). According to the results, the DR isotherm model had the best agreement with the experimental data. The effect of temperature on adsorption of human serum proteins (HSP) onto the synthesized nano-HA was studied. The experimental results indicated that temperature increase generally causes an increase in the adsorption of HSP onto the nano-HA. This is basically due to the effect of temperature on the HSP activity and its diffusion rate on HA surfaces.

  11. Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Escherichia coli.

    Science.gov (United States)

    Nyström, T; Neidhardt, F C

    1992-11-01

    The response of non-differentiating bacteria to nutrient starvation is complex and includes the sequential synthesis of starvation-inducible proteins. Although starvation for different individual nutrients generally provokes unique and individual patterns of protein expression, some starvation stimulons share member proteins. Two-dimensional polyacrylamide gel electrophoresis revealed that the synthesis of a small (13.5 kDa) cytoplasmic protein in Escherichia coli was greatly increased during growth inhibition caused by the exhaustion of any of a variety of nutrients (carbon, nitrogen, phosphate, sulphate, required amino acid) or by the presence of a variety of toxic agents including heavy metals, oxidants, acids and antibiotics. To determine further the mode of regulation of the protein designated UspA (universal stress protein A) we cloned the gene encoding the protein by the technique of reverse genetics. We isolated the protein from a preparative two-dimensional polyacrylamide gel, determined its N-terminal amino acid sequence, and used this sequence to construct a degenerate oligonucleotide probe. Two phages of the Kohara library were found to contain the gene which then was subcloned from the DNA in the overlapping region of these two clones. The amino acid sequence, deduced from the nucleotide sequence of the uspA gene, shows no significant homology with any other known protein. The uspA gene maps at 77 min on the E. coli W3110 chromosome, and is transcribed in a clockwise direction. The increase in the level of UspA during growth arrest was found to be primarily a result of transcriptional activation of the corresponding gene. The induction was independent of the RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB and H-NS proteins during stress conditions that are known to induce or activate these global regulators. The -10 and -35 regions upstream of the transcriptional start site of the uspA gene are characteristic of a sigma 70-dependent promoter.

  12. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

    Directory of Open Access Journals (Sweden)

    Juan C Marini

    Full Text Available Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20 on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L, and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

  13. Oxidative stress impairs the heat stress response and delays unfolded protein recovery.

    Directory of Open Access Journals (Sweden)

    Masaaki Adachi

    2009-11-01

    Full Text Available Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability.Pretreatment of hydrogen peroxide (H(2O(2 specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. H(2O(2 exposure impaired the HSP40/HSP70 induction as heat shock response (HSR and the unfolded protein recovery, and enhanced eIF2alpha phosphorylation and/or XBP1 splicing, land marks of ER stress. These H(2O(2-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked H(2O(2-mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1 -/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for H(2O(2-mediated enhanced heat sensitivity.H(2O(2 blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stress as a crucial factor affecting heat tolerance.

  14. RNA-processing protein TDP-43 regulates FOXO-dependent protein quality control in stress response.

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2014-10-01

    Full Text Available Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43 is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.

  15. Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

    Directory of Open Access Journals (Sweden)

    Bozhena Jhas

    Full Text Available Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

  16. Protein-poly(amino acid) precipitation stabilizes a therapeutic protein l-asparaginase against physicochemical stress.

    Science.gov (United States)

    Maruyama, Takuya; Izaki, Shunsuke; Kurinomaru, Takaaki; Handa, Kenji; Kimoto, Tomoaki; Shiraki, Kentaro

    2015-12-01

    Long-term storage in aqueous solution has been demanded for the practical application of therapeutic proteins. Recently, a precipitation-redissolution method was proposed to prepare salt-dissociable protein-polyelectrolyte complex (PPC). To elucidate the utility of the complex for storage of proteins, we investigated the stress tolerance of PPC precipitates containing l-asparaginase (ASNase) and poly-l-lysine (polyK). PPC precipitate containing ASNase and polyK was prepared by precipitation-redissolution method. The sample was treated to three types of stress, i.e., heat, shaking, and oxidation. The protein concentration, enzyme activity, and CD spectrum of the supernatants of samples were measured after stressed. PPC precipitate consisting of ASNase and polyK showed tolerance against thermal and shaking stress compared to the native solution. In addition, PPC precipitate protected ASNase from inactivation by oxidation. PPC precipitate of ASNase/polyK complex successfully stabilized ASNase against physicochemical stresses. These results suggest that the PPC precipitate has great potential as a storage method in aqueous solution for unstable proteins. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. Overexpression of agouti protein and stress responsiveness in mice.

    Science.gov (United States)

    Harris, R B; Zhou, J; Shi, M; Redmann, S; Mynatt, R L; Ryan, D H

    2001-07-01

    Ectopic overexpression of agouti protein, an endogenous antagonist of melanocortin receptors' linked to the beta-actin promoter (BAPa) in mice, produces a phenotype of yellow coat color, Type II diabetes, obesity and increased somatic growth. Spontaneous overexpression of agouti increases stress-induced weight loss. In these experiments, other aspects of stress responsiveness were tested in 12-week-old male wild-type mice and BAPa mice. Two hours of restraint on three consecutive days produced greater increases in corticosterone and post-stress weight loss in BAPa than wild-type mice. In Experiment 2, anxiety-type behavior was measured immediately after 12 min of restraint. This mild stress did not produce many changes indicative of anxiety, but BAPa mice spent more time in the dark side of a light-dark box and less time in the open arms of an elevated plus maze than restrained wild-type mice. In a defensive withdrawal test, grooming was increased by restraint in all mice, but the duration of each event was substantially shorter in BAPa mice, possibly due to direct antagonism of the MC4-R by agouti protein. Thus, BAPa mice showed exaggerated endocrine and energetic responses to restraint stress with small differences in anxiety-type behavior compared with wild-type mice. These results are consistent with observations in other transgenic mice in which the melanocortin system is disrupted, but contrast with reports that acute blockade of central melanocortin receptors inhibits stress-induced hypophagia. Thus, the increased stress responsiveness in BAPa mice may be a developmental compensation for chronic inhibition of melanocortin receptors.

  18. Abscisic acid refines the synthesis of chloroplast proteins in maize (Zea mays in response to drought and light.

    Directory of Open Access Journals (Sweden)

    Xiuli Hu

    Full Text Available To better understand abscisic acid (ABA regulation of the synthesis of chloroplast proteins in maize (Zea mays L. in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry (MS. After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C(4 plants.

  19. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors...... involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls....

  20. Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA binding protein.

    Science.gov (United States)

    Dai, Haixia; Choe, Woo-Seok; Thai, Corrine K; Sarikaya, Mehmet; Traxler, Beth A; Baneyx, François; Schwartz, Daniel T

    2005-11-09

    We show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable (nonequilibrium) conditions, reproducing a key feature of biological hard-tissue growth and assembly. The nonequilibrium synthesis of Cu(2)O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein TraI in a room-temperature precursor electrolyte. The functional TraI derivative (TraIi1753::CN225) is engineered to possess a cysteine-constrained 12-residue Cu(2)O binding sequence, designated CN225, that is inserted into a permissive site in TraI. When TraIi1753::CN225 is included in the precursor electrolyte, stable Cu(2)O nanoparticles form, even though the concentrations of [Cu(+)] and [OH(-)] are at 5% of the solubility product (K(sp,Cu2O)). Negative control experiments verify that Cu(2)O formation is controlled by inclusion of the CN225 binding sequence. Transmission electron microscopy and electron diffraction reveal a core-shell structure for the nonequilibrium nanoparticles: a 2 nm Cu(2)O core is surrounded by an adsorbed protein shell. Quantitative protein adsorption studies show that the unexpected stability of Cu(2)O is imparted by the nanomolar surface binding affinity of TraIi1753::CN225 for Cu(2)O (K(d) = 1.2 x 10(-)(8) M), which provides favorable interfacial energetics (-45 kJ/mol) for the core-shell configuration. The protein shell retains the DNA-binding traits of TraI, as evidenced by the spontaneous organization of nanoparticles onto circular double-stranded DNA.

  1. Petit-High Pressure Carbon Dioxide stress increases synthesis of S-Adenosylmethionine and phosphatidylcholine in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Niu, Liyuan; Nomura, Kazuki; Iwahashi, Hitoshi; Matsuoka, Hiroyuki; Kawachi, Satoshi; Suzuki, Yoshihisa; Tamura, Katsuhiro

    2017-12-01

    Petit-High Pressure Carbon Dioxide (p-HPCD) is a promising nonthermal technology for foods pasteurization. Cluster analysis of gene expression profiles of Saccharomyces cerevisiae exposed to various stresses exhibited that gene expression profile for p-HPCD stress (0.5MPa, 25°C) was grouped into a cluster including profiles for Sodium Dodecyl Sulfate and Roundup herbicide. Both are detergents that can disorder membrane structurally and functionally, which suggests that cell membrane may be a target of p-HPCD stress to cause cell growth inhibition. Through metabolomic analysis, amount of S-Adenosylmethionine (AdoMet) that is used as methyl donor to participate in phosphatidylcholine synthesis via phosphatidylethanolamine (PE) methylation pathway, was increased after p-HPCD treatment for 2h. The key gene OPI3 encoding phospholipid methyltransferase that catalyzes the last two steps in PE methylation pathway was confirmed significantly induced by RT-PCR. Transcriptional expression of genes (MET13, MET16, MET10, MET17, MET6 and SAM2) related to AdoMet biosynthesis was also significantly induced. Choline as the PC precursor and ethanolamine as PE precursor in Kennedy pathway were also found increased under p-HPCD condition. We also found that amounts of most of amino acids involving protein synthesis were found decreased after p-HPCD treatment for 2h. Moreover, morphological changes on cell surface were observed by scanning electron microscope. In conclusion, the effects of p-HPCD stress on cell membrane appear to be a very likely cause of yeast growth inhibition and the enhancement of PC synthesis could contribute to maintain optimum structure and functions of cell membrane and improve cell resistance to inactivation. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Protein Thiols as an Indication of Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Yousef Rezaei Chianeh

    2014-06-01

    Full Text Available Thiol is an organic compound that contain sulphhydryl group that have a critical role in preventing any involvement of oxidative stress in the cell. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment.The process of protection of the cell against an oxidative damage occur by thiol and cystein residue that has a low molecular weight. These residue are present in the active sites of a protein like, peroxiredoxin and thioredoxin. Apart from intracellular antioxidant defense mechanism by protein thiol, there are presence of thiol in outer surface of protein that are not involved with the function of protein, even though they can interact with intracellular part of the cell. [Archives Medical Review Journal 2014; 23(3.000: 443-456

  3. Protein synthesis in a solitary benthic cephalopod, the Southern dumpling squid (Euprymna tasmanica).

    Science.gov (United States)

    Carter, Chris G; Lynch, Kerri A; Moltschaniwskyj, Natalie A

    2009-06-01

    Rates of protein synthesis were measured in the whole body and tissues of southern dumpling squid Euprymna tasmanica to validate the use of a flooding-dose of (3)H phenylalanine for the measurement of protein synthesis with different size squid and to make a preliminary investigation into the effects of feeding regime. In smaller (2.8+/-0.5 g, mean+/-SE) and larger (14.8+/-2.2 g) squid whole body fractional rates of protein synthesis were 9.45+/-1.21 and 1.49+/-0.29% d(-1), respectively. Differences in total whole body protein content meant there was no difference in absolute rates of whole body protein synthesis between the larger and smaller squid. In larger squid, fractional rates of protein synthesis were significantly higher in the digestive gland (9.24+/-1.63% d(-1)) than in the arm tissue (1.43+/-0.31% d(-1)), which were significantly higher than in the anterior (0.56+/-0.13% d(-1)) and posterior (0.36+/-0.04% d(-1)) mantle. In smaller squid there were no differences in protein synthesis between tissues and high individual variation, due to differences in feeding, was a likely cause. Consequently, the effect of feeding regime on protein synthesis was compared between two groups of individually held squid: daily-feeding and minimal-feeding squid. The daily-feeding squid had significantly higher feed intake, gained mass and had a significantly higher growth rate than the minimal-feeding squid which lost mass. Whole body protein synthesis was significantly higher in the daily-feeding squid as was the protein content of the digestive gland, anterior and posterior mantle. There were few other differences in indices of protein metabolism. Individual squid showed differences in growth and protein metabolism, and there were significant relationships between growth rate and both rates of protein synthesis and protein degradation. Thus, higher individual growth was a consequence of increased protein synthesis, decreased protein degradation and, therefore, increased

  4. A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

    Science.gov (United States)

    Banerjee, Arka; Adolph, Ramona S.; Gopalakrishnapai, Jayashree; Kleinboelting, Silke; Emmerich, Christiane; Steegborn, Clemens; Visweswariah, Sandhya S.

    2015-01-01

    Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as “sinks” for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell. PMID:25802331

  5. Masseter muscle myofibrillar protein synthesis and degradation in an experimental critical illness myopathy model.

    Science.gov (United States)

    Akkad, Hazem; Corpeno, Rebeca; Larsson, Lars

    2014-01-01

    Critical illness myopathy (CIM) is a debilitating common consequence of modern intensive care, characterized by severe muscle wasting, weakness and a decreased myosin/actin (M/A) ratio. Limb/trunk muscles are primarily affected by this myopathy while cranial nerve innervated muscles are spared or less affected, but the mechanisms underlying these muscle-specific differences remain unknown. In this time-resolved study, the cranial nerve innervated masseter muscle was studied in a unique experimental rat intensive care unit (ICU) model, where animals were exposed to sedation, neuromuscular blockade (NMB), mechanical ventilation, and immobilization for durations varying between 6 h and 14d. Gel electrophoresis, immunoblotting, RT-PCR and morphological staining techniques were used to analyze M/A ratios, myofiber size, synthesis and degradation of myofibrillar proteins, and levels of heat shock proteins (HSPs). Results obtained in the masseter muscle were compared with previous observations in experimental and clinical studies of limb muscles. Significant muscle-specific differences were observed, i.e., in the masseter, the decline in M/A ratio and muscle fiber size was small and delayed. Furthermore, transcriptional regulation of myosin and actin synthesis was maintained, and Akt phosphorylation was only briefly reduced. In studied degradation pathways, only mRNA, but not protein levels of MuRF1, atrogin-1 and the autophagy marker LC3b were activated by the ICU condition. The matrix metalloproteinase MMP-2 was inhibited and protective HSPs were up-regulated early. These results confirm that the cranial nerve innervated masticatory muscles is less affected by the ICU-stress response than limb muscles, in accordance with clinical observation in ICU patients with CIM, supporting the model' credibility as a valid CIM model.

  6. A novel pulse-chase SILAC strategy measures changes in protein decay and synthesis rates induced by perturbation of proteostasis with an Hsp90 inhibitor.

    Directory of Open Access Journals (Sweden)

    Ivo Fierro-Monti

    Full Text Available Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc variant of SILAC (stable isotope labeling by amino acids in cell culture. pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

  7. Tropical legume supplementation influences microbial protein synthesis and rumen ecology.

    Science.gov (United States)

    Phesatcha, K; Wanapat, M

    2017-06-01

    Four rumen-fistulated male swamp buffaloes, 5-year-old with initiated live weight at 360 ± 12 kg, were randomly assigned according to a 4 × 4 Latin square design to investigate the effect of feeding high level of dried Leucaena leaf (DLL) on feed intake, fermentation efficiency and microbial protein synthesis. The dietary treatments were the feeding levels of DLL at 0, 2, 4 and 6 kg/head/day. All buffaloes were supplemented with concentrate mixtures at 0.1% of body weight, and rice straw was fed ad libitum with the availability of water and mineral block at all time. The results revealed that the total feed intake and nutrient digestibility were significantly improved with the increasing levels of DLL feeding, and the highest was in the buffaloes consuming DLL at 6 kg/head/day. Feeding high levels of DLL did not affect on ruminal pH and temperature, while ammonia nitrogen, blood urea nitrogen and volatile fatty acid concentration were significantly enhanced. Moreover, methane production was dramatically reduced by increasing levels of DLL feeding. Total direct counts of the micro-organism population were increased with the increasing levels of DLL feeding. According to the application of quantitative PCR to quantity cellulolytic bacteria (16S rRNA) targets, it was found that the population of total bacteria and Fibrobactor succinogenes was affected by treatments, while Ruminococcus flavefaciens and methanogen population were significantly decreased as buffaloes were fed with DLL. The nitrogen balance and microbial nitrogen supply were remarkably improved with the increasing levels of DLL feeding. Based on this study, it could be concluded that high levels of DLL feeding at 6 kg/head/day could enhance feed intake, nutrient digestibility, rumen fermentation efficiency and microbial protein synthesis in swamp buffaloes fed on rice straw without any adverse effect. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

  8. Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

    Science.gov (United States)

    O'Brien, Jeffrey; Shea, Kenneth J

    2016-06-21

    Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is

  9. Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

    DEFF Research Database (Denmark)

    Mundy, John; Hejgaard, Jørn; Hansen, Annette

    1986-01-01

    RNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2......, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment......To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using m...

  10. DNA packaging in mouse spermatids: synthesis of protamine variants and four-transition proteins

    Energy Technology Data Exchange (ETDEWEB)

    Balhorn, R.; Weston, S.; Thomas, C.; Wyrobek, A.J.

    1984-01-01

    A comparison of the protein compositions of mouse late-step spermatids and cauda epididymal sperm has revealed that the relative distribution of the two amino acid sequence variants of mouse protamine differ markedly in spermatids and sperm. Sonication-resistant spermatids contain the two variants in a ratio of 1:1, while the ratio of these two proteins in cauda epididymal sperm is approx. 2:1. Labeling studies in vivo have shown that this difference is due, in part, to an asynchrony in the time of synthesis of the two protamine variants. Both proteins are synthesized in late-step spermatids, but synthesis of the tyrosine variant in sperm chromatin begins approximately one day before synthesis of the more predominant histidine variant. Analyses of the time of synthesis of protamine and the four transition proteins in late-step spermatids allowed us to estimate the spermatid stage in which these proteins are deposited on DNA and relate these events to the onset of sonication resistance in maturing spermatids. These results indicate that: (1) synthesis and deposition of protamine begins coincident with the onset of sonication resistance in early step 12 spermatids; (2) protamine deposition is complete by mid-step 15; and (3) synthesis of the transition proteins occurs coincident with protamine synthesis.

  11. No effect of menstrual cycle on myofibrillar and connective tissue protein synthesis in contracting skeletal muscle

    DEFF Research Database (Denmark)

    Miller, Benjamin F; Hansen, Mette; Olesen, Jens L

    2006-01-01

    in the follicular (FP, n=8) or the luteal phase (LP, n=7, n=1 out of phase) 24 h after an acute bout of one-legged exercise (60 min of kicking at 67% W(max)), samples being taken from the vastus lateralis in both the exercised and resting legs. Rates of synthesis of myofibrillar and muscle collagen proteins were...... measured by incorporation of [(13)C]leucine. Myofibrillar protein synthesis (means+/-SD; rest FP: 0.053+/-0.009%/h, LP: 0.055+/-0.013%/h) was increased at 24-h postexercise (FP: 0.131+/-0.018%/h, Pmuscle collagen synthesis...... of exercise, on myofibrillar protein synthesis and muscle collagen synthesis in women....

  12. Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1.

    Science.gov (United States)

    Chen, Ming-Ju; Tang, Hsin-Yu; Chiang, Ming-Lun

    2017-09-01

    Lactobacillus kefiranofaciens M1 is a probiotic strain isolated from Taiwanese kefir grains. The present study evaluated the effects of heat, cold, acid and bile salt adaptations on the stress tolerance of L. kefiranofaciens M1. The regulation of protein expression of L. kefiranofaciens M1 under these adaptation conditions was also investigated. The results showed that adaptation of L. kefiranofaciens M1 to heat, cold, acid and bile salts induced homologous tolerance and cross-protection against heterologous challenge. The extent of induced tolerance varied depending on the type and condition of stress. Proteomic analysis revealed that 27 proteins exhibited differences in expression between non-adapted and stress-adapted L. kefiranofaciens M1 cells. Among these proteins, three proteins involved in carbohydrate metabolism (triosephosphate isomerase, enolase and NAD-dependent glycerol-3-phosphate dehydrogenase), two proteins involved in pH homeostasis (ATP synthase subunits AtpA and AtpB), two stress response proteins (chaperones DnaK and GroEL) and one translation-related protein (30S ribosomal protein S2) were up-regulated by three of the four adaptation treatments examined. The increased synthesis of these stress proteins might play a critical protective role in the cellular defense against heat, cold, acid and bile salt stresses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Design and modular parallel synthesis of a MCR derived α-helix mimetic protein-protein interaction inhibitor scaffold

    NARCIS (Netherlands)

    Antuch, Walfrido; Menon, Sanjay; Chen, Quin-Zene; Lu, Yingchun; Sakamuri, Sukumar; Beck, Barbara; Schauer-Vukašinović, Vesna; Agarwal, Seema; Hess, Sibylle; Dömling, Alexander

    2006-01-01

    A terphenyl α-helix mimetic scaffold recognized to be capable of disrupting protein-protein interactions was structurally morphed into an easily amenable and versatile multicomponent reaction (MCR) backbone. The design, modular in-parallel library synthesis, initial cell based biological data, and

  14. Enhanced green fluorescent protein-mediated synthesis of biocompatible graphene.

    Science.gov (United States)

    Gurunathan, Sangiliyandi; Woong Han, Jae; Kim, Eunsu; Kwon, Deug-Nam; Park, Jin-Ki; Kim, Jin-Hoi

    2014-10-03

    Graphene is the 2D form of carbon that exists as a single layer of atoms arranged in a honeycomb lattice and has attracted great interest in the last decade in view of its physical, chemical, electrical, elastic, thermal, and biocompatible properties. The objective of this study was to synthesize an environmentally friendly and simple methodology for the preparation of graphene using a recombinant enhanced green fluorescent protein (EGFP). The successful reduction of GO to graphene was confirmed using UV-vis spectroscopy, and FT-IR. DLS and SEM were employed to demonstrate the particle size and surface morphology of GO and EGFP-rGO. The results from Raman spectroscopy suggest the removal of oxygen-containing functional groups from the surface of GO and formation of graphene with defects. The biocompatibility analysis of GO and EGFP-rGO in human embryonic kidney (HEK) 293 cells suggests that GO induces significant concentration-dependent cell toxicity in HEK cells, whereas graphene exerts no adverse effects on HEK cells even at a higher concentration (100 μg/mL). Altogether, our findings suggest that recombinant EGFP can be used as a reducing and stabilizing agent for the preparation of biocompatible graphene. The novelty and originality of this work is that it describes a safe, simple, and environmentally friendly method for the production of graphene using recombinant enhanced green fluorescent protein. Furthermore, the synthesized graphene shows excellent biocompatibility with HEK cells; therefore, biologically synthesized graphene can be used for biomedical applications. To the best of our knowledge, this is the first and novel report describing the synthesis of graphene using recombinant EGFP.

  15. Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis.

    Science.gov (United States)

    Eustice, D C; Wilhelm, J M

    1984-01-01

    Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C] lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B. PMID:6433789

  16. Proline-rich antimicrobial peptides targeting protein synthesis.

    Science.gov (United States)

    Graf, Michael; Mardirossian, Mario; Nguyen, Fabian; Seefeldt, A Carolin; Guichard, Gilles; Scocchi, Marco; Innis, C Axel; Wilson, Daniel N

    2017-07-01

    Covering: up to 2017The innate immune system employs a broad array of antimicrobial peptides (AMPs) to attack invading microorganisms. While most AMPs act by permeabilizing the bacterial membrane, specific subclasses of AMPs have been identified that pass through membranes and inhibit bacterial growth by targeting fundamental intracellular processes. One such subclass is the proline-rich antimicrobial peptides (PrAMPs) that bind to the ribosome and interfere with the process of protein synthesis. A diverse range of PrAMPs have been identified in insects, such as bees, wasps and beetles, and crustaceans, such as crabs, as well as in mammals, such as cows, sheep, goats and pigs. Mechanistically, the best-characterized PrAMPs are the insect oncocins, such as Onc112, and bovine bactenecins, such as Bac7. Biochemical and structural studies have revealed that these PrAMPs bind within the ribosomal exit tunnel with a reverse orientation compared to a nascent polypeptide chain. The PrAMPs allow initiation but prevent the transition into the elongation phase of translation. Insight into the interactions of PrAMPs with their ribosomal target provides the opportunity to further develop these peptides as novel antimicrobial agents.

  17. PGD2 stimulates osteoprotegerin synthesis via AMP-activated protein kinase in osteoblasts: Regulation of ERK and SAPK/JNK.

    Science.gov (United States)

    Kainuma, Shingo; Tokuda, Haruhiko; Kuroyanagi, Gen; Yamamoto, Naohiro; Ohguchi, Reou; Fujita, Kazuhiko; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

    2015-10-01

    AMP-activated protein kinase (AMPK), a key enzyme sensing cellular energy metabolism, is currently known to regulate multiple metabolic pathways. Osteoprotegerin plays a pivotal role in the regulation of bone metabolism by inhibiting osteoclast activation. We have previously reported that prostaglandin D2 (PGD2) stimulates the synthesis of osteoprotegerin through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. On the basis of these findings, we herein investigated the implication of AMPK in PGD2-stimulated osteoprotegerin synthesis in these cells. PGD2 induced the phosphorylation of AMPKα (Thr-172) and AMPKβ (Ser-108), and the phosphorylation of acetyl-coenzyme A carboxylase, a direct AMPK substrate. Compound C, an AMPK inhibitor, which suppressed the phosphorylation of acetyl-coenzyme A carboxylase, significantly attenuated both the release and the mRNA levels of osteoprotegerin stimulated by PGD2. The PGD2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK but not p38 MAP kinase were markedly inhibited by compound C. These results strongly suggest that AMPK regulates the PGD2-stimulated osteoprotegerin synthesis at a point upstream of p44/p42 MAP kinase and SAPK/JNK in osteoblasts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Proteasome inhibition induces DNA damage and reorganizes nuclear architecture and protein synthesis machinery in sensory ganglion neurons.

    Science.gov (United States)

    Palanca, Ana; Casafont, Iñigo; Berciano, María T; Lafarga, Miguel

    2014-05-01

    Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.

  19. PCR-based gene synthesis to produce recombinant proteins for crystallization

    Directory of Open Access Journals (Sweden)

    Byrne-Steele Miranda L

    2008-04-01

    Full Text Available Abstract Background Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves. Results A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA. The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis. Conclusion We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.

  20. Targeting Oncogenic Protein-Protein Interactions by Diversity Oriented Synthesis and Combinatorial Chemistry Approaches

    Directory of Open Access Journals (Sweden)

    Andreas G. Tzakos

    2011-05-01

    Full Text Available We are currently witnessing a decline in the development of efficient new anticancer drugs, despite the salient efforts made on all fronts of cancer drug discovery. This trend presumably relates to the substantial heterogeneity and the inherent biological complexity of cancer, which hinder drug development success. Protein-protein interactions (PPIs are key players in numerous cellular processes and aberrant interruption of this complex network provides a basis for various disease states, including cancer. Thus, it is now believed that cancer drug discovery, in addition to the design of single-targeted bioactive compounds, should also incorporate diversity-oriented synthesis (DOS and other combinatorial strategies in order to exploit the ability of multi-functional scaffolds to modulate multiple protein-protein interactions (biological hubs. Throughout the review, we highlight the chemistry driven approaches to access diversity space for the discovery of small molecules that disrupt oncogenic PPIs, namely the p53-Mdm2, Bcl-2/Bcl-xL-BH3, Myc-Max, and p53-Mdmx/Mdm2 interactions.

  1. Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals

    National Research Council Canada - National Science Library

    Mitsunori Miyazaki; Karyn A. Esser

    2009-01-01

    .... In this review, we discuss the animal and cell culture models used and the signaling mechanisms identified in understanding regulation of protein synthesis in response to mechanical loading/resistance exercise...

  2. Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome

    National Research Council Canada - National Science Library

    Osterweil, Emily K; Chuang, Shih-Chieh; Chubykin, Alexander A; Sidorov, Michael; Bianchi, Riccardo; Wong, Robert K S; Bear, Mark F

    2013-01-01

    .... We discovered that lovastatin, a drug that is widely prescribed for the treatment of high cholesterol, can correct excess hippocampal protein synthesis in the mouse model of FXS and can prevent one...

  3. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis...

  4. Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate

    Science.gov (United States)

    Corazzari, Marco; Gagliardi, Mara; Fimia, Gian Maria; Piacentini, Mauro

    2017-01-01

    Perturbation of endoplasmic reticulum (ER) homeostasis results in a stress condition termed “ER stress” determining the activation of a finely regulated program defined as unfolded protein response (UPR) and whose primary aim is to restore this organelle’s physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s) regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch. PMID:28491820

  5. Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate

    Directory of Open Access Journals (Sweden)

    Marco Corazzari

    2017-04-01

    Full Text Available Perturbation of endoplasmic reticulum (ER homeostasis results in a stress condition termed “ER stress” determining the activation of a finely regulated program defined as unfolded protein response (UPR and whose primary aim is to restore this organelle’s physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch.

  6. Oxidative Stress, Unfolded Protein Response, and Apoptosis in Developmental Toxicity

    Science.gov (United States)

    Kupsco, Allison; Schlenk, Daniel

    2016-01-01

    Physiological development requires precise spatiotemporal regulation of cellular and molecular processes. Disruption of these key events can generate developmental toxicity in the form of teratogenesis or mortality. The mechanism behind many developmental toxicants remains unknown. While recent work has focused on the unfolded protein response (UPR), oxidative stress, and apoptosis in the pathogenesis of disease, few studies have addressed their relationship in developmental toxicity. Redox regulation, UPR, and apoptosis are essential for physiological development and can be disturbed by a variety of endogenous and exogenous toxicants to generate lethality and diverse malformations. This review examines the current knowledge of the role of oxidative stress, UPR, and apoptosis in physiological development as well as in developmental toxicity, focusing on studies and advances in vertebrates model systems. PMID:26008783

  7. A Chrysanthemum Heat Shock Protein Confers Tolerance to Abiotic Stress

    Science.gov (United States)

    Song, Aiping; Zhu, Xirong; Chen, Fadi; Gao, Haishun; Jiang, Jiafu; Chen, Sumei

    2014-01-01

    Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD) activity, higher proline content and inhibited malondialdehyde (MDA) content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect. PMID:24663057

  8. A Chrysanthemum Heat Shock Protein Confers Tolerance to Abiotic Stress

    Directory of Open Access Journals (Sweden)

    Aiping Song

    2014-03-01

    Full Text Available Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD activity, higher proline content and inhibited malondialdehyde (MDA content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect.

  9. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

  10. [Influence of different adhesive composition on sporulation and protein synthesis by Bacillus thuringiensis collection strains].

    Science.gov (United States)

    Krut', V V; Dankevych, L A; Votselko, S K; Patyka, V P

    2014-01-01

    The influence of different sticky-gene composition on sporulation and protein synthesis by B. thuringiensis collection strains has been investigated. It has been detemined that the most effective according this characteristics were B. thuringiensis collection strains 0293 and 98. It has been shown that the best on protein synthesis processes and sporulation by investigated B. thuringiensis strains influences adding to the culture medium sticky-gene compositions A and E in a concentration of from 10 to 15%.

  11. Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling

    OpenAIRE

    Drummond, Micah J.; Dreyer, Hans C.; Fry, Christopher S.; Glynn, Erin L.; Rasmussen, Blake B

    2009-01-01

    In this review we discuss current findings in the human skeletal muscle literature describing the acute influence of nutrients (leucine-enriched essential amino acids in particular) and resistance exercise on muscle protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) signaling. We show that essential amino acids and an acute bout of resistance exercise independently stimulate human skeletal muscle protein synthesis. It also appears that ingestion of essential amino acids fo...

  12. Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis

    DEFF Research Database (Denmark)

    Doessing, Simon; Heinemeier, Katja; Holm, Lars

    2010-01-01

    matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue.......In skeletal muscle and tendon the extracellular matrix confers important tensile properties and is crucially important for tissue regeneration after injury. Musculoskeletal tissue adaptation is influenced by mechanical loading, which modulates the availability of growth factors, including growth...... young individuals. rhGH administration caused an increase in serum GH, serum IGF-I, and IGF-I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9-fold and 1.3-fold, respectively (P muscle collagen I m...

  13. Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis.

    Science.gov (United States)

    Kunst, F; Rapoport, G

    1995-05-01

    Growth under conditions of salt stress has important effects on the synthesis of degradative enzymes in Bacillus subtilis. Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold). It is suggested that the DegS-DegU two-component system is involved in sensing salt stress. Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.

  14. New insights into ferritin synthesis and function highlight a link between iron homeostasis and oxidative stress in plants.

    Science.gov (United States)

    Briat, Jean-Francois; Ravet, Karl; Arnaud, Nicolas; Duc, Céline; Boucherez, Jossia; Touraine, Brigitte; Cellier, Francoise; Gaymard, Frederic

    2010-05-01

    Iron is an essential element for both plant productivity and nutritional quality. Improving plant iron content was attempted through genetic engineering of plants overexpressing ferritins. However, both the roles of these proteins in plant physiology, and the mechanisms involved in the regulation of their expression are largely unknown. Although the structure of ferritins is highly conserved between plants and animals, their cellular localization differs. Furthermore, regulation of ferritin gene expression in response to iron excess occurs at the transcriptional level in plants, in contrast to animals which regulate ferritin expression at the translational level. In this review, an overview of our knowledge of bacterial and mammalian ferritin synthesis and functions is presented. Then the following will be reviewed: (a) the specific features of plant ferritins; (b) the regulation of their synthesis during development and in response to various environmental cues; and (c) their function in plant physiology, with special emphasis on the role that both bacterial and plant ferritins play during plant-bacteria interactions. Arabidopsis ferritins are encoded by a small nuclear gene family of four members which are differentially expressed. Recent results obtained by using this model plant enabled progress to be made in our understanding of the regulation of the synthesis and the in planta function of these various ferritins. Studies on plant ferritin functions and regulation of their synthesis revealed strong links between these proteins and protection against oxidative stress. In contrast, their putative iron-storage function to furnish iron during various development processes is unlikely to be essential. Ferritins, by buffering iron, exert a fine tuning of the quantity of metal required for metabolic purposes, and help plants to cope with adverse situations, the deleterious effects of which would be amplified if no system had evolved to take care of free reactive iron.

  15. Prenatal stress alters Fos protein expression in hippocampus and locus coeruleus stress-related brain structures.

    Science.gov (United States)

    Viltart, Odile; Mairesse, Jérôme; Darnaudéry, Muriel; Louvart, Hélène; Vanbesien-Mailliot, Christel; Catalani, Assia; Maccari, Stefania

    2006-07-01

    Prenatal stress (PS) durably influences responses of rats from birth throughout life by inducing deficits of the hypothalamo-pituitary-adrenal (HPA) axis feedback. The neuronal mechanisms sustaining such alterations are still unknown. The purpose of the present study was to determine whether in PS and control rats, the exposure to a mild stressor differentially induces Fos protein in hippocampus and locus coeruleus, brain areas involved in the feedback control of the HPA axis. Moreover, Fos protein expression was also evaluated in the hypothalamic paraventricular nucleus (PVN) that reflect the magnitude of the hormonal response to stress. Basal plasma corticosterone levels were not different between the groups, while, PS rats exhibited higher number of Fos-immunoreactive neurons than controls, in the hippocampus and locus coeruleus in basal condition. A higher basal expression of a marker of GABAergic synapses, the vGAT, was also observed in the hypothalamus of PS rats. Fifteen minutes after the end of the exposure to the open arm of the elevated plus-maze (mild stress) a similar increased plasma corticosterone levels was observed in both groups in parallel with an increased number of Fos-immunoreactive neurons in the PVN. Return to basal plasma corticosterone values was delayed only in the PS rats. On the contrary, after stress, no changes in Fos-immunoreactivity were observed in the hippocampus and locus coeruleus of PS rats compared to basal condition. After stress, only PS rats presented an elevation of the number of activated catecholaminergic neurons in the locus coeruleus. In conclusion, these results suggest for the first time that PS alters the neuronal activation of hippocampus and locus coeruleus implicated in the feedback mechanism of the HPA axis. These data give anatomical substrates to sustain the HPA axis hyperactivity classically described in PS rats after stress exposure.

  16. Pregnancy-associated reduction in vascular protein kinase C activity rebounds during inhibition of NO synthesis.

    Science.gov (United States)

    Kanashiro, C A; Cockrell, K L; Alexander, B T; Granger, J P; Khalil, R A

    2000-02-01

    Vascular reactivity has been shown to be reduced during pregnancy and to be enhanced during chronic inhibition of nitric oxide (NO) synthesis in pregnant rats; however, the cellular mechanisms involved are unclear. The purpose of this study was to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of vascular protein kinase C (PKC). Active stress as well as the amount and activity of PKC was measured in deendothelialized thoracic aortic strips from virgin and pregnant rats untreated or treated with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In virgin rats, the PKC activator phorbol 12,13-dibutyrate (PDBu, 10(-6) M) and the alpha-adrenergic agonist phenylephrine (Phe, 10(-5) M) caused significant increases in active stress and PKC activity that were inhibited by the PKC inhibitors staurosporine and calphostin C. Western blot analysis in aortic strips of virgin rats showed significant amount of the alpha-PKC isoform. Both PDBu and Phe caused significant translocation of alpha-PKC from the cytosolic to the particulate fraction. Compared with virgin rats, the PDBu- and Phe-stimulated active stress and PKC activity as well as the amount and the PDBu- and Phe-induced translocation of alpha-PKC were significantly reduced in late pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. The PDBu- and Phe-induced changes in active stress and the amount, distribution, and activity of alpha-PKC in virgin rats treated with L-NAME were not significantly different from that in virgin rats, whereas the changes in pregnant rats treated with L-NAME + the NO synthase substrate L-arginine were not significantly different from that in pregnant rats. These results provide evidence that a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced during chronic inhibition of NO synthesis. The results

  17. Regulation of protein synthesis by amino acids in muscle of neonates

    Science.gov (United States)

    Suryawan, Agus; Davis, Teresa A.

    2011-01-01

    The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed. PMID:21196241

  18. Acute phase proteins in cattle after exposure to complex stress

    DEFF Research Database (Denmark)

    Lomborg, S. R.; Nielsen, L. R.; Heegaard, Peter M. H.

    2008-01-01

    Abstract Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels...... concentrations of SAA and haptoglobin increased significantly in response to the stressors (P...... in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin...

  19. Physiological responses of salt stress and osmoprotection with ...

    African Journals Online (AJOL)

    Yomi

    2011-12-21

    Dec 21, 2011 ... Keywords: Salt stress, bacterial growth, osmoregulation, proline, stress protein synthesis. INTRODUCTION. Bacterial cells had ... under osmotic stress increase the proline pool of endo- genous origin by regulation of ... synthesis of proteins involved in the protection and repair of macromolecules. As with ...

  20. The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing.

    Science.gov (United States)

    Snijder, E J; Decroly, E; Ziebuhr, J

    2016-01-01

    Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26-32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7-nsp10) act as crucial cofactors of these enzymes and contribute to the emerging "nsp interactome." Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies. © 2016 Elsevier Inc. All rights reserved.

  1. A statistical view of protein chemical synthesis using NCL and extended methodologies.

    Science.gov (United States)

    Agouridas, Vangelis; El Mahdi, Ouafâa; Cargoët, Marine; Melnyk, Oleg

    2017-09-15

    Native chemical ligation and extended methodologies are the most popular chemoselective reactions for protein chemical synthesis. Their combination with desulfurization techniques can give access to small or challenging proteins that are exploited in a large variety of research areas. In this report, we have conducted a statistical review of their use for protein chemical synthesis in order to provide a flavor of the recent trends and identify the most popular chemical tools used by protein chemists. To this end, a protein chemical synthesis (PCS) database (http://pcs-db.fr) was created by collecting a set of relevant data from more than 450 publications covering the period 1994-2017. A preliminary account of what this database tells us is presented in this report. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Flexible programming of cell-free protein synthesis using magnetic bead-immobilized plasmids.

    Directory of Open Access Journals (Sweden)

    Ka-Young Lee

    Full Text Available The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis.

  3. Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis.

    Science.gov (United States)

    Tudor, Jennifer C; Davis, Emily J; Peixoto, Lucia; Wimmer, Mathieu E; van Tilborg, Erik; Park, Alan J; Poplawski, Shane G; Chung, Caroline W; Havekes, Robbert; Huang, Jiayan; Gatti, Evelina; Pierre, Philippe; Abel, Ted

    2016-04-26

    Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. Copyright © 2016, American Association for the Advancement of Science.

  4. Studies on protein synthesis by protoplasts of saccharomyces carlsbergensis III. Studies on the specificity and the mechanism of the action of ribonuclease on protein synthesis

    NARCIS (Netherlands)

    Kloet, S.R. de; Dam, G.J.W. van; Koningsberger, V.V.

    1962-01-01

    In this paper, the experimental results are presented of a continued study on the specificity and the mechanism of the inhibition by ribonuclease of protein synthesis in protoplasts of Saccharomyces carlsbergensis. By comparing the effects of native pancreatic ribonuclease with those of

  5. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Jacobsen, Susanne; Hammer, Karin

    1997-01-01

    pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min, GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min, The class of slowly induced......, by comparison of prototrophic wild-type strains and auxotrophic domesticated (daily) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L,. lactis subsp. cremoris...... laboratory strain MG1363, which was originally derived from a dairy strain, After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shack repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase...

  6. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    Science.gov (United States)

    2016-07-01

    AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...1 July 2015- 30 June 2016 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a... chronic kidney disease , diabetic nephropathy, calreticulin, TGF-beta, ER stress, ultrasound, tubulointerstitial fibrosis 5 3. ACCOMPLISHMENTS a. WHAT

  7. Differential synthesis of glyceraldehyde-3-phosphate dehydrogenase polypeptides in stressed yeast cells.

    Science.gov (United States)

    Boucherié, H; Bataille, N; Fitch, I T; Perrot, M; Tuite, M F

    1995-01-15

    Three unlinked genes, TDH1, TDH2 and TDH3, encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (triose-phosphate dehydrogenase; TDH) in the yeast Saccharomyces cerevisiae. We demonstrate that the synthesis of the three encoded TDH polypeptides (TDHa, TDHb and TDHc, respectively) is not co-ordinately regulated and that TDHa is only synthesised as cells enter stationary phase, due to glucose starvation, or in heat-shocked cells. Furthermore, the synthesis of TDHb, but not TDHc, is strongly repressed by a heat shock. Hence, the TDHa enzyme may play a cellular role, distinct from glycolysis, that is required by stressed cells.

  8. Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Yan-Long Jia

    2016-01-01

    Full Text Available Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE was used to investigate the expression of halotolerant proteins under high (3 M NaCl and low (0.75 M NaCl salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

  9. Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis

    Directory of Open Access Journals (Sweden)

    J. K. Seo

    2013-03-01

    Full Text Available A series of in vitro studies were carried out to determine i the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS in in vitro experiments. Untreated corn (C and enzyme-treated corn (EC were combined with soy bean meal with (ES and without (S enzyme treatment or formaldehyde treatment (FS. Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS. The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity.

  10. [Exogenous NO mediated GSH-PCs synthesis pathway in tomato under copper stress].

    Science.gov (United States)

    Wang, Jian; Yu, Shi-Xin; Zhang, Min; Cui, Xiu-Min

    2014-09-01

    Nitric oxide (NO), as a biologically active molecule, widely involved in the biotic and abiotic stresses. By using solution culture, this paper reported the dynamic changes in enzyme activity and metabolites related to GSH-PCs synthesis way mediated by exogenous NO in tomato (Lycopersicon esculentum). The results showed that exogenous NO could affect the metabolic pathway of GSH-PCs in tomato seedlings under copper stress. Compared with CK, the activity of γ-ECS and GS was significantly activated, consequently resulting in a sharp rise in GSH and PCs contents in tomato root. Moreover, γ-ECS and GS activity, GSH and PCs contents constantly rise with the extension of processing time under copper stress. Adding exogenous SNP could further improve γ-ECS and GS activity in tomato, and promote the production of GSH and PCs, which contributed to enhancing the ability of removing superoxide and chelating excess Cu2+ to reduce its biological toxicity. To a certain extent, GSH-PCs metabolic changes in leaf lagged behind that in roots. Exogenous BSO could significantly inhibit γ-ECS activity, and applying SNP could significantly reverse the inhibition on GSH and PCs synthesis by BSO. BSO had little effects on PCs content in leaf. Under copper stress, exogenous NO may initiate a signal mechanism and reduce the biotoxicity and oxidative damage caused by excessive Cu2+ by activating or enhancing the enzymatic and non-enzymatic systems in the GSH-PCs synthesis path.

  11. Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis.

    Science.gov (United States)

    Quast, Robert B; Mrusek, Devid; Hoffmeister, Christian; Sonnabend, Andrei; Kubick, Stefan

    2015-07-08

    Over the last years protein engineering using non-standard amino acids has gained increasing attention. As a result, improved methods are now available, enabling the efficient and directed cotranslational incorporation of various non-standard amino acids to equip proteins with desired characteristics. In this context, the utilization of cell-free protein synthesis is particularly useful due to the direct accessibility of the translational machinery and synthesized proteins without having to maintain a vital cellular host. We review prominent methods for the incorporation of non-standard amino acids into proteins using cell-free protein synthesis. Furthermore, a list of non-standard amino acids that have been successfully incorporated into proteins in cell-free systems together with selected applications is provided. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Sepsis and development impede muscle protein synthesis in neonatal pigs by different ribosomal mechanisms

    Science.gov (United States)

    In muscle, sepsis reduces protein synthesis (MPS) by restraining translation in neonates and adults. Even though protein accretion decreases with development as neonatal MPS rapidly declines by maturation, the changes imposed by development on the sepsis-associated decrease in MPS have not been desc...

  13. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

  14. A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

  15. Developmental changes in insulin- and amino acid-induced mTOR signalling regulate muscle protein synthesis in neonatal pigs

    Science.gov (United States)

    The enhanced efficiency, with which dietary protein is used for growth in the neonate, is due to the ability of neonatal muscle to markedly increase protein synthesis in response to feeding (Davis "et al.", 1996). The stimulation of protein synthesis by feeding in neonatal muscle is independently m...

  16. Expression of the α-tocopherol transfer protein gene is regulated by oxidative stress and common single-nucleotide polymorphisms.

    Science.gov (United States)

    Ulatowski, Lynn; Dreussi, Cara; Noy, Noa; Barnholtz-Sloan, Jill; Klein, Eric; Manor, Danny

    2012-12-15

    Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in most animal species. By controlling the secretion of vitamin E from the liver, the α-tocopherol transfer protein regulates whole-body distribution and levels of this vital nutrient. However, the mechanism(s) that regulates the expression of this protein is poorly understood. Here we report that transcription of the TTPA gene in immortalized human hepatocytes is induced by oxidative stress and by hypoxia, by agonists of the nuclear receptors PPARα and RXR, and by increased cAMP levels. The data show further that induction of TTPA transcription by oxidative stress is mediated by an already-present transcription factor and does not require de novo protein synthesis. Silencing of the cAMP response element-binding (CREB) transcription factor attenuated transcriptional responses of the TTPA gene to added peroxide, suggesting that CREB mediates responses of this gene to oxidative stress. Using a 1.9-kb proximal segment of the human TTPA promoter together with a site-directed mutagenesis approach, we found that single-nucleotide polymorphisms that are commonly found in healthy humans dramatically affect promoter activity. These observations suggest that oxidative stress and individual genetic makeup contribute to vitamin E homeostasis in humans. These findings may explain the variable responses to vitamin E supplementation observed in human clinical trials. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Is there any relationship between decreased AgNOR protein synthesis and human hair loss?

    Science.gov (United States)

    Eroz, R; Tasdemir, S; Dogan, H

    2012-11-01

    Argyrophilic nucleolar organizing region associated proteins (AgNORs) play roles in cell proliferation and a variety of diseases. We attempted to determine whether decreased NOR protein synthesis causes human hair loss. We studied 21 healthy males who suffered hair loss on the frontal/vertex portion of the head. Hair root cells from normal and hair loss sites were stained for AgNOR. One hundred nuclei per site were evaluated and the AgNOR number and NORa/TNa proportions of individual cells were determined using a computer program. The cells from normal sites had significantly higher AgNOR counts than those from hair loss sites. Also, the cells from the normal sites had significantly higher NORa/TNa than cells from the hair loss sites. In the normal sites, the cells demonstrated more NOR protein synthesis than cells in hair loss sites. Therefore, decreased NOR protein synthesis appears to be related to hair loss in humans.

  18. Formation of taste aversion and preference in protein synthesis inhibition in rats.

    Science.gov (United States)

    Serova, O N; Solov'ea, N A; Lagutina, L V; Obukhova, M F

    1996-01-01

    The investigation was devoted to the role of the synthesis of protein and peptide factors during the formation of chemosensory memory in rats. Two models of gustatory memorization were used: conditioned taste aversion (CTA), induced by the association of the taste of saccharine with a toxic injection of lithium chloride, and enhanced taste preference (ETP), induced by the influence of preliminary drinking of a saccharine solution on its repeat consumption. It was found that, under conditions of the inhibition of protein synthesis in the brain of 43% by cycloheximide and of 59% by 8-azaguanine, CTA does not form. ETP does not form under the influence of cycloheximide, but not [sic] of 8-azaguanine. A hypothesis was advanced regarding the participation of a varied spectrum of protein and peptide substances in the formation of taste aversion and preference. An influence of protein synthesis blockers on the process of retrieval of gustatory memory was not found.

  19. ER Stress and Unfolded Protein Response in Amyotrophic Lateral Sclerosis – A Controversial Role of Protein Disulphide Isomerase

    Directory of Open Access Journals (Sweden)

    Merja eJaronen

    2014-12-01

    Full Text Available Accumulation of proteins in aberrant conformation occurs in many neurodegenerative diseases. Furthermore, dysfunctions in protein handling in endoplasmic reticulum (ER and the following ER stress have been implicated in a vast number of diseases, such as amyotrophic lateral sclerosis (ALS. During excessive ER stress unfolded protein response (UPR is activated to return ER to its normal physiological balance. The exact mechanisms of protein misfolding, accumulation and the following ER stress could lead to neurodegeneration and the question whether UPR is a beneficial compensatory mechanism slowing down the neurodegenerative processes are of interest. Protein disulphide isomerase (PDI is a disulfide bond-modulating ER chaperone, which can also facilitate the ER-associated degradation (ERAD of misfolded proteins. In this review we discuss the recent findings of ER stress, UPR and especially the role of PDI in ALS.

  20. Immediate and residual effects of heat stress and restricted intake on milk protein and casein composition and energy metabolism.

    Science.gov (United States)

    Cowley, F C; Barber, D G; Houlihan, A V; Poppi, D P

    2015-04-01

    The effects of heat stress on dairy production can be separated into 2 distinct causes: those effects that are mediated by the reduced voluntary feed intake associated with heat stress, and the direct physiological and metabolic effects of heat stress. To distinguish between these, and identify their effect on milk protein and casein concentration, mid-lactation Holstein-Friesian cows (n = 24) were housed in temperature-controlled chambers and either subjected to heat stress [HS; temperature-humidity index (THI) ~78] or kept in a THIIntake accounted for only part of the effects of heat stress. Heat stress reduced the milk protein concentration, casein number, and casein concentration and increased the urea concentration in milk beyond the effects of restriction of intake. Under HS, the proportion in total casein of αS1-casein increased and the proportion of αS2-casein decreased. Because no effect of HS on milk fat or lactose concentration was found, these effects appeared to be the result of specific downregulation of mammary protein synthesis, and not a general reduction in mammary activity. No residual effects were found of HS or TN-R on milk production or composition after THIintake were restored. Heat-stressed cows had elevated blood concentrations of urea and Ca, compared with TN-R and TN-AL. Cows in TN-R had higher serum nonesterified fatty acid concentrations than cows in HS. It was proposed that HS and TN-R cows may mobilize different tissues as endogenous sources of energy. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise.

    Science.gov (United States)

    West, Daniel W D; Burd, Nicholas A; Coffey, Vernon G; Baker, Steven K; Burke, Louise M; Hawley, John A; Moore, Daniel R; Stellingwerff, Trent; Phillips, Stuart M

    2011-09-01

    Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis. This trial was registered at clinicaltrials.gov as NCT01319513.

  2. Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis.

    Science.gov (United States)

    Inda, Mari Carmen; Delgado-García, José María; Carrión, Angel Manuel

    2005-02-23

    Memory, as measured by changes in an animal's behavior some time after learning, is a reflection of many processes. Here, using a trace paradigm, in mice we show that de novo protein synthesis is required for acquisition, consolidation, reconsolidation, and extinction of classically conditioned eyelid responses. Two critical periods of protein synthesis have been found: the first, during training, the blocking of which impaired acquisition; and the second, lasting the first 4 h after training, the blocking of which impaired consolidation. The process of reconsolidation was sensitive to protein synthesis inhibition if anisomycin was injected before or just after the reactivation session. Furthermore, extinction was also dependent on protein synthesis, following the same temporal course as that followed during acquisition and consolidation. This last fact reinforces the idea that extinction is an active learning process rather than a passive event of forgetting. Together, these findings demonstrate that all of the different stages of memory formation involved in the classical conditioning of eyelid responses are dependent on protein synthesis.

  3. Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

    Science.gov (United States)

    Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

    2017-08-01

    Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

  4. Intra-axonal protein synthesis - a new target for neural repair?

    Directory of Open Access Journals (Sweden)

    Jeffery L Twiss

    2016-01-01

    Full Text Available Although initially argued to be a feature of immature neurons with incomplete polarization, there is clear evidence that neurons in the peripheral nervous system retain the capacity for intra-axonal protein synthesis well into adulthood. This localized protein synthesis has been shown to contribute to injury signaling and axon regeneration in peripheral nerves. Recent works point to potential for protein synthesis in axons of the vertebrate central nervous system. mRNAs and protein synthesis machinery have now been documented in lamprey, mouse, and rat spinal cord axons. Intra-axonal protein synthesis appears to be activated in adult vertebrate spinal cord axons when they are regeneration-competent. Rat spinal cord axons regenerating into a peripheral nerve graft contain mRNAs and markers of activated translational machinery. Indeed, levels of some growth-associated mRNAs in these spinal cord axons are comparable to the regenerating sciatic nerve. Markers of active translation tend to decrease when these axons stop growing, but can be reactivated by a second axotomy. These emerging observations raise the possibility that mRNA transport into and translation within axons could be targeted to facilitate regeneration in both the peripheral and central nervous systems.

  5. Deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation.

    Science.gov (United States)

    Sekhar, Rajagopal V; Patel, Sanjeet G; Guthikonda, Anuradha P; Reid, Marvin; Balasubramanyam, Ashok; Taffet, George E; Jahoor, Farook

    2011-09-01

    Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. Eight elderly and 8 younger subjects received stable-isotope infusions of [2H(2)]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F(2)-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 μmol/L; P oxidative stress (304 ± 16 compared with 346 ± 20 Carratelli units; P oxidative stress and F(2)-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and concentrations and lowers levels of oxidative stress and oxidant damages. These findings suggest a practical and effective approach to decreasing oxidative stress in aging.

  6. Endoplasmic reticulum stress inhibits collagen synthesis independent of collagen-modifying enzymes in different chondrocyte populations and dermal fibroblasts

    NARCIS (Netherlands)

    Vonk, Lucienne A.; Doulabi, Behrouz Zandieh; Huang, Chun-Ling; Helder, Marco N.; Everts, Vincent; Bank, Ruud A.

    Chondrocytes respond to glucose deprivation with a decreased collagen synthesis due to disruption of a proper functioning of the endoplasmic reticulum (ER): ER stress. Since the mechanisms involved in the decreased synthesis are unknown, we have investigated whether chaperones and collagen-modifying

  7. Endoplasmic reticulum stress inhibits collagen synthesis independent of collagen-modifying enzymes in different chondrocyte populations and dermal fibroblasts

    NARCIS (Netherlands)

    Vonk, L.A.; Doulabi, B.Z.; Huang, C.L.; Helder, M.N.; Everts, V.; Bank, R.A.

    2010-01-01

    Chondrocytes respond to glucose deprivation with a decreased collagen synthesis due to disruption of a proper functioning of the endoplasmic reticulum (ER): ER stress. Since the mechanisms involved in the decreased synthesis are unknown, we have investigated whether chaperones and collagen-modifying

  8. Inhibiting corticosterone synthesis during fear memory formation exacerbates cued fear extinction memory deficits within the single prolonged stress model.

    Science.gov (United States)

    Keller, Samantha M; Schreiber, William B; Stanfield, Briana R; Knox, Dayan

    2015-01-01

    Using the single prolonged stress (SPS) animal model of post-traumatic stress disorder (PTSD), previous studies suggest that enhanced glucocorticoid receptor (GR) expression leads to cued fear extinction retention deficits. However, it is unknown how the endogenous ligand of GRs, corticosterone (CORT), may contribute to extinction retention deficits in the SPS model. Given that CORT synthesis during fear learning is critical for fear memory consolidation and SPS enhances GR expression, CORT synthesis during fear memory formation could strengthen fear memory in SPS rats by enhancing GR activation during fear learning. In turn, this could lead to cued fear extinction retention deficits. We tested the hypothesis that CORT synthesis during fear learning leads to cued fear extinction retention deficits in SPS rats by administering the CORT synthesis inhibitor metyrapone to SPS and control rats prior to fear conditioning, and observed the effect this had on extinction memory. Inhibiting CORT synthesis during fear memory formation in control rats tended to decrease cued freezing, though this effect never reached statistical significance. Contrary to our hypothesis, inhibiting CORT synthesis during fear memory formation disrupted extinction retention in SPS rats. This finding suggests that even though SPS exposure leads to cued fear extinction memory deficits, CORT synthesis during fear memory formation enhances extinction retention in SPS rats. This suggests that stress-induced CORT synthesis in previously stressed rats can be beneficial. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Effect of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis.

    Science.gov (United States)

    Larsen, M; Røntved, C M; Theil, P K; Khatun, M; Lauridsen, C; Kristensen, N B

    2017-05-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin β (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P < 0.01) and mRNA expression

  10. Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting.

    Science.gov (United States)

    Lang, Kathrin

    2018-01-18

    The power of CPS, live! More than 90 attendees from around the world came together in Haifa to present and hear about cutting-edge science in protein chemistry, from advances in synthetic methods to applications in biology and medicine. The meeting was a powerful demonstration that chemical protein synthesis can provide otherwise unattainable insights into protein structure and function. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli, most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis-acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times.IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli, synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In this

  12. Quantity of dietary protein intake, but not pattern of intake, affects net protein balance primarily through differences in protein synthesis in older adults.

    Science.gov (United States)

    Kim, Il-Young; Schutzler, Scott; Schrader, Amy; Spencer, Horace; Kortebein, Patrick; Deutz, Nicolaas E P; Wolfe, Robert R; Ferrando, Arny A

    2015-01-01

    To examine whole body protein turnover and muscle protein fractional synthesis rate (MPS) following ingestions of protein in mixed meals at two doses of protein and two intake patterns, 20 healthy older adult subjects (52-75 yr) participated in one of four groups in a randomized clinical trial: a level of protein intake of 0.8 g (1RDA) or 1.5 g·kg(-1)·day(-1) (∼2RDA) with uneven (U: 15/20/65%) or even distribution (E: 33/33/33%) patterns of intake for breakfast, lunch, and dinner over the day (1RDA-U, 1RDA-E, 2RDA-U, or 2RDA-E). Subjects were studied with primed continuous infusions of L-[(2)H5]phenylalanine and L-[(2)H2]tyrosine on day 4 following 3 days of diet habituation. Whole body protein kinetics [protein synthesis (PS), breakdown, and net balance (NB)] were expressed as changes from the fasted to the fed states. Positive NB was achieved at both protein levels, but NB was greater in 2RDA vs. 1RDA (94.8 ± 6.0 vs. 58.9 ± 4.9 g protein/750 min; P = 0.0001), without effects of distribution on NB. The greater NB was due to the higher PS with 2RDA vs. 1RDA (15.4 ± 4.8 vs. -18.0 ± 8.4 g protein/750 min; P = 0.0018). Consistent with PS, MPS was greater with 2RDA vs. 1RDA, regardless of distribution patterns. In conclusion, whole body net protein balance was greater with protein intake above recommended dietary allowance (0.8 g protein·kg(-1)·day(-1)) in the context of mixed meals, without demonstrated effects of protein intake pattern, primarily through higher rates of protein synthesis at whole body and muscle levels. Copyright © 2015 the American Physiological Society.

  13. Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways.

    Science.gov (United States)

    Quinlan, Margaret P

    2004-08-01

    Transformation progression of epithelial cells involves alterations in their morphology, polarity, and adhesive characteristics, all of which are associated with the loss and/or reorganization of actin structures. To identify the underlying mechanism of formation of the adhesion-dependent, circumferential actin network, the expression and localization of the actin binding and regulating proteins (ABPs), vinculin, VASP, and profilin were evaluated. Experimental depolarization of epithelial cells results in the loss of normal F-actin structures and the transient upregulation of vinculin, VASP, and profilin. This response is due to the loss of cell-cell, and not cell-substrate interactions, since cells that no longer express focal adhesions or stress fibers are still sensitive to changes in adhesion and manifest this in the altered profile of expression of these ABPs. Transient upregulation is dependent upon de novo protein synthesis, and protein kinase-, but not phosphatase-sensitive signal transduction pathway(s). Inhibition of the synthesis of these proteins is accompanied by dephosphorylation of the ribosomal S6 protein, but does not involve inhibition of the PI3-kinase-Akt-mTOR pathway. Constitutive expression of VASP results in altered cell morphology and adhesion and F-actin and vinculin structures. V12rac1 expressing epithelial cells are constitutively nonadhesive, malignantly transformed, and constitutively express high levels of these ABPs, with altered subcellular localizations. Transformation suppression is accompanied by the restoration of normal levels of the three ABPs, actin structures, adhesion, and epithelial morphology. Thus, vinculin, VASP, and profilin are coordinately regulated by signal transduction pathways that effect a translational response. Additionally, their expression profile maybe indicative of the adhesion and transformation status of epithelial cells. Copyright 2004 Wiley-Liss, Inc.

  14. Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis.

    Science.gov (United States)

    Hoffmann, Frank; Weber, Jan; Rinas, Ursula

    2002-11-05

    The metabolic burden and the stress load resulting from temperature-induced production of human basic fibroblast growth factor is connected to an increase in the respiratory activity of recombinant Escherichia coli, thereby reducing the biomass yield. To study the underlying changes in metabolic enzyme synthesis rates, the radiolabeled proteom was subjected to two-dimen- sional gel electrophoresis. After temperature-induction, the cAMP-CRP controlled dehydrogenases of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle (LpdA and SdhA) were induced four times, reaching a maximum 1 h after the temperature upshift. The more abundant tricarboxylic acid cycle dehydrogenases (Icd and Mdh) were initially produced at reduced rates but regained preshift rates within 30 min. The adenylate energy charge dropped immediately after the temperature upshift but recovered within 1 h. Similar profiles in dehydrogenase synthesis rates and adenylate energy charge were found in a control cultivation of a strain carrying the "empty" parental expression vector. Although both strains exhibited significant differences in growth pattern and respiration rates after the temperature upshift, the adaptation of the energetic state of the cells and the synthesis of enzymes from the energy-generating catabolic pathway did not seem to be affected by the strong overproduction of the recombinant growth factor. In contrast, the synthesis rates of enzymes belonging to the biosynthetic machinery, e.g., translational elongation factors, decreased more strongly in the culture synthesizing the recombinant protein. In control and producing culture, synthesis rates of elongation factors paralleled the respective growth rate profiles. Thus, cells seem to readjust their metabolic activities according to their energetic requirements and, if necessary, at the cost of their biosynthetic capabilities. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 313-319, 2002.

  15. Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system.

    Science.gov (United States)

    Wang, Dong; Vo, Nam V; Sowa, Gwendolyn A; Hartman, Robert A; Ngo, Kevin; Choe, So Ra; Witt, William T; Dong, Qing; Lee, Joon Y; Niedernhofer, Laura J; Kang, James D

    2011-02-01

    Bupivacaine is a local anesthetic commonly used for back pain management in interventional procedures. Cytotoxic effects of bupivacaine have been reported in articular cartilage and, recently, in intervertebral disc cell culture. However, the relevance of these effects to discs in vivo remains unclear. This study examines the effect of bupivacaine on disc cell metabolism using an organotypic culture model system that mimics the in vivo environment. To assess the effect of bupivacaine on disc cell viability and matrix protein synthesis using an organotypic model system and to determine whether this anesthetic has toxic effects. Mouse intervertebral discs were isolated and maintained ex vivo in an organotypic culture then exposed to clinically relevant concentrations of bupivacaine, and the impact on disc cell viability and matrix proteoglycan (PG) and collagen syntheses were measured in the presence and absence of the drug. Mouse functional spine units (FSUs) were isolated from the lumbar spines of 10-week-old mice. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Total PG and collagen syntheses were determined by measuring the incorporation of radioactive (35)S-sulfate and (3)H-l-proline into PG and collagen, respectively. Organotypic cultures of mouse FSUs were exposed to different concentrations (0%-0.5%) of bupivacaine for variable amounts of time (0-2 hours). Cell viability within disc tissue was quantified by MTT staining and histologic assay. Matrix protein synthesis was measured by incorporation of radioactive (35)S-sulfate (for PG synthesis) and (3)H-l-proline (for collagen synthesis). Untreated mouse disc organs were maintained in culture for up to 1 month with minimal changes in tissue histology, cell viability, and matrix protein synthesis. Exposure to bupivacaine decreased cell viability in a dose- and time-dependent manner. Exposure to bupivacaine at concentrations less than or equal to 0.25% did

  16. The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance

    DEFF Research Database (Denmark)

    Hyun, Tae Kyung; van der Graaff, Eric; Albacete, Alfonso

    2014-01-01

    Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain...... and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination...... of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions...

  17. Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

    National Research Council Canada - National Science Library

    Mason, Alexander F; Thordarson, Pall

    2016-01-01

    The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs...

  18. Induction of Glutathione Synthesis and Glutathione Reductase Activity by Abiotic Stresses in Maize and Wheat

    Directory of Open Access Journals (Sweden)

    Gábor Kocsy

    2002-01-01

    Full Text Available The effect of different abiotic stresses (extreme temperatures and osmotic stress on the synthesis of glutathione and hydroxymethylglutathione, on the ratio of the reduced to oxidised forms of these thiols (GSH/GSSG, hmGSH/hmGSSG, and on the glutathione reductase (GR activity was studied in maize and wheat genotypes having different sensitivity to low temperature stress. Cold treatment induced a greater increase in total glutathione (TG content and in GR activity in tolerant genotypes of both species than in sensitive ones. The GSH/GSSG and hmGSH/hmGSSG ratios were increased by this treatment only in the frost-tolerant wheat variety. High-temperature stress increased the TG content and the GSH/GSSG ratio only in the chilling-sensitive maize genotype, but GR activity was greater after this treatment in both maize genotypes. Osmotic stress resulted in a great increase in the TG content in wheat and the GR activity in maize. The amount of total hydroxymethylglutathione increased following all stress treatments. These results indicate the involvement of these antioxidants in the stress responses of wheat and maize.

  19. Role for protein synthesis in the neurotoxic effects of methamphetamine in mice and rats.

    Science.gov (United States)

    Finnegan, K T; Karler, R

    1992-09-18

    The mechanism by which the amphetamines damage selectively nigrostriatal dopaminergic neurons in experimental animals remains uncertain. The observation that neuronal cell death during embryogenesis involves an activation of gene expression and new protein synthesis, coupled with recent reports indicating that the amphetamines are capable of inducing neuropeptide biosynthesis, offers a possible clue as to their neurotoxic mechanism of action. Based on these considerations, we evaluated the effects of two different inhibitors of protein synthesis, cycloheximide and anisomycin, on the long-term, amine-depleting effects of methamphetamine (METH) in mice and rats. Both inhibitors were found to block the amine-depleting effects of METH in these species. In other experiments, cycloheximide did not affect the functional integrity of dopaminergic or glutamatergic neurons, transmitter systems previously implicated in the neurotoxic mechanism of action of METH. These findings raise the possibility that the neuronal-damaging effects of METH are mediated via a synthesis of 'neurotoxic' proteins.

  20. TORC1-regulated protein kinase Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis.

    Science.gov (United States)

    Shimobayashi, Mitsugu; Oppliger, Wolfgang; Moes, Suzette; Jenö, Paul; Hall, Michael N

    2013-03-01

    The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.

  1. Long-term olfactory memories are stabilised via protein synthesis in Camponotus fellah ants

    DEFF Research Database (Denmark)

    Guerrieri, Fernando Javier; D'Ettorre, Patrizia; Deveaud, J-M.

    2011-01-01

    to conditioning. Cycloheximide did not impair acquisition of either short-term memory (10¿min) or early and late mid-term memories (1 or 12¿h). These results show that, upon olfactory learning, ants form different memories with variable molecular bases. While short- and mid-term memories do not require protein......-chain hydrocarbons, one paired with sucrose and the other with quinine solution. Differential conditioning leads to the formation of a long-term memory retrievable at least 72¿h after training. Long-term memory consolidation was impaired by the ingestion of cycloheximide, a protein synthesis blocker, prior...... synthesis, long-term memories are stabilised via protein synthesis. Our behavioural protocol opens interesting research avenues to explore the cellular and molecular bases of olfactory learning and memory in ants....

  2. The effects of volatile microbial secondary metabolites on protein synthesis in Serpula lacrymans.

    Science.gov (United States)

    Humphris, Sonia N; Bruce, Alan; Buultjens, Eldridge; Wheatley, Ron E

    2002-05-07

    The effects of volatile secondary metabolites produced by Trichoderma pseudokoningii, Trichoderma viride and Trichoderma aureoviride on growth rate and protein synthesis in two Serpula lacrymans isolates were investigated. Mycelial growth was affected to differing degrees, depending on the specific interactive microbial couplet involved. Protein synthesis by both S. lacrymans (Forfar) and S. lacrymans (H28) was affected by the volatile secondary metabolites of T. aureoviride and T. viride, but not by those of T. pseudokoningii. Mycelial growth and the original pattern of protein synthesis resumed when the antagonists were removed. It is probable that volatile secondary metabolites have played an important role during the evolution of microorganisms in the context of community, population and functional dynamics.

  3. Synthesis of the major storage protein, hordein, in barley

    DEFF Research Database (Denmark)

    Giese, Nanna Henriette; Andersen, B.; Doll, Hans

    1983-01-01

    A liquid culture system for culturing detached spikes of barley (Hordeum vulgare L.) at different nutritional levels was established. The synthesis of hordein polypeptides was studied by pulse-labeling with [14C]sucrose at different stages of development and nitrogen (N) nutrition. All polypeptid...

  4. Proteomic Analysis of Copper-Binding Proteins in Excess Copper-Stressed Roots of Two Rice (Oryza sativa L. Varieties with Different Cu Tolerances.

    Directory of Open Access Journals (Sweden)

    Chen Chen

    Full Text Available To better understand the mechanisms involved in the heavy metal stress response and tolerance in plants, a proteomic approach was used to investigate the differences in Cu-binding protein expression in Cu-tolerant and Cu-sensitive rice varieties. Cu-binding proteins from Cu-treated rice roots were separated using a new IMAC method in which an IDA-sepharose column was applied prior to the Cu-IMAC column to remove metal ions from protein samples. More than 300 protein spots were reproducibly detected in the 2D gel. Thirty-five protein spots exhibited changes greater than 1.5-fold in intensity compared to the control. Twenty-four proteins contained one or more of nine putative metal-binding motifs reported by Smith et al., and 19 proteins (spots contained one to three of the top six motifs reported by Kung et al. The intensities of seven protein spots were increased in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195 (p<0.05 and six protein spots were markedly up-regulated in B1139, but not detectable in B1195. Four protein spots were significantly up-regulated in B1139, but unchanged in B1195 under Cu stress. In contrast, two protein spots were significantly down-regulated in B1195, but unchanged in B1139. These Cu-responsive proteins included those involved in antioxidant defense and detoxification (spots 5, 16, 21, 22, 28, 29 and 33, pathogenesis (spots 5, 16, 21, 22, 28, 29 and 33, regulation of gene transcription (spots 8 and 34, amino acid synthesis (spots 8 and 34, protein synthesis, modification, transport and degradation (spots 1, 2, 4, 10, 15, 19, 30, 31, 32 and 35, cell wall synthesis (spot 14, molecular signaling (spot 3, and salt stress (spots 7, 9 and 27; together with other proteins, such as a putative glyoxylate induced protein, proteins containing dimeric alpha-beta barrel domains, and adenosine kinase-like proteins. Our results suggest that these proteins, together with related physiological processes, play

  5. Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

    Directory of Open Access Journals (Sweden)

    Ghazala eMUSTAFA

    2014-11-01

    Full Text Available Flooding stress has a negative impact on soybean cultivation because it severely impairs growth and development. To understand the flooding responsive mechanism in early stage soybeans, a glycoproteomic technique was used. Two-day-old soybeans were treated with flooding for 2 days and roots were collected. Globally, the accumulation level of glycoproteins, as revealed by cross-reaction with concanavalin A decreased by 2 days of flooding stress. Glycoproteins were enriched from total protein extracts using concanavalin A lectin resin and analyzed using a gel-free proteomic technique. One-hundred eleven and 69 glycoproteins were identified without and with 2 days of flooding stress, respectively. Functional categorization of these identified glycoproteins indicated that the accumulation level of proteins related to protein degradation, cell wall, and glycolysis increased, while stress-related proteins decreased under flooding stress. Also the accumulation level of glycoproteins localized in the secretory pathway (e.g., peroxidases and plycosyl hydrolases decreased under flooding stress. Out of 23 common glycoproteins between control and flooding conditions, peroxidases and glycosyl hydrolases were decreased by 2 days of flooding stress. mRNA expression levels of proteins in the endoplasmic reticulum and N-glycosylation related proteins were downregulated by flooding stress. These results suggest that flooding might negatively affect the process of N-glycosylation of proteins related to stress and protein degradation; however glycoproteins involved in glycolysis are activated.

  6. Effects of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis

    DEFF Research Database (Denmark)

    Larsen, Mogens; Røntved, Christine Maria; Theil, Peter Kappel

    2017-01-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL......]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli...... supply seem to enhance vital body functions as interpreted from increased liver synthesis of albumin, increased rumen papillae proliferation, and stabilized the ex vivo inflammatory responsiveness of leukocytes. Further studies are needed to enlighten the importance of increased postpartum protein supply...

  7. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling.

    Science.gov (United States)

    Lee, Elaine Choung-Hee; Strange, Kevin

    2012-12-15

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.

  8. Streptococcus mutans protein synthesis during mixed-species biofilm development by high-throughput quantitative proteomics.

    Directory of Open Access Journals (Sweden)

    Marlise I Klein

    Full Text Available Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA and glucan-binding (gbpB during this transition (P<0.05. Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism, and molecular chaperones (GroEL. Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms demonstrating fundamental differences in the matrix assembly, survival and biofilm

  9. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    Science.gov (United States)

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (Pmutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other

  10. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

    Science.gov (United States)

    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  11. Endoplasmic reticulum stress sensing in the unfolded protein response

    National Research Council Canada - National Science Library

    Gardner, Brooke M; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M; Walter, Peter

    2013-01-01

    Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation...

  12. Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons.

    Science.gov (United States)

    Kar, Amar N; MacGibeny, Margaret A; Gervasi, Noreen M; Gioio, Anthony E; Kaplan, Barry B

    2013-04-24

    Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.

  13. Protein Synthesis during Germination: Shedding New Light on a Classical Question.

    Science.gov (United States)

    Boone, Tyler; Driks, Adam

    2016-12-15

    Despite over a century of research into the mystery of bacterial spore dormancy and germination, a key question remains unresolved: is protein synthesis required for germination? The development of more sophisticated techniques for assessing and preventing protein synthesis has renewed interest in this long-standing question in recent years. In this issue, Korza et al. (G. Korza, B. Setlow, L. Rao, Q. Li, and P. Setlow, J. Bacteriol 198:3254-3264, 2016, http://dx.doi.org/10.1128/JB.00583-16) address this with a novel approach. We discuss their results in the context of recently published data. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. Effect of transcutaneous electrical muscle stimulation on postoperative muscle mass and protein synthesis

    DEFF Research Database (Denmark)

    Vinge, O; Edvardsen, L; Jensen, F

    1996-01-01

    In an experimental study, 13 patients undergoing major elective abdominal surgery were given postoperative transcutaneous electrical muscle stimulation (TEMS) to the quadriceps femoris muscle on one leg; the opposite leg served as control. Changes in cross-sectional area (CSA) and muscle protein...... synthesis were assessed by computed tomography and ribosome analysis of percutaneous muscle biopsies before surgery and on the sixth postoperative day. The percentage of polyribosomes in the ribosome suspension decreased significantly (P ... muscle protein synthesis and muscle mass after abdominal surgery and should be evaluated in other catabolic states with muscle wasting....

  15. Physiological responses of salt stress and osmoprotection with ...

    African Journals Online (AJOL)

    ... correct folding of new proteins and prevents aggregation of proteins altered, the SDS PAGE analysis of the proteins contents of two strains CHT1, CHT4, indicated the appearance of high and low molecular mass new proteins. Keywords: Salt stress, bacterial growth, osmoregulation, proline, stress protein synthesis.

  16. Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

    Science.gov (United States)

    Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

    2014-08-25

    When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Generalization of fear inhibition by disrupting hippocampal protein synthesis-dependent reconsolidation process.

    Science.gov (United States)

    Yang, Chih-Hao; Huang, Chiung-Chun; Hsu, Kuei-Sen

    2011-09-01

    Repetitive replay of fear memories may precipitate the occurrence of post-traumatic stress disorder and other anxiety disorders. Hence, the suppression of fear memory retrieval may help prevent and treat these disorders. The formation of fear memories is often linked to multiple environmental cues and these interconnected cues may act as reminders for the recall of traumatic experiences. However, as a convenience, a simple paradigm of one cue pairing with the aversive stimulus is usually used in studies of fear conditioning in animals. Here, we built a more complex fear conditioning model by presenting several environmental stimuli during fear conditioning and characterize the effectiveness of extinction training and the disruption of reconsolidation process on the expression of learned fear responses. We demonstrate that extinction training with a single-paired cue resulted in cue-specific attenuation of fear responses but responses to other cures were unchanged. The cue-specific nature of the extinction persisted despite training sessions combined with D-cycloserine treatment reveals a significant weakness in extinction-based treatment. In contrast, the inhibition of the dorsal hippocampus (DH) but not the basolateral amygdala (BLA)-dependent memory reconsolidation process using either protein synthesis inhibitors or genetic disruption of cAMP-response-element-binding protein-mediated transcription comprehensively disrupted the learned connections between fear responses and all paired environmental cues. These findings emphasize the distinct role of the DH and the BLA in the reconsolidation process of fear memories and further indicate that the disruption of memory reconsolidation process in the DH may result in generalization of fear inhibition.

  18. Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2.

    Directory of Open Access Journals (Sweden)

    Brooke M Baker

    Full Text Available Cells respond to defects in mitochondrial function by activating signaling pathways that restore homeostasis. The mitochondrial peptide exporter HAF-1 and the bZip transcription factor ATFS-1 represent one stress response pathway that regulates the transcription of mitochondrial chaperone genes during mitochondrial dysfunction. Here, we report that GCN-2, an eIF2α kinase that modulates cytosolic protein synthesis, functions in a complementary pathway to that of HAF-1 and ATFS-1. During mitochondrial dysfunction, GCN-2-dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans. Reactive oxygen species (ROS generated from dysfunctional mitochondria are required for GCN-2-dependent eIF2α phosphorylation but not ATFS-1 activation. Simultaneous deletion of ATFS-1 and GCN-2 compounds the developmental defects associated with mitochondrial stress, while stressed animals lacking GCN-2 display a greater dependence on ATFS-1 and stronger induction of mitochondrial chaperone genes. These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPR(mt.

  19. Proteomic Analysis of Copper-Binding Proteins in Excess Copper-Stressed Roots of Two Rice (Oryza sativa L.) Varieties with Different Cu Tolerances.

    Science.gov (United States)

    Chen, Chen; Song, Yufeng; Zhuang, Kai; Li, Lu; Xia, Yan; Shen, Zhenguo

    2015-01-01

    To better understand the mechanisms involved in the heavy metal stress response and tolerance in plants, a proteomic approach was used to investigate the differences in Cu-binding protein expression in Cu-tolerant and Cu-sensitive rice varieties. Cu-binding proteins from Cu-treated rice roots were separated using a new IMAC method in which an IDA-sepharose column was applied prior to the Cu-IMAC column to remove metal ions from protein samples. More than 300 protein spots were reproducibly detected in the 2D gel. Thirty-five protein spots exhibited changes greater than 1.5-fold in intensity compared to the control. Twenty-four proteins contained one or more of nine putative metal-binding motifs reported by Smith et al., and 19 proteins (spots) contained one to three of the top six motifs reported by Kung et al. The intensities of seven protein spots were increased in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195 (pprotein spots were markedly up-regulated in B1139, but not detectable in B1195. Four protein spots were significantly up-regulated in B1139, but unchanged in B1195 under Cu stress. In contrast, two protein spots were significantly down-regulated in B1195, but unchanged in B1139. These Cu-responsive proteins included those involved in antioxidant defense and detoxification (spots 5, 16, 21, 22, 28, 29 and 33), pathogenesis (spots 5, 16, 21, 22, 28, 29 and 33), regulation of gene transcription (spots 8 and 34), amino acid synthesis (spots 8 and 34), protein synthesis, modification, transport and degradation (spots 1, 2, 4, 10, 15, 19, 30, 31, 32 and 35), cell wall synthesis (spot 14), molecular signaling (spot 3), and salt stress (spots 7, 9 and 27); together with other proteins, such as a putative glyoxylate induced protein, proteins containing dimeric alpha-beta barrel domains, and adenosine kinase-like proteins. Our results suggest that these proteins, together with related physiological processes, play an important role in the

  20. Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein.

    Science.gov (United States)

    Ayub, Maximiliano Juri; Nyambega, Benson; Simonetti, Leandro; Duffy, Tomas; Longhi, Silvia A; Gómez, Karina A; Hoebeke, Johan; Levin, Mariano J; Smulski, Cristian R

    2012-01-01

    The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

  1. Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein.

    Directory of Open Access Journals (Sweden)

    Maximiliano Juri Ayub

    Full Text Available The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5 directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

  2. Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

    Science.gov (United States)

    Huang, Tengfang; Jander, Georg

    2017-07-01

    Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

  3. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis

    Science.gov (United States)

    Briz, Victor; Hsu, Yu-Tien; Li, Yi; Lee, Erin; Bi, Xiaoning; Baudry, Michel

    2013-01-01

    Memory consolidation has been suggested to be protein synthesis-dependent. Recent data indicate that BDNF-induced dendritic protein synthesis is a key event in memory formation through activation of the mammalian target of rapamycin (mTOR) pathway. BDNF also activates calpain, a calcium-dependent cysteine protease, which has been shown to play a critical role in learning and memory. This study was therefore directed at testing the hypothesis that calpain activity is required for BDNF-stimulated local protein synthesis, and at identifying the underlying molecular mechanism. In rat hippocampal slices, cortical synaptoneurosomes, and cultured neurons, BDNF-induced mTOR pathway activation and protein translation were blocked by calpain inhibition. BDNF treatment rapidly reduced levels of hamartin and tuberin, negative regulators of mTOR, in a calpain-dependent manner. Treatment of brain homogenates with purified calpain-1 and calpain-2 truncated both proteins. BDNF treatment increased phosphorylation of both Akt and ERK, but only the effect on Akt was blocked by calpain inhibition. Levels of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a phosphatase that inactivates Akt, were decreased following BDNF treatment, and calpain inhibition reversed this effect. Calpain-2 but not calpain-1 treatment of brain homogenates resulted in PTEN degradation. In cultured cortical neurons, knock-down of calpain-2 but not calpain-1 by siRNA completely suppressed the effect of BDNF on mTOR activation. Our results reveal a critical role for calpain-2 in BDNF-induced mTOR signaling and dendritic protein synthesis via PTEN, hamartin and tuberin degradation. This mechanism therefore provides a link between proteolysis and protein synthesis that might contribute to synaptic plasticity. PMID:23467348

  4. The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1.

    Science.gov (United States)

    Kathage, Barbara; Gehlert, Sebastian; Ulbricht, Anna; Lüdecke, Laura; Tapia, Victor E; Orfanos, Zacharias; Wenzel, Daniela; Bloch, Wilhelm; Volkmer, Rudolf; Fleischmann, Bernd K; Fürst, Dieter O; Höhfeld, Jörg

    2017-01-01

    The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Feldherr, C.M.; Hodges, P. (Univ. of Florida, Gainesville (USA)); Paine, P.L. (Michigan Cancer Foundation, Detroit (USA))

    1988-12-01

    Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with ({sup 3}H)leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.

  6. The application of 2H2O to measure skeletal muscle protein synthesis

    Directory of Open Access Journals (Sweden)

    Fluckey James D

    2010-04-01

    Full Text Available Abstract Skeletal muscle protein synthesis has generally been determined by the precursor:product labeling approach using labeled amino acids (e.g., [13C]leucine or [13C]-, [15N]-, or [2H]phenylalanine as the tracers. Although reliable for determining rates of protein synthesis, this methodological approach requires experiments to be conducted in a controlled environment, and as a result, has limited our understanding of muscle protein renewal under free-living conditions over extended periods of time (i.e., integrative/cumulative assessments. An alternative tracer, 2H2O, has been successfully used to measure rates of muscle protein synthesis in mice, rats, fish and humans. Moreover, perturbations such as feeding and exercise have been included in these measurements without exclusion of common environmental and biological factors. In this review, we discuss the principle behind using 2H2O to measure muscle protein synthesis and highlight recent investigations that have examined the effects of feeding and exercise. The framework provided in this review should assist muscle biologists in designing experiments that advance our understanding of conditions in which anabolism is altered (e.g., exercise, feeding, growth, debilitating and metabolic pathologies.

  7. Identification of proteins involved in excess boron stress in roots of ...

    African Journals Online (AJOL)

    Plants are constantly challenged by various biotic and abiotic stresses in nature. Boron toxicity have become one of the important abiotic stress factor for plants. Boron toxicity responses of plants is reflected by alterations in protein expression level, activity, location and concentration. In this study, we identified the proteins ...

  8. Inhibitory effects of different fractions of Nepeta satureioides on melanin synthesis through reducing oxidative stress.

    Science.gov (United States)

    Emami, Seyed Ahmad; Yazdian-Robati, Rezvan; Sadeghi, Mohammad; Baharara, Javad; Amini, Elaheh; Salek, Farzaneh; Tayarani-Najaran, Zahra

    2017-04-01

    Nepeta satureioides Boiss. has been used in traditional medicine of eastern countries and is famous for its medicinal properties. The aim of this study was to evaluate the effect of methanol (MeOH), n-hexane and dichloromethane (CH2Cl2) fractions of the extract on melanin synthesis and oxidative stress in B16F10 melanoma cell line. The B16F10 cell line viability after treatment with increasing concentrations of different fractions of the plant (5-60 μg/mL) was measured using MTT assay. The inhibitory effect on synthesis of melanin, mushroom tyrosinase activity, cellular tyrosinase and oxidative stress were determined by the colorimetric and fluorometric methods. The data showed that at concentrations below 60 μg/mL, fractions did not show significant toxicity on melanoma cells. The amount of melanin synthesis by MeOH and CH2Cl2 fractions and mushroom tyrosinase activity by the MeOH fraction declined in B16F10 cells. In addition to the capacity of MeOH, n-hexane and CH2Cl2 fractions in decreasing the amount of reactive oxygen species (ROS) in melanoma cells, all fractions revealed remarkable antioxidant activity. The melanogenesis inhibitory and antioxidant effects of N. satureioides on B16F10 cells may suggest this plant as a new pharmaceutical agent in reducing skin pigment and skin aging in cosmetic industry.

  9. Proteomic screening of glucose-responsive and glucose non-reponsive MIN-6 beta cells reveals differential expression of protein involved in protein folding, secretion and oxidative stress

    DEFF Research Database (Denmark)

    Dowling, P.; O´Driscoll, L.; O´Sullivan, F.

    2006-01-01

    .8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum......-6 cells to successfully fold, modify or secrete proteins and counteract the problems associated with oxidative stress...

  10. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems.

    Science.gov (United States)

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-11-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  11. Effects of drought stress on seed sink strength and leaf protein ...

    African Journals Online (AJOL)

    Quantitative and qualitative changes in leaf protein patterns was assessed using 2D-gel electrophoresis. A drought-resistant inbred line (SEA 15) and a droughtsusceptible cultivar (BrSp) were grown under non-stress and drought stress conditions in a vegetation hall during the summer of 2004. Drought stress commenced ...

  12. Identification of 30 protein species involved in replicative senescence and stress-induced premature senescence

    DEFF Research Database (Denmark)

    Dierick, Jean François; Kalume, Dário E; Wenders, Frédéric

    2002-01-01

    Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level...... of protein expression, stress-induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress-induced premature senescence....... These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways....

  13. Deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation1234

    Science.gov (United States)

    Patel, Sanjeet G; Guthikonda, Anuradha P; Reid, Marvin; Balasubramanyam, Ashok; Taffet, George E; Jahoor, Farook

    2011-01-01

    Background: Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. Objective: We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. Design: Eight elderly and 8 younger subjects received stable-isotope infusions of [2H2]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F2-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. Results: Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 μmol/L; P glutathione (2.08 ± 0.12 compared with 1.12 ± 0.18 mmol/L RBCs; P glutathione fractional (83.14 ± 6.43% compared with 45.80 ± 5.69%/d; P glutathione concentration, a 78.8% higher fractional synthesis rate, a 230.9% higher absolute synthesis rate, and significantly lower plasma oxidative stress and F2-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. Conclusions: Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and concentrations and lowers levels of oxidative stress and oxidant damages. These findings suggest a practical and effective approach to decreasing oxidative stress in aging. PMID:21795440

  14. Protein synthesis, growth and energetics in larval herring (Clupea harengus) at different feeding regimes

    DEFF Research Database (Denmark)

    Houlihan, D F; Pedersen, B H; Steffensen, J F

    1995-01-01

    . Larvae were bathed in (3)H phenylalanine for several hours and the free pool and protein-bound phenylalanine specific radioactivities were determined.Fractional rates of protein synthesis increased 5 to 11 fold with feeding after a period of fasting. Efficiencies of retention of synthesized protein were...... approximately 50% during rapid growth. Rapid growth in herring larvae thus appears to be characterized by moderate levels of protein turnover similar to those obtained for larger fish. Increases in growth rate occurred without changes in RNA concentration, i.e., the larvae increased the efficiency of RNA...

  15. Arenavirus Z protein controls viral RNA synthesis by locking a polymerase-promoter complex.

    Science.gov (United States)

    Kranzusch, Philip J; Whelan, Sean P J

    2011-12-06

    Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.

  16. Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

    Science.gov (United States)

    Kranzusch, Philip J.; Whelan, Sean P. J.

    2011-01-01

    Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z–L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression. PMID:22106304

  17. Antioxidant defence and stress protein induction following heat stress in the Mediterranean snail Xeropicta derbentina.

    Science.gov (United States)

    Troschinski, Sandra; Dieterich, Andreas; Krais, Stefanie; Triebskorn, Rita; Köhler, Heinz-R

    2014-12-15

    The Mediterranean snail Xeropicta derbentina (Pulmonata, Hygromiidae), being highly abundant in Southern France, has the need for efficient physiological adaptations to desiccation and over-heating posed by dry and hot environmental conditions. As a consequence of heat, oxidative stress manifests in these organisms, which, in turn, leads to the formation of reactive oxygen species (ROS). In this study, we focused on adaptations at the biochemical level by investigation of antioxidant defences and heat shock protein 70 (Hsp70) induction, both essential mechanisms of the heat stress response. We exposed snails to elevated temperature (25, 38, 40, 43 and 45°C) in the laboratory and measured the activity of the antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx), determined the Hsp70 level and quantified lipid peroxidation. In general, we found a high constitutive level of CAT activity in all treatments, which may be interpreted as a permanent protection against ROS, i.e. hydrogen peroxide. CAT and GPx showed temperature-dependent activity: CAT activity was significantly increased in response to high temperatures (43 and 45°C), whereas GPx exhibited a significantly increased activity at 40°C, probably in response to high levels of lipid peroxides that occurred in the 38°C treatment. Hsp70 showed a maximum induction at 40°C, followed by a decrease at higher temperatures. Our results reveal that X. derbentina possesses a set of efficient mechanisms to cope with the damaging effects of heat. Furthermore, we demonstrated that, besides the well-documented Hsp70 stress response, antioxidant defence plays a crucial role in the snails' competence to survive extreme temperatures. © 2014. Published by The Company of Biologists Ltd.

  18. Use of theoretical efficiencies of protein and fat synthesis to ...

    African Journals Online (AJOL)

    The main objection against conventional net energy systems is that owing to variation in gain composition and the different energy contents of protein and fat, the efficiency of energy gain cannot be regarded as a growth constant. The present approach shows that the separate accommodation of protein and fat in predicting ...

  19. The cascade of inflammatory cytokines regulating synthesis of acute phase proteins.

    Science.gov (United States)

    Koj, A; Magielska-Zero, D; Bereta, J; Kurdowska, A; Rokita, H; Gauldie, J

    1988-12-01

    The acute phase cytokines: interleukin 1, tumor necrosis factor alpha (cachectin) and beta (lymphotoxin), hepatocyte stimulating factor and several interferons, all belong to the family of endotoxin-inducible, low molecular weight proteins. Their synthesis in macrophages, fibroblasts, lymphocytes, epithelial and some tumor cells is enhanced by the same cytokines, often in the autocrine manner, and suppressed by dexamethasone. The principal hepatocyte stimulating factor (HSF) regulating synthesis of acute phase proteins is probably identical with IFN-beta 2/BSF-2/IL-6, but other inflammatory cytokines (IL-1, TNF alpha, IFN-gamma) are able to induce distinct sets of acute phase proteins, or to modulate the final response pattern. The effect of hrIFN-gamma on production of acute phase proteins by human hepatoma Hep G2 cells is discussed in detail. It is concluded that the cascades of inflammatory cytokines in different tissues represent amplification and regulatory pathways controlling the development of acute phase response in vivo.

  20. Contraction intensity and feeding affect collagen and myofibrillar protein synthesis rates differently in human skeletal muscle

    DEFF Research Database (Denmark)

    Holm, Lars; van Hall, Gerrit; Rose, Adam

    2010-01-01

    with the fasting condition. The Rp-s6k-4E-binding protein-1 (BP1) and the mitogen-activated protein kinase (MAPk) pathways were activated by the HL intensity and were suggested to be responsible for regulating myofibrillar FSR in response to adequate contractile activity. Feeding predominantly affected Rp-s6k...... to contractile activity, whereas elongation mainly was found to respond to feeding. Furthermore, although functionally linked, the contractile and the supportive matrix structures upregulate their protein synthesis rate quite differently in response to feeding and contractile activity and intensity.......Exercise stimulates muscle protein fractional synthesis rate (FSR), but the importance of contractile intensity and whether it interplays with feeding is not understood. This was investigated following two distinct resistance exercise (RE) contraction intensities using an intrasubject design...

  1. Shiga Toxins as Multi-Functional Proteins: Induction of Host Cellular Stress Responses, Role in Pathogenesis and Therapeutic Applications

    Directory of Open Access Journals (Sweden)

    Moo-Seung Lee

    2016-03-01

    Full Text Available Shiga toxins (Stxs produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are primary virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications, such as hemolytic uremic syndrome and central nervous system abnormalities. Current therapeutic options to treat patients infected with toxin-producing bacteria are limited. The structures of Stxs, toxin-receptor binding, intracellular transport and the mode of action of the toxins have been well defined. However, in the last decade, numerous studies have demonstrated that in addition to being potent protein synthesis inhibitors, Stxs are also multifunctional proteins capable of activating multiple cell stress signaling pathways, which may result in apoptosis, autophagy or activation of the innate immune response. Here, we briefly present the current understanding of Stx-activated signaling pathways and provide a concise review of therapeutic applications to target tumors by engineering the toxins.

  2. Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

    Science.gov (United States)

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

    Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Effect of acute maternal starvation on tyrosine metabolism and protein synthesis in fetal sheep

    Energy Technology Data Exchange (ETDEWEB)

    Krishnamurti, C.R.; Schaefer, A.L.

    To determine the effects of acute maternal starvation on intrauterine growth, tyrosine concentration and specific activity values in plasma, intracellular free and protein bound pools were determined in catheterized ovine fetuses following an 8 h continuous infusion of L-(2,3,5,6 /sup 3/H) or L-(U-/sup 14/C) tyrosine into the ewe and fetus respectively at 115-125 days of gestation. From the kinetic data the rates of whole body and tissue fractional protein synthesis were calculated. Although placental protein synthesis was not significantly changed as a result of acute maternal starvation, fetal whole body protein synthesis was reduced from 63 g/d/kg in the fed to 25 g/d/kg in the starved condition. There was also a 10 fold reduction in the net placental transfer of tyrosine to the fetus in the starved ewes. In addition, a three fold increase was observed in the quantity of tyrosine used for oxidation by the fetuses of starved ewes, changing from 5.2% of tyrosine net utilization in the fed to 13.7% in the starved condition. Significant reductions in tissue fractional protein synthesis rates were also seen in the liver, brain, lung kidney and GIT tissues from 78, 37, 65, 45 and 71%/d respectively in the fed to 12, 10, 23, 22 and 35%/d in the fetuses of starved ewes. The data indicate that during acute maternal starvation the sheep fetus utilizes more tyrosine for oxidation and less for anabolic purposes which is reflected in a decrease both in whole body and tissue fractional rates of protein synthesis.

  4. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Application of proteomics to investigate stress-induced proteins for improvement in crop protection.

    Science.gov (United States)

    Afroz, Amber; Ali, Ghulam Muhammad; Mir, Asif; Komatsu, Setsuko

    2011-05-01

    Proteomics has contributed to defining the specific functions of genes and proteins involved in plant-pathogen interactions. Proteomic studies have led to the identification of many pathogenicity and defense-related genes and proteins expressed during phytopathogen infections, resulting in the collection of an enormous amount of data. However, the molecular basis of plant-pathogen interactions remains an intensely active area of investigation. In this review, the role of differential analysis of proteins expressed during fungal, bacterial, and viral infection is discussed, as well as the role of JA and SA in the production of stress related proteins. Resistance acquired upon induction of stress related proteins in intact plant leaves is mediated by potentiation of pathogens via signal elicitors. Stress related genes extensively used in biotechnology had been cited. Stress related proteins identified must be followed through for studying the molecular mechanism for plant defense against pathogens.

  6. Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

    Directory of Open Access Journals (Sweden)

    Pushpika eUdawat

    2016-04-01

    Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

  7. [Inhibition of aggregation of dissociated embryonic Amphibian cells by inhibitors of protein and RNA synthesis].

    Science.gov (United States)

    Grunz, Horst

    1969-06-01

    1. Aggregation of embryonic Amphibian cells dissociated by Trypsin or EDTA is supressed reversibly by actidion (cycloheximid 0.5-2 Μg/ml), an inhibitor of protein synthesis. Protein synthesis is inhibited more than 90% at these concentrations. The inhibition by actidion was still reversible after 48 hours, when the cells were transferred to normal medium. Actidion added to cells, cultured in Flickinger solution for 3 h, was no longer able to stop further aggregation. 2. Puromycin reversibly suppresses aggregation at a concentration of 20 (Μg/ml, when protein synthesis is inhibited to 66 %. 3. After EDTA-dissociation the formation of initial small clusters of 5-10 cells (primary aggregation) occurred even in the presence of inhibitors of protein synthesis. The primary aggregation is however suppressed if actidion or puromycin are already added during the dissociation procedure. 4. In the presence of actinomycin-D (1-2 Μg/ml) the cells continue to aggregate like the controls. After 30 h further aggregation is stopped and the aggregates loose single cells. 5. Differences in the aggregation of cells after EDTA and Trypsin treatment are discussed.

  8. Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

    Directory of Open Access Journals (Sweden)

    Yazmín Ramiro-Cortés

    Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

  9. Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

    DEFF Research Database (Denmark)

    Brøndsted Nielsen, Mogens; Andersen, Lars Henrik; Rinza, Tomás Rocha

    2011-01-01

    The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase...

  10. Dose-dependent differential effect of hemin on protein synthesis and ...

    Indian Academy of Sciences (India)

    Permanent link: http://www.ias.ac.in/article/fulltext/jbsc/026/02/0225-0231. Keywords. Hemin chloride; hsp90; promastigotes; protein synthesis; protozoan parasite; -tubulin. Abstract. Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes ...

  11. Degradation and de novo synthesis of D1 protein and psbA ...

    Indian Academy of Sciences (India)

    synthesis than the visible radiation (Jones and Kok 1966). As compared to vast literature available on visible ..... 1997). The degradation of other PS II core proteins including CP 47 and change in heterogeneity can not be ruled out. The apparent photosynthesis that observed in. UV-B treated C. reinhardtii is the net result of ...

  12. Inhibition of prefrontal protein synthesis following recall does not disrupt memory for trace fear conditioning

    Directory of Open Access Journals (Sweden)

    Dash Pramod K

    2006-10-01

    Full Text Available Abstract Background The extent of similarity between consolidation and reconsolidation is not yet fully understood. One of the differences noted is that not every brain region involved in consolidation exhibits reconsolidation. In trace fear conditioning, the hippocampus and the medial prefrontal cortex (mPFC are required for consolidation of long-term memory. We have previously demonstrated that trace fear memory is susceptible to infusion of the protein synthesis inhibitor anisomycin into the hippocampus following recall. In the present study, we examine whether protein synthesis inhibition in the mPFC following recall similarly results in the observation of reconsolidation of trace fear memory. Results Targeted intra-mPFC infusions of anisomycin or vehicle were performed immediately following recall of trace fear memory at 24 hours, or at 30 days, following training in a one-day or a two-day protocol. The present study demonstrates three key findings: 1 trace fear memory does not undergo protein synthesis dependent reconsolidation in the PFC, regardless of the intensity of the training, and 2 regardless of whether the memory is recent or remote, and 3 intra-mPFC inhibition of protein synthesis immediately following training impaired remote (30 days memory. Conclusion These results suggest that not all structures that participate in memory storage are involved in reconsolidation. Alternatively, certain types of memory-related information may reconsolidate, while other components of memory may not.

  13. Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2008-01-01

    The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

  14. Solid-phase synthesis of an apoptosis-inducing tetrapeptide mimicking the Smac protein

    DEFF Research Database (Denmark)

    Le Quement, Sebastian Thordal; Ishøy, Mette; Petersen, Mette Terp

    2011-01-01

    An approach for the solid-phase synthesis of apoptosis-inducing Smac peptidomimetics is presented. Using a Rink linker strategy, tetrapeptides mimicking the N-4-terminal residue of the Smac protein [(N-Me)AVPF sequence] were synthesized on PEGA resin in excellent purities and yields. Following two...

  15. Dose-dependent differential effect of hemin on protein synthesis and ...

    Indian Academy of Sciences (India)

    Unknown

    Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has ...

  16. In Profile: Models of Ribosome Biogenesis Defects and Regulation of Protein Synthesis

    NARCIS (Netherlands)

    Essers, P.B.M.

    2013-01-01

    Ribosomes are the mediators of protein synthesis in the cell and therefore crucial to proper cell function. In addition, ribosomes are highly abundant, with ribosomal RNA making up 80% of the RNA in the cell. A large amount of resources go into maintaining this pool of ribosomes, so ribosome

  17. Inhibition of protein synthesis in vitro by a lectin from Momordica charantia and by other haemagglutinins.

    Science.gov (United States)

    Barbieri, L; Lorenzoni, E; Stirpe, F

    1979-08-15

    Protein synthesis by a rabbit reticulocyte lysate is inhibited by the haemagglutinating lectins from Momordica charantia and Crotalaria juncea seeds and from the roe of Rutilus rutilus, and by a commercial preparation of the mitogenic lectin from Phytolacca americana. The haemagglutinins from the seeds of Ricinus communis and of Vicia cracca acquired inhibitory activity after their reduction with 2-mercaptoethanol.

  18. Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants.

    Science.gov (United States)

    Menzies, Karensa K; Lee, Heather J; Lefèvre, Christophe; Ormandy, Christopher J; Macmillan, Keith L; Nicholas, Kevin R

    2010-03-01

    Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes.

  19. Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis

    Energy Technology Data Exchange (ETDEWEB)

    Hatch, T.P.; Miceli, M.; Silverman, J.A.

    1985-06-01

    Synthesis of protein by the obligate intracellular parasitic bacteria Chlamydia psittaci (6BC) and Chlamydia trachomatis (serovar L2) isolated from host cells (host-free chlamydiae) was demonstrated for the first time. Incorporation of (/sup 35/S)methionine and (/sup 35/S)cysteine into trichloroacetic acid-precipitable material by reticulate bodies of chlamydiae persisted for 2 h and was dependent upon a exogenous source of ATP, an ATP-regenerating system, and potassium or sodium ions. Magnesium ions and amino acids stimulated synthesis; chloramphenicol, rifampin, oligomycin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a proton ionophore) inhibited incorporation. Ribonucleoside triphosphates (other than ATP) had little stimulatory effect. The optimum pH for host-free synthesis was between 7.0 and 7.5. The molecular weights of proteins synthesized by host-free reticulate bodies closely resembled the molecular weights of proteins synthesized by reticulate bodies in an intracellular environment, and included outer membrane proteins. Elementary bodies of chlamydiae were unable to synthesize protein even when incubated in the presence of 10 mM dithiothreitol, a reducing agent which converted the highly disulfide bond cross-linked major outer membrane protein to monomeric form.

  20. Control of protein synthesis in yeast mitochondria: the concept of translational activators.

    Science.gov (United States)

    Herrmann, Johannes M; Woellhaf, Michael W; Bonnefoy, Nathalie

    2013-02-01

    Mitochondria contain their own genome which codes for a small number of proteins. Most mitochondrial translation products are part of the membrane-embedded reaction centers of the respiratory chain complexes. In the yeast Saccharomyces cerevisiae, the expression of these proteins is regulated by translational activators that bind mitochondrial mRNAs, in most cases to their 5'-untranslated regions, and each mitochondrial mRNA appears to have its own translational activator(s). Recent studies showed that these translational activators can be part of feedback control loops which only permit translation if the downstream assembly of nascent translation products can occur. In several cases, the accumulation of a non-assembled protein prevents further synthesis of this protein but not translation in general. These control loops prevent the synthesis of potentially harmful assembly intermediates of the reaction centers of mitochondrial enzymes. Since such regulatory feedback loops only work if translation occurs in the compartment in which the complexes of the respiratory chain are assembled, these control mechanisms require the presence of a translation machinery in mitochondria. This might explain why eukaryotic cells maintained DNA in mitochondria during the last two billion years of evolution. This review gives an overview of the mitochondrial translation system and summarizes the current knowledge on translational activators and their role in the regulation of mitochondrial protein synthesis. This article is part of a Special Issue entitled: Protein import and quality control in mitochondria and plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. PI3Kδ activates E2F1 synthesis in response to mRNA translation stress.

    Science.gov (United States)

    Gnanasundram, Sivakumar Vadivel; Pyndiah, Slovénie; Daskalogianni, Chrysoula; Armfield, Kate; Nylander, Karin; Wilson, Joanna B; Fåhraeus, Robin

    2017-12-13

    The c-myc oncogene stimulates ribosomal biogenesis and protein synthesis to promote cellular growth. However, the pathway by which cells sense and restore dysfunctional mRNA translation and how this is linked to cell proliferation and growth is not known. We here show that mRNA translation stress in cis triggered by the gly-ala repeat sequence of Epstein-Barr virus (EBV)-encoded EBNA1, results in PI3Kδ-dependent induction of E2F1 mRNA translation with the consequent activation of c-Myc and cell proliferation. Treatment with a specific PI3Kδ inhibitor Idelalisib (CAL-101) suppresses E2F1 and c-Myc levels and causes cell death in EBNA1-induced B cell lymphomas. Suppression of PI3Kδ prevents E2F1 activation also in non-EBV-infected cells. These data illustrate an mRNA translation stress-response pathway for E2F1 activation that is exploited by EBV to promote cell growth and proliferation, offering new strategies to treat EBV-carrying cancers.

  2. Proteomics applied on plant abiotic stresses: role of heat shock proteins (HSP).

    Science.gov (United States)

    Timperio, Anna Maria; Egidi, Maria Giulia; Zolla, Lello

    2008-10-07

    The most crucial function of plant cell is to respond against stress induced for self-defence. This defence is brought about by alteration in the pattern of gene expression: qualitative and quantitative changes in proteins are the result, leading to modulation of certain metabolic and defensive pathways. Abiotic stresses usually cause protein dysfunction. They have an ability to alter the levels of a number of proteins which may be soluble or structural in nature. Nowadays, in higher plants high-throughput protein identification has been made possible along with improved protein extraction, purification protocols and the development of genomic sequence databases for peptide mass matches. Thus, recent proteome analysis performed in the vegetal Kingdom has provided new dimensions to assess the changes in protein types and their expression levels under abiotic stress. As reported in this review, specific and novel proteins, protein-protein interactions and post-translational modifications have been identified, which play a role in signal transduction, anti-oxidative defence, anti-freezing, heat shock, metal binding etc. However, beside specific proteins production, plants respond to various stresses in a similar manner by producing heat shock proteins (HSPs), indicating a similarity in the plant's adaptive mechanisms; in plants, more than in animals, HSPs protect cells against many stresses. A relationship between ROS and HSP also seems to exist, corroborating the hypothesis that during the course of evolution, plants were able to achieve a high degree of control over ROS toxicity and are now using ROS as signalling molecules to induce HSPs.

  3. Stress tolerances of nullmutants of function-unknown genes encoding menadione stress-responsive proteins in Aspergillus nidulans.

    Science.gov (United States)

    Leiter, Éva; Bálint, Mihály; Miskei, Márton; Orosz, Erzsébet; Szabó, Zsuzsa; Pócsi, István

    2016-07-01

    A group of menadione stress-responsive function-unkown genes of Aspergillus nidulans (Locus IDs ANID_03987.1, ANID_06058.1, ANID_10219.1, and ANID_10260.1) was deleted and phenotypically characterized. Importantly, comparative and phylogenetic analyses of the tested A. nidulans genes and their orthologs shed light only on the presence of a TANGO2 domain with NRDE protein motif in the translated ANID_06058.1 gene but did not reveal any recognizable protein-encoding domains in other protein sequences. The gene deletion strains were subjected to oxidative, osmotic, and metal ion stress and, surprisingly, only the ΔANID_10219.1 mutant showed an increased sensitivity to 0.12 mmol l(-1) menadione sodium bisulfite. The gene deletions affected the stress sensitivities (tolerances) irregularly, for example, some strains grew more slowly when exposed to various oxidants and/or osmotic stress generating agents, meanwhile the ΔANID_10260.1 mutant possessed a wild-type tolerance to all stressors tested. Our results are in line with earlier studies demonstrating that the deletions of stress-responsive genes do not confer necessarily any stress-sensitivity phenotypes, which can be attributed to compensatory mechanisms based on other elements of the stress response system with overlapping functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Differential analysis of protein expression in RNA-binding-protein transgenic and parental rice seeds cultivated under salt stress.

    Science.gov (United States)

    Nakamura, Rika; Nakamura, Ryosuke; Adachi, Reiko; Hachisuka, Akiko; Yamada, Akiyo; Ozeki, Yoshihiro; Teshima, Reiko

    2014-02-07

    Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.

  5. Differential Analysis of Protein Expression in RNA-Binding-Protein Transgenic and Parental Rice Seeds Cultivated under Salt Stress

    Science.gov (United States)

    2015-01-01

    Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds. PMID:24410502

  6. The differential role of cortical protein synthesis in taste memory formation and persistence

    Science.gov (United States)

    Levitan, David; Gal-Ben-Ari, Shunit; Heise, Christopher; Rosenberg, Tali; Elkobi, Alina; Inberg, Sharon; Sala, Carlo; Rosenblum, Kobi

    2016-05-01

    The current dogma suggests that the formation of long-term memory (LTM) is dependent on protein synthesis but persistence of the memory trace is not. However, many of the studies examining the effect of protein synthesis inhibitors (PSIs) on LTM persistence were performed in the hippocampus, which is known to have a time-dependent role in memory storage, rather than the cortex, which is considered to be the main structure to store long-term memories. Here we studied the effect of PSIs on LTM formation and persistence in male Wistar Hola (n⩾5) rats by infusing the protein synthesis inhibitor, anisomycin (100 μg, 1 μl), into the gustatory cortex (GC) during LTM formation and persistence in conditioned taste aversion (CTA). We found that local anisomycin infusion to the GC before memory acquisition impaired LTM formation (P=8.9E-5), but had no effect on LTM persistence when infused 3 days post acquisition (P=0.94). However, when we extended the time interval between treatment with anisomycin and testing from 3 days to 14 days, LTM persistence was enhanced (P=0.01). The enhancement was on the background of stable and non-declining memory, and was not recapitulated by another amnesic agent, APV (10 μg, 1 μl), an N-methyl-D-aspartate receptor antagonist (P=0.54). In conclusion, CTA LTM remains sensitive to the action of PSIs in the GC even 3 days following memory acquisition. This sensitivity is differentially expressed between the formation and persistence of LTM, suggesting that increased cortical protein synthesis promotes LTM formation, whereas decreased protein synthesis promotes LTM persistence.

  7. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  8. Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

    KAUST Repository

    Ruchti, E.

    2016-10-08

    The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

  9. Duodenal juice total protein and pancreatic enzyme synthesis, turnover, and secretion in patients after acute pancreatitis.

    Science.gov (United States)

    Ogden, J M; O'Keefe, S J; Louw, J A; Adams, G; Marks, I N

    1993-09-01

    It is controversial whether acute pancreatitis has longterm effects on pancreatic function. Pancreatic enzyme synthesis, turnover, and secretion were measured in 10 patients in clinical remission who had had one or more (one to six) attacks of acute alcoholic pancreatitis. The studies were done between two and 29 months after the most recent attack. A control group included five patients with no evidence of pancreatic disease. A four hour primed/continuous intravenous infusion of [14C]L-leucine tracer was given with secretin (2 U/kg/h) and cholecystokinin (0.5 U/kg/h) and secreted duodenal juice aspirated. Amylase and trypsin were extracted from duodenal juice by affinity chromatography, permitting measurement of the rate of isotope incorporation into total protein, amylase, and trypsin. The results showed non-parallel changes in enzyme synthesis and turnover with decreases in total enzyme protein and amylase synthesis and turnover but preservation of trypsin synthesis and turnover. The low turnover rates may be ascribed to continuing pancreatic cell malfunction after recovery from acute alcoholic pancreatitis and suggest that the decreased amylase secretion rates are partly a consequence of impaired amylase synthesis and not simply because of loss of pancreatic tissue.

  10. THE ROLE OF POST-EXERCISE NUTRIENT ADMINISTRATION ON MUSCLE PROTEIN SYNTHESIS AND GLYCOGEN SYNTHESIS

    OpenAIRE

    Chris Poole; Colin Wilborn; Lem Taylor; Chad Kerksick

    2010-01-01

    Nutrient administration following an exercise bout vastly affects anabolic processes within the human body, irrespective of exercise mode. Of particular importance are protein and carbohydrates whereby these two macronutrients portray distinct functions as anabolic agents. It has been confirmed that protein and/or amino acid ingestion following resistance training is required to reach a positive protein/nitrogen balance, and carbohydrate intake during recovery is the most important considerat...

  11. review article: Phospholemman: A Novel Cardiac Stress Protein

    Science.gov (United States)

    Cheung, Joseph Y.; Zhang, Xue‐Qian; Song, Jianliang; Gao, Erhe; Rabinowitz, Joseph E.; Chan, Tung O.; Wang, JuFang

    2010-01-01

    Abstract Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co‐localizes and co‐immunoprecipitates with Na+‐K+‐ATPase, Na+/Ca2+ exchanger, and L‐type Ca2+ channel. Functionally, when phosphorylated at serine68, PLM stimulates Na+‐K+‐ATPase but inhibits Na+/Ca2+ exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L‐type Ca2+ channel. Therefore, PLM occupies a key modulatory role in intracellular Na+ and Ca2+ homeostasis and is intimately involved in regulation of excitation–contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na+ overload and simultaneously preserves inotropy by inhibiting Na+/Ca2+ exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na+‐K+‐ATPase, Na+/Ca2+ exchanger, and potentially L‐type Ca2+ channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189–196 PMID:20718822

  12. The heat shock protein/chaperone network and multiple stress resistance

    KAUST Repository

    Jacob, Pierre

    2016-11-15

    Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone

  13. Multienzyme Modification of Hemp Protein for Functional Peptides Synthesis

    Directory of Open Access Journals (Sweden)

    Ranjana Das

    2015-01-01

    Full Text Available Functional foods and nutraceuticals are of special importance, particularly for their impact on human health and prevention of certain chronic diseases. Consequently, the production and properties of bioactive peptides have received an increasing scientific interest over past few years. Present work intends to compare the competence of metalloendopeptidases (“Protease N” and “Protease A” with papain for getting functional peptides from hemp seed meal, which is an obligatory waste of hemp fiber production industry. As a measure of the functional potential hemp protein hydrolysates were analyzed for their antiradical properties in DPPH system. “Protease N” modified protein hydrolysate exhibited comparatively superior radical scavenging activity in DPPH system. Overall findings represent the importance of “Protease N,” as endopeptidase in getting peptides of good antiradical properties from various protein sources.

  14. Expedient total synthesis of small to medium-sized membrane proteins via Fmoc chemistry.

    Science.gov (United States)

    Zheng, Ji-Shen; Yu, Mu; Qi, Yun-Kun; Tang, Shan; Shen, Fei; Wang, Zhi-Peng; Xiao, Liang; Zhang, Longhua; Tian, Chang-Lin; Liu, Lei

    2014-03-05

    Total chemical synthesis provides a unique approach for the access to uncontaminated, monodisperse, and more importantly, post-translationally modified membrane proteins. In the present study we report a practical procedure for expedient and cost-effective synthesis of small to medium-sized membrane proteins in multimilligram scale through the use of automated Fmoc chemistry. The key finding of our study is that after the attachment of a removable arginine-tagged backbone modification group, the membrane protein segments behave almost the same as ordinary water-soluble peptides in terms of Fmoc solid-phase synthesis, ligation, purification, and mass spectrometry characterization. The efficiency and practicality of the new method is demonstrated by the successful preparation of Ser64-phosphorylated M2 proton channel from influenza A virus and the membrane-embedded domain of an inward rectifier K(+) channel protein Kir5.1. Functional characterizations of these chemically synthesized membrane proteins indicate that they provide useful and otherwise-difficult-to-access materials for biochemistry and biophysics studies.

  15. ROLE OF NEUROTRANSMITTERS AND PROTEIN SYNTHESIS IN SHORT- AND LONG-TERM MEMORY

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, E.L.; Rosenzweig, M.R.; Flood, J.F.

    1978-10-01

    Anisomycin is an effective inhibitor of cerebral protein synthesis in mice and is also an effective amnestic agent for both passive and active behavioral tasks. From use of anisomycin in combination with a variety of stimulant and depressant drugs, we conclude that the level of arousal following acquisition plays an important role in determining the duration and the rate of the biosynthetic phase of memory formation. While we have interpreted the experiments with anisomycin as evidence for an essential role of protein in memory storage, others have suggested that side effects of inhibitors of protein synthesis on catecholamine metabolism are the main cause of amnesia. Several experiments were therefore done to compare the effects of anisemycin and catecholamine inhibitors on memory. We conclude that anisomycin's principal amnestic mechanism does not involve inhibition of the catecholamine system. The results strengthen our conclusion that protein synthesis is an essential component for longterm memory trace formation. Also, it is suggested that proteins synthesized in the neuronal cell body are used, in conjunction with other molecules, to produce permanent and semi-permanent anatomical changes.

  16. Increased protein synthesis by cells exposed to a 1,800-MHz radio-frequency mobile phone electromagnetic field, detected by proteome profiling.

    Science.gov (United States)

    Gerner, Christopher; Haudek, Verena; Schandl, Ulla; Bayer, Editha; Gundacker, Nina; Hutter, Hans Peter; Mosgoeller, Wilhelm

    2010-08-01

    To investigate whether or not low intensity radio frequency electromagnetic field exposure (RF-EME) associated with mobile phone use can affect human cells, we used a sensitive proteome analysis method to study changes in protein synthesis in cultured human cells. Four different cell kinds were exposed to 2 W/kg specific absorption rate in medium containing 35S-methionine/cysteine, and autoradiography of 2D gel spots was used to measure the increased synthesis of individual proteins. While short-term RF-EME did not significantly alter the proteome, an 8-h exposure caused a significant increase in protein synthesis in Jurkat T-cells and human fibroblasts, and to a lesser extent in activated primary human mononuclear cells. Quiescent (metabolically inactive) mononuclear cells, did not detectably respond to RF-EME. Since RF exposure induced a temperature increase of less than 0.15 degrees C, we suggest that the observed cellular response is a so called "athermal" effect of RF-EME. Our finding of an association between metabolic activity and the observed cellular reaction to low intensity RF-EME may reconcile conflicting results of previous studies. We further postulate that the observed increased protein synthesis reflects an increased rate of protein turnover stemming from protein folding problems caused by the interference of radio-frequency electromagnetic fields with hydrogen bonds. Our observations do not directly imply a health risk. However, vis-a-vis a synopsis of reports on cells stress and DNA breaks, after short and longer exposure, on active and inactive cells, our findings may contribute to the re-evaluation of previous reports.

  17. Effect of Estradiol-17beta on protein synthesis and degradation rates in fused bovine satellite cell cultures.

    Science.gov (United States)

    Kamanga-Sollo, E; White, M E; Hathaway, M R; Weber, W J; Dayton, W R

    2010-07-01

    Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Further, in vivo studies indicate that estradiol (E2) affects muscle protein synthesis and/or degradation, but in vitro results are inconsistent. We have examined the effects of E2 treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, to learn more about the mechanisms involved in E2-enhanced muscle growth, we have examined the effects of compounds that interfere with binding of E2 or insulin-like growth factor (IGF)-1 to their respective receptors on E2-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in protein degradation rate. The pure estrogen antagonist ICI 182 780 suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation in fused BSC cultures. The G-protein coupled receptor (GPR)-30 agonist G1 does not affect either synthesis or degradation rate, which establishes that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-1 binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation. In summary, our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical estrogen receptor (ER) and IGFR-1.

  18. Identification of specific changes in the pattern of brain protein synthesis after training.

    Science.gov (United States)

    Shashoua, V E

    1976-09-24

    Double labeling studies with [3H]valine and [14C]valine were used to investigate the pattern of protein synthesis in the brains of goldfish. The protein fractions in three bands (alpha, beta, and gamma) on sodium dodecyl sulfate-polyacrylamide gels indicate that more valine was incorporated in the brains of goldfish that had been trained in a vestibular conditioning task than in the brains of untrained fish or fish trained in a variety of control behavioral situation. Changes in the pattern of labeling were localized in the cytoplasmic fraction of the brain; no increases in labeling occurred in either the nuclear or synaptosomal components. The results suggest that a specific change occurs in the pattern of protein synthesis in the brain after the acquistion of a new behavior.

  19. MATHEMATICAL AND COMPUTATIONAL MODELLING OF RIBOSOMAL MOVEMENT AND PROTEIN SYNTHESIS: AN OVERVIEW

    Directory of Open Access Journals (Sweden)

    Tobias von der Haar

    2012-04-01

    Full Text Available Translation or protein synthesis consists of a complex system of chemical reactions, which ultimately result in decoding of the mRNA and the production of a protein. The complexity of this reaction system makes it difficult to quantitatively connect its input parameters (such as translation factor or ribosome concentrations, codon composition of the mRNA, or energy availability to output parameters (such as protein synthesis rates or ribosome densities on mRNAs. Mathematical and computational models of translation have now been used for nearly five decades to investigate translation, and to shed light on the relationship between the different reactions in the system. This review gives an overview over the principal approaches used in the modelling efforts, and summarises some of the major findings that were made.

  20. Role of Oxidative Stress in Modulating Unfolded Protein Response Activity in Chronic Myeloid Leukemia Cell Line.

    Science.gov (United States)

    Bazi, Ali; Keramati, Mohammad Reza; Gholamin, Mehran

    2016-01-01

    Recently, it has been revealed that tyrosine kinase inhibitors (TKIs) act through inducing both oxidative and endoplasmic reticulum (ER) stress in chronic myeloid leukemia cells. However, ER stress signaling triggers both apoptotic and survival processes within cells. Nevertheless, mechanisms by which TKIs avoid the pro-survival effects are not clear. The aim of this study was to evaluate the potential role of oxidative stress in activity of unfolded protein response (UPR) survival pathway within K562 cell line. The expression of UPR survival target genes, Xbp1, and Grp94 (glucose requiring protein 94) was studied in single and combined exposure to oxidative and ER stress in K562 cell line by quantitative and qualitative PCR. The expression of UPR-related survival gene Grp94 was hampered by exposing to oxidative stress in cell induced with ER stress. Interaction of oxidative and ER stress may role as a mediator influencing UPR signaling activity.

  1. Comparison of cellular stress levels and green-fluorescent-protein expression in several Escherichia coli strains.

    Science.gov (United States)

    Seo, Jeong Hyun; Kang, Dong Gyun; Cha, Hyung Joon

    2003-04-01

    Constructs comprising stress-gene promoter elements from rpoH (Sigma 32), clpB or dnaK linked to a green-fluorescent-protein (GFP) expression vector were previously used as non-invasive "stress probes" in Escherichia coli. We compared cellular stress responses in four E. coli strains: production hosts JM105 and BL21, and cloning hosts HB101 and TOP10. When GFP was also used as a model for foreign protein production, we generally observed that the level of expression was inversely proportional to the level of cellular stress. JM105 showed the highest cellular stress level and very low GFP expression, while BL21 exhibited the lowest cellular stress level and the highest GFP expression, in both normal and heat-shock stress environments.

  2. The sensitivity of memory consolidation and reconsolidation to inhibitors of protein synthesis and kinases: Computational analysis

    Science.gov (United States)

    Zhang, Yili; Smolen, Paul; Baxter, Douglas A.; Byrne, John H.

    2010-01-01

    Memory consolidation and reconsolidation require kinase activation and protein synthesis. Blocking either process during or shortly after training or recall disrupts memory stabilization, which suggests the existence of a critical time window during which these processes are necessary. Using a computational model of kinase synthesis and activation, we investigated the ways in which the dynamics of molecular positive-feedback loops may contribute to the time window for memory stabilization and memory maintenance. In the models, training triggered a transition in the amount of kinase between two stable states, which represented consolidation. Simulating protein synthesis inhibition (PSI) from before to 40 min after training blocked or delayed consolidation. Beyond 40 min, substantial (>95%) PSI had little effect despite the fact that the elevated amount of kinase was maintained by increased protein synthesis. However, PSI made established memories labile to perturbations. Simulations of kinase inhibition produced similar results. In addition, similar properties were found in several other models that also included positive-feedback loops. Even though our models are based on simplifications of the actual mechanisms of molecular consolidation, they illustrate the practical difficulty of empirically measuring “time windows” for consolidation. This is particularly true when consolidation and reconsolidation of memory depends, in part, on the dynamics of molecular positive-feedback loops. PMID:20736337

  3. Influence of the liposomal preparation Butaintervite on protein synthesis function in the livers of rats under the influence of carbon tetrachloride poisoning

    Directory of Open Access Journals (Sweden)

    M. I. Hariv

    2016-09-01

    Full Text Available This article presents the results of research into the influence of the complex liposomal preparation Butaintervit on protein synthesis function in the livers of rats under the influence of carbon tetrachloride poisoning. Intramuscular injection of carbon tetrachloride into rats at a dose of 0.25 ml per100 gof body weight causes antigenic load on the body and leads to disruption of protein synthesis function in the liver. This is shown by reduction in blood levels of total protein and its fractions. Thus, the level of albumin in the serum of rats under the conditions of oxidative stress was 70% lower than in clinically healthy animals. However, the level of total protein in the serum was only 10% lower. This is because, along with the decrease of albumin content in the serum, the levels of globulin protein fraction increased by 8.8%. This has led to albumin/globulin disparities in the serum of sick animals. As a result, the value of A/G coefficient was 0.28 ± 0.03, compared to 0.52 ±0.02 inclinically healthy rats. For the normalization of functional state of the liver under oxidative stress it is advisable to apply the liposomal preparation Butaintervite, which in its structure contains butafosfan, interferon, thistle and vitamins A, D and E. Under conditions of oxidative stress and under the action of the liposomal preparation in the rats from the second experimental group we have found significant increase in the levels of total protein and albumins and a decrease in serum globulin in the animals on the fifth and tenth days of the experiment. On the fourteenth day of the experiment under the conditions of oxidative stress and under the action of the liposomal preparation in the rats from the second experimental group the normalization of protein synthesis function in the liver was observed. The level of indicators of total protein, albumin, globulin and the coefficient of albumin/globulin compared with the control group of animals were

  4. Contraction mode and whey protein intake affect the synthesis rate of intramuscular connective tissue

    DEFF Research Database (Denmark)

    Holm, Lars; Klejs Rahbek, Stine; Farup, Jean

    2016-01-01

    IntroductionIn this study we investigated the impact of whey protein hydrolysate and maltodextrin (WPH) intake on intramuscular connective tissue (IMCT) protein fractional synthesis rate (FSR) after maximal shortening and lengthening contractions. Methods Twenty young men were randomized to receive...... either WPH or maltodextrin [carbohydrate (CHO)] immediately after completion of unilateral shortening and lengthening knee extensions. Ring-13C6-phenylalanine was infused, and muscle biopsies were obtained. IMCT protein FSR was measured at 1–5, as well as 1–3 and 3–5 hours after contractions and nutrient...

  5. Myofibrillar protein synthesis following ingestion of soy protein isolate at rest and after resistance exercise in elderly men

    Directory of Open Access Journals (Sweden)

    Yang Yifan

    2012-06-01

    Full Text Available Abstract Background Increased amino acid availability stimulates muscle protein synthesis, however, aged muscle appears less responsive to the anabolic effects of amino acids when compared to the young. We aimed to compare changes in myofibrillar protein synthesis (MPS in elderly men at rest and after resistance exercise following ingestion of different doses of soy protein and compare the responses to those we previously observed with ingestion of whey protein isolate. Methods Thirty elderly men (age 71 ± 5 y completed a bout of unilateral knee-extensor resistance exercise prior to ingesting no protein (0 g, or either 20 g or 40 g of soy protein isolate (0, S20, and S40 respectively. We compared these responses to previous responses from similar aged men who had ingested 20 g and 40 g of whey protein isolate (W20 and W40. A primed constant infusion of L-[1-13 C]leucine and L-[ring-13 C6]phenylalanine and skeletal muscle biopsies were used to measure whole-body leucine oxidation and MPS over 4 h post-protein consumption in both exercised and non-exercised legs. Results Whole-body leucine oxidation increased with protein ingestion and was significantly greater for S20 vs. W20 (P = 0.003. Rates of MPS for S20 were less than W20 (P = 0.02 and not different from 0 g (P = 0.41 in both exercised and non-exercised leg muscles. For S40, MPS was also reduced compared with W40 under both rested and post-exercise conditions (both P P = 0.04. Conclusions The relationship between protein intake and MPS is both dose and protein source-dependent, with isolated soy showing a reduced ability, as compared to isolated whey protein, to stimulate MPS under both rested and post-exercise conditions. These differences may relate to the lower postprandial leucinemia and greater rates of amino acid oxidation following ingestion of soy versus whey protein.

  6. Enteral leucine and protein synthesis in skeletal and cardiac muscle

    Science.gov (United States)

    There are three members of the Branch Chain Amino Acids: leucine, isoleucine, and valine. As essential amino acids, these amino acids have important functions which include a primary role in protein structure and metabolism. It is intriguing that the requirement for BCAA in humans comprise about 40–...

  7. Acute propranolol infusion stimulates protein synthesis in rabbit skin wound.

    Science.gov (United States)

    Zhang, Xiao-Jun; Meng, Chengyue; Chinkes, David L; Finnerty, Celeste C; Aarsland, Asle; Jeschke, Marc G; Herndon, David N

    2009-05-01

    Propranolol administration has been demonstrated to improve cardiac work, decrease energy expenditure, and attenuate lipolysis in burned patients; however, its effect on wound healing has not been reported. In rabbits, a partial-thickness skin donor site wound was created on the back, and catheters were placed in the carotid artery and jugular vein. A nasogastric feeding tube was placed for enteral feeding. On day 5 after injury, stable isotope tracers were infused to determine protein and DNA kinetics in the wound. Propranolol hydrochloride was injected in 1 group during the tracer infusion to decrease heart rate, and the other group without propranolol injection served as a control. The propranolol infusion decreased heart rate by 21%. The protein fractional synthetic rate in the wound was greater in the propranolol group (8.6 +/- 0.9 vs 6.1 +/- 0.5%/day, P cascades in local wounds were not affected after propranolol treatment. Propranolol infusion increased wound protein synthetic rate and tended to increase wound protein deposition rate, which might be beneficial to wound healing. These changes might reflect a systemic response to the beta-adrenergic blockade.

  8. Visible light induced fast synthesis of protein-polymer conjugates: controllable polymerization and protein activity.

    Science.gov (United States)

    Li, Xin; Wang, Lei; Chen, Gaojian; Haddleton, David M; Chen, Hong

    2014-06-21

    Herein visible light is used to induce RAFT polymerization from protein for preparing protein-polymer conjugates at ambient temperature. Polymerization is fast and can be conveniently controlled with irradiation time. By site-specific polymerization of NIPAm to protein, the protein activity is maintained and in certain cases it presents an efficient on-off-switchable property.

  9. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae.

    Science.gov (United States)

    Venkata Mohan, S; Devi, M Prathima

    2014-08-01

    Influence of salinity as a stress factor to harness biodiesel was assessed during dual mode cultivation of microalgae by integrating biomass growth phase (BGP) and salinity induced lipid induction phase (LIP). BGP was evaluated in mixotrophic mode employing nutrients (NPK) and carbon (glucose) source while LIP was operated under stress environment with varying salt concentrations (0, 0.5, 1 and 2gNaCl/l). Salinity stress triggered both biomass growth and lipid synthesis in microalgae significantly. BGP showed higher increments in biomass growth (2.55g/l) while LIP showed higher lipid productivity (1gNaCl/l; total/neutral lipid, 23.4/9.2%) than BGP (total/neutral lipid, 15.2/6%). Lower concentrations of salinity showed positive influence on the process while higher concentrations showed marked inhibition. Salinity stress also facilitated in maintaining saturated fatty acid methyl esters in higher amounts which associates with the improved fuel properties. Efficient wastewater treatment was observed during BGP operation indicating the assimilation of carbon/nutrients by microalgae. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

  11. Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver.

    Science.gov (United States)

    León-López, Liliana; Márquez-Mota, Claudia C; Velázquez-Villegas, Laura A; Gálvez-Mariscal, Amanda; Arrieta-Báez, Daniel; Dávila-Ortiz, Gloria; Tovar, Armando R; Torres, Nimbe

    2015-09-01

    Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition.

  12. Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1.

    Science.gov (United States)

    Schaffrath, Raffael; Klassen, Roland

    2017-01-02

    Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm5s2U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm5s2U and Ψ38 in tRNAGlnUUG was shown to mediate efficient synthesis of the Q/N rich [PIN+] prion forming protein Rnq1. 1 In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNAGlnUUG, but not its isoacceptor tRNAGlnCUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm5s2U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.

  13. Influence of Protoplasmic Water Loss on the Control of Protein Synthesis in the Desiccation-Tolerant Moss Tortula ruralis 1

    Science.gov (United States)

    Oliver, Melvin J.

    1991-01-01

    Desiccation tolerance of the moss Tortula ruralis is characterized by a desiccation-induced change in gene expression that becomes evident upon rehydration. As reported earlier, this change in gene expression is apparently brought about by a change in the control of translation and does not include a major shift in mRNA abundance. A full qualitative and quantitative analysis of the alteration in gene expression, which is characterized by the loss of (or greater than fivefold decrease in) the synthesis of 25 hydration (h) proteins and initiation (or greater than fivefold increase) of the synthesis of 74 rehydration (r) proteins, is given in this report. Exposure to a desiccating atmosphere, for times that result in varying levels of water loss, enabled the determination that the control of synthesis of r proteins is different from the control of synthesis of h proteins. The r and h protein synthesis responses are internally coordinate, however. Similarly, the return to normal levels of h protein synthesis differs from that of the r proteins. The return to normal synthetic levels for all h proteins is synchronous, but the rate of loss of r protein synthesis varies with each individual r protein. Run-off translation of polysomes isolated from gametophytes during the drying phase demonstrates that there are no novel mRNAs recruited and no particular mRNA is favored for translation during desiccation. These findings add credence to the argument that translational control is the major component of the desiccation-induced alteration in gene expression in this plant, as discussed. Aspects of the response of protein synthesis to desiccation are consistent with the hypothesis that T. ruralis exhibits a repair-based mechanism of desiccation tolerance. ImagesFigure 2Figure 3Figure 5Figure 6Figure 7 PMID:16668577

  14. Plant Desiccation and Protein Synthesis. IV. RNA Synthesis, Stability, and Recruitment of RNA into Protein Synthesis during Desiccation and Rehydration of the Desiccation-Tolerant Moss, Tortula ruralis1

    Science.gov (United States)

    Oliver, Melvin J.; Bewley, J. Derek

    1984-01-01

    Upon rehydration of desiccated Tortula ruralis, RNA synthesis is immediately resumed; this resumption is quicker in moss recovering from slow drying than from rapid drying. Newly synthesized RNA enters the protein synthetic complex almost immediately upon rehydration, reaching control steady state levels within 2 hours after slow drying and 6 hours after rapid drying. RNA synthesized in the 1st hour following the readdition of water enters into polysomes much earlier after slow drying than after rapid drying. The RNA components of the protein synthetic complex are stable to desiccation at either slow or rapid speeds, although more so following the former drying regime. Immediately upon rehydration, these conserved RNA are readily utilized for protein synthesis, and continue to be so at least 4 hours thereafter. Hence, the speed of desiccation affects the rate at which RNA is synthesized upon subsequent rehydration, as well as the mode of utilization of that RNA. PMID:16663379

  15. Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress

    Science.gov (United States)

    Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ)induce oxidative stress by redox cycling, which generates hydrogen peroxide (H202). Cysteinylthio...

  16. Impact of Post-Translational Modifications of Crop Proteins under Abiotic Stress.

    Science.gov (United States)

    Hashiguchi, Akiko; Komatsu, Setsuko

    2016-12-21

    The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs) can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future.

  17. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response inSaccharomyces cerevisiae.

    Science.gov (United States)

    Kawazoe, Nozomi; Kimata, Yukio; Izawa, Shingo

    2017-01-01

    Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae , it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER) and unfolded protein response (UPR) has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v). Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1 Δ and hac1 Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid) and mild ethanol stress (5% ethanol) induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.

  18. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae

    Science.gov (United States)

    Kawazoe, Nozomi; Kimata, Yukio; Izawa, Shingo

    2017-01-01

    Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER) and unfolded protein response (UPR) has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v). Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid) and mild ethanol stress (5% ethanol) induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH. PMID:28702017

  19. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Nozomi Kawazoe

    2017-06-01

    Full Text Available Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER and unfolded protein response (UPR has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v. Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid and mild ethanol stress (5% ethanol induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.

  20. Translational control of protein synthesis: the early years.

    Science.gov (United States)

    Lodish, Harvey F

    2012-10-19

    For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and β-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments.

  1. Oxidative stress and mitochondrial protein quality control in aging.

    Science.gov (United States)

    Lionaki, Eirini; Tavernarakis, Nektarios

    2013-10-30

    Mitochondrial protein quality control incorporates an elaborate network of chaperones and proteases that survey the organelle for misfolded or unfolded proteins and toxic aggregates. Repair of misfolded or aggregated protein and proteolytic removal of irreversibly damaged proteins are carried out by the mitochondrial protein quality control system. Initial maturation and folding of the nuclear or mitochondrial-encoded mitochondrial proteins are mediated by processing peptidases and chaperones that interact with the protein translocation machinery. Mitochondrial proteins are subjected to cumulative oxidative damage. Thus, impairment of quality control processes may cause mitochondrial dysfunction. Aging has been associated with a marked decline in the effectiveness of mitochondrial protein quality control. Here, we present an overview of the chaperones and proteases involved in the initial folding and maturation of new, incoming precursor molecules, and the subsequent repair and removal of oxidized aggregated proteins. In addition, we highlight the link between mitochondrial protein quality control mechanisms and the aging process. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.

    Science.gov (United States)

    Signer, Robert A J; Qi, Le; Zhao, Zhiyu; Thompson, David; Sigova, Alla A; Fan, Zi Peng; DeMartino, George N; Young, Richard A; Sonenberg, Nahum; Morrison, Sean J

    2016-08-01

    Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis. © 2016 Signer et al.; Published by Cold Spring Harbor Laboratory Press.

  3. The stress response against denatured proteins in the deletion of cytosolic chaperones SSA1/2 is different from heat-shock response in Saccharomyces cerevisiae

    Science.gov (United States)

    Matsumoto, Rena; Akama, Kuniko; Rakwal, Randeep; Iwahashi, Hitoshi

    2005-01-01

    Background A yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. We investigated the genomic response at the level of mRNA expression to the deletion of SSA1/2 in comparison with the mild heat-shocked wild-type using cDNA microarray. Results Yeast cDNA microarray analysis revealed that genes involved in the stress response, including molecular chaperones, were up-regulated in a similar manner in both the ssa1/2 deletion mutant and the mild heat-shocked wild-type. Genes involved in protein synthesis were up-regulated in the ssa1/2 deletion mutant, but were markedly suppressed in the mild heat-shocked wild-type. The genes involved in ubiquitin-proteasome protein degradation were also up-regulated in the ssa1/2 deletion mutant, whereas the unfolded protein response (UPR) genes were highly expressed in the mild heat-shocked wild-type. RT-PCR confirmed that the genes regulating protein synthesis and cytosolic protein degradation were up-regulated in the ssa1/2 deletion mutant. At the translational level, more ubiquitinated proteins and proteasomes were detected in the ssa1/2 deletion mutant, than in the wild-type, confirming that ubiquitin-proteasome protein degradation was up-regulated by the deletion of SSA1/2. Conclusion These results suggest that the mechanism for rescue of denatured proteins in the ssa1/2 deletion mutant is different from that in the mild heat-shocked wild-type: Activated protein synthesis in the ssa1/2 deletion mutant supplies a deficiency of proteins by their degradation, whereas mild heat-shock induces UPR. PMID:16209719

  4. Cocaine-but not methamphetamine-associated memory requires de novo protein synthesis.

    Science.gov (United States)

    Kuo, Yu-Min; Liang, Keng Chen; Chen, Hsiang-Hua; Cherng, Chianfang G; Lee, Hsueh-Te; Lin, Yinchiu; Huang, A-Min; Liao, Ruey-Ming; Yu, Lung

    2007-01-01

    Context-induced drug craving and continuous drug use manifest the critical roles of specific memory episodes associated with the drug use experiences. Drug-induced conditioned place preference (CPP) in C57BL/6J mouse model, in this regard, is an appropriate behavioral paradigm to study such drug use-associated memories. Requirement of protein synthesis in various forms of long-term memory formation and storage has been phylogenetically demonstrated. This study was undertaken to study the requirement of protein synthesis in the learning and memory aspect of the conditioned place preference induced by cocaine and methamphetamine, two abused drugs of choice in local area. Since pCREB has been documented as a candidate substrate for mediating the drug-induced neuroadaptation, the pCREB level in hippocampus, nucleus accumbens, and prefrontal cortex was examined for its potential participation in the formation of CPP caused by these psychostimulants. We found that cocaine (2.5 and 5.0 mg/kg/dose)-induced CPP was abolished by the pretreatment of anisomycin (50 mg/kg/dose), a protein synthesis inhibitor, whereas methamphetamine (0.5 or 1.0 mg/kg/dose)-induced CPP was not affected by the anisomycin pretreatment. Likewise, cocaine-induced CPP was mitigated by another protein synthesis inhibitor, cycloheximide (15 mg/kg/injection) pretreatment, whereas methamphetamine-induced CPP remained intact by such pretreatment. Moreover, anisomycin treatment 2h after each drug-place pairing disrupted the cocaine-induced CPP, whereas the same treatment did not affect methamphetamine-induced CPP. An increase of accumbal pCREB level was found to associate with the learning phase of cocaine, but not with the learning phase of methamphetamine. We further found that intraaccumbal CREB antisense oligodeoxynucleotide infusion diminished cocaine-induced CPP, whereas did not affect the methamphetamine-induced CPP. Taken together, these data suggest that protein synthesis and accumbal CREB

  5. WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses

    Directory of Open Access Journals (Sweden)

    Aditya Banerjee

    2015-01-01

    Full Text Available WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research.

  6. WRKY proteins: signaling and regulation of expression during abiotic stress responses.

    Science.gov (United States)

    Banerjee, Aditya; Roychoudhury, Aryadeep

    2015-01-01

    WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research.

  7. WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses

    Science.gov (United States)

    Banerjee, Aditya

    2015-01-01

    WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research. PMID:25879071

  8. P-B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site-Specific Deuteration.

    Science.gov (United States)

    Jin, Kang; Li, Tianlu; Chow, Hoi Yee; Liu, Han; Li, Xuechen

    2017-11-13

    Cysteine-mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3 H; TCEP=tris(2-carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation-desulfurization approach. This method, termed P-B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2 O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post-translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ-synuclein, and histone H2A. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Change detection in the dynamics of an intracellular protein synthesis model using nonlinear Kalman filtering.

    Science.gov (United States)

    Rigatos, Gerasimos G; Rigatou, Efthymia G; Djida, Jean Daniel

    2015-10-01

    A method for early diagnosis of parametric changes in intracellular protein synthesis models (e.g. the p53 protein - mdm2 inhibitor model) is developed with the use of a nonlinear Kalman Filtering approach (Derivative-free nonlinear Kalman Filter) and of statistical change detection methods. The intracellular protein synthesis dynamic model is described by a set of coupled nonlinear differential equations. It is shown that such a dynamical system satisfies differential flatness properties and this allows to transform it, through a change of variables (diffeomorphism), to the so-called linear canonical form. For the linearized equivalent of the dynamical system, state estimation can be performed using the Kalman Filter recursion. Moreover, by applying an inverse transformation based on the previous diffeomorphism it becomes also possible to obtain estimates of the state variables of the initial nonlinear model. By comparing the output of the Kalman Filter (which is assumed to correspond to the undistorted dynamical model) with measurements obtained from the monitored protein synthesis system, a sequence of differences (residuals) is obtained. The statistical processing of the residuals with the use of x2 change detection tests, can provide indication within specific confidence intervals about parametric changes in the considered biological system and consequently indications about the appearance of specific diseases (e.g. malignancies).

  10. The Staphylococcus aureus Membrane Protein SA2056 Interacts with Peptidoglycan Synthesis Enzymes

    Directory of Open Access Journals (Sweden)

    Brigitte Berger-Bächi

    2013-01-01

    Full Text Available The yet uncharacterized membrane protein SA2056 belongs to the ubiquitous RND (Resistance-Nodulation-cell Division family of transmembrane efflux transporters. The sa2056 gene is located downstream of femX, the gene encoding the essential, non-ribosomal peptidyl-transferase adding the first glycine in the staphylococcal cell wall pentaglycine interpeptide. Due to its proximity to and weak co-transcription with femX, we assumed that sa2056 may somehow be involved in peptidoglycan synthesis. Specific antibodies against SA2056 showed that this protein is expressed during growth and present in the membrane fraction of cell preparations. Using a bacterial two hybrid system, SA2056 was shown to interact (i with itself, (ii with FemB, which adds glycines 4 and 5 to the peptidoglycan interpeptide and (iii with the essential penicillin binding proteins, PBP1 and PBP2, required for cell division and incorporation of the peptidoglycan into the cell wall. Unexpectedly, deletion of sa2056 led to no phenotype regarding growth, antibiotic resistances or cell morphology; nor did sa2056 deletion in combination with femB inactivation alter b-lactam and lysostaphin sensitivity and resistance, respectively, pointing to possible redundancy in the cell wall synthesis pathway. These results suggest an accessory role of SA2056 in S. aureus peptidoglycan synthesis, broadening the range of biological functions of RND proteins.

  11. Resistance exercise increases leg muscle protein synthesis and mTOR signalling independent of sex

    Science.gov (United States)

    Dreyer, Hans C.; Fujita, Satoshi; Glynn, Erin L.; Drummond, Micah J.; Volpi, Elena; Rasmussen, Blake B.

    2010-01-01

    Aim Sex differences are evident in human skeletal muscle as the cross-sectional area of individual muscle fibres is greater in men as compared to women. We have recently shown that resistance exercise stimulates 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, 9 male and 8 female, were studied in the fasted state before, during and for two hours following a bout of high-intensity leg resistance exercise. Mixed muscle protein fractional synthetic rate (FSR) 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 (P0.05). Akt phosphorylation increased in both groups at 1 hr post-exercise (P0.05). 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 hrs 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 be playing a role in the sexual dimorphism of leg skeletal muscle in young men and women. PMID:20070283

  12. Stress inhibition of melatonin synthesis in the pineal organ of rainbow trout (Oncorhynchus mykiss) is mediated by cortisol.

    Science.gov (United States)

    López-Patiño, Marcos A; Gesto, Manuel; Conde-Sieira, Marta; Soengas, José L; Míguez, Jesús M

    2014-04-15

    Cortisol has been suggested to mediate the effect of stress on pineal melatonin synthesis in fish. Therefore, we aimed to determine how pineal melatonin synthesis is affected by exposing rainbow trout to different stressors, such as hypoxia, chasing and high stocking density. In addition, to test the hypothesis that cortisol is a mediator of such stress-induced effects, a set of animals were intraperitoneally implanted with coconut oil alone or containing cortisol (50 mg kg(-1) body mass) and sampled 5 or 48 h post-injection at midday and midnight. The specificity of such effect was also assessed in cultured pineal organs exposed to cortisol alone or with the general glucocorticoid receptor antagonist, mifepristone (RU486). Stress (in particular chasing and high stocking density) affected the patterns of plasma and pineal organ melatonin content during both day and night, with the greatest reduction occurring at night. The decrease in nocturnal melatonin levels in the pineal organ of stressed fish was accompanied by increased serotonin content and decreased AANAT2 enzymatic activity and mRNA abundance. Similar effects on pineal melatonin synthesis to those elicited by stress were observed in trout implanted with cortisol for either 5 or 48 h. These data indicate that stress negatively influences the synthesis of melatonin in the pineal organ, thus attenuating the day-night variations of circulating melatonin. The effect might be mediated by increased cortisol, which binds to trout pineal organ-specific glucocorticoid receptors to modulate melatonin rhythms. Our results in cultured pineal organs support this. Considering the role of melatonin in the synchronization of daily and annual rhythms, the results suggest that stress-induced alterations in melatonin synthesis could affect the availability of fish to integrate rhythmic environmental information.

  13. Endoplasmic reticulum proteins quality control and the unfolded protein response: the regulative mechanism of organisms against stress injuries.

    Science.gov (United States)

    Fu, Xi Ling; Gao, Dong Sheng

    2014-01-01

    The endoplasmic reticulum is the cellular compartment in which secretory proteins are synthesized and folded. Perturbations of endoplasmic reticulum homeostasis lead to the accumulation of unfolded proteins. The activation of the unfolded protein response during endoplasmic reticulum stress transmits information about the status of protein folding to the cytosol and nucleus. The unfolded protein response leads to the upregulation of genes encoding endoplasmic reticulum chaperones, attenuation of translation, and initiation of the endoplasmic reticulum quality control system to restore endoplasmic reticulum homeostasis. When the unfolded protein response is insufficient to rebuild the steady state in endoplasmic reticulum, the programmed cell death or apoptosis would be initiated, by triggering cell injuries, even to cell death through apoptosis signals. In this review, we briefly outline research on the chaperones and foldases conserved in eukaryotes and plants, and describe the general principles and mechanisms of the endoplasmic reticulum quality control and the unfolded protein response. We describe the current models for the molecular mechanism of the unfolded protein response in plants, and emphasize the role of inositol requiring enzyme-1-dependent network in the unfolded protein response. Finally, we give a general overview of the directions for future research on the unfolded protein response in plants and its role in the response to environmental stresses. © 2014 International Union of Biochemistry and Molecular Biology.

  14. Bringing the science of proteins into the realm of organic chemistry: total chemical synthesis of SEP (synthetic erythropoiesis protein).

    Science.gov (United States)

    Kent, Stephen B H

    2013-11-11

    Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as "arguably the most successful drug spawned by the revolution in recombinant DNA technology". Recently, the EPO glycoprotein molecule has re-emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this "synthetic erythropoiesis protein" was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption.

    Directory of Open Access Journals (Sweden)

    Kei Fujiwara

    Full Text Available Cell-free protein synthesis (CFPS is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20-30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL.

  16. Exercise & NSAID: Effect on muscle protein synthesis in knee osteoarthritis patients?

    DEFF Research Database (Denmark)

    Petersen, S.G.; Miller, Ben F; Hansen, M

    2011-01-01

    the exercise-induced response of muscle contractile protein FSR. However, we cannot exclude that a minor inhibition of muscle sarcoplasmic proteins may have been present with NSAID treatment. This study suggests that muscle hypertrophy after long-term training is not influenced by NSAIDs.......PURPOSE:The purpose of this study was to determine muscle and tendon protein fractional synthesis rates (FSR) at rest and after a one-legged kicking exercise in patients with knee osteoarthritis (OA) receiving either placebo or nonsteroidal anti-inflammatory drugs (NSAIDs).METHODS:Twenty patients...... the contralateral leg remained rested. Twenty-four hours after exercise, we determined circulating concentrations of inflammatory parameters and measured FSR of myofibrillar and sarcoplasmic protein fractions of vastus lateralis muscle and patellar tendon collagen protein by the direct incorporation method using...

  17. Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1.

    Science.gov (United States)

    Loeser, Richard F; Gandhi, Uma; Long, David L; Yin, Weihong; Chubinskaya, Susan

    2014-08-01

    To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50-100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4. Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1- and OP-1-stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1-stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser(206) inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1-stimulated nuclear translocation of Smad4. These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling. Copyright © 2014 by the American College of Rheumatology.

  18. A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based

    Directory of Open Access Journals (Sweden)

    Hillebrecht Jason R

    2008-07-01

    Full Text Available Abstract Background Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications. Results Protein synthesis was investigated in vitro in a reconstituted prokaryotic system, a S30 extract-based system and a eukaryotic system. Compared to the S30 system, protein synthesis in the reconstituted system resulted in a reduced yield, and was more cold-sensitive. Supplementing the reconstituted system with fractions from a size-exclusion separation of the S30 extract significantly increased the yield and activity, to a level close to that of the S30 system. Though protein synthesis in both prokaryotic and eukaryotic systems showed no significant differences for eukaryotic reporter proteins, drastic differences were observed when an artificial fusion protein was synthesized in vitro. The prokaryotic systems failed to synthesize and correctly fold a significant amount of the full-length fusion protein, even when supplemented with the eukaryotic lysate. The active full-length fusion protein was synthesized only in the eukaryotic system. Conclusion The reconstituted bacterial system is sufficient but not efficient in protein synthesis. The S30 system by comparison contains additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate templates into active proteins.

  19. Top-down synthesis of versatile polyaspartamide linkers for single-step protein conjugation to materials.

    Science.gov (United States)

    Cha, Chaenyung; Jeong, Jae Hyun; Tang, Xin; Zill, Andrew T; Prakash, Y S; Zimmerman, Steven C; Saif, Taher A; Kong, Hyunjoon

    2011-12-21

    Materials used in various biological applications are often modified with proteins to regulate biomolecular and cellular adhesion. Conventional strategies of protein conjugation accompany monovalent bifunctional protein linkers, which present several limitations in molecular synthesis and protein conjugation. Herein, we present a new strategy of preparing multivalent polyaspartamide linkers in a simple top-down manner, and also demonstrate that the resulting polymer linkers allow us to readily conjugate proteins to both organic and inorganic materials. The top-down synthesis of polyaspartamide linkers was performed by partially opening succinimidyl ring moieties of polysuccinimide (PSI) with the controlled number of nucleophiles reactive to photo-cross-linked hydrogel or gold-coated inorganic materials: (1) Poly(2-hydroxyethyl-co-2-methacryloxyethyl aspartamide) (PHMAA) presenting methacrylate was used to micropattern fibronectin or collagen on a hydrogel in order to regulate cell adhesion and growth area on a micrometer scale. (2) Poly(2-hydroxyethyl-co-2-mercaptoethyl aspartamide) (PHMCA) presenting thiol functional groups was used to link fibronectin to a gold-coated silicon microelectromechanical probe designed to measure cell traction force. Overall, these multivalent polyaspartamide protein linkers will greatly assist efforts to analyze and regulate the cellular adhesion to and phenotypic activities of a wide array of substrates and devices.

  20. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  1. Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

    DEFF Research Database (Denmark)

    Ngara, Rudo; Ndimba, Roya; Borch-Jensen, Jonas

    2012-01-01

    sorghum variety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination...... with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p...

  2. Synthesis of nanoparticles with frog foam nest proteins

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hyo-Jick, E-mail: choihc@ucmail.uc.edu; Ebersbacher, Charles F. [University of Cincinnati, School of Energy, Environmental, Biological and Medical Engineering (United States); Myung, Nosang V. [University of California, Riverside, Department of Chemical and Environmental Engineering (United States); Montemagno, Carlo D., E-mail: montemcd@ucmail.uc.edu [University of Cincinnati, School of Energy, Environmental, Biological and Medical Engineering (United States)

    2012-09-15

    Microemulsions provide an efficient means of synthesizing monodispersed nanoparticles. Recent studies have demonstrated potential problems of surfactant due to the interaction with nanoparticles/precursors. To solve the problems, various types of chemical surfactants have been tested, but natural biosurfactants have not received a great deal of attention in engineering application. Here, we report the formation of microemulsions using frog foam nest protein, ranaspumin-2 (RSN-2), based on the hypothesis that RSN-2 assembles at the water-oil interface as a result of conformational change into an extended form. Fluorescence spectroscopic studies showed that RSN-2 undergoes a reversible transition between extended and globular conformation in foams/microemulsions and aqueous solution, respectively. Microemulsions were formulated with RSN-2 to synthesize 8-10 nm superparamagnetic iron oxide nanoparticles by mixing precursor-containing microemulsions with base-containing microemulsions. RSN-2 proteins were recovered from microemulsions and found to be recycled to make foams and microemulsions. Fluorescence spectroscopic analyses showed that RSN-2 maintained its mechanical agitation-induced amphiphilicity throughout multiple foaming/defoaming processes. These results suggest that conformational flexibility and structural stability of RSN-2 in aggressive environments enable the recycled use of RSN-2, elucidating the cost-effective advantage.

  3. Identification of nucleoid associated proteins (NAPs) under oxidative stress in Staphylococcus aureus.

    Science.gov (United States)

    Ushijima, Yuri; Ohniwa, Ryosuke L; Morikawa, Kazuya

    2017-10-02

    Bacterial nucleoid consists of genome DNA, RNA, and hundreds of nucleoid-associated proteins (NAPs). Escherichia coli nucleoid is compacted towards the stationary phase, replacing most log-phase NAPs with the major stationary-phase nucleoid protein, Dps. In contrast, Staphylococcus aureus nucleoid sustains the fiber structures throughout the growth. Instead, the Dps homologue, MrgA, expresses under oxidative stress conditions to clump the nucleoid, but the composition of the clumped nucleoid was elusive. The staphylococcal nucleoid under oxidative stress was isolated by sucrose gradient centrifugation, and the proteins were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We identified 299 proteins in the nucleoid under oxidative stress, including 113 csNAPs (contaminant-subtracted NAPs). Comparison with the previously identified csNAPs in log- and stationary phase indicated that one fifth of the csNAPs under oxidative stress were the constitutive nucleoid components; importantly, several factors including HU, SarA, FabZ, and ribosomes were sustained under oxidative stress. Some factors (e.g. SA1663 and SA0092/SA0093) with unknown functions were included in the csNAPs list specifically under oxidative stress condition. Nucleoid constitutively holds Hu, SarA, FabG, and ribosomal proteins even under the oxidative stress, reflecting the active functions of the clumped nucleoid, unlikely to the dormant E. coli nucleoid compacted in the stationary phase or starvation.

  4. Identification of proteins ınvolved in excess boron stress response in ...

    African Journals Online (AJOL)

    Aylin Eşiz Dereboylu

    2011-11-07

    Nov 7, 2011 ... Plants are constantly challenged by various biotic and abiotic stresses in nature. Boron toxicity have become one of the important abiotic stress factor for plants. Boron toxicity responses of plants is reflected by alterations in protein expression level, activity, location and concentration. In this study,.

  5. Overexpression of an abiotic-stress inducible plant protein in the ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-09-17

    Sep 17, 2008 ... The aim of our work was the overexpression of the abiotic stress-inducible dehydrin protein, namely. RAB16A, from rice in the BL21 ... various abiotic stress like water deficit, high salinity and low temperature or .... tomato Adh2 gene and Adh2 pseudogenes, and a study of Adh2 gene expression in fruit.

  6. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    Energy Technology Data Exchange (ETDEWEB)

    Klinefelter, G.R.; Hamilton, D.W.

    1985-11-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-(35S)methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-(35S)methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB(3H)4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.

  7. Coping with complexity: machine learning optimization of cell-free protein synthesis.

    Science.gov (United States)

    Caschera, Filippo; Bedau, Mark A; Buchanan, Andrew; Cawse, James; de Lucrezia, Davide; Gazzola, Gianluca; Hanczyc, Martin M; Packard, Norman H

    2011-09-01

    Biological systems contain complex metabolic pathways with many nonlinearities and synergies that make them difficult to predict from first principles. Protein synthesis is a canonical example of such a pathway. Here we show how cell-free protein synthesis may be improved through a series of iterated high-throughput experiments guided by a machine-learning algorithm implementing a form of evolutionary design of experiments (Evo-DoE). The algorithm predicts fruitful experiments from statistical models of the previous experimental results, combined with stochastic exploration of the experimental space. The desired experimental response, or evolutionary fitness, was defined as the yield of the target product, and new experimental conditions were discovered to have ∼ 350% greater yield than the standard. An analysis of the best experimental conditions discovered indicates that there are two distinct classes of kinetics, thus showing how our evolutionary design of experiments is capable of significant innovation, as well as gradual improvement. Copyright © 2011 Wiley Periodicals, Inc.

  8. Comparison of whole animal costs of protein synthesis among polar and temperate populations of the same species of gammarid amphipod.

    Science.gov (United States)

    Rastrick, S P S; Whiteley, N M

    2017-05-01

    Protein synthesis can account for a substantial proportion of metabolic rate. Energetic costs of protein synthesis, should in theory, be the same in marine invertebrates from a range of thermal habitats, and yet direct measurements using inhibitors produce widely differing values, especially in the cold. The present study aimed to remove any potential confounding interspecific effects by determining costs of protein synthesis in two latitudinally separated populations of the same species (amphipod, Gammarus oceanicus) living in two different thermal regimes; polar vs cold-temperate. Costs of protein synthesis were determined in summer acclimatised G. oceanicus from Svalbard (79°N) at 5°C and from Scotland (58°N) at 13°C. Amphipods were injected with the protein synthesis inhibitor, cycloheximide (CHX), at 9mmoll-1 in crab saline to give a tissue concentration of 0.05mgCHXg-1FW and left for 60min before the injection of [3H] phenylalanine. After incubation for 120min (180min in total from initial injection), both whole-animal rates of oxygen uptake and absolute rates of protein synthesis were significantly reduced in CHX-treated amphipods vs controls injected with saline. Both populations exhibited similar costs of protein synthesis of ~7μmolO2mg-1protein which is close to the estimated theoretical minimum for peptide bond formation, and similar to the values obtained in cell-free systems. The study demonstrates that in G. oceanicus, costs of protein synthesis rates were not elevated in the cold but were fixed among polar and cold-temperate populations. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    Science.gov (United States)

    Santiquet, Nicolas; Sasseville, Maxime; Laforest, Martin; Guillemette, Christine; Gilchrist, Robert B; Richard, François J

    2014-08-01

    The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian

  10. Oil and Protein Accumulation in Soybean Grains under Salinity Stress

    Directory of Open Access Journals (Sweden)

    Kazem GHASSEMI-GOLEZANI

    2010-06-01

    Full Text Available Two factorial experiments based on randomized complete block design (RCBD with three replications were conducted in 2007 and 2008, to evaluate grain development (four harvests and rate and duration of oil and protein accumulation in three soybean cultivars (Williams, Zan and L17 under a non-saline (control and three saline (3, 6 and 9 ds/m NaCl conditions. Six seeds were sown in each pot filled with 900 g perlite, using 144 pots for each experiment. After emergence, seedlings were thinned and 4 plants were kept in each pot. Rate of oil accumulation up to maturity was not significantly affected by salinity. With increasing salinity, rate and percentage of protein accumulation, duration of oil and protein accumulation and oil and protein content per grain decreased, but oil percentage increased. Oil and protein yields per plant decreased as salinity increased. These reductions were mainly attributed to the short duration of protein and oil accumulation and grain yield per plant under saline conditions. Williams had the highest rate and duration of protein accumulation and rate of oil accumulation, but L17 had the highest grain yield per plant. Consequently, differences in protein and oil yields per plant between these two cultivars were not statistically significant. However, Zan had the lowest protein and oil yields, due to the lowest grain yield per plant.

  11. Oil and Protein Accumulation in Soybean Grains under Salinity Stress

    Directory of Open Access Journals (Sweden)

    Kazem GHASSEMI-GOLEZANI

    2010-06-01

    Full Text Available Two factorial experiments based on randomized complete block design (RCBD with three replications were conducted in 2007 and 2008, to evaluate grain development (four harvests and rate and duration of oil and protein accumulation in three soybean cultivars (�Williams�, �Zan� and �L17� under a non-saline (control and three saline (3, 6 and 9 ds/m NaCl conditions. Six seeds were sown in each pot filled with 900 g perlite, using 144 pots for each experiment. After emergence, seedlings were thinned and 4 plants were kept in each pot. Rate of oil accumulation up to maturity was not significantly affected by salinity. With increasing salinity, rate and percentage of protein accumulation, duration of oil and protein accumulation and oil and protein content per grain decreased, but oil percentage increased. Oil and protein yields per plant decreased as salinity increased. These reductions were mainly attributed to the short duration of protein and oil accumulation and grain yield per plant under saline conditions. �Williams� had the highest rate and duration of protein accumulation and rate of oil accumulation, but �L17� had the highest grain yield per plant. Consequently, differences in protein and oil yields per plant between these two cultivars were not statistically significant. However, �Zan� had the lowest protein and oil yields, due to the lowest grain yield per plant.

  12. Oxidized mitochondrial protein degradation and repair in aging and oxidative stress.

    Science.gov (United States)

    Ugarte, Nicolas; Petropoulos, Isabelle; Friguet, Bertrand

    2010-08-15

    Proteins are main targets for oxidative damage that occurs during aging and in oxidative stress situations. Since the mitochondria is a major source of reactive oxygen species, mitochondrial proteins are especially exposed to oxidative modification, and elimination of oxidized proteins is crucial for maintaining the integrity of this organelle. Hence, enzymatic reversal of protein oxidation and protein degradation is critical for protein homeostasis while protein maintenance failure has been implicated in the age-related accumulation of oxidized proteins. Within the mitochondrial matrix, the ATP-stimulated mitochondrial Lon protease is believed to play an important role in the degradation of oxidized protein, and age-associated impairment of Lon-like protease activity has been suggested to contribute to oxidized protein buildup in the mitochondria. Oxidized protein repair is limited to certain oxidation products of the sulfur-containing amino acids cysteine and methionine. Oxidized protein repair systems, thioredoxin/thioredoxin reductase or glutaredoxin/glutathione/glutathione reductase that catalytically reduce disulfide bridges or sulfenic acids, and methionine sulfoxide reductase that reverses methionine sulfoxide back to methionine within proteins, are present in the mitochondrial matrix. Thus, the role of the mitochondrial Lon protease and the oxidized protein repair system methionine sulfoxide reductase is further addressed in the context of oxidative stress and aging.

  13. Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney.

    Science.gov (United States)

    Harris, Peter S; Roy, Samantha R; Coughlan, Christina; Orlicky, David J; Liang, Yongliang; Shearn, Colin T; Roede, James R; Fritz, Kristofer S

    2015-12-01

    In this study, we present the novel findings that chronic ethanol consumption induces mitochondrial protein hyperacetylation in the kidney and correlates with significantly increased renal oxidative stress. A major proteomic footprint of alcoholic liver disease (ALD) is an increase in hepatic mitochondrial protein acetylation. Protein hyperacetylation has been shown to alter enzymatic function of numerous proteins and plays a role in regulating metabolic processes. Renal mitochondrial targets of hyperacetylation include numerous metabolic and antioxidant pathways, such as lipid metabolism, oxidative phosphorylation, and amino acid metabolism, as well as glutathione and thioredoxin pathways. Disruption of protein lysine acetylation has the potential to impair renal function through metabolic dysregulation and decreased antioxidant capacity. Due to a significant elevation in ethanol-mediated renal oxidative stress, we highlight the acetylation of superoxide dismutase, peroxiredoxins, glutathione reductase, and glutathione transferase enzymes. Since oxidative stress is a known factor in ethanol-induced nephrotoxicity, we examined biochemical markers of protein hyperacetylation and oxidative stress. Our results demonstrate increased protein acetylation concurrent with depleted glutathione, altered Cys redox potential, and the presence of 4-HNE protein modifications in our 6-week model of early-stage alcoholic nephrotoxicity. These findings support the hypothesis that ethanol metabolism causes an influx of mitochondrial metabolic substrate, resulting in mitochondrial protein hyperacetylation with the potential to impact mitochondrial metabolic and antioxidant processes. Copyright © 2015. Published by Elsevier B.V.

  14. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

    Directory of Open Access Journals (Sweden)

    Sara Landeras-Bueno

    2016-04-01

    Full Text Available Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection.

  15. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

    Science.gov (United States)

    Loll-Krippleber, Raphael; Brown, Grant W

    2017-09-15

    mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

  16. Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes.

    Science.gov (United States)

    Anavi, Sarit; Ni, Zhixu; Tirosh, Oren; Fedorova, Maria

    2015-01-01

    Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes

    Directory of Open Access Journals (Sweden)

    Sarit Anavi

    2015-04-01

    Full Text Available Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA complex (1 mM, 2:1 oleic and palmitic acids. In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2. Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP. 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment.

  18. Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

    OpenAIRE

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf; Douthwaite, Stephen

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC50], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleo...

  19. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Downs, S.M. (Marquette Univ., Milwaukee, WI (USA))

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  20. Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J. [Queensland Univ., St. Lucia, QLD (Australia). Dept. of Chemistry

    1996-12-31

    The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6-{sup 3}H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

  1. Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells.

    Science.gov (United States)

    Kong, Xiangfeng; Wang, Xiaoqiu; Yin, Yulong; Li, Xilong; Gao, Haijun; Bazer, Fuller W; Wu, Guoyao

    2014-11-01

    Insufficient placental growth is a major factor contributing to intrauterine growth retardation in mammals. There is growing evidence that putrescine produced from arginine (Arg) and proline via ornithine decarboxylase is a key regulator of angiogenesis, embryogenesis, as well as placental and fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that putrescine stimulates protein synthesis by activating the mechanistic target of rapamycin (mTOR) signaling pathway in porcine trophectoderm cell line 2 cells. The cells were cultured for 2 to 4 days in customized Arg-free Dulbecco modified Eagle Ham medium containing 0, 10, 25, or 50 μM putrescine or 100 μM Arg. Cell proliferation, protein synthesis, and degradation, as well as the abundance of total and phosphorylated mTOR, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. Our results indicate that putrescine promotes cell proliferation and protein synthesis in a dose- and time-dependent manner, which was inhibited by difluoro-methylornithine (an inhibitor of ornithine decarboxylase). Moreover, supplementation of culture medium with putrescine increased the abundance of phosphorylated mTOR and its downstream targets, 4EBP1 and p70 S6K1 proteins. Collectively, these findings reveal a novel and important role for putrescine in regulating the mTOR signaling pathway in porcine placental cells. We suggest that dietary supplementation with or intravenous administration of putrescine may provide a new and effective strategy to improve survival and growth of embryos/fetuses in mammals. © 2014 by the Society for the Study of Reproduction, Inc.

  2. Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

    Directory of Open Access Journals (Sweden)

    Chen Guo

    2010-05-01

    Full Text Available Abstract Background PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation. Results Fusion protein PhaR-intein-target protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP, maltose binding protein (MBP and β-galactosidase (lacZ, were successfully purified using the PhaR based protein purification method. Conclusion The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.

  3. Feedback Control of Snf1 Protein and Its Phosphorylation Is Necessary for Adaptation to Environmental Stress*

    Science.gov (United States)

    Hsu, Hsiang-En; Liu, Tzu-Ning; Yeh, Chung-Shu; Chang, Tien-Hsien; Lo, Yi-Chen; Kao, Cheng-Fu

    2015-01-01

    Snf1, a member of the AMP-activated protein kinase family, plays a critical role in metabolic energy control in yeast cells. Snf1 activity is activated by phosphorylation of Thr-210 on the activation loop of its catalytic subunit; following activation, Snf1 regulates stress-responsive transcription factors. Here, we report that the level of Snf1 protein is dramatically decreased in a UBP8- and UBP10-deleted yeast mutant (ubp8Δ ubp10Δ), and this is independent of transcriptional regulation and proteasome-mediated degradation. Surprisingly, most Snf1-mediated functions, including glucose limitation regulation, utilization of alternative carbon sources, stress responses, and aging, are unaffected in this strain. Snf1 phosphorylation in ubp8Δ ubp10Δ cells is hyperactivated upon stress, which may compensate for the loss of the Snf1 protein and protect cells against stress and aging. Furthermore, artificial elevation of Snf1 phosphorylation (accomplished through deletion of REG1, which encodes a protein that regulates Snf1 dephosphorylation) restored Snf1 protein levels and the regulation of Snf1 activity in ubp8Δ ubp10Δ cells. Our results reveal the existence of a feedback loop that controls Snf1 protein level and its phosphorylation, which is masked by Ubp8 and Ubp10 through an unknown mechanism. We propose that this dynamic modulation of Snf1 phosphorylation and its protein level may be important for adaptation to environmental stress. PMID:25947383

  4. Amnesia produced by altered release of neurotransmitters after intraamygdala injections of a protein synthesis inhibitor

    Science.gov (United States)

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2007-01-01

    Amnesia produced by protein synthesis inhibitors such as anisomycin provides major support for the prevalent view that the formation of long-lasting memories requires de novo protein synthesis. However, inhibition of protein synthesis might disrupt other neural functions to interfere with memory formation. Intraamygdala injections of anisomycin before inhibitory avoidance training impaired memory in rats tested 48 h later. Release of norepinephrine (NE), dopamine (DA), and serotonin, measured at the site of anisomycin infusions, increased quickly by ≈1,000–17,000%, far above the levels seen under normal conditions. NE and DA release later decreased far below baseline for several hours before recovering at 48 h. Intraamygdala injections of a β-adrenergic receptor antagonist or agonist, each timed to blunt effects of increases and decreases in NE release after anisomycin, attenuated anisomycin-induced amnesia. In addition, similar to the effects on memory seen with anisomycin, intraamygdala injections of a high dose of NE before training impaired memory tested at 48 h after training. These findings suggest that altered release of neurotransmitters may mediate amnesia produced by anisomycin and, further, raise important questions about the empirical bases for many molecular theories of memory formation. PMID:17640910

  5. A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

    Science.gov (United States)

    Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko; Nitharwal, Ram G.; Takase, Ryuichi; Han, Kyu Young; Leslie, Benjamin J.; Liu, Cuiping; Gamper, Howard; Ha, Taekjip; Sanyal, Suparna

    2017-01-01

    Abstract Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering. PMID:27956502

  6. Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

    Directory of Open Access Journals (Sweden)

    Mark A Johnson

    Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

  7. Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

    Science.gov (United States)

    Pearce, Sarah F.; Rorbach, Joanna; He, Jiuya; Brea-Calvo, Gloria; Minczuk, Michal; Reyes, Aurelio; Holt, Ian J.; Spinazzola, Antonella

    2014-01-01

    Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation. PMID:24718614

  8. Effects of alimet on nutrient digestibility, bacterial protein synthesis, and ruminal disappearance during continuous culture.

    Science.gov (United States)

    Vázquez-Añón, M; Cassidy, T; McCullough, P; Varga, G A

    2001-01-01

    A dual effluent continuous culture system was used to investigate the effects of inclusion of Alimet (Novus International, Inc., St. Louis, MO) feed supplement [an 88% aqueous solution of dl, 2-hydroxy-4-(methylthio) butanoic acid (HMB)] in the diet on nutrient digestibility, bacterial protein synthesis and ruminal disappearance of HMB. Four fermenters were fed three times daily a basal diet that consisted of 50% grain mixture and 50% forage for 9 d. In experiment 1, four concentrations of HMB (0, 0.20, 0.77, and 1.43% DM basis) were added to the diet and fed to the fermenters twice daily. In experiment 2, two concentrations of dietary HMB (0 and 0.88% DM basis) were fed twice daily and evaluated with two solids retention times (16.7 vs. 25.0 h) and two liquid dilution rates (0.15 vs. 0.125 h(-1)). Increasing the amount of HMB in the diet did not affect nutrient digestibility, volatile fatty acid concentrations, or ruminal escape of HMB. Bacterial protein synthesis was improved with the addition of HMB during high and low retention times. The extent of HMB escaping ruminal degradation ranged from 21.6 to 43.2% and was highest at the lower retention time. It can be concluded that a fraction of HMB survives rumen microbial degradation and, therefore, provides a rumen-protected form of methionine at the same time that it improves bacterial protein synthesis.

  9. Impact of osmotic stress on protein diffusion in Lactococcus lactis

    NARCIS (Netherlands)

    Mika, Jacek; Schavemaker, Paul; Krasnikov, Viktor; Poolman, Bert

    2014-01-01

    We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane

  10. The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

    Directory of Open Access Journals (Sweden)

    Proud Christopher G

    2002-05-01

    Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

  11. Global analysis of protein folding using massively parallel design, synthesis, and testing.

    Science.gov (United States)

    Rocklin, Gabriel J; Chidyausiku, Tamuka M; Goreshnik, Inna; Ford, Alex; Houliston, Scott; Lemak, Alexander; Carter, Lauren; Ravichandran, Rashmi; Mulligan, Vikram K; Chevalier, Aaron; Arrowsmith, Cheryl H; Baker, David

    2017-07-14

    Proteins fold into unique native structures stabilized by thousands of weak interactions that collectively overcome the entropic cost of folding. Although these forces are "encoded" in the thousands of known protein structures, "decoding" them is challenging because of the complexity of natural proteins that have evolved for function, not stability. We combined computational protein design, next-generation gene synthesis, and a high-throughput protease susceptibility assay to measure folding and stability for more than 15,000 de novo designed miniproteins, 1000 natural proteins, 10,000 point mutants, and 30,000 negative control sequences. This analysis identified more than 2500 stable designed proteins in four basic folds-a number sufficient to enable us to systematically examine how sequence determines folding and stability in uncharted protein space. Iteration between design and experiment increased the design success rate from 6% to 47%, produced stable proteins unlike those found in nature for topologies where design was initially unsuccessful, and revealed subtle contributions to stability as designs became increasingly optimized. Our approach achieves the long-standing goal of a tight feedback cycle between computation and experiment and has the potential to transform computational protein design into a data-driven science. Copyright © 2017, American Association for the Advancement of Science.

  12. Protein mediated synthesis of fluorescent Au-nanoclusters for metal sensory coatings

    Energy Technology Data Exchange (ETDEWEB)

    Vogel, Manja; Raff, Johannes [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biogeochemistry

    2017-06-01

    Fluorescent Au-nanocluster were successfully synthesized and used for the selective detection of Cu{sup 2} {sup +}. The synthesized Au-BSA-nanoclusters remain functional also after immobilization and show high thermal stability. Additionally, the transfer of the protein mediated Au-nanocluster synthesis route to S-layer proteins was achieved. (The presented work is part of the project BIONEWS dealing with long-term stable cells for the set-up and regeneration of sensor and actor materials for strategic relevant metals, in particular rare earth elements).

  13. Differential Analysis of Protein Expression in RNA-Binding-Protein Transgenic and Parental Rice Seeds Cultivated under Salt Stress

    OpenAIRE

    Nakamura, Rika; Nakamura, Ryosuke; Adachi, Reiko; Hachisuka, Akiko; Yamada, Akiyo; Ozeki, Yoshihiro; Teshima, Reiko

    2014-01-01

    Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in pr...

  14. CCM proteins control endothelial β1 integrin dependent response to shear stress

    Directory of Open Access Journals (Sweden)

    Zuzana Macek Jilkova

    2014-11-01

    Full Text Available Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that β1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated “shear-stress-like” phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.

  15. COMPARISON OF STRESS PROTEINS PARTICIPATION IN ADAPTATION MECHANISMS OF BAIKALIAN AND PALEARCTIC AMPHIPOD (AMPHIPODA; CRUSTACEA SPECIES

    Directory of Open Access Journals (Sweden)

    Timofeyev M.A

    2006-03-01

    Full Text Available The aim of the present study was a study of the influence different stressful factor on syntheses and activity of the stress proteins (HSP70, sHSP and peroxidase of freshwater organism. Six freshwater amphipod species were investigated: Eulimnogammarus cyaneus (Dyb., E verrucosus (Gerstf., E vittatus (Dyb. - endemic species from Lake Baikal which were compared with Palearctic species - Gammarus lacustris Sars., G tigrinus (Sexton, Chaetogammarus ischnus (Stebbins. It was shown expression of sHSP by heat and toxic stresses for all amphipods species. Oxidative stress induced HSP70 for Palearctic species G tigrinus and C ischnus but not for baikalian species. Heat stress did not caused the increase of HSP70 level for Baikalian species of amphipods. The activity of the peroxidase was decrease by heat and toxic stresses. Oxidative stress caused the increase of peroxidase activity for Palearctic species, and the decrease for Baikalian once.

  16. Proteomic quantification and identification of carbonylated proteins upon oxidative stress and during cellular aging.

    Science.gov (United States)

    Baraibar, Martin A; Ladouce, Romain; Friguet, Bertrand

    2013-10-30

    Increased protein carbonyl content is a hallmark of cellular and organismal aging. Protein damage leading to the formation of carbonyl groups derives from direct oxidation of several amino acid side chains but can also derive through protein adducts formation with lipid peroxidation products and dicarbonyl glycating compounds. All these modifications have been implicated during oxidative stress, aging and age-related diseases. However, in most cases, the proteins targeted by these deleterious modifications as well as their consequences have not yet been clearly identified. Indeed, this is essential to determine whether and how these modified proteins are impacting on cellular function, on the development of the senescent phenotype and the pathogenesis of age-related diseases. In this context, protein modifications occurring during aging and upon oxidative stress as well as main proteomic methods for detecting, quantifying and identifying oxidized proteins are described. Relevant proteomics studies aimed at monitoring the extent of protein carbonylation and identifying the targeted proteins in the context of aging and oxidative stress are also presented. Proteomics approaches, i.e. fluorescent based 2D-gel electrophoresis and mass spectrometry methods, represent powerful tools for monitoring at the proteome level the extent of protein oxidative and related modifications and for identifying the targeted proteins. Accumulation of damaged macromolecules, including oxidatively damaged (carbonylated) proteins, is a hallmark of cellular and organismal aging. Since protein carbonyls are the most commonly used markers of protein oxidation, different methods have been developed for the detection and quantification of carbonylated proteins. The identification of these protein targets is of valuable interest in order to understand the mechanisms by which damaged proteins accumulate and potentially affect cellular functions during oxidative stress, cellular senescence and

  17. Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes.

    Directory of Open Access Journals (Sweden)

    Noriko Umegaki-Arao

    Full Text Available Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2 expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

  18. [Influence of different sticky-gene composition on sporulation and protein synthesis by Bacillus thuringiensis collection strains].

    Science.gov (United States)

    2014-01-01

    The influence of different sticky-gene composition on sporulation and protein synthesis by B. thuringiensis collection strains has been investigated. It has been detemined that the most effective according this characteristics were B. thuringiensis collection strains 0293 and 98. It has been shown that the best on protein synthesis processes and sporulation by investigated B. thuringiensis strains influences adding to the culture medium sticky-gene compositions A and E in a concentration of from 10 to 15%.

  19. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    Science.gov (United States)

    Li, Shikuo; Shen, Yuhua; Xie, Anjian; Yu, Xuerong; Zhang, Xiuzhen; Yang, Liangbao; Li, Chuanhao

    2007-10-01

    We describe the formation of amorphous selenium (α-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO32-) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO32- ions to Se0, but also controls the nucleation and growth of Se0, and even participates in the formation of α-Se/protein composites. The size and shell thickness of the α-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO32- ions by Capsicum annuum L extract.

  20. A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

    KAUST Repository

    Zhu, Jianhua

    2010-04-16

    Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress. © 2010 Blackwell Publishing Ltd.

  1. The homeodomain protein ladybird late regulates synthesis of milk proteins during pregnancy in the tsetse fly (Glossina morsitans.

    Directory of Open Access Journals (Sweden)

    Geoffrey M Attardo

    2014-04-01

    Full Text Available Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina, the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1 by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This

  2. Sodium nitrate co-ingestion with protein does not augment postprandial muscle protein synthesis rates in older, type 2 diabetes patients.

    Science.gov (United States)

    Kouw, Imre W K; Cermak, Naomi M; Burd, Nicholas A; Churchward-Venne, Tyler A; Senden, Joan M; Gijsen, Annemarie P; van Loon, Luc J C

    2016-08-01

    The age-related anabolic resistance to protein ingestion is suggested to be associated with impairments in insulin-mediated capillary recruitment and postprandial muscle tissue perfusion. The present study investigated whether dietary nitrate co-ingestion with protein improves muscle protein synthesis in older, type 2 diabetes patients. Twenty-four men with type 2 diabetes (72 ± 1 yr, 26.7 ± 1.4 m/kg(2) body mass index, 7.3 ± 0.4% HbA1C) received a primed continuous infusion of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine and ingested 20 g of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled protein with (PRONO3) or without (PRO) sodium nitrate (0.15 mmol/kg). Blood and muscle samples were collected to assess protein digestion and absorption kinetics and postprandial muscle protein synthesis rates. Upon protein ingestion, exogenous phenylalanine appearance rates increased in both groups (P nitrate co-ingestion with protein does not modulate protein digestion and absorption kinetics, nor does it further increase postprandial muscle protein synthesis rates or the incorporation of dietary protein-derived amino acids into de novo myofibrillar protein in older, type 2 diabetes patients. Copyright © 2016 the American Physiological Society.

  3. Endogenous stress proteins as targets for anti-inflammatory T cells

    NARCIS (Netherlands)

    Wieten, L.

    2009-01-01

    Stress proteins such as heat shock proteins (Hsp) are important controllers of both cellular and immune homeostasis. Enhanced Hsp expression can be observed in virtually every inflammatory condition and has been proposed by us and others to lead to local activation of Hsp-specific anti-inflammatory

  4. Mechanisms of autoprotection and the role of stress-proteins in natural defenses, autoprotection, and salutogenesis

    NARCIS (Netherlands)

    Schaefer, J; Nierhaus, KH; Lohff, B; Peters, T; Schaefer, T; Vos, R

    We hypothesize that in all physiotherapeutically oriented procedures of naturotherapy - such as helio-, climate-, thalasso- or hydrotherapy or certain forms of physical exercise - the transient expression of stress-proteins (heat-shock proteins, HSPs) is an important element of salutogenesis. These

  5. GCN2 has inhibitory effect on human immunodeficiency virus-1 protein synthesis and is cleaved upon viral infection.

    Directory of Open Access Journals (Sweden)

    Javier del Pino

    Full Text Available The reversible phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses, including viral infection. Beside PKR, the eIF2alpha kinase GCN2 participates in the cellular response against viral infection by RNA viruses with central nervous system tropism. PKR has also been involved in the antiviral response against HIV-1, although this antiviral effect is very limited due to the distinct mechanisms evolved by the virus to counteract PKR action. Here we report that infection of human cells with HIV-1 conveys the proteolytic cleavage of GCN2 and that purified HIV-1 and HIV-2 proteases produce direct proteolysis of GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Transfection of distinct cell lines with a plasmid encoding an HIV-1 cDNA clone competent for a single round of replication resulted in the activation of GCN2 and the subsequent eIF2alpha phosphorylation. Moreover, transfection of GCN2 knockout cells or cells with low levels of phosphorylated eIF2alpha with the same HIV-1 cDNA clone resulted in a marked increase of HIV-1 protein synthesis. Also, the over-expression of GCN2 in cells led to a diminished viral protein synthesis. These findings suggest that viral RNA produced during HIV-1 infection activates GCN2 leading to inhibition of viral RNA translation, and that HIV-1 protease cleaves GCN2 to overcome its antiviral effect.

  6. Amino acids stimulate leg muscle protein synthesis in peripheral arterial disease.

    Science.gov (United States)

    Killewich, Lois A; Tuvdendorj, Demidmaa; Bahadorani, John; Hunter, Glenn C; Wolfe, Robert R

    2007-03-01

    Older patients with peripheral arterial disease (PAD) and intermittent claudication have impaired walking ability resulting from reduced lower extremity blood flow. Evidence suggests that leg muscle abnormalities may also contribute to walking intolerance in claudicants. In healthy elderly people, leg muscle protein synthesis can be augmented by nutritional supplementation with amino acids; preliminary data suggest that this increases muscle mass, walking ability, and functional status. In this study, we investigated whether amino acid supplementation would improve leg muscle protein synthesis in elderly PAD subjects, given that reduced leg blood flow might restrict the availability of amino acids to muscle. Two groups participated in the study: a group of 11 claudicants (mean age, 62 years; mean ankle-brachial index, 0.62; 46% male) and a group of 9 age- and sex-matched healthy controls (mean ankle-brachial index, 1.1). Both groups underwent measurement of leg blood flow by using strain gauge plethysmography, as well as measurement of baseline and amino acid-stimulated protein synthesis in leg muscle. Protein synthesis was quantified from calf muscle biopsy samples by measurement of the fractional synthetic rate (FSR) of protein, by using the incorporation of the stable isotope l-[ring-(2)H(5)]-phenylalanine into muscle protein. Total protein was extracted from muscle samples, and gas chromatography/mass spectroscopy methodology was used to measure incorporation rates. After measurement of basal FSR, all subjects were given an oral drink of 15 g of essential amino acids, and the measurements of FSR were repeated. Data are expressed as mean +/- SD; statistical analysis of differences between the two groups (with and without amino acid supplementation) was performed by using analysis of variance with repeated measures. Calf blood flow was reduced in the PAD subjects compared with controls (1.44 +/- 0.53 mL/min per 100 mg of tissue vs 2.40 +/- 0.57 mL/min per 100 mg

  7. Removable Backbone Modification Method for the Chemical Synthesis of Membrane Proteins.

    Science.gov (United States)

    Li, Jia-Bin; Tang, Shan; Zheng, Ji-Shen; Tian, Chang-Lin; Liu, Lei

    2017-05-16

    Chemical synthesis can produce water-soluble globular proteins bearing specifically designed modifications. These synthetic molecules have been used to study the biological functions of proteins and to improve the pharmacological properties of protein drugs. However, the above advances notwithstanding, membrane proteins (MPs), which comprise 20-30% of all proteins in the proteomes of most eukaryotic cells, remain elusive with regard to chemical synthesis. This difficulty stems from the strong hydrophobic character of MPs, which can cause considerable handling issues during ligation, purification, and characterization steps. Considerable efforts have been made to improve the solubility of transmembrane peptides for chemical ligation. These methods can be classified into two main categories: the manipulation of external factors and chemical modification of the peptide. This Account summarizes our research advances in the development of chemical modification especially the two generations of removable backbone modification (RBM) strategy for the chemical synthesis of MPs. In the first RBM generation, we install a removable modification group at the backbone amide of Gly within the transmembrane peptides. In the second RBM generation, the RBM group can be installed into all primary amino acid residues. The second RBM strategy combines the activated intramolecular O-to-N acyl transfer reaction, in which a phenyl group remains unprotected during the coupling process, which can play a catalytic role to generate the activated phenyl ester to assist in the formation of amide. The key feature of the RBM group is its switchable stability in trifluoroacetic acid. The stability of these backbone amide N-modifications toward TFA can be modified by regulating the electronic effects of phenol groups. The free phenol group is acylated to survive the TFA deprotection step, while the acyl phenyl ester will be quantitatively hydrolyzed in a neutral aqueous solution, and the free

  8. Reciprocal regulation of protein synthesis and carbon metabolism for thylakoid membrane biogenesis.

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    Alexandra-Viola Bohne

    Full Text Available Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2, a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC, which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.

  9. Natural thermal adaptation increases heat shock protein levels and decreases oxidative stress

    Directory of Open Access Journals (Sweden)

    Niku K.J. Oksala

    2014-01-01

    Full Text Available Heat shock proteins (HSPs, originally identified as heat-inducible gene products, are a family of highly conserved proteins that respond to a wide variety of stress including oxidative stress. Although both acute and chronic oxidative stress have been well demonstrated to induce HSP responses, little evidence is available whether increased HSP levels provide enhanced protection against oxidative stress under elevated yet sublethal temperatures. We studied relationships between oxidative stress and HSPs in a physiological model by using Garra rufa (doctor fish, a fish species naturally acclimatized to different thermal conditions. We compared fish naturally living in a hot spring with relatively high water temperature (34.4±0.6 °C to those living in normal river water temperature (25.4±4.7 °C, and found that levels of all the studied HSPs (HSP70, HSP60, HSP90, HSC70 and GRP75 were higher in fish living in elevated water temperature compared with normal river water temperature. In contrast, indicators of oxidative stress, including protein carbonyls and lipid hydroperoxides, were decreased in fish living in the elevated temperature, indicating that HSP levels are inversely associated with oxidative stress. The present results provide evidence that physiologically increased HSP levels provide protection against oxidative stress and enhance cytoprotection.

  10. Evaluation of differential protein expression in Haliclona aquarius and sponge-associated microorganisms under cadmium stress.

    Science.gov (United States)

    Wanick, Rodrigo Cunha; de Sousa Barbosa, Herbert; Frazão, Leonardo Revoredo; Santelli, Ricardo Erthal; Arruda, Marco Aurélio Zezzi; Coutinho, Cristiano Carvalho

    2013-09-01

    A comparative proteomic approach was used to assess differentially expressed proteins in marine sponges after 36 h of exposure to cadmium (Cd). After separation performed by 2-D polyacrylamide gel electrophoresis, 46 protein spots indicated differential expression, and 17 of these proteins were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. From the proteins identified, 76% were attributed to sponge-associated microorganisms (fungi and bacteria), and 24% were attributed to Haliclona aquarius. Some of the proteins that were identified may be related to cell proliferation and differentiation or processes of oxidative stress repair and energy procurement. An integrated evaluation based on spot expression levels and the postulated functions of these proteins allowed a more accurate evaluation of the stress caused to the sponge holobiont system by cadmium exposure. This study could provide new insights into the use of a proteomic approach in the marine sponge to assess the effects of Cd pollution in a marine environment.

  11. Production of Functional Proteins: Balance of Shear Stress and Gravity

    Science.gov (United States)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Haysen, James Howard (Inventor)

    2005-01-01

    The present invention provides for a method of culturing cells and inducing the expression of at least one gene in the cell culture. The method provides for contacting the cell with a transcription factor decoy oligonucleotide sequence directed against a nucleotide sequence encoding a shear stress response element.

  12. Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

    DEFF Research Database (Denmark)

    Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M

    2015-01-01

    In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome...

  13. Sulfide induction of synthesis of a periplasmic protein in the cyanobacterium Oscillatoria limnetica.

    Science.gov (United States)

    Arieli, B; Binder, B; Shahak, Y; Padan, E

    1989-02-01

    Two proteins which may play a role in the induction of anoxygenic photosynthesis in Oscillatoria limnetica have been demonstrated by comparing the pattern of labeling during pulses of [35S]methionine of cells incubated under inducing conditions [anaerobic conditions plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea, light, and sulfide) with that of cells incubated under noninducing conditions (without sulfide). The major inducible protein has an apparent molecular mass of 11.5 kilodaltons and is associated with a less strongly labeled 12.5-kilodalton protein. The synthesis of both proteins commences within the first 30 min of induction and continues throughout the 2-h induction period. Since these proteins are not synthesized in the presence of dithionite without sulfide, low redox potential alone is insufficient as an inducer of these proteins. Lysozyme treatment and/or osmotic shock of intact cells results in the release of the sulfide-induced proteins. Our data thus indicate that these proteins are located in the periplasmic space of the cells.

  14. Abiotic stress responses in plants: roles of calmodulin-regulated proteins

    Science.gov (United States)

    Virdi, Amardeep S.; Singh, Supreet; Singh, Prabhjeet

    2015-01-01

    Intracellular changes in calcium ions (Ca2+) in response to different biotic and abiotic stimuli are detected by various sensor proteins in the plant cell. Calmodulin (CaM) is one of the most extensively studied Ca2+-sensing proteins and has been shown to be involved in transduction of Ca2+ signals. After interacting with Ca2+, CaM undergoes conformational change and influences the activities of a diverse range of CaM-binding proteins. A number of CaM-binding proteins have also been implicated in stress responses in plants, highlighting the central role played by CaM in adaptation to adverse environmental conditions. Stress adaptation in plants is a highly complex and multigenic response. Identification and characterization of CaM-modulated proteins in relation to different abiotic stresses could, therefore, prove to be essential for a deeper understanding of the molecular mechanisms involved in abiotic stress tolerance in plants. Various studies have revealed involvement of CaM in regulation of metal ions uptake, generation of reactive oxygen species and modulation of transcription factors such as CAMTA3, GTL1, and WRKY39. Activities of several kinases and phosphatases have also been shown to be modulated by CaM, thus providing further versatility to stress-associated signal transduction pathways. The results obtained from contemporary studies are consistent with the proposed role of CaM as an integrator of different stress signaling pathways, which allows plants to maintain homeostasis between different cellular processes. In this review, we have attempted to present the current state of understanding of the role of CaM in modulating different stress-regulated proteins and its implications in augmenting abiotic stress tolerance in plants. PMID:26528296

  15. Sm-like protein-mediated RNA metabolism is required for heat stress tolerance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Masanori Okamoto

    2016-07-01

    Full Text Available Sm-like proteins play multiple functions in RNA metabolism, which is essential for biological processes such as stress responses in eukaryotes. The Arabidopsis thaliana sad1 mutant has a mutation of sm-like protein 5 (LSM5 and shows impaired drought and salt stress tolerances. The lsm5/sad1 mutant also showed hypersensitivity to heat stress. GFP-fused LSM5/SAD1 was localized in the nucleus under optimal growth conditions. After heat stress treatment, GFP-fused LSM5/SAD1 fluorescence was also observed as small cytoplasmic dots, in addition to nuclear localization. Whole genome transcriptome analysis revealed that many genes in Arabidopsis were drastically changed in response to heat stress. More heat-responsive genes were highly expressed in lsm5/sad1 mutant at both 2 h and 6 h after heat stress treatment. Additionally, intron-retained and capped transcripts accumulated in the lsm5/sad1 mutant after heat stress treatment. In this study, we also identified non-Arabidopsis Genome Initiative (AGI transcripts that were expressed from unannotated regions. Most of these transcripts were antisense transcripts, and many capped non-AGI transcripts accumulated in the lsm5/sad1 mutant during heat stress treatment. These results indicated that LSM5/SAD1 functions to degrade aberrant transcripts through appropriate mRNA splicing and decapping, and precise RNA metabolic machinery is required for heat stress tolerance.

  16. Elevated C-reactive protein in adolescents: roles of stress and coping.

    Science.gov (United States)

    Low, Carissa A; Matthews, Karen A; Hall, Martica

    2013-06-01

    Psychological stress can up-regulate inflammatory processes and increase disease risk. In the context of stress, differences in how individuals cope might have implications for health. The goal of this study was to evaluate associations among stress, coping, and inflammation in a sample of African American and white adolescents. Adolescents (n = 245) completed self-report measures of stressful life events and coping, provided daily diary reports of interpersonal conflict for 7 days, and provided fasting blood samples for assessment of C-reactive protein (CRP). In regression analyses adjusted for age, sex, race, body mass index, smoking, and socioeconomic status, there were no significant associations between stress and CRP, but significant interactions between stress and coping emerged. For adolescents reporting more unpleasant stressful life events in the past 12 months, positive engagement coping was inversely associated with CRP (β= -.19, p adolescents reporting fewer stressful life events. Positive engagement coping was significantly and inversely associated with CRP in the context of interpersonal stress, whether measured as stressful life events reflecting interpersonal conflict (e.g., arguments with parents or siblings, conflict between adults in the home, and friendship ended) or frequency of arguments with others reported in daily diaries. Disengagement coping was unrelated to CRP. Findings suggest that positive engagement coping is associated with lower levels of inflammation, but only when adolescents are challenged by significant stress.

  17. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Kamolrat, Torkamol [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom); Gray, Stuart R., E-mail: s.r.gray@abdn.ac.uk [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom)

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  18. Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome

    Science.gov (United States)

    Liu, Zhong-Hua; Huang, Tianjian; Smith, Carolyn Beebe

    2012-01-01

    Individuals with fragile X syndrome (FXS), an inherited form of cognitive disability, have a wide range of symptoms including hyperactivity, autistic behavior, seizures and learning deficits. FXS is caused by silencing of FMR1 and the consequent absence of fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that associates with polyribosomes and negatively regulates translation. In a previous study of a mouse model of FXS (Fmr1 knockout (KO)) we demonstrated that in vivo rates of cerebral protein synthesis (rCPS) were elevated in selective brain regions suggesting that the absence of FMRP in FXS may result in dysregulation of cerebral protein synthesis. Lithium, a drug used clinically to treat bipolar disorder, has been used to improve mood dysregulation in individuals with FXS. We reported previously that in the Fmr1 KO mouse chronic dietary lithium treatment reversed or ameliorated both behavioral and morphological abnormalities. Herein we report that chronic dietary lithium treatment reversed the increased rCPS in Fmr1 KO mice with little effect on wild type mice. We also report our results of analyses of key signaling molecules involved in regulation of mRNA translation. Our analyses indicate that neither effects on the PI3K/Akt nor the MAPK/ERK 1/2 pathway fully account for the effects of lithium treatment on rCPS. Collectively our findings and those from other laboratories on the efficacy of lithium treatment in animal models support further studies in patients with FXS. PMID:22227453

  19. Influence of Auxin and Gibberellin on in Vivo Protein Synthesis during Early Pea Fruit Growth.

    Science.gov (United States)

    Van Huizen, R.; Ozga, J. A.; Reinecke, D. M.

    1996-09-01

    Developing pea fruits (Pisum sativum L.) offer a unique opportunity to study growth and development in a tissue that is responsive to both gibberellins (GAs) and auxin (4-chloroindole-3-acetic acid[4-CI-IAA]). To begin a molecular analysis of the interaction of GAs and auxins in pea fruit development, in vivo labeling with [35S]methionine coupled with two-dimensional gel electrophoresis were used to characterize de novo synthesis of proteins during gibberellic acid (GA3)-, 4-CI-indoleacetic acid-, and seed-induced pea pericarp growth. The most significant and reproducible polypeptide changes were observed between molecular weights of 20 and 60. Comparing about 250 de novo synthesized proteins revealed that seed removal changed the pattern substantially. We identified one class of polypeptides that was uniquely seed induced and five classes that were affected by hormone treatment. The latter included 4-CI-IAA-induced, GA3-induced, GA3- and 4-CI-IAA-induced, 4-CI-IAA-repressed, and GA3- and 4-CI-IAA-repressed polypeptides. Similar patterns of protein expression were associated with both hormone treatments; however, changes unique to GA3 or 4-CI-IAA treatment also indicate that the effects of GA3 and 4-CI-IAA on this process are not equivalent. In general, application of 4-CI-IAA plus GA3 replaced the seed effects on pericarp protein synthesis, supporting our hypothesis that both hormones are involved in pea pericarp development.

  20. Phenotypical temperature adaptation of protein synthesis in wheat seedlings: time curves for readaptation.

    Science.gov (United States)

    Weidner, M; Combrink, G

    1979-07-01

    Optimum temperature and temperature coefficient of protein synthesis in young wheat plants exhibit phenotypical temperature adaptation. In plants grown for 2 days at either chilling (4 C), medium (20 C), or high (36 C) temperature the respective values are: 27 C and 14.2 kilocalories per mole, 31 C and 18.2 kilocalories per mole, 35 C and 23.6 kilocalories per mole, based on in vivo [(14)C]leucine incorporation into total protein. The validity of the [(14)C]leucine incubation method has been confirmed by double-labeling experiments. Readaptation time curves are complex: the optimum temperature parameter readjusts within approximately 4 hours to an altered temperature regime, whereas the temperature coefficient needs between 4 and 96 hours for complete readaptation-depending on the temperature conditions prior to the temperature shift. Heat-preadapted plants need a recovery period at medium temperature to regain their cold adaptability with respect to optimum temperature. Cycloheximide (30 micrograms per milliliter) reduces [(14)C]leucine incorporation into protein by 85%, thus indicating that predominantly the cytoplasmic 80S system of protein synthesis is involved in temperature adaptation.

  1. From endosymbiont to host-controlled organelle: the hijacking of mitochondrial protein synthesis and metabolism.

    Directory of Open Access Journals (Sweden)

    Toni Gabaldón

    2007-11-01

    Full Text Available Mitochondria are eukaryotic organelles that originated from the endosymbiosis of an alpha-proteobacterium. To gain insight into the evolution of the mitochondrial proteome as it proceeded through the transition from a free-living cell to a specialized organelle, we compared a reconstructed ancestral proteome of the mitochondrion with the proteomes of alpha-proteobacteria as well as with the mitochondrial proteomes in yeast and man. Overall, there has been a large turnover of the mitochondrial proteome during the evolution of mitochondria. Early in the evolution of the mitochondrion, proteins involved in cell envelope synthesis have virtually disappeared, whereas proteins involved in replication, transcription, cell division, transport, regulation, and signal transduction have been replaced by eukaryotic proteins. More than half of what remains from the mitochondrial ancestor in modern mitochondria corresponds to translation, including post-translational modifications, and to metabolic pathways that are directly, or indirectly, involved in energy conversion. Altogether, the results indicate that the eukaryotic host has hijacked the proto-mitochondrion, taking control of its protein synthesis and metabolism.

  2. Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

    Science.gov (United States)

    Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

    2017-09-29

    Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium.

    Directory of Open Access Journals (Sweden)

    Stuart Smith

    Full Text Available A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.

  4. Protein engineering through chemical synthesis: design and synthesis of betabellins 4 and 5

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, S.B.; Bobe, F.W.; Reddy, P.A.; Richardson, J.S.; Richardson, D.C.; Erickson, B.W.

    1986-05-01

    Betabellin (beta-barrel bell-shaped protein) was designed to provide a framework for the construction of binding and active sites. Its two identical peptide chains are COOH-terminally cross-linked by a symmetric diamino acid. Each chain should fold into an amphiphilic 4-stranded anti-parallel beta-pleated sheet. Each 32-residue chain of betabellin 4 is shown below, where Abu = 4-aminobutyric acid and Phi = 4-iodophenylalanine. Cross-linker X is 3,5-bis(aminoethyl)benzoic acid (Bab). A disulfide bridge between the two Cys-21 residues increases structural rigidity. Betabellin 5 contains D-amino acids at six positions in each chain (Pro, Asn). Betabellins 4 and 5 were synthesized from Boc/sub 2/Bab-Abu-NH-resin by simultaneous solid-phase assembly of both chains and were purified by gel filtration and centrifugal partition chromatography. CD and NMR studies indicate substantial beta-sheet structure.

  5. Protein oxidation in plant mitochondria as a stress indicator

    DEFF Research Database (Denmark)

    Møller, I.M.; Kristensen, B.K.

    2004-01-01

    Plant mitochondria produce reactive oxygen species (ROS) as an unavoidable side product of aerobic metabolism, but they have mechanisms for regulating this production such as the alternative oxidase. Once produced, ROS can be removed by several different enzyme systems. Finally, should the first...... oxidation of cysteine and methionine side chains is an important mechanism for regulating enzyme activity. Mitochondria from both mammalian and plant tissues contain a number of oxidised proteins, but the relative abundance of these post-translationally modified forms is as yet unknown......, as are the consequences of the modification for the properties and turnover time of the proteins. Specific proteins appear to be particularly vulnerable to oxidative carbonylation in the matrix of plant mitochondria; these include several enzymes of the Krebs cycle, glycine decarboxylase, superoxide dismutase and heat...

  6. Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

    Directory of Open Access Journals (Sweden)

    Brian M. G. Janssen

    2015-06-01

    Full Text Available The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR. Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

  7. Heat Stress Decreases Levels of Nutrient-Uptake and -Assimilation Proteins in Tomato Roots

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    Anju Giri

    2017-01-01

    Full Text Available Global warming will increase root heat stress, which is already common under certain conditions. Effects of heat stress on root nutrient uptake have rarely been examined in intact plants, but the limited results indicate that heat stress will decrease it; no studies have examined heat-stress effects on the concentration of nutrient-uptake proteins. We grew Solanum lycopersicum (tomato at 25 °C/20 °C (day/night and then transferred some plants for six days to 35 °C /30 °C (moderate heat or 42 °C/37 °C (severe heat (maximum root temperature = 32 °C or 39 °C, respectively; plants were then moved back to control conditions for seven days to monitor recovery. In a second experiment, plants were grown for 15 days at 28 °C/23 °C, 32 °C/27 °C, 36 °C/31 °C, and 40 °C/35 °C (day/night. Concentrations of nutrient-uptake and -assimilation proteins in roots were determined using protein-specific antibodies and ELISA (enzyme-linked immunosorbent assay. In general, (1 roots were affected by heat more than shoots, as indicated by decreased root:shoot mass ratio, shoot vs. root %N and C, and the level of nutrient metabolism proteins vs. less sensitive photosynthesis and stomatal conductance; and (2 negative effects on roots were large and slow-to-recover only with severe heat stress (40 °C–42 °C. Thus, short-term heat stress, if severe, can decrease total protein concentration and levels of nutrient-uptake and -assimilation proteins in roots. Hence, increases in heat stress with global warming may decrease crop production, as well as nutritional quality, partly via effects on root nutrient relations.

  8. Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines*

    Science.gov (United States)

    Müller, Alexandra; Eller, Jakob; Albrecht, Frank; Prochnow, Pascal; Kuhlmann, Katja; Bandow, Julia Elisabeth; Slusarenko, Alan John

    2016-01-01

    Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines. PMID:27008862

  9. Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines.

    Science.gov (United States)

    Müller, Alexandra; Eller, Jakob; Albrecht, Frank; Prochnow, Pascal; Kuhlmann, Katja; Bandow, Julia Elisabeth; Slusarenko, Alan John; Leichert, Lars Ingo Ole

    2016-05-27

    Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Invitro Synthesis of Barley Endosperm Proteins on Wild Type and Mutant Templates

    DEFF Research Database (Denmark)

    Brandt, A.; Ingversen, J.

    1976-01-01

    Membrane bound and free polyribosomes were isolated from 20 day old barley endosperms. Sucrose gradient analysis revealed distinct polysomal peaks up to heptamers. The isolated polysomes were active in a cell-free protein synthesizing system employing wheat germ extract. SDS-polyacrylamide gel...... electrophoresis showed that proteins with molecular weights ranging from 200,000 to 10,000 daltons were synthesized. A substantial part of the polypeptides coded for by the template associated with the membrane bound polysomes was identified as hordeins by their solubility in 55% isopropanol and by their co......-migration with native hordein on SDS-polyacrylamide gels. Membrane bound endosperm polysomes from a barley mutant defective in hordein synthesis produced in the cell-free protein synthesizing system only a small amount of hordein. Conversely membrane bound polysomes from the endosperm of a mutant giving rise...

  11. Antigenic protein synthesis of Campylobacter jejuni in contact with chicken cells

    DEFF Research Database (Denmark)

    Vegge, Christina Skovgaard; Bang, Dang D.; Li, Yiping

    to the environment of the avian gastrointestinal tract. Consequently, the most important reservoir for C. jejuni is the gut of chickens, which are colonized efficiently without causing disease in the birds. Upon co-cultivation with mammalian cells, C. jejuni secrete specific Cia proteins, which are required...... for internalization into host cells. However, the pathogenic lifestyle of C. jejuni in the human intestine is different from the commensal colonization of the chicken gut, and it was therefore hypothesized that different proteins are secreted during chicken colonization. This hypothesis was tested by analyzing...... the synthesis of antigenic C. jejuni proteins upon cultivation with chicken cells. Two strains of C. jejuni (the human isolate NCTC11168 and the chicken isolate DVI-SC11) were incubated with primary intestinal chicken cells and subsequently used to raise antisera in rabbits. Negative controls were carried out...

  12. Antigenic protein synthesis of Campylobacter jejuni in contact with chicken cells

    DEFF Research Database (Denmark)

    Vegge, Christina Skovgaard; Bang, Dang D.; Li, Yiping

    to the environment of the avian gastrointestinal tract. Consequently, the most important reservoir for C. jejuni is the gut of chickens, which are colonized efficiently without causing disease in the birds. Upon co-cultivation with mammalian cells, C. jejuni secrete specific Cia proteins, which are required...... for internalization into host cells. However, the pathogenic lifestyle of C. jejuni in the human intestine is different from the commensal colonization of the chicken gut, and it was therefore hypothesized that different proteins are secreted during chicken colonization. This hypothesis was tested by analyzing...... the synthesis of antigenic C. jejuni proteins upon cultivation with chicken cells. Two strains of C. jejuni (the human isolate NCTC11168 and the chicken isolate DVI-SC11) were incubated with primary intestinal chicken cells and subsequently used to raise antisera in rabbits. Negative controls were carried out...

  13. Disruption of the serine/threonine protein kinase H affects phthiocerol dimycocerosates synthesis in Mycobacterium tuberculosis

    Science.gov (United States)

    Gómez-Velasco, Anaximandro; Bach, Horacio; Rana, Amrita K.; Cox, Liam R.; Bhatt, Apoorva; Besra, Gurdyal S.

    2013-01-01

    Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell-wall components. PMID:23412844

  14. Synthesis of fluorescent dipeptidomimetics and their ribosomal incorporation into green fluorescent protein.

    Science.gov (United States)

    Chowdhury, Sandipan Roy; Maini, Rumit; Dedkova, Larisa M; Hecht, Sidney M

    2015-11-01

    The synthesis and incorporation into position 66 of green fluorescent protein (GFP) by in vitro protein translation of novel oxazole and thiazole based dipeptidomimetics are described. The compounds may be regarded as GFP chromophore analogues, and are strongly fluorescent. An α-amido-β-ketoester intermediate was obtained via bisacylation of a protected glycine. The intermediate underwent dehydrative cyclization to afford the 1,3-oxazole and was treated with Lawesson's reagent to furnish the 1,3-thiazole. When these fluorophores were introduced into position 66 of GFP in place of Tyr66, the resulting GFP analogues exhibited fluorescence emission several-fold greater than wild-type GFP; the emission was also shifted to shorter wavelength. It may be noted that compared to the typical fluorophores formed in the natural and modified fluorescent proteins, the oxazole and thiazole fluorophores are completely stable and do not require activation by posttranslational modification to exhibit fluorescence. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Ferreira, Natalia Santos; Engelsby, Hanne; Neess, Ditte

    2017-01-01

    and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very...... of ACBP(-/-) mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial...

  16. The proteome and transcriptome of proteins responsive to salt stress ...

    African Journals Online (AJOL)

    acer

    2011-11-16

    Nov 16, 2011 ... proteins were identified, mainly representing gene products involved in photosynthesis, carbon assimilation and the ... elevated transcript level of genes needed for salt tolerance, the posttranscriptional mechanisms controlling ... tional and molecular breeding of cultivars with desirable culinary qualities that ...

  17. Leucine content of dietary proteins is a determinant of postprandial skeletal muscle protein synthesis in adult rats

    Directory of Open Access Journals (Sweden)

    Norton Layne E

    2012-07-01

    Full Text Available Abstract Background Leucine (Leu regulates muscle protein synthesis (MPS producing dose-dependent plasma Leu and MPS responses from free amino acid solutions. This study examined the role of Leu content from dietary proteins in regulation of MPS after complete meals. Methods Experiment 1 examined 4 protein sources (wheat, soy, egg, and whey with different Leu concentrations (6.8, 8.0, 8.8, and 10.9% (w/w, respectively on the potential to increase plasma Leu, activate translation factors, and stimulate MPS. Male rats (~250 g were trained for 14 day to eat 3 meals/day consisting of 16/54/30% of energy from protein, carbohydrates and fats. Rats were killed on d14 either before or 90 min after consuming a 4 g breakfast meal. Experiment 2 compared feeding wheat, whey, and wheat + Leu to determine if supplementing the Leu content of the wheat meal would yield similar anabolic responses as whey. Results In Experiment 1, only whey and egg groups increased post-prandial plasma Leu and stimulated MPS above food-deprived controls. Likewise, greater phosphorylation of p70 S6 kinase 1 (S6K1 and 4E binding protein-1 (4E-BP1 occurred in whey and egg groups versus wheat and soy groups. Experiment 2 demonstrated that supplementing wheat with Leu to equalize the Leu content of the meal also equalized the rates of MPS. Conclusion These findings demonstrate that Leu content is a critical factor for evaluating the quantity and quality of proteins necessary at a meal for stimulation of MPS.

  18. Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man

    Energy Technology Data Exchange (ETDEWEB)

    Gelfand, R.A.; Barrett, E.J.

    1987-07-01

    Although insulin stimulates protein synthesis and inhibits protein breakdown in skeletal muscle in vitro, the actual contribution of these actions to its anabolic effects in man remains unknown. Using the forearm perfusion method together with systemic infusion of L-(ring-2,6-3H)phenylalanine and L-(1-/sup 14/C)leucine, we measured steady state amino acid exchange kinetics across muscle in seven normal males before and in response to a 2-h intraarterial infusion of insulin. Postabsorptively, the muscle disposal (Rd) of phenylalanine (43 +/- 5 nmol/min per 100 ml forearm) and leucine (113 +/- 13) was exceeded by the concomitant muscle production (Ra) of these amino acids (57 +/- 5 and 126 +/- 9 nmol/min per dl, respectively), resulting in their net release from the forearm (-14 +/- 4 and -13 +/- 5 nmol/min per dl, respectively). In response to forearm hyperinsulinemia (124 +/- 11 microU/ml), the net balance of phenylalanine and leucine became positive (9 +/- 3 and 61 +/- 8 nmol/min per dl, respectively (P less than 0.005 vs. basal). Despite the marked increase in net balance, the tissue Rd for both phenylalanine (42 +/- 2) and leucine (124 +/- 9) was unchanged from baseline, while Ra was markedly suppressed (to 33 +/- 5 and 63 +/- 9 nmol/min per dl, respectively, P less than 0.01). Since phenylalanine is not metabolized in muscle (i.e., its only fates are incorporation into or release from protein) these results strongly suggest that in normal man, physiologic elevations in insulin promote net muscle protein anabolism primarily by inhibiting protein breakdown, rather than by stimulating protein synthesis.

  19. Synthesis of Kojidextrins and Their Protein Conjugates. Incidence of Steric Mismatch in Oligosaccharide Synthesis.

    Science.gov (United States)

    Pozsgay, Vince; Dubois, Eric P.; Pannell, Lewis

    1997-05-02

    Kojidextrins are biologically important oligosaccharides that are involved in many physiological processes including protein glycosylation and bacterial growth. As part of our project to explore the role kojidextrins may play in bacterial pathogenesis, here we report synthetic routes to kojibiose (54), -triose (58), -tetraose (64), and -pentaose (69) equipped with alpha-linked (hydrazinocarbonyl)pentyl aglycon, using linear and convergent strategies. In the search for a rapid convergent strategy for the construction of extended kojidextrins, four kojibiose donors (1-4) were synthesized that contain acyl- and ether-type protecting groups in various ratios. These were tested to probe the influence of diverse protecting group assemblies on their glycosyl donor ability. Attempted condensation of these donors with kojitriose and -tetraose acceptors failed to give the desired products apparently because of steric mismatch between the donor and the acceptor moieties. A one-pot procedure was developed for the covalent attachment of the synthetic saccharides through their hydrazido group to human serum albumin (HSA) using Tietze's squarate method to give neoglycoproteins containing up to 28 saccharide units per HSA.

  20. Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5.

    Science.gov (United States)

    Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul; Wu, Jianguo; He, Biao

    2015-12-01

    Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of

  1. The LuxR regulator protein controls synthesis of polyhydroxybutyrate in Vibrio harveyi.

    Science.gov (United States)

    Miyamoto, C M; Sun, W; Meighen, E A

    1998-05-19

    The LuxR regulatory protein of Vibrio harveyi has been shown to control synthesis of polyhydroxybutyrate (PHB) as well as luminescence so as to occur at high cell density, suggesting that it is a general regulatory protein. Mutants defective in the production of LuxR (D1, D34, and MR1130) were found to be missing PHB, whose synthesis could be restored by complementation with luxR. Triparental mating with a V. harveyi genomic library revealed the presence of three genomic clones (G1, G2 and G3) that could also restore PHB synthesis and luminescence to cells which express low levels of luxR (D1 and D34) but not to luxR- cells (MR1130) suggesting that luxR expression was being stimulated. Analyses of luxR mRNA levels by mRNA dot blot hybridization and by primer extension confirmed that luxR mRNA levels were increased 4 to 7-fold in the D1 and D34 cells by the G1, G2 and G3 fragments and show that expression of a single genomic copy of luxR is sufficient to restore synthesis of PHB. The results demonstrate that V. harveyi LuxR controls the induction of a process not intimately involved in the bioluminescence system and clearly distinguishes its role in V. harveyi from that of LuxR from Vibrio (Photobacterium) fischeri, which has only been associated with regulation of light emission.

  2. Effect of osmotic stress on in vitro translational capacity of polysomes and on the composition of polysome-associated proteins in germinating seeds of pea (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    Wioletta Brosowska-Arendt

    2012-10-01

    Full Text Available Plant growth throughout the world is often limited by unfavourable environmental conditions. This paper reports results of a study on long- and short-term osmotic stress (−0.5 MPa followed by a recovery on in vitro translational capacity of polysomes and on the composition of polysome-associated proteins in germinating pea (Pisum sativum L. seeds. Here we show that, under osmotic stress, cytoskeleton-bound polysomes were charaterized by the highest translation activity, which may be indicative of an important role that this population of polysomes plays in the synthesis of the so-called “stress proteins”. We also find out that in response to osmotic stress, new proteins (22.01, 96.47 and 105.3 kDa, absent in the unstressed sample, associated with the total pool of polysomes, whereas the protein of 22.95 kDa, which was present in the embryonic tissue of seeds germinating under unstressed conditions, disappeared. These changes may have affected both the stability and the translational capacity of polysomes.

  3. Essential Role of X-Box Binding Protein-1 during Endoplasmic Reticulum Stress in Podocytes.

    Science.gov (United States)

    Hassan, Hossam; Tian, Xuefei; Inoue, Kazunori; Chai, Nathan; Liu, Chang; Soda, Keita; Moeckel, Gilbert; Tufro, Alda; Lee, Ann-Hwee; Somlo, Stefan; Fedeles, Sorin; Ishibe, Shuta

    2016-04-01

    Podocytes are terminally differentiated epithelial cells that reside along the glomerular filtration barrier. Evidence suggests that after podocyte injury, endoplasmic reticulum stress response is activated, but the molecular mechanisms involved are incompletely defined. In a mouse model, we confirmed that podocyte injury induces endoplasmic reticulum stress response and upregulated unfolded protein response pathways, which have been shown to mitigate damage by preventing the accumulation of misfolded proteins in the endoplasmic reticulum. Furthermore, simultaneous podocyte-specific genetic inactivation of X-box binding protein-1 (Xbp1), a transcription factor activated during endoplasmic reticulum stress and critically involved in the untranslated protein response, and Sec63, a heat shock protein-40 chaperone required for protein folding in the endoplasmic reticulum, resulted in progressive albuminuria, foot process effacement, and histology consistent with ESRD. Finally, loss of both Sec63 and Xbp1 induced apoptosis in podocytes, which associated with activation of the JNK pathway. Collectively, our results indicate that an intact Xbp1 pathway operating to mitigate stress in the endoplasmic reticulum is essential for the maintenance of a normal glomerular filtration barrier. Copyright © 2016 by the American Society of Nephrology.

  4. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Atsushi eSato

    2014-09-01

    Full Text Available Triacylglycerol (TG synthesis is induced for energy and carbon storage in algal cells under nitrogen(N-starved conditions, and helps prevent reactive oxygen species production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P-starved cells. S- and N-starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N-starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of diacylglycerol acyltransferase genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  5. Heat Stress Reduces Sperm Motility via Activation of Glycogen Synthase Kinase-3α and Inhibition of Mitochondrial Protein Import

    Directory of Open Access Journals (Sweden)

    Yabin Gong

    2017-09-01

    Full Text Available The adverse effects of high environmental temperature exposure on animal reproductive functions have been concerned for many decades. However, the molecular basis of heat stress (HS-induced decrease of sperm motility has not been entirely elucidated. We hypothesized that the deteriorate effects of HS may be mediated by damage of mitochondrial function and ATP synthesis. To test this hypothesis, we use mature boar sperm as model to explore the impacts of HS on mitochondrial function and sperm motility. A 6 h exposure to 42°C (HS induced significant decrease in sperm progressive motility. Concurrently, HS induced mitochondrial dysfunction that is indicated by decreased of membrane potential, respiratory chain complex I and IV activities and adenosine triphosphate (ATP contents. Exogenous ATP abolished this effect suggesting that reduced of ATP synthesis is the committed step in HS-induced reduction of sperm motility. At the molecular level, the mitochondrial protein contents were significantly decreased in HS sperm. Notably, the cytochrome c oxidase subunit 4, which was synthesized in cytoplasm and translocated into mitochondria, was significantly lower in mitochondria of HS sperm. Glycogen synthase kinase-3α (GSK3α, a negative regulator of sperm motility that is inactivated by Ser21 phosphorylation, was dephosphorylated after HS. The GSK3α inhibitor CHIR99021 was able to abolish the effects of HS on sperm and their mitochondria. Taken together, our results demonstrate that HS affects sperm motility through downregulation of mitochondrial activity and ATP synthesis yield, which involves dephosphorylation of GSK3α and interference of mitochondrial remodeling.

  6. Effect of administration of oral contraceptives on the synthesis and breakdown of myofibrillar proteins in young women

    DEFF Research Database (Denmark)

    Hansen, M; Langberg, H.; Holm, Lars

    2011-01-01

    ±4 years n=12) performed one-legged kicking exercise. The next day, the myofibrillar protein fractional synthesis rate (FSR) was measured using stable isotopic tracers ((13)C-proline) while the subjects were fed standardized nutrient drinks. Simultaneously, a marker for myofibrillar protein breakdown, 3...

  7. A Novel 43-kDa Protein as a Negative Regulatory Component of Phenoloxidase-induced Melanin Synthesis

    National Research Council Canada - National Science Library

    Mingyi Zhao; Irene Söderhäll; Ji Won Park; Young Gerl Ma; Tsukusa Osaki; Nam-Chul Ha; Chun Fu Wu; Kenneth Söderhäll; Bok Luel Lee

    2005-01-01

    ...(s) of melanin synthesis may exist in hemolymph. Here, we report the purification and cloning of a cDNA of a novel 43-kDa protein, from the meal-worm Tenebrio molitor , which functions as a melanization-inhibiting protein (MIP...

  8. Oxidative Stress in Cardiovascular Diseases and Obesity: Role of p66Shc and Protein Kinase C

    Science.gov (United States)

    Baldassari, Federica; Wieckowski, Mariusz R.

    2013-01-01

    Reactive oxygen species (ROS) are a byproduct of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis. An imbalance between ROS production and the cellular antioxidant defence system leads to oxidative stress. Environmental factors and genetic interactions play key roles in oxidative stress mediated pathologies. In this paper, we focus on cardiovascular diseases and obesity, disorders strongly related to each other; in which oxidative stress plays a fundamental role. We provide evidence of the key role played by p66Shc protein and protein kinase C (PKC) in these pathologies by their intracellular regulation of redox balance and oxidative stress levels. Additionally, we discuss possible therapeutic strategies aimed at attenuating the oxidative damage in these diseases. PMID:23606925

  9. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  10. Aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis

    Directory of Open Access Journals (Sweden)

    Fry Christopher S

    2011-03-01

    Full Text Available Abstract Background Sarcopenia, the loss of skeletal muscle mass during aging, increases the risk for falls and dependency. Resistance exercise (RE training is an effective treatment to improve muscle mass and strength in older adults, but aging is associated with a smaller amount of training-induced hypertrophy. This may be due in part to an inability to stimulate muscle-protein synthesis (MPS after an acute bout of RE. We hypothesized that older adults would have impaired mammalian target of rapamycin complex (mTORC1 signaling and MPS response compared with young adults after acute RE. Methods We measured intracellular signaling and MPS in 16 older (mean 70 ± 2 years and 16 younger (27 ± 2 years subjects. Muscle biopsies were sampled at baseline and at 3, 6 and 24 hr after exercise. Phosphorylation of regulatory signaling proteins and MPS were determined on successive muscle biopsies by immunoblotting and stable isotopic tracer techniques, respectively. Results Increased phosphorylation was seen only in the younger group (PP >0.05. After exercise, MPS increased from baseline only in the younger group (PP 0.05. Conclusions We conclude that aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. These age-related differences may contribute to the blunted hypertrophic response seen after resistance-exercise training in older adults, and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.

  11. Protein misfolding and oxidative stress promote glial-mediated neurodegeneration in an Alexander disease model.

    Science.gov (United States)

    Wang, Liqun; Colodner, Kenneth J; Feany, Mel B

    2011-02-23

    Although alterations in glial structure and function commonly accompany death of neurons in neurodegenerative diseases, the role glia play in modulating neuronal loss is poorly understood. We have created a model of Alexander disease in Drosophila by expressing disease-linked mutant versions of glial fibrillary acidic protein (GFAP) in fly glia. We find aggregation of mutant human GFAP into inclusions bearing the hallmarks of authentic Rosenthal fibers. We also observe significant toxicity of mutant human GFAP to glia, which is mediated by protein aggregation and oxidative stress. Both protein aggregation and oxidative stress contribute to activation of a robust autophagic response in glia. Toxicity of mutant GFAP to glial cells induces a non-cell-autonomous stress response and subsequent apoptosis in neurons, which is dependent on glial glutamate transport. Our findings thus establish a simple genetic model of Alexander disease and further identify cellular pathways critical for glial-induced neurodegeneration.

  12. Study of GOLPH3: a potential stress-inducible protein from Golgi apparatus.

    Science.gov (United States)

    Li, Ting; You, Hong; Zhang, Jie; Mo, Xiaoye; He, Wenfang; Chen, Yang; Tang, Xiangqi; Jiang, Zheng; Tu, Ranran; Zeng, Liuwang; Lu, Wei; Hu, Zhiping

    2014-06-01

    Although the Golgi apparatus has been studied extensively for over 100 years, the complex structure-function relationships have yet to be elucidated. It is well known that the Golgi complex plays an important role in the transport, processing, sorting, and targeting of numerous proteins and lipids destined for secretion, plasma membrane, and lysosomes. Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream effector of stress signals in cell death pathways. It undergoes disassembly and fragmentation in several neurological disorders. Recent studies indicate that Golgi phosphoprotein 3 (GOLPH3 also known as GPP34/GMx33/MIDAS), a peripheral membrane protein of trans-Golgi network, represents an exciting new class of oncoproteins involved in cell signal transduction and is potentially mobilized by stress. In this review, we focus on the importance of GOLPH3 in vesicular trafficking, Golgi architecture maintenance, receptor sorting, protein glycosylation, and further discuss its potential in signal sensing in stress response.

  13. Changes in rubisco, cysteine-rich proteins and antioxidant system of spinach (Spinacia oleracea L.) due to sulphur deficiency, cadmium stress and their combination.

    Science.gov (United States)

    Bagheri, Rita; Ahmad, Javed; Bashir, Humayra; Iqbal, Muhammad; Qureshi, M Irfan

    2017-03-01

    Sulphur (S) deficiency, cadmium (Cd) toxicity and their combinations are of wide occurrence throughout agricultural lands. We assessed the impact of short-term (2 days) and long-term (4 days) applications of cadmium (40 μg/g soil) on spinach plants grown on sulphur-sufficient (300 μM SO42-) and sulphur-deficient (30 μM SO42-) soils. Compared with the control (+S and -Cd), oxidative stress was increased by S deficiency (-S and -Cd), cadmium (+S and +Cd) and their combination stress (-S and +Cd) in the order of (S deficiency) leaf proteins showed a high vulnerability of rubisco large subunit (RbcL) to S deficiency. Rubisco small subunit (RbcS) was particularly sensitive to Cd as well as dual stress (+Cd and -S) but increased with Cd in the presence of S. Cysteine content in low molecular weight proteins/peptide was also affected, showing a significant increase under cadmium treatment. Components of ascorbate-glutathione antioxidant system altered their levels, showing the maximum decline in ascorbate (ASA), dehydroascorbate (DHA), total ascorbate (ASA + DHA, hereafter TA), glutathione (GSH) and total glutathione (GSH + GSSG, hereafter TG) under S deficiency. However, total ascorbate and total glutathione increased, besides a marginal increase in their reduced and oxidized forms, when Cd was applied in the presence of sufficient S. Sulphur supply also helped in increasing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) under Cd stress. However, their activity suffered by S deficiency and by Cd stress during S deficiency. Each stress declined the contents of soluble protein and photosynthetic pigments; the highest decline in contents of protein and pigments occurred under S deficiency and dual stress respectively. The fresh and dry weights, although affected adversely by every stress, declined most under dual stress. It may be concluded that an optimal level of S is required during Cd stress

  14. Synthesis and characterization of an elastin-mimetic amphiphilic block copolymer protein

    Science.gov (United States)

    Lee, Terrence Anita-Talley

    2000-10-01

    The overall goal in material science is to be able to control the molecular architecture of a material and thus its end properties. There is no method that offers greater control than the biological synthesis of proteins. From the DNA sequence to the final synthesized protein, the entire process is finitely controlled. This present work describes methods developed and used to synthesize protein polymers by manipulating this process. From the initial DNA sequence chosen, the end properties that the protein polymer will have are dictated. An amphiphilic diblock copolymer was designed based on environmentally responsive elastin-mimetic peptide sequences [(Val/Ile)-Pro-Gly-Xaa-Gly] (Xaa = Ala or Glu for the hydrophilic block, Val or Phe for the hydrophobic block) and synthesized using a genetic engineering approach. Differential scanning calorimetry measurements in aqueous solution revealed that reversible hydrophobic folding and assembly of the copolymer occurs above the inverse temperature transition, Tt, of the hydrophobic block. This process results in the formation of 50 nm protein-based micellar aggregates, which were characterized by electron microscopy and temperature-dependent dynamic light scattering techniques. The distribution of micellar aggregates can be altered reproducibly through variation of environmental conditions including pH and temperature. The uniform and defined macromolecular architecture of this protein copolymer permits greater control over the physical properties of the micelles, which therefore may facilitate applications in controlled release of small molecules.

  15. New synthesis of a platelet-specific protein: platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system

    OpenAIRE

    1983-01-01

    The site of synthesis of platelet-specific proteins remains to be established. With the use of short-term megakaryocyte-enriched cultures, direct evidence was obtained to show that megakaryocytes synthesize the platelet-specific protein, platelet factor 4. A megakaryocyte-enriched fraction of rabbit bone marrow for culture was obtained by centrifugal elutriation and cultured with [3H]leucine. Newly synthesized 3H-platelet factor 4 was sought by copurification with added carrier rabbit platele...

  16. Local NSAID infusion does not affect protein synthesis and gene expression in human muscle after eccentric exercise

    DEFF Research Database (Denmark)

    Mikkelsen, U R; Schjerling, P.; Langberg, Henning

    2011-01-01

    models, and inhibit the exercise-induced satellite cell proliferation and protein synthesis in humans. However, the cellular mechanisms eliciting these responses remain unknown. Eight healthy male volunteers performed 200 maximal eccentric contractions with each leg. To block prostaglandin synthesis......Unaccustomed exercise leads to satellite cell proliferation and increased skeletal muscle protein turnover. Several growth factors and cytokines may be involved in the adaptive responses. Non-steroidal anti-inflammatory drugs (NSAIDs) negatively affect muscle regeneration and adaptation in animal...... locally in the skeletal muscle, indomethacin (NSAID) was infused for 7.5 h via microdialysis catheters into m. vastus lateralis of one leg. Protein synthesis was determined by the incorporation of 1,2-(13) C(2) leucine into muscle protein from 24 to 28 h post-exercise. Furthermore, mRNA expression...

  17. Prominent increase in synthesis of a nuclear protein is an early signal associated with mitogenesis of B cells

    Energy Technology Data Exchange (ETDEWEB)

    Feuerstein, N.; Mond, J.J.

    1987-05-01

    Activation of murine splenic B lymphocytes with various mitogens was found to be associated with a prominent increase in synthesis and abundance of a 40 KDa/pI 5.0 nuclear protein (p40). Subcellular fractionation revealed that this protein is not found in the cytosol fraction and cannot be solubilized by DNAse or RNAse digestion, indication that the protein is mainly associated with the nuclear matrix. The increase in synthesis of p40 was detected at early G1 phase, 60 min following activation of the cells by the mitogen, reached a peak at 16h and declined to control level at 48h (during S phase). Activation of the cells with non mitogenic stimuli such as BSF-1, A23187, and PMA, induced an increase in synthesis of discrete nuclear proteins, but failed to affect the synthesis of p40, suggesting that the increase in synthesis of p40 is specifically associated with the signal of mitogenic stimuli but not with this of non mitogenic stimuli. Inhibition of the mitogenic effect of anti-Ig by PMA treatment of the cells was associated with specific inhibition in the synthesis of p40, while overcoming this inhibition by addition of 8-mercaptoguanosine was associated with restoration of the mitogenic effect of anti-Ig. These results suggest that p40 may have an important role in early induction of mitogenesis in B cells.

  18. Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses.

    Science.gov (United States)

    Kim, Eun Yu; Park, Ki Youl; Seo, Young Sam; Kim, Woo Taek

    2016-04-01

    Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. Influence of natural thermal gradients on whole animal rates of protein synthesis in marine gammarid amphipods.

    Directory of Open Access Journals (Sweden)

    Samuel P S Rastrick

    Full Text Available Although temperature is known to have an important effect on protein synthesis rates and growth in aquatic ectotherms held in the laboratory, little is known about the effects of thermal gradients on natural populations in the field. To address this issue we determined whole-animal fractional rates of protein synthesis (ks in four dominant species of gammarid amphipods with different distributions along the coasts of Western Europe from arctic to temperate latitudes. Up to three populations of each species were collected in the summer and ks measured within 48 h. Summer ks values were relatively high in the temperate species, Gammarus locusta, from Portugal (48°N and Wales (53°N and were maintained across latitudes by the conservation of translational efficiency. In sharp contrast, summer ks remained remarkably low in the boreal/temperate species G. duebeni from Wales, Scotland (58°N and Tromsø (70°N, probably as a temporary energy saving strategy to ensure survival in rapidly fluctuating environments of the high intertidal. Values for ks increased in acclimated G. duebeni from Scotland and Tromsø showing a lack of compensation with latitude. In the subarctic/boreal species, G. oceanicus, summer ks remained unchanged in Scotland and Tromsø but fell significantly in Svalbard (79°N at 5°C, despite a slight increase in RNA content. At 79°N, mean ks was 4.5 times higher in the circumpolar species G. setosus than in G. oceanicus due to a doubling in RNA content. The relationship between whole-animal protein synthesis rates and natural thermal gradients is complex, varies between species and appears to be associated with local temperatures and their variability, as well as changes in other environmental factors.

  20. Protein synthesis of eucaryotic cells could be decreased by antisense-DNA of the multi KH domain protein vigilin.

    Science.gov (United States)

    Schuh, Antje; Assmuth, Karen; Hilgendorf, Inken; Mueller, Peter K; Kruse, Charli

    2003-07-01

    Vigilin, a member of the KH protein family, is exceptional among these proteins as it contains 14 KH domains in consecutive order. Vigilin is present in the nucleus and the cytoplasm of all eucaryotic cells studied so far and has apparently high affinity to tRNA and mRNA. There is circumstantial evidence that vigilin expression parallels high translational activity as demonstrated for pancreatic cells in vitro and in vivo as well as for carcinoma cell lines. On a molecular level we have recently demonstrated that vigilin promotes in vitro the export of tRNA from the nucleus to the translational machinery in the cytoplasm and may hence function as an intercompartimental conveyor. In the present study we show that exposure to a vigilin antisense oligo DNA (VAOD) expectedly resulted in a decrease of vigilin-expression, and was concomitant to lower amylase- and trypsin synthesis in freshly isolated pancreatic cells. In addition, carcinoma cells reacted with an increased mortality under exposure to VAOD giving further support for the notion that vigilin participates in cellular life-sustaining processes such as protein translation.

  1. The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

    KAUST Repository

    Ambrosone, Alfredo

    2015-03-17

    Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

  2. Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation

    Directory of Open Access Journals (Sweden)

    Seo Kang

    2011-06-01

    Full Text Available Abstract Background In vitro maturation (IVM of mammalian oocytes is divided into the GV (germinal vesicle stage, MI (metaphase I stage and MII (metaphase II stage stages, and only fully mature oocytes have acquired the ability to be fertilized and initiate zygotic development. These observations have been mostly based on morphological evaluations, but the molecular events governing these processes are not fully understood. The aim of the present study was to better understand the processes involved in the molecular regulation of IVM using 2-DE analysis followed by mass spectrometry to identify proteins that are differentially expressed during oocyte IVM. Result A total of 16 up-regulated and 12 down-regulated proteins were identified. To investigate the IVM process, we specifically focused on the proteins that were up-regulated during the MII stage when compared with the GV stage, which included PRDX 2, GST, SPSY, myomegalin, PED4D, PRKAB 1, and DTNA. These up-regulated proteins were functionally involved in redox regulation and the cAMP-dependent pathway, which are essential for the intracellular signaling involved in oocyte maturation. Interestingly, the PDE4D and its partner, myomegalin, during the MII stage was consistently confirmed up-regulation by western blot analyses. Conclusion These results could be used to better understand some aspects of the molecular mechanisms underlying porcine oocyte maturation. This study identified some regulatory proteins that may have important roles in the molecular events involved in porcine oocyte maturation, particularly with respect to the regulation of oocyte meiotic resumption, MII arrest and oocyte activation. In addition, this study may have beneficial applications not only to basic science with respect to the improvement of oocyte culture conditions but also to mammalian reproductive biotechnology with potential implications.

  3. Overexpression of a Pathogenesis-Related Protein 10 Enhances Biotic and Abiotic Stress Tolerance in Rice

    Directory of Open Access Journals (Sweden)

    Jingni Wu

    2016-12-01

    Full Text Available Pathogenesis-related proteins play multiple roles in plant development and biotic and abiotic stress tolerance. Here, we characterize a rice defense related gene named “jasmonic acid inducible pathogenesis-related class 10” (JIOsPR10 to gain an insight into its functional properties. Semi-quantitative RT-PCR analysis showed up-regulation of JIOsPR10 under salt and drought stress conditions. Constitutive over-expression JIOsPR10 in rice promoted shoot and root development in transgenic plants, however, their productivity was unaltered. Further experiments exhibited that the transgenic plants showed reduced susceptibility to rice blast fungus, and enhanced salt and drought stress tolerance as compared to the wild type. A comparative proteomic profiling of wild type and transgenic plants showed that overexpression of JIOsPR10 led to the differential modulation of several proteins mainly related with oxidative stresses, carbohydrate metabolism, and plant defense. Taken together, our findings suggest that JIOsPR10 plays important roles in biotic and abiotic stresses tolerance probably by activation of stress related proteins.

  4. Protein Phosphatase 2A Mediates Oxidative Stress Induced Apoptosis in Osteoblasts.

    Science.gov (United States)

    Huang, Chong-xin; Lv, Bo; Wang, Yue

    2015-01-01

    Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis.

  5. Plant lipid-associated fibrillin proteins condition jasmonate production under photosynthetic stress.

    Science.gov (United States)

    Youssef, Abir; Laizet, Yec'han; Block, Maryse A; Maréchal, Eric; Alcaraz, Jean-Pierre; Larson, Tony R; Pontier, Dominique; Gaffé, Joël; Kuntz, Marcel

    2010-02-01

    The role of a subfamily of lipid globule-associated proteins, referred to as plant fibrillins (FIB1a, -1b, -2), was determined using a RNA interference (RNAi) strategy. We show that Arabidopsis plants with reduced levels of these plastid structural proteins are impaired in long-term acclimation to environmental constraint, namely photooxidative stress imposed by high light combined with cold. As a result, their photosynthetic apparatus is inefficiently protected. This leads to the prevalence of an abnormal granal and stromal membrane arrangement, as well as higher photosystem II photoinhibition under stress. The visible phenotype of FIB1-2 RNAi lines also includes retarded shoot growth and a deficit in anthocyanin accumulation under stress. All examined phenotypic effects of lower FIB levels are abolished by jasmonate (JA) treatment. An atypical expression pattern of several JA-induced genes was observed in RNAi plants. A JA-deficient mutant was found to share similar stress phenotypic characteristics with FIB RNAi plants. We conclude a new physiological role for JA, namely acclimation of chloroplasts, and that light/cold stress-related JA biosynthesis is conditioned by the accumulation of plastoglobule-associated FIB1-2 proteins. Consistent correlative data suggest that this FIB effect is mediated by plastoglobule (and triacylglycerol) accumulation as the potential site for initiating the chloroplast stress-related JA biosynthesis.

  6. Oxidative Stress-Associated Protein Tyrosine Kinases and Phosphatases in Fanconi Anemia

    Science.gov (United States)

    Pang, Qishen

    2014-01-01

    Abstract Significance: Fanconi anemia (FA) is a genetic disorder featuring chromosomal instability, developmental defects, progressive bone marrow failure, and predisposition to cancer. Besides the predominant role in DNA damage response and/or repair, many studies have linked FA proteins to oxidative stress. Oxidative stress, defined as imbalance in pro-oxidant and antioxidant homeostasis, has been considered to contribute to disease development, including FA. Recent Advances: A variety of signaling pathways may be influenced by oxidative stress, particularly the equilibrium between protein kinases and phosphatases, consequently leading to an aberrant phosphorylation state of cellular proteins. Dysfunction of kinases/phosphatases has been implicated in the pathophysiology of human diseases. In FA, evidence is emerging that links abnormal phosphorylation/de-phosphorylation of signaling molecules to clinical complications and malformations. Critical Issues: In this study, we review the recent findings on the oxidative stress-related kinases and phosphatases, particularly tyrosine phosphatases in FA. Future Directions: Understanding the role of oxidative stress-related kinases and phosphatases in FA may provide unique and generic possibilities for the future development of therapeutic strategies by targeting the dysregulated protein kinases and phosphatases in a clinical setting. Antioxid. Redox Signal. 20, 2290–2301. PMID:24206276

  7. Protein synthesis as a function of depth in slices of rat hippocampus.

    Science.gov (United States)

    Hesse, G W; Shashoua, V E

    1990-02-05

    Physiologically viable slices of rat hippocampus were incubated in radiolabeled valine, then cut into 20 microns serial sections to evaluate the profile of protein synthesis through the depth of the slice. Maximum radiolabel incorporation was observed near the center of the slice, while at the upper (gas interface) and lower (liquid interface) surfaces radiolabel incorporation per section was reduced by about 30% and 90%, respectively. The results suggest that in properly slices damage due to slicing may be less important to cell viability than are limits on oxygen diffusion into the tissue.

  8. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides.

    Science.gov (United States)

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf; Douthwaite, Stephen

    2012-11-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC(50)], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site.

  9. The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.

    Science.gov (United States)

    Rojas-Benítez, Diego; Ibar, Consuelo; Glavic, Álvaro

    2013-01-01

    The TOR signaling pathway is crucial in the translation of nutritional inputs into the protein synthesis machinery regulation, allowing animal growth. We recently identified the Bud32 (yeast)/PRPK (human) ortholog in Drosophila, Prpk (p53-related protein kinase), and found that it is required for TOR kinase activity. Bud32/PRPK is an ancient and atypical kinase conserved in evolution from Archeae to humans, being essential for Archeae. It has been linked with p53 stabilization in human cell culture and its absence in yeast causes a slow-growth phenotype. This protein has been associated to KEOPS (kinase, putative endopeptidase and other proteins of small size) complex together with Kae1p (ATPase), Cgi-121 and Pcc1p. This complex has been implicated in telomere maintenance, transcriptional regulation, bud site selection and chemical modification of tRNAs (tRNAs). Bud32p and Kae1p have been related with N6-threonylcarbamoyladenosine (t (6)A) synthesis, a particular chemical modification that occurs at position 37 of tRNAs that pair A-starting codons, required for proper translation in most species. Lack of this modification causes mistranslations and open reading frame shifts in yeast. The core constituents of the KEOPS complex are present in Drosophila, but their physical interaction has not been reported yet. Here, we present a review of the findings regarding the function of this complex in different organisms and new evidence that extends our recent observations of Prpk function in animal growth showing that depletion of Kae1 or Prpk, in accordance with their role in translation in yeast, is able to induce the unfolded protein response (UPR) in Drosophila. We suggest that EKC/KEOPS complex could be integrating t (6)A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth.

  10. Aniline-induced nitrosative stress in rat spleen: proteomic identification of nitrated proteins.

    Science.gov (United States)

    Fan, Xiuzhen; Wang, Jianling; Soman, Kizhake V; Ansari, G A S; Khan, M Firoze

    2011-08-15

    Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Protein biogenesis in Archaea: addressing translation initiation using an in vitro protein synthesis system for Haloferax volcanii.

    Science.gov (United States)

    Ring, Gabriela; Londei, Paola; Eichler, Jerry

    2007-05-01

    Translation initiation in Archaea combines aspects of the parallel process in Eukarya and Bacteria alongside traits unique to this domain. To better understand translation initiation in Archaea, an in vitro translation system from the haloarchaeon Haloferax volcanii has been developed. The ability to translate individual mRNAs both under the conditions used in previously developed poly(U)-dependent poly(Phe) synthesis systems as well as under physiological conditions was shown. Using the H. volcanii system, mRNAs proceeded by either 'strong' or 'weak' Shine-Dalgarno (SD) motifs, or completely lacking leader sequences were effectively translated. The in vitro haloarchaeal system also successfully translated mRNA from Bacteria, again either presenting a SD initiation motif or completely lacking a leader sequence. Thus, the ability to translate individual mRNAs in vitro offers a system to address translation initiation as well as other aspects of protein biogenesis in Archaea.

  12. Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice.

    Science.gov (United States)

    Xu, Enshun; Chen, Mingming; He, Hui; Zhan, Chengfang; Cheng, Yanhao; Zhang, Hongsheng; Wang, Zhoufei

    2016-01-01

    Enhancement of salinity tolerance during seed germination is very important for direct seeding in rice. In this study, the salt-tolerant japonica landrace Jiucaiqing was used to determine the regulators that are involved in seed imbibition under salt stress. Briefly, the comparative proteomic analysis was conducted between dry (0 h) and imbibed (24 h) seeds with 150 mM NaCl. Under salt stress, the uptake of water increased rapidly before 24 h imbibition (Phase I), followed by a plateau of seed imbibition from 24 to 96 h imbibition (Phase II). We identified 14 proteins involved in seed imbibition, in which the majority of these proteins were involved in energy supply and storage protein. The early imbibition process was mediated by protein cat