Science.gov

Sample records for active protein synthesis

  1. Amiloride, protein synthesis, and activation of quiescent cells.

    PubMed

    Lubin, M; Cahn, F; Coutermarsh, B A

    1982-11-01

    Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.

  2. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    NASA Technical Reports Server (NTRS)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  3. Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking.

    PubMed

    Lopez, Joëlle; Gamache, Karine; Schneider, Rilla; Nader, Karim

    2015-02-11

    Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA23Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA23Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval.

  4. Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes.

    PubMed

    Salto, Rafael; Vílchez, José D; Cabrera, Elena; Guinovart, Joan J; Girón, María D

    2014-06-27

    The balance between the rates of protein synthesis and degradation in muscle is regulated by PI3K/Akt signaling. Here we addressed the effect of ERK activation by sodium tungstate on protein turnover in rat L6 myotubes. Phosphorylation of ERK by this compound increased protein synthesis by activating MTOR and prevented dexamethasone-induced protein degradation by blocking FoxO3a activity, but it did not alter Akt phosphorylation. Thus, activation of ERK by tungstate improves protein turnover in dexamethasone-treated cells. On the basis of our results, we propose that tungstate be considered an alternative to IGF-I and its analogs in the prevention of skeletal muscle atrophy.

  5. Sodium tungstate activates glycogen synthesis through a non-canonical mechanism involving G-proteins.

    PubMed

    Zafra, Delia; Nocito, Laura; Domínguez, Jorge; Guinovart, Joan J

    2013-01-31

    Tungstate treatment ameliorates experimental diabetes by increasing liver glycogen deposition through an as yet unidentified mechanism. The signalling mechanism of tungstate was studied in CHOIR cells and primary cultured hepatocytes. This compound exerted its pro-glycogenic effects through a new G-protein-dependent and Tyr-Kinase Receptor-independent mechanism. Chemical or genetic disruption of G-protein signalling prevented the activation of the Ras/ERK cascade and the downstream induction of glycogen synthesis caused by tungstate. Thus, these findings unveil a novel non-canonical signalling pathway that leads to the activation of glycogen synthesis and that could be exploited as an approach to treat diabetes.

  6. M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis.

    PubMed

    Arion, D; Meijer, L

    1989-08-01

    Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation. H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity. H1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation. Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K. These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation.

  7. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    PubMed

    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

  8. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.

    PubMed

    Kir, Serkan; Beddow, Sara A; Samuel, Varman T; Miller, Paul; Previs, Stephen F; Suino-Powell, Kelly; Xu, H Eric; Shulman, Gerald I; Kliewer, Steven A; Mangelsdorf, David J

    2011-03-25

    Fibroblast growth factor (FGF) 19 is an enterokine synthesized and released when bile acids are taken up into the ileum. We show that FGF19 stimulates hepatic protein and glycogen synthesis but does not induce lipogenesis. The effects of FGF19 are independent of the activity of either insulin or the protein kinase Akt and, instead, are mediated through a mitogen-activated protein kinase signaling pathway that activates components of the protein translation machinery and stimulates glycogen synthase activity. Mice lacking FGF15 (the mouse FGF19 ortholog) fail to properly maintain blood concentrations of glucose and normal postprandial amounts of liver glycogen. FGF19 treatment restored the loss of glycogen in diabetic animals lacking insulin. Thus, FGF19 activates a physiologically important, insulin-independent endocrine pathway that regulates hepatic protein and glycogen metabolism.

  9. Impaired translation initiation activation and reduced protein synthesis in weaned piglets fed a low-protein diet.

    PubMed

    Deng, Dun; Yao, Kang; Chu, Wuying; Li, Tiejun; Huang, Ruiling; Yin, Yulong; Liu, Zhiqiang; Zhang, Jianshe; Wu, Guoyao

    2009-07-01

    Weanling mammals (including infants) often experience intestinal dysfunction when fed a high-protein diet. Recent work with the piglet (an animal model for studying human infant nutrition) shows that reducing protein intake can improve gut function during weaning but compromises the provision of essential amino acids (EAA) for muscle growth. The present study was conducted with weaned pigs to test the hypothesis that supplementing deficient EAA (Lys, Met, Thr, Trp, Leu, Ile and Val) to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in tissues. Pigs were weaned at 21 days of age and fed diets containing 20.7, 16.7 or 12.7% crude protein (CP), with the low-CP diets supplemented with EAA to achieve the levels in the high-CP diet. On Day 14 of the trial, tissue protein synthesis was determined using the phenylalanine flooding dose method. Reducing dietary CP levels decreased protein synthesis in pancreas, liver, kidney and longissimus muscle. A low-CP diet reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) in skeletal muscle and liver while increasing the formation of an inactive eIF4E.4E-BP1 complex in muscle. Dietary protein deficiency also decreased the phosphorylation of mammalian target of rapamycin (mTOR) and the formation of an active eIF4E.eIF4G complex in liver. These results demonstrate for the first time that chronic feeding of a low-CP diet suppresses protein synthesis in animals partly by inhibiting mTOR signaling. Additionally, our findings indicate that supplementing deficient EAA to low-protein diets is not highly effective in restoring protein synthesis or whole-body growth in piglets. We suggest that conditionally essential amino acids (e.g., glutamine and arginine) may be required to maintain the activation of translation initiation factors and optimal protein synthesis in neonates.

  10. RNA and protein synthesis is required for Ancylostoma caninum larval activation.

    PubMed

    Dryanovski, Dilyan I; Dowling, Camille; Gelmedin, Verena; Hawdon, John M

    2011-06-30

    The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 μM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 μM), cycloheximide (IC(50)=50 μM), and anisomycin (IC(50)=200 μM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h. None of the RNA or protein synthesis inhibitors prevented secretion of the activation-associated protein ASP-1, despite nearly complete inhibition of feeding. The results indicate that unlike dauer recovery in C. elegans, de novo gene expression is required for hookworm larval activation, and the critical genes are expressed within 12h of exposure to activating stimuli. However, secretion of infection-associated proteins is independent of gene expression, indicating that the proteins are pre-synthesized and stored for rapid release during the initial stages of infection. The genes that are inhibited

  11. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

    PubMed

    Henin, N; Vincent, M F; Gruber, H E; Van den Berghe, G

    1995-04-01

    AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

  12. Activation of the beta interferon promoter by paramyxoviruses in the absence of virus protein synthesis.

    PubMed

    Killip, M J; Young, D F; Precious, B L; Goodbourn, S; Randall, R E

    2012-02-01

    Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-β promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.

  13. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

  14. Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

    PubMed

    Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

    2014-05-01

    Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases.

  15. Total synthesis and structure-activity relationship studies of a series of selective G protein inhibitors

    NASA Astrophysics Data System (ADS)

    Xiong, Xiao-Feng; Zhang, Hang; Underwood, Christina R.; Harpsøe, Kasper; Gardella, Thomas J.; Wöldike, Mie F.; Mannstadt, Michael; Gloriam, David E.; Bräuner-Osborne, Hans; Strømgaard, Kristian

    2016-11-01

    G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins; however, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure-activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.

  16. Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

    PubMed

    Lee, Hak Joo; Mariappan, Meenalakshmi M; Feliers, Denis; Cavaglieri, Rita C; Sataranatarajan, Kavithalakshmi; Abboud, Hanna E; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

    2012-02-10

    Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

  17. Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

    PubMed Central

    Wang, Gang; Sun, Xiaoliang; Wang, Guifeng; Wang, Fei; Gao, Qiang; Sun, Xin; Tang, Yuanping; Chang, Chong; Lai, Jinsheng; Zhu, Lihuang; Xu, Zhengkai; Song, Rentao

    2011-01-01

    In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds. PMID:21954158

  18. Chemical Synthesis of Proteins

    PubMed Central

    Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

    2010-01-01

    Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

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

    PubMed Central

    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

  20. High-throughput cell-free systems for synthesis of functionally active proteins.

    PubMed

    Spirin, Alexander S

    2004-10-01

    Continuous cell-free translation systems with perpetual supply of consumable substrates and removal of reaction products made the process of in vitro synthesis of individual proteins sustainable and productive. Improvements of cell-free reaction mixtures, including new ways for efficient energy generation, had an additional impact on progress in cell-free protein synthesis technology. The requirement for gene-product identification in genomic studies, the development of high-throughput structural proteomics, the need for protein engineering without cell constraints (including the use of unnatural amino acids), and the need to produce cytotoxic, poorly expressed and unstable proteins have caused increased interest in cell-free protein synthesis technologies for molecular biologists, biotechnologists and pharmacologists.

  1. Influence of Nrf2 activators on subcellular skeletal muscle protein and DNA synthesis rates after 6 weeks of milk protein feeding in older adults.

    PubMed

    Konopka, Adam R; Laurin, Jaime L; Musci, Robert V; Wolff, Christopher A; Reid, Justin J; Biela, Laurie M; Zhang, Qian; Peelor, Fredrick F; Melby, Christopher L; Hamilton, Karyn L; Miller, Benjamin F

    2017-03-10

    In older adults, chronic oxidative and inflammatory stresses are associated with an impaired increase in skeletal muscle protein synthesis after acute anabolic stimuli. Conjugated linoleic acid (CLA) and Protandim have been shown to activate nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor for the antioxidant response element and anti-inflammatory pathways. This study tested the hypothesis that compared to a placebo control (CON), CLA and Protandim would increase skeletal muscle subcellular protein (myofibrillar, mitochondrial, cytoplasmic) and DNA synthesis in older adults after 6 weeks of milk protein feeding. CLA decreased oxidative stress and skeletal muscle oxidative damage with a trend to increase messenger RNA (mRNA) expression of a Nrf2 target, NAD(P)H dehydrogenase quinone 1 (NQO1). However, CLA did not influence other Nrf2 targets (heme oxygenase-1 (HO-1), glutathione peroxidase 1 (Gpx1)) or protein or DNA synthesis. Conversely, Protandim increased HO-1 protein content but not the mRNA expression of downstream Nrf2 targets, oxidative stress, or skeletal muscle oxidative damage. Rates of myofibrillar protein synthesis were maintained despite lower mitochondrial and cytoplasmic protein syntheses after Protandim versus CON. Similarly, DNA synthesis was non-significantly lower after Protandim compared to CON. After Protandim, the ratio of protein to DNA synthesis tended to be greater in the myofibrillar fraction and maintained in the mitochondrial and cytoplasmic fractions, emphasizing the importance of measuring both protein and DNA synthesis to gain insight into proteostasis. Overall, these data suggest that Protandim may enhance proteostatic mechanisms of skeletal muscle contractile proteins after 6 weeks of milk protein feeding in older adults.

  2. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

    PubMed

    Manjarín, Rodrigo; Columbus, Daniel A; Suryawan, Agus; Nguyen, Hanh V; Hernandez-García, Adriana D; Hoang, Nguyet-Minh; Fiorotto, Marta L; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.

  3. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs

    PubMed Central

    Suryawan, Agus; Nguyen, Hanh V.; Hernandez-García, Adriana D.; Hoang, Nguyet-Minh; Fiorotto, Marta L.; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight−1·day−1) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E·eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs. PMID:26334346

  4. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    PubMed

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  5. Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning.

    PubMed

    Alkon, Daniel L; Epstein, Herman; Kuzirian, Alan; Bennett, M Catherine; Nelson, Thomas J

    2005-11-08

    Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo prolonged activation after associative learning. Under normal conditions, two training events with paired visual and vestibular stimuli cause short-term memory of the mollusc Hermissenda that lasts approximately 7 min. However, after 4-h exposures to Bryo (0.25 ng/ml) on two preceding days, the same two training events produced long-term conditioning that lasted >1 week and that was not blocked by anisomycin (1 mug/ml). Anisomycin, however, eliminated long-term memory lasting at least 1 week after nine training events. Both the nine training events alone and two Bryo exposures plus two training event regimens caused comparably increased levels of the PKC alpha-isozyme substrate calexcitin in identified type B neurons and enhanced PKC activity in the membrane fractions. Furthermore, Bryo increased overall protein synthesis in cultured mammalian neurons by up to 60% for >3 days. The specific PKC antagonist Ro-32-0432 blocked much of this Bryo-induced protein synthesis as well as the Bryo-induced enhancement of the behavioral conditioning. Thus, Bryo-induced PKC activation produces those proteins necessary and sufficient for long-term memory on days in advance of the training events themselves.

  6. Design, Synthesis and Affinity Properties of Biologically Active Peptide and Protein Conjugates of Cotton Cellulose

    SciTech Connect

    Edwards, J. V.; Goheen, Steven C.

    2002-11-30

    The formation of peptide and protein conjugates of cellulose on cotton fabrics provides promising leads for the development of wound healing, antibacterial, and decontaminating textiles. An approach to the design, synthesis, and analysis of bioconjugates containing cellulose peptide and protein conjugates includes: 1) computer graphic modeling for a rationally designed structure; 2) attachment of the peptide or protein to cotton cellulose through a linker amino acid, and 3) characterization of the resulting bioconjugate. Computer graphic simulation of protein and peptide cellulose conjugates gives a rationally designed biopolymer to target synthetic modifications to the cotton cellulose. Techniques for preparing these types of conjugates involve both sequential assembly of the peptide on the fabric and direct crosslinking of the peptide or protein as cellulose bound esters or carboxymethylcellulose amides.

  7. Cellular mechanisms by which oxytocin mediates ovine endometrial prostaglandin F2alpha synthesis: role of G(i) proteins and mitogen-activated protein kinases.

    PubMed

    Burns, P D; Mendes, J O; Yemm, R S; Clay, C M; Nelson, S E; Hayes, S H; Silvia, W J

    2001-10-01

    Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2alpha synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2alpha synthesis. The objective of experiment 1 was to determine whether G(i) proteins mediate oxytocin-induced PGF2alpha synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of G(i) proteins, had no effect on the ability of oxytocin to induce PGF2alpha synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF(2alpha) synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2alpha synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF(2alpha synthesis in a dose

  8. Effects of Inhibitors of RNA and Protein Synthesis on Aspartate Transcarbamylase Activity in Etiolated Plant Tissue 1

    PubMed Central

    Johnson, Lowell B.; Niblett, Charles L.; Lee, Richard F.

    1976-01-01

    Aspartate transcarbamylase (ATCase) activity declines in etiolated cowpea (Vigna unguiculata L. Walp.) and soybean (Glycine max L. Merr.) hypocotyls between 3 and 11 days after planting. Treating cow-pea hypocotyls with cycloheximide (CH), actinomycin D (AMD), 6-methyl purine (6-MP), or cordycepin increases ATCase activity up to 740, 350, 465, and 305%, respectively, over water-treated controls 48 to 72 hours after treatment. In contrast erythromycin had no effect, and d-threo-chloramphenicol (CHL) reduced ATCase activity nearly 40%. CH, AMD, and CHL, whose effects were further characterized, each markedly reduced total RNA synthesis and protein synthesis. Respiration was stimulated by CH and AMD and reduced by CHL. In soybean, CHL-treated tissues and water-treated controls had comparable ATCase activities 48 hours after treatment, while AMD, 6-MP, and CH treatments reduced activities 29, 37, and 78%, respectively. The results suggest that the level of ATCase activity in etiolated cowpea hypocotyls is regulated by a mechanism or mechanisms that are interfered with by inhibition of RNA and protein synthesis. Possibly the mechanism is absent from etiolated soybean hypocotyls. PMID:16659653

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. One-pot synthesis of protein-embedded metal-organic frameworks with enhanced biological activities.

    PubMed

    Lyu, Fengjiao; Zhang, Yifei; Zare, Richard N; Ge, Jun; Liu, Zheng

    2014-10-08

    Protein molecules were directly embedded in metal-organic frameworks (MOFs) by a coprecipitation method. The protein molecules majorly embedded on the surface region of MOFs display high biological activities. As a demonstration of the power of such materials, the resulting Cyt c embedded in ZIF-8 showed a 10-fold increase in peroxidase activity compared to free Cyt c in solution and thus gave convenient, fast, and highly sensitive detection of trace amounts of explosive organic peroxides in solution.

  11. Leucine stimulation of skeletal muscle protein synthesis

    SciTech Connect

    Layman, D.K.; Grogan, C.K.

    1986-03-01

    Previous work in this laboratory has demonstrated a stimulatory effect of leucine on skeletal muscle protein synthesis measured in vitro during catabolic conditions. Studies in other laboratories have consistently found this effect in diaphragm muscle, however, studies examining effects on nitrogen balance or with in vivo protein synthesis in skeletal muscle are equivocal. This experiment was designed to determine the potential of leucine to stimulate skeletal muscle protein synthesis in vivo. Male Sprague-Dawley rats weighing 200 g were fasted for 12 hrs, anesthetized, a jugular cannula inserted, and protein synthesis measured using a primed continuous infusion of /sup 14/C-tyrosine. A plateau in specific activity was reached after 30 to 60 min and maintained for 3 hrs. The leucine dose consisted of a 240 umole priming dose followed by a continuous infusion of 160 umoles/hr. Leucine infusion stimulated protein synthesis in the soleus muscle (28%) and in the red (28%) and white portions (12%) of the gastrocnemius muscle compared with controls infused with only tyrosine. The increased rates of protein synthesis were due to increased incorporation of tyrosine into protein and to decreased specific activity of the free tyrosine pool. These data indicate that infusion of leucine has the potential to stimulate in vivo protein synthesis in skeletal muscles.

  12. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

    PubMed

    Tao, Weikang; South, Victoria J; Diehl, Ronald E; Davide, Joseph P; Sepp-Lorenzino, Laura; Fraley, Mark E; Arrington, Kenneth L; Lobell, Robert B

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.

  13. SYNTHESIS AND MIGRATION OF PROTEINS IN THE CELLS OF THE EXOCRINE PANCREAS AS REVEALED BY SPECIFIC ACTIVITY DETERMINATION FROM RADIOAUTOGRAPHS

    PubMed Central

    Warshawsky, H.; Leblond, C. P.; Droz, B.

    1963-01-01

    Radioautographs of pancreatic acinar cells were prepared in rats and mice sacrificed at various times after injection of leucine-, glycine-, or methionine-H3. Measurements of radioactivity concentration (number of silver grains per unit area) and relative protein concentration (by microspectrophotometry of Millon-treated sections) yielded the mean specific activity of proteins in various regions of the acinar cells. The 2 to 5 minute radioautographs as well as the specific activity time curves demonstrate protein synthesis in ergastoplasm. From there, most newly synthesized proteins migrate to and accumulate in the Golgi zone. Then they spread to the whole zymogen region and, finally, enter the excretory ducts. An attempt at estimating turnover times indicated that two classes of proteins are synthesized in the ergastoplasm: "sedentary" with a slow turnover (62.5 hours) and "exportable" with rapid turnover (4.7 minutes). It is estimated that the exportable proteins spend approximately 11.7 minutes in the Golgi zone where they are built up into zymogen granules, and thereafter 36.0 minutes as fully formed zymogen granules, before they are released outside the acinar cell as pancreatic secretion. The mean life span of a zymogen granule in the cell is estimated to be 47.7 minutes. PMID:13999005

  14. New learning while consolidating memory during sleep is actively blocked by a protein synthesis dependent process

    PubMed Central

    Levy, Roi; Levitan, David; Susswein, Abraham J

    2016-01-01

    Brief experiences while a memory is consolidated may capture the consolidation, perhaps producing a maladaptive memory, or may interrupt the consolidation. Since consolidation occurs during sleep, even fleeting experiences when animals are awakened may produce maladaptive long-term memory, or may interrupt consolidation. In a learning paradigm affecting Aplysia feeding, when animals were trained after being awakened from sleep, interactions between new experiences and consolidation were prevented by blocking long-term memory arising from the new experiences. Inhibiting protein synthesis eliminated the block and allowed even a brief, generally ineffective training to produce long-term memory. Memory formation depended on consolidative proteins already expressed before training. After effective training, long term memory required subsequent transcription and translation. Memory formation during the sleep phase was correlated with increased CREB1 transcription, but not CREB2 transcription. Increased C/EBP transcription was a correlate of both effective and ineffective training and of treatments not producing memory. DOI: http://dx.doi.org/10.7554/eLife.17769.001 PMID:27919318

  15. Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids.

    PubMed

    Dickinson, Jared M; Fry, Christopher S; Drummond, Micah J; Gundermann, David M; Walker, Dillon K; Glynn, Erin L; Timmerman, Kyle L; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2011-05-01

    The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor 4E binding protein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

  16. Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation*

    PubMed Central

    Eichelbaum, Katrin; Krijgsveld, Jeroen

    2014-01-01

    Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes on a rapid time scale, as evidenced by the detection of 4852 newly synthesized proteins after only a 20-min SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome, and secretome analyses, producing an integrative analysis of the first 3 h of lipopolysaccharide-induced macrophage activation. We observed the rapid induction of multiple processes well known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not previously associated with immune response. By correlating transcriptional, translational, and secretory events, we derived novel mechanistic principles of processes specifically induced by lipopolysaccharides, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed azidohomoalanine and pulsed SILAC permits the detailed characterization of proteomic events on a rapid time scale. We anticipate that this approach will be very useful in probing the immediate effects of cellular stimuli and will provide mechanistic insight into cellular perturbation in multiple biological systems. The data have been deposited

  17. Protein synthesis is not required for acquisition, consolidation, and extinction of high foot-shock active avoidance training.

    PubMed

    González-Salinas, Sofía; Medina, Andrea C; Marín-Vignando, Vera; Ruiz-López, Clyo X; Quirarte, Gina L; Prado-Alcalá, Roberto A

    2015-01-01

    Long-term memory of active avoidance in mice is not disturbed by administration of protein synthesis inhibitors (PSIs) when relatively high levels of training are used, whereas a detrimental effect is produced with lower levels of training. PSIs also disrupt extinction of avoidance behaviors in rodents, but it is not clear whether PSIs also affect this form of learning when the behavior to be extinguished was produced by a high level of training. Experiment 1 demonstrated that rats treated with the PSI cycloheximide (CXM) 30 min before training developed normal acquisition after training with either high or low foot-shock stimulation, but that memory consolidation was hindered only after low foot-shock training. Experiment 2 demonstrated that CXM disrupted extinction when administered before the first of a series of extinction sessions when low foot-shock intensity was used during training; in contrast, after training with a higher foot-shock, the PSI treatment only interfered transiently with extinction. These results indicate that acquisition, consolidation, and extinction of active avoidance learning produced by high aversive stimulation are not dependent on protein synthesis and that these processes are governed by mechanisms different from those underlying moderate forms of learning.

  18. Cooperation of taurine uptake and dopamine D1 receptor activation facilitates the induction of protein synthesis-dependent late LTP.

    PubMed

    Suárez, Luz M; Bustamante, Julián; Orensanz, Luís M; Martín del Río, Rafael; Solís, José M

    2014-04-01

    Co-activation of NMDA and dopamine receptors is required for the induction of the late phase of LTP (L-LTP) that is dependent on new protein synthesis. Other neuromodulatory substances may also contribute to this process. Here, we examined whether taurine is one of the neuromodulators contributing to L-LTP induction, since it is known that taurine uptake induces a long-lasting synaptic potentiation dependent on protein synthesis, and taurine uptake inhibition blocks L-LTP induced by tetanization. Experiments were conducted using rat hippocampal slices where field synaptic potentials were evoked and recorded in CA3-CA1 synapses. Taurine (1 mM) applied 10 min before a high frequency stimulation (HFS) train converted a transitory early-LTP (E-LTP) into an L-LTP dependent on protein synthesis. This taurine effect was blocked by a taurine uptake inhibitor. A facilitation of L-LTP induction was also obtained by pre-application of SKF38393, a D1/D5 dopamine receptor (D1R) agonist. In this case, LTP facilitation was not affected by the taurine uptake inhibitor. Nevertheless, when taurine and SKF38393 were simultaneously pre-applied at a concentration that individually did not modify E-LTP, they produced a synergistic mechanism that facilitated the induction of L-LTP with a sole HFS train. This facilitation of L-LTP was blocked by inhibiting either taurine uptake or D1R activation. Taurine and SKF38393 activated different signaling pathways to transform E-LTP into L-LTP. Taurine-induced L-LTP facilitation required MAPK activation, while D1R-agonist-induced facilitation depended mainly on PKA activation and partially on MAPK activation. On the other hand, the synergistic mechanisms induced by the cooperative action of taurine and SKF38393 were impaired by inhibitors against MAPK, PKA and PI3-K. This pharmacological profile resembles that displayed by L-LTP induced by three HFS trains at 10-min intervals. These results indicate that taurine uptake is necessary and

  19. The Synthesis, Characterization, and Application of 13C-Methyl Isocyanide as an NMR Probe of Heme Protein Active Sites

    PubMed Central

    McCullough, Christopher; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy; Sem, Daniel

    2014-01-01

    The cytochromes P450 (CYPs) play a central role in a variety of important biological oxidations, such as steroid synthesis and the metabolism of xenobiotic compounds, including most drugs. Because CYPs are frequently assayed as drug targets or as anti-targets, tools that provide confirmation of active-site binding and information on binding orientation would be of great utility. Of greatest value are assays that are reasonably high throughput. Other heme proteins, too—such as the nitric oxide synthases (NOSs), with their importance in signaling, regulation of blood pressure, and involvement in the immune response—often display critical roles in the complex functions of many higher organisms, and also require improved assay methods. To this end, we have developed an analog of cyanide, with a 13CH3-reporter group attached to make methyl isocyanide. We describe the synthesis and use of 13C-methyl isocyanide as a probe of both bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. The 13C-methyl isocyanide probe can be used in a relatively high-throughput 1-D experiment to identify binders, but it can also be used to detect structural changes in the active site based on chemical shift changes, and potentially nuclear Overhauser effects between probe and inhibitor. PMID:23475666

  20. Evidence for intrathecal synthesis of alternative pathway complement activation proteins in experimental meningitis.

    PubMed Central

    Stahel, P. F.; Frei, K.; Fontana, A.; Eugster, H. P.; Ault, B. H.; Barnum, S. R.

    1997-01-01

    Complement has been shown to contribute to intrathecal inflammation in bacterial meningitis. However, the cellular source of complement in the infected central nervous system has not been determined. In this study, we analyzed protein and mRNA expression of two alternative pathway complement activation proteins, C3 and factor B, in the brains of mice with Listeria monocytogenes meningitis. Complement protein levels were found elevated in the cerebrospinal fluid of infected mice, compared with mock-infected animals. In the course of the disease, enhanced C3 and factor B mRNA expression was detected on pyramidal neurons and Purkinje cells within 6 hours, peaking at 12 hours and then gradually decreasing by 72 hours after infection. In addition, leukocytes infiltrating the subarachnoid space, within 12 to 24 hours, expressed mRNA for C3 and factor B. The cellular infiltration increased dramatically up to 72 hours. Intraperitoneal injection of tumor necrosis factor (TNF)-alpha up-regulated C3 and factor B mRNA expression on neurons in normal mice, suggesting that TNF-alpha may represent one cytokine regulating complement expression in this model of bacterial meningitis. However, additional mediators may be involved in regulation of intrathecal complement expression, as infected mice deficient of TNF/lymphotoxin-alpha genes did not demonstrate attenuated complement expression in the brain. Images Figure 1 Figure 2 Figure 3 PMID:9327721

  1. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC-ERK1/2-mTOR signaling pathway.

    PubMed

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W

    2015-03-17

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

  2. Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sepsis disrupts skeletal muscle proteostasis and mitigates the anabolic response to leucine (Leu) in muscle of mature animals. We have shown that Leu stimulates muscle protein synthesis (PS) in healthy neonatal piglets. To determine if supplemental Leu can stimulate PS and reduce protein degradation...

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

    PubMed Central

    Lee, Elaine Choung-Hee

    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 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. PMID:23076791

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

    PubMed

    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.

  5. Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway.

    PubMed

    Solaz-Fuster, Maria Carmen; Gimeno-Alcañiz, José Vicente; Ros, Susana; Fernandez-Sanchez, Maria Elena; Garcia-Fojeda, Belen; Criado Garcia, Olga; Vilchez, David; Dominguez, Jorge; Garcia-Rocha, Mar; Sanchez-Piris, Maribel; Aguado, Carmen; Knecht, Erwin; Serratosa, Jose; Guinovart, Joan Josep; Sanz, Pascual; Rodriguez de Córdoba, Santiago

    2008-03-01

    Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

  6. "In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

    ERIC Educational Resources Information Center

    Higgins, Pamela J.

    2005-01-01

    This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

  7. Rapamycin blocks leucine-induced protein synthesis by suppressing mTORC1 activation in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine (Leu). To elucidate the molecular mechanism by which Leu stimulates protein synthesis in neonatal muscle, overnight fasted 7-day-old piglets were...

  8. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  9. Protein synthesis inhibitor from potato tuber

    SciTech Connect

    Romaen, R. )

    1989-04-01

    A protein fraction capable of inhibit in vitro protein synthesis was found in potato tubers in fresh and wounded tissue. Inhibitor activity from fresh tissue decays with wounding. Inhibition activity was detected absorbed to ribsomal fraction and cytosol of potato tuber tissue by a partially reconstituted in vitro system from potato tuber and wheat germ. Adsorbed ribosomal fraction was more suitable of purification. This fraction was washed from ribosomes with 0.3M KCl, concentrated with ammonium sulfate precipitation and purified through sephadex G100 and sephadex G-75 columns chromatography. After 61 fold purification adsorbed protein fraction can inhibit germination of maize, wheat and sesame seeds, as well as {sup 3}H-leucine incorporation into protein by imbibed maize embryos. Inhibition activity was lost by temperature, alkali and protease-K hydrolysis. Preliminar analysis could not show presence of reductor sugars. Physiological role of this inhibitor in relation to rest and active tissue remains to be studied.

  10. The evolution of the protein synthesis system. I - A model of a primitive protein synthesis system

    NASA Technical Reports Server (NTRS)

    Mizutani, H.; Ponnamperuma, C.

    1977-01-01

    A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

  11. Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

    PubMed

    Sampieri, Clara L; Nuttall, Robert K; Young, David A; Goldspink, Deborah; Clark, Ian M; Edwards, Dylan R

    2008-03-01

    The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.

  12. Modulation of protein synthesis by polyamines.

    PubMed

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

    Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis.

  13. Oxazin-5-Ones as a Novel Class of Penicillin Binding Protein Inhibitors: Design, Synthesis and Structure Activity Relationship

    PubMed Central

    Onoabedje, Efeturi Abraham; Ibezim, Akachukwu; Okafor, Sunday Nwankwor; Onoabedje, Ufuoma Shalom; Okoro, Uchechukwu Chris

    2016-01-01

    Penicillin binding proteins (PBPs) are normal constituents of bacterial which are absent in mammalian cells. The theoretical binding modes of known oxazin-5-ones toward the protein were used as a guide to synthesis new inhibitors. Structural studies of protein-ligand complexes revealed that conformational discrepancies of the derivatives in the protein’s binding site gave rise to the variation in their inhibition constant which ranged from 68.58 μM to 2.04 mM. Biological assay results further confirmed the antibiotic potencies of the studied compounds. Although the outcome of biological screening does not parallel computational predictions, the results obtained from both methods suggest that the oxazin-5-one derivatives are potential PBP inhibitors, hence interesting antibiotic lead agents. PMID:27749913

  14. Differential effects of leucine on translation initiation factor activation and protein synthesis in skeletal muscle, renal and adipose tissues of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In adult rats, protein synthesis in skeletal muscle and adipose tissue increases in response to pharmacological doses of leucine (Leu) administered orally. In neonatal pigs, a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle without increasing hepatic protein...

  15. A Working Model of Protein Synthesis Using Lego(TM) Building Blocks.

    ERIC Educational Resources Information Center

    Templin, Mark A.; Fetters, Marcia K.

    2002-01-01

    Uses Lego building blocks to improve the effectiveness of teaching about protein synthesis. Provides diagrams and pictures for a 2-3 day student activity. Discusses mRNA, transfer RNA, and a protein synthesis model. (MVL)

  16. Mechanism and regulation of eukaryotic protein synthesis.

    PubMed Central

    Merrick, W C

    1992-01-01

    This review presents a description of the numerous eukaryotic protein synthesis factors and their apparent sequential utilization in the processes of initiation, elongation, and termination. Additionally, the rare use of reinitiation and internal initiation is discussed, although little is known biochemically about these processes. Subsequently, control of translation is addressed in two different settings. The first is the global control of translation, which is effected by protein phosphorylation. The second is a series of specific mRNAs for which there is a direct and unique regulation of the synthesis of the gene product under study. Other examples of translational control are cited but not discussed, because the general mechanism for the regulation is unknown. Finally, as is often seen in an active area of investigation, there are several observations that cannot be readily accommodated by the general model presented in the first part of the review. Alternate explanations and various lines of experimentation are proposed to resolve these apparent contradictions. PMID:1620067

  17. Protein chemical synthesis in drug discovery.

    PubMed

    Liu, Fa; Mayer, John P

    2015-01-01

    The discovery of novel therapeutics to combat human disease has traditionally been among the most important goals of research chemists. After a century of innovation, state-of-the-art chemical protein synthesis is now capable of efficiently assembling proteins of up to several hundred residues in length from individual amino acids. By virtue of its unique ability to incorporate non-native structural elements, chemical protein synthesis has been seminal in the recent development of several novel drug discovery technologies. In this chapter, we review the key advances in peptide and protein chemistry which have enabled our current synthetic capabilities. We also discuss the synthesis of D-proteins and their applications in mirror image phage-display and racemic protein crystallography, the synthesis of enzymes for structure-based drug discovery, and the direct synthesis of homogenous protein pharmaceuticals.

  18. Chronological protein synthesis in regenerating rat liver.

    PubMed

    He, Jinjun; Hao, Shuai; Zhang, Hao; Guo, Fuzheng; Huang, Lingyun; Xiao, Xueyuan; He, Dacheng

    2015-07-01

    Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration.

  19. Extinction of Conditioned Taste Aversion Depends on Functional Protein Synthesis but Not on NMDA Receptor Activation in the Ventromedial Prefrontal Cortex

    ERIC Educational Resources Information Center

    Akirav, Irit; Khatsrinov, Vicktoria; Vouimba, Rose-Marie; Merhav, Maayan; Ferreira, Guillaume; Rosenblum, Kobi; Maroun, Mouna

    2006-01-01

    We investigated the role of the ventromedial prefrontal cortex (vmPFC) in extinction of conditioned taste aversion (CTA) by microinfusing a protein synthesis inhibitor or N-methyl-d-asparate (NMDA) receptors antagonist into the vmPFC immediately following a non-reinforced extinction session. We found that the protein synthesis blocker anisomycin,…

  20. Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

    PubMed Central

    Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

    2011-01-01

    Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

  1. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

    Clements, Lee Ann J.; Jackson, Karen E.

    1998-01-01

    Describes an interactive game designed to help students see and understand the dynamic relationship between DNA, RNA, and proteins. Appropriate for either a class or laboratory setting, following a lecture session about protein synthesis. (DDR)

  2. Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

    PubMed Central

    Pabla, Ravinder; Weyrich, Andrew S.; Dixon, Dan A.; Bray, Paul F.; McIntyre, Thomas M.; Prescott, Stephen M.; Zimmerman, Guy A.

    1999-01-01

    Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell–cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin αIIbβ3 by fibrinogen and platelets deficient in this integrin, we found that αIIbβ3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin αIIbβ3 is engaged by a conformation-altering antibody against integrin αIIbβ3. Thus, outside-in signals delivered by integrin αIIbβ3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin α2β1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression. PMID:9885253

  3. Quest for the chemical synthesis of proteins.

    PubMed

    Engelhard, Martin

    2016-05-01

    The chemical synthesis of proteins has been the wish of chemists since the early 19th century. There were decisive methodological steps necessary to accomplish this aim. Cornerstones were the introduction of the Z-protecting group of Bergmann and Zervas, the development of Solid-phase Peptide Synthesis of Merrifield, and the establishment of Native Chemical Ligation by Kent. Chemical synthesis of proteins has now become generally applicable technique for the synthesis of proteins with tailor made properties which can be applied not only in vitro but also in vivo .Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  4. Oestrogen requires the insulin-like growth factor-I receptor for stimulation of prolactin synthesis via mitogen-activated protein kinase.

    PubMed

    Arroba, A I; Frago, L M; Argente, J; Chowen, J A

    2005-02-01

    Sex steroids and growth factors interact at the intracellular level in a variety of tissues to control numerous physiological functions. Oestrogen is known to stimulate prolactin synthesis and secretion, but the effect of insulin-like growth factor (IGF)-I is less clear. We used GH3 cells, a somatolactotroph cell line, to study the interaction of 17beta-oestradiol (E(2)) and IGF-I on prolactin protein levels and the intracellular mechanisms involved. Cell cultures were treated with E(2) (10 nM) and/or IGF-I (10 ng/ml) for 8 h. The real-time reverse transcriptase-polymerase chain reaction, Western blot and enzyme-immunoassay were used to determine changes in prolactin mRNA and protein levels. At this time-point, there were no significant changes in cell number, prolactin mRNA expression, or the amount of secreted prolactin. However, E(2) increased intracellular prolactin concentrations. IGF-I alone had no effect, but blocked the stimulatory effect of E(2). MAPK (ERK1/2) activation, as determined by Western blot analysis, increased with both E(2) and IGF-I, but not with the combination of these factors. The MAPK inhibitor PD98059 blocked the ability of E(2) to increase intracellular prolactin concentrations. Similarly, the IGF-I receptor antagonist, JB1, blocked the effect of E(2) on prolactin synthesis and MAPK activation, as did the oestrogen receptor antagonist ICI182 780. These results suggest that, to stimulate prolactin synthesis, E(2) activates the MAPK cascade and that this requires the presence of both oestrogen and IGF-I receptors.

  5. Engineering the prion protein using chemical synthesis.

    PubMed

    Ball, H L; King, D S; Cohen, F E; Prusiner, S B; Baldwin, M A

    2001-11-01

    In recent years, the technology of solid-phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared several modified and wild-type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, alpha-helical cellular protein (PrPc) into a beta-sheet-rich pathogenic isoform (PrP(Sc)). The ability to form PrP(Sc) in transgenic mice is retained by a 106 residue 'mini-prion' (PrP106), with the deletions 23-88 and 141-176. Synthetic PrP106 (sPrP106) and a His-tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N-(2-chlorobenzyloxycarbonyloxy) succinimide. A single reversed-phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90-200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t-Boc chemistry. To mimic the glycosylphosphatidyl inositol (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surface, where the conversion of PrPc is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences. These groups enabled sPrP to be immobilized on either the cell surface or a streptavidin-coated ELISA plate, respectively, in an orientation analogous to that of membrane-bound, GPI-anchored PrPc. The chemical manipulation of such

  6. Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates.

    PubMed

    Wurm, Frederik; Dingels, Carsten; Frey, Holger; Klok, Harm-Anton

    2012-04-09

    Polymer-protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer-protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG-lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure-activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight.

  7. Inadequacy of prebiotic synthesis as origin of proteinous amino acids.

    PubMed

    Wong, J T; Bronskill, P M

    1979-07-18

    The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

  8. [The synthesis of proteins in unnucleated blood platelets].

    PubMed

    Bijak, Michał; Saluk, Joanna; Ponczek, Michał Błażej Ponczek; Nowak, Paweł; Wachowicz, Barbara

    2013-07-23

    Platelets are the smallest, unnucleated blood cells that play a key role in maintaining normal hemostasis. In the human body about 1x1011 platelets are formed every day, as a the result of complex processes of differentiation, maturation and fragmentation of megakaryocytes. Studies done over 4 decades ago demonstrated that circulating in blood mature platelets can synthesize proteins. Recent discoveries confirm protein synthesis by unnucleated platelets in response to activation. Moreover, protein synthesis alters the phenotype and function of platelets. Platelets synthesize several proteins involved in hemostasis (COX, αIIbβ3, TF PAI-1, Factor XI, protein C inhibitor) and in inflammatory process (IL-1β, CCL5/RANTES). In spite of lack of transcription platelets have a stable mRNA transcripts with a long life correlated with platelet life span. Platelets also show expression of two important key regulators of translation eIF4E and EIF-2α and have a variety of miRNA molecules responsible for translational regulation. This article describes the historical overview of research on protein synthesis by platelets and presents the molecular mechanisms of protein synthesis in activated platelets (and synthesis of the most important platelet proteins).

  9. Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Gerwins, P; Johnson, G L; Henson, P M

    1997-04-01

    Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.

  10. Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

    PubMed Central

    Coleclough, C; Kuhn, L; Lefkovits, I

    1990-01-01

    Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides. Images PMID:2308934

  11. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    SciTech Connect

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  12. Chemical protein synthesis (CPS) meeting 2013.

    PubMed

    Metanis, Norman

    2013-07-22

    Building bonds in Vienna: The Chemical Protein Synthesis meeting recently took place at the University of Vienna, Austria. This report describes the event and highlights the science presented over the four days.

  13. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC–ERK1/2–mTOR signaling pathway

    PubMed Central

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W.

    2015-01-01

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases. PMID:25733860

  14. Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin.

    PubMed Central

    Mendez, R; Kollmorgen, G; White, M F; Rhoads, R E

    1997-01-01

    The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression. PMID:9271396

  15. Remotely activated protein-producing nanoparticles.

    PubMed

    Schroeder, Avi; Goldberg, Michael S; Kastrup, Christian; Wang, Yingxia; Jiang, Shan; Joseph, Brian J; Levins, Christopher G; Kannan, Sneha T; Langer, Robert; Anderson, Daniel G

    2012-06-13

    The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.

  16. Determination of in vivo protein synthesis in human palatine tonsil.

    PubMed

    Januszkiewicz, Anna; Klaude, Maria; Loré, Karin; Andersson, Jan; Ringdén, Olle; Rooyackers, Olav; Wernerman, Jan

    2005-02-01

    The palatine tonsils are constantly exposed to ingested or inhaled antigens which, in turn, lead to a permanent activation of tonsillar immune cells, even in a basic physiological state. The aim of the present study was to investigate if the immunological activation of the human palatine tonsil is reflected by a high metabolic activity, as determined by in vivo measurement of protein synthesis. The protein synthesis rate of the tonsil was also compared with that of the circulating T-lymphocytes, the total blood mononuclear cells and the whole population of blood leucocytes. Phenotypic characterization of immune-competent cells in tonsil tissue and blood was performed by flow cytometry. Pinch tonsil biopsies were taken after induction of anaesthesia in healthy adult patients (n=12) scheduled for ear surgery, uvulopalatopharyngoplasty or nose surgery. Protein synthesis was quantitatively determined during a 90-min period by a flooding-dose technique. The in vivo protein synthesis rate in the palatine tonsils was 22.8+/-5.7%/24 h (mean+/-S.D.), whereas protein synthesis in the circulating T-lymphocytes was 10.7+/-3.4%/24 h, in mononuclear cells was 10.8+/-2.8%/24 h and in leucocytes was 3.2+/-1.2%/24 h. CD3+ lymphocytes were the most abundant cell population in the tonsil. The in vivo protein synthesis rate in human tonsils was higher compared with the circulating immune cells. This high metabolic rate may reflect the permanent immunological activity present in human tonsils, although cell phenotypes and activity markers do not explain the differences.

  17. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    NASA Astrophysics Data System (ADS)

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  18. Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

    PubMed

    Anderson, Mark J; Stark, Jessica C; Hodgman, C Eric; Jewett, Michael C

    2015-07-08

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.

  19. The global transcriptional activator of Saccharomyces cerevisiae, Gcr1p, mediates the response to glucose by stimulating protein synthesis and CLN-dependent cell cycle progression.

    PubMed Central

    Willis, Kristine A; Barbara, Kellie E; Menon, Balaraj B; Moffat, Jason; Andrews, Brenda; Santangelo, George M

    2003-01-01

    Growth of Saccharomyces cerevisiae requires coordination of cell cycle events (e.g., new cell wall deposition) with constitutive functions like energy generation and duplication of protein mass. The latter processes are stimulated by the phosphoprotein Gcr1p, a transcriptional activator that operates through two different Rap1p-mediated mechanisms to boost expression of glycolytic and ribosomal protein genes, respectively. Simultaneous disruption of both mechanisms results in a loss of glucose responsiveness and a dramatic drop in translation rate. Since a critical rate of protein synthesis (CRPS) is known to mediate passage through Start and determine cell size by modulating levels of Cln3p, we hypothesized that GCR1 regulates cell cycle progression by coordinating it with growth. We therefore constructed and analyzed gcr1delta cln3delta and gcr1delta cln1delta cln2delta strains. Both strains are temperature and cold sensitive; interestingly, they exhibit different arrest phenotypes. The gcr1delta cln3delta strain becomes predominantly unbudded with 1N DNA content (G1 arrest), whereas gcr1delta cln1delta cln2delta cells exhibit severe elongation and apparent M phase arrest. Further analysis demonstrated that the Rap1p/Gcr1p complex mediates rapid growth in glucose by stimulating both cellular metabolism and CLN transcription. PMID:14668361

  20. Adrenergic activation of melatonin secretion in ovine pineal explants in short-term superfusion culture occurs via protein synthesis independent and dependent phenomena.

    PubMed

    Lewczuk, Bogdan; Ziółkowska, Natalia; Prusik, Magdalena; Przybylska-Gornowicz, Barbara

    2014-01-01

    The ovine pineal is generally considered as an interesting model for the study on adrenergic regulation of melatonin secretion due to some functional similarities with this gland in the human. The present investigations, performed in the superfusion culture of pineal explants, demonstrated that the norepinephrine-induced elevation of melatonin secretion in ovine pinealocytes comprised of two subsequent periods: a rapid increase phase and a slow increase phase. The first one included the quick rise in release of N-acetylserotonin and melatonin, occurring parallel to elevation of NE concentration in the medium surrounding explants. This rapid increase phase was not affected by inhibition of translation. The second, slow increase phase began after NE level had reached the maximum concentration in the culture medium and lasted about two hours. It was completely abolished by the treatment with translation inhibitors. The obtained results showed for the first time that the regulation of N-acetylserotonin synthesis in pinealocytes of some species like the sheep involves the on/off mechanism, which is completely independent of protein synthesis and works very fast. They provided strong evidence pointing to the need of revision of the current opinion that arylalkylamines N-acetyltransferase activity in pinealocytes is controlled exclusively by changes in enzyme abundance.

  1. Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

    PubMed

    Kazuta, Yasuaki; Matsuura, Tomoaki; Ichihashi, Norikazu; Yomo, Tetsuya

    2014-11-01

    In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies.

  2. Synthesis, molecular structure, theoretical calculation, DNA/protein interaction and cytotoxic activity of manganese(III) complex with 8-hydroxyquinoline.

    PubMed

    Thamilarasan, V; Sengottuvelan, N; Sudha, A; Srinivasan, P; Siva, A

    2015-01-01

    Manganese(III) complex (1) [Mn(8-hq)3] (where 8-hq=8-hydroxyquinoline) has been synthesized and characterized by elemental, spectral (UV-vis, FT-IR) and thermal analysis. The structure of complex (1) has been determined by single crystal X-ray diffraction studies and the configuration around manganese(III) ion was elongated octahedral coordination geometry. Density functional theory calculations were performed for ligand and its complex. Binding studies of ligand and complex 1 with calf thymus DNA (CT-DNA) was investigated by absorption, fluorescence, circular dichroic (CD) spectroscopy and viscosity measurements. Absorption spectral studies revealed that ligand and complex 1 binds to DNA groove and its intrinsic binding strength has been found to be 2.57×10(4) and 2.91×10(4)M(-1). A molecular docking study confirm that the complex 1 is a minor groove binder and was stabilized through hydrogen bonding interactions. Complex 1 exhibits a good binding propensity to bovine serum albumin (BSA) protein. The in vitro cytotoxicity study of complex 1 on breast cancer cell line (MCF-7) indicate that it has the potential to act as effective anticancer drug, with IC50 values of 3.25μM. The ligand and its complex have been screened for antimicrobial activities and the complex showed better antimicrobial activity than the free ligand.

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

    PubMed

    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.

  4. Prostaglandin A1 inhibits avian influenza virus replication at a postentry level: Effect on virus protein synthesis and NF-κB activity.

    PubMed

    Carta, Stefania; La Frazia, Simone; Donatelli, Isabella; Puzelli, Simona; Rossi, Antonio; Santoro, M Gabriella

    2014-12-01

    Influenza A viruses (IAV) have the potential to cause devastating pandemics. In recent years, the emergence of new avian strains able to infect humans represents a serious threat to global human health. The increase in drug-resistant IAV strains underscores the need for novel approaches to anti-influenza chemotherapy. Herein we show that prostaglandin-A1 (PGA1) possesses antiviral activity against avian IAV, including H5N9, H7N1 and H1N1 strains, acting at a level different from the currently available anti-influenza drugs. PGA1 acts at postentry level, causing dysregulation of viral protein synthesis and preventing virus-induced disassembly of host microtubular network and activation of pro-inflammatory factor NF-κB. The antiviral activity is dependent on the presence of a cyclopentenone ring structure and is associated with activation of a cytoprotective heat shock response in infected cells. The results suggest that cyclopentenone prostanoids or prostanoids-derived molecules may represent a new tool to combat avian influenza virus infection.

  5. Studies with Hydroxyurea VII. Hydroxyurea and the Synthesis of Functional Proteins

    PubMed Central

    Rosenkranz, Herbert S.; Winshell, Elaine B.; Mednis, Aiga; Carr, Howard S.; Ellner, Cornelia J.

    1967-01-01

    Hydroxyurea affected neither the synthesis nor the degradation of bacterial messenger-ribonucleic acid. The proteins made by hydroxyurea-treated cells were structurally intact and fully functional. Since the expression of the lethal action of hydroxyurea requires active protein production, the data indicate that treated cells do not die as the result of the synthesis of abnormal proteins. Images PMID:4963772

  6. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    PubMed

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  7. Protein synthesis in geostimulated root caps

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.

    1982-01-01

    A study is presented of the processes occurring in the root cap of corn which are requisite for the formation of root cap inhibitor and which can be triggered or modulated by both light and gravity. The results of this study indicate the importance of protein synthesis for light-induced gravitropic bending in roots. Root caps in which protein synthesis is prevented are unable to induce downward bending. This suggests that light acts by stimulating proteins which are necessary for the translation of the gravitropic stimulus into a growth response (downward bending). The turnover of protein with time was also examined in order to determine whether light acts by stimulating the synthesis of unique proteins required for downward growth. It is found that auxin in combination with light allows for the translation of the gravitropic stimulus into a growth response at least in part through the modification of protein synthesis. It is concluded that unique proteins are stimulated by light and are involved in promoting the downward growth in roots which are responding to gravity.

  8. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes.

  9. In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure.

    PubMed Central

    Schultze, M; Staehelin, C; Röhrig, H; John, M; Schmidt, J; Kondorosi, E; Schell, J; Kondorosi, A

    1995-01-01

    The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior. Images Fig. 5 PMID:7708710

  10. Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

    NASA Technical Reports Server (NTRS)

    Tucker, K. R.; Seider, M. J.; Booth, F. W.

    1981-01-01

    Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.

  11. Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

    PubMed

    Xu, Y J; Yau, L; Yu, L P; Elimban, V; Zahradka, P; Dhalla, N S

    1996-12-13

    Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

  12. Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

    SciTech Connect

    Nair, M.P.N.; Pottathil, R.; Heimer, E.P.; Schwartz, S.A.

    1988-09-01

    Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by (/sup 3/H)thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3/sup +/ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3/sup /minus// lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection.

  13. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    PubMed

    He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

    2012-07-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

  14. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    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.

  15. The synthesis of active biomaterials through nanofabrication and sol-gel encapsulation of liposomes and membrane proteins

    NASA Astrophysics Data System (ADS)

    Soong, Ricky Kai

    The following dissertation reveals the latest advancements in developing self-sustaining hybrid nano-systems. Three areas of research were initiated: (1) Dielectrophoretic (DEP) mediation of hybrid assembly, (2) Solar powered proton pumping films, and (3) Silica materials with biochemical output for integration with nano-devices. The first topic of research was devoted to creating reliable hybridization platforms. This was achieved by implementing AC electric-field forces. One of the primary considerations in utilizing DEP is buffer conductivity. The initial medium used to preserve biomotor functionality was too conductive and AC field effects were significantly reduced. Subsequent testing with lower ionic strength indicated that the biomolecules were repelled from field intense regions. Hence, nano-electrode arrays were reconfigured to trap device components. Initial results showed promising potential but current lithographic limitations require new nanofabrication methodologies to obtain the desired electrode design. The second research project was focused on creating solar powered biomaterials. Liposomes containing bR proton pumping proteins and pyranine fluorescent dye into phospholipid vesicles were encapsulated within a silica matrix. The characteristic 402/456 nm pyranine peaks blue shifted upon acidification by bR. The proteoliposomes were mixed in a 3:1 ratio with tetramethyl orthosilicate (TMOS) sol respectively to provide a solar powered thin proteogel films. Ultimately, the ability to prepare these proteogels enabled the establishment of a proton gradient, and therefore opportunities to use these materials for biologically based power generation. The third research project involved engineering nanobiochemical reaction environments within a three-dimensional construct. The goal here was to recruit encapsulated enzymes to actively synthesize biochemical compounds. These compounds were subsequently collected and used as a fuel source for integrated nano

  16. [Protein nutrition and physical activity].

    PubMed

    Navarro, M P

    1992-09-01

    The relationship between physical exercise and diet in order to optimize performance is getting growing interest. This review examines protein needs and protein intakes as well as the role of protein in the body and the metabolic changes occurring at the synthesis and catabolic levels during exercise. Protein synthesis in muscle or liver, amino acids oxidation, glucose production via gluconeogenesis from amino acids, etc., are modified, and consequently plasma and urinary nitrogen metabolites are affected. A brief comment on the advantages, disadvantages and forms of different protein supplements for sportsmen is given.

  17. Viral protein R of HIV type-1 induces retrotransposition and upregulates glutamate synthesis by the signal transducer and activator of transcription 1 signaling pathway.

    PubMed

    Doi, Akihiro; Iijima, Kenta; Kano, Shigeyuki; Ishizaka, Yukihito

    2015-07-01

    Viral protein R (Vpr) of HIV-1 plays an important role in viral replication in macrophages. Various lines of evidence suggest that expression of Vpr in macrophages causes immunopathogenesis; however, the underlying mechanism is not yet fully understood. In this study, it was shown that recombinant Vpr (rVpr) induces retrotransposition of long interspersed element-1 in RAW264.7, a macrophage-like cell line, and activates reverse transcriptase-dependent immunotoxic cascades including production of IFN-β and phosphorylation of signal transducer and activator of transcription 1 (STAT1). Knockout experiments based on the CRISPR/Cas9 nickase system further demonstrated that cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and stimulator of interferon gene (STING) are responsible for IFN-β production and STAT1 phosphorylation, respectively. Moreover, rVpr was found to increase production of glutaminase C, a regulator of glutamate synthesis, which is also dependent on the cGAS-STING pathway. Taken together with reports that glutaminase C is involved in the pathogenesis of HIV-associated neurocognitive disorder (HAND) and that Vpr is detectable in the cerebrospinal fluid of HIV-1-positive patients, a possible role of Vpr-induced L1-RTP and immunotoxic cascades in the development of HAND is discussed.

  18. Origins of the protein synthesis cycle

    NASA Technical Reports Server (NTRS)

    Fox, S. W.

    1981-01-01

    Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

  19. Cell-free protein synthesis and assembly on a biochip

    NASA Astrophysics Data System (ADS)

    Heyman, Yael; Buxboim, Amnon; Wolf, Sharon G.; Daube, Shirley S.; Bar-Ziv, Roy H.

    2012-06-01

    Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.

  20. HSP-72 Synthesis is Promoted by Increase in (Ca2+)i or Activation of G Proteins but not pHi of cAMP

    DTIC Science & Technology

    1994-01-01

    p.M fura 2 -AM plus 0.2% pluronic F-127 at 37°C shift assays. Confluent cells in 150-cm 2 flasks were trypsinized for 60 min. Cells were then washed...synthesis was measured 8 h after trimethoxybenzoate (TMB-8), fura 2 -AM, BAPTA-AM, and the first heating. The level of HSP-72 in the cell after the BCECF...Loss of a calcium requirement for protein synthesis in Price and Calderwood (42) reported that both Ca 2 + and pituitary cells following thermal or

  1. Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ.

    PubMed

    Li, Xin; Sheng, Juzheng; Huang, Guihua; Ma, Ruixin; Yin, Fengxin; Song, Di; Zhao, Can; Ma, Shutao

    2015-06-05

    In an attempt to discover potential antibacterial agents against the increasing bacterial resistance, novel cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their antibacterial activity against nine significant pathogens using broth microdilution method, and their cell division inhibitory activity against four representative strains. In the in vitro antibacterial activity, the newly synthesized compounds generally displayed better efficacy against Staphylococcus aureus ATCC25923 than the others. In particular, compounds 3, 8 and 10 exerted superior or comparable activity to all the reference drugs. In the cell division inhibitory activity, all the compounds showed the same trend as their in vitro antibacterial activity, exhibiting better activity against S. aureus ATCC25923 than the other strains. Additionally, compounds 3, 6, 7 and 8 displayed potent cell division inhibitory activity with an MIC value of below 1 μg/mL, over 256-fold better than all the reference drugs.

  2. Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec).

    PubMed

    Wang, Y; McAllister, T A; Rode, L M; Beauchemin, K A; Morgavi, D P; Nsereko, V L; Iwaasa, A D; Yang, W

    2001-03-01

    The effects of an exogenous enzyme preparation, the application method and feed type on ruminal fermentation and microbial protein synthesis were investigated using the rumen simulation technique (Rusitec). Steam-rolled barley grain and chopped alfalfa hay were sprayed with water (control, C), an enzyme preparation with a predominant xylanase activity (EF), or autoclaved enzyme (AEF) 24 h prior to feeding, or the enzyme was supplied in the buffer infused into the Rusitec (EI). Microbial N incorporation was measured using (15NH4)2SO4 in the buffer. Spent feed bags were pummelled mechanically in buffer to segregate the feed particle-associated (FPA) and feed particle-bound (FPB) bacterial fractions. Enzymes applied to feed reduced neutral-detergent fibre content, and increased the concentration of reducing sugars in barley grain, but not alfalfa hay. Ruminal cellulolytic bacteria were more numerous with EF than with C. Disappearance of DM from barley grain was higher with EF than with C, but alfalfa was unaffected by EF. Treatment EF increased incorporation of 15N into FPA and FPB fractions at 24 and 48 h. In contrast, AEF reduced the 24 h values, relative to C; AEF and C were similar at 48 h. Infused enzyme (EI) did not affect 15N incorporation. Xylanase activity in effluent was increased by EF and EI, compared to C, but not by AEF. Xylanase activity in FPA was higher at 48 h than at 24 h with all treatments; it was higher with EF than C at 24 and 48 h, but was not altered by AEF or EI. Applying enzymes onto feeds before feeding was more effective than dosing directly into the artificial rumen for increasing ruminal fibrolytic activity.

  3. Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Kapeli, Katannya; Hurlin, Peter J.

    2011-01-01

    Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-RasG12V) in very different ways. H-RasG12V promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-RasG12V-mediated destabilization of N-Myc. We show that H-RasG12V-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis. PMID:21908617

  4. Green synthesis of peptide-templated gold nanoclusters as novel fluorescence probes for detecting protein kinase activity.

    PubMed

    Song, Wei; Liang, Ru-Ping; Wang, Ying; Zhang, Li; Qiu, Jian-Ding

    2015-06-21

    A green method was employed for synthesizing peptide-templated nanoclusters without requiring strong reducing agents. Using synthetic peptide-gold nanoclusters as fluorescence probes, a novel assay for detecting protein kinase is developed based on phosphorylation against carboxypeptidase Y digestion.

  5. Perylenequinones: Isolation, Synthesis, and Biological Activity

    PubMed Central

    Mulrooey, Carol A.; O'Brien, Erin M.; Morgan, Barbara J.

    2013-01-01

    The perylenequinones are a novel class of natural products characterized by pentacyclic conjugated chromophore giving rise to photoactivity. Potentially useful light-activated biological activity, targeting protein kinase C (PKC), has been identified for several of the natural products. Recently discovered new members of this class of compound, as well as several related phenanthroperylenequinones, are reviewed. Natural product modifications that improve biological profiles, and avenues for the total synthesis of analogs, which are not available from the natural product series, are outlined. An overview of structure/function relationships is provided. PMID:24039544

  6. Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

    PubMed

    Corbett, Grant T; Roy, Avik; Pahan, Kalipada

    2013-03-22

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

  7. A microplate assay for the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis.

    PubMed

    Kumar, Vidya P; Basavannacharya, Chandrakala; de Sousa, Sunita M

    2014-07-18

    A microplate, scintillation proximity assay to measure the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[(3)H]N-acetylglucosamine in the presence of 40 μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-D-ala-D-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a 'neutral' combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.

  8. Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

    NASA Technical Reports Server (NTRS)

    Raghavan, V.

    1991-01-01

    Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

  9. Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal...

  10. Prolonged leucine infusion differentially affects tissue protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine (Leu) acutely stimulates protein synthesis by activating the mammalian target of rapamycin complex 1 (mTORC1) pathway. To determine whether Leu can stimulate protein synthesis in muscles of different fiber types and visceral tissues of the neonate for a prolonged period and to determine the ...

  11. Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

    ERIC Educational Resources Information Center

    Wolf, Gerald; Engelmann, Mario; Richter, Karin

    2005-01-01

    Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was…

  12. Feeding rapidly stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing translation initiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food consumption increases protein synthesis in most tissues by promoting translation initiation, and in the neonate, this increase is greatest in skeletal muscle. In this study, we aimed to identify the currently unknown time course of changes in the rate of protein synthesis and the activation of ...

  13. Haematopoietic stem cells require a highly regulated protein synthesis rate.

    PubMed

    Signer, Robert A J; Magee, Jeffrey A; Salic, Adrian; Morrison, Sean J

    2014-05-01

    Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

  14. Affinity labeling of protein synthesis factors

    SciTech Connect

    Anthony, D.D.; Dever, T.E.; Abramson, R.D.; Lobur, M.; Merrick, W.C.

    1986-05-01

    The authors laboratory is interested in determining those eukaryotic protein synthesis factors which interact with nucleotides and mRNA. To study the binding the authors have used the nucleotides, their analogs, and mRNA analogs as listed below: (1) UV cross-linking with normal (/sup 32/P)XTP; (2) Oxidized GTP; (3) 3'p-azido benzoyl GDP (GTP); (4) 5'p-fluoro sulfonyl benzoyl guanosine; (5) 5'p-fluoro sulfonyl benzoyl adenosine; (6) oxidized mRNA. Currently, they are continuing their efforts to specifically label the proteins, and they are also trying to isolate a single labeled tryptic peptide from the proteins.

  15. Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

    USGS Publications Warehouse

    Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

    2002-01-01

    Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

  16. Pin1 and PKMζ Sequentially Control Dendritic Protein Synthesis

    PubMed Central

    Westmark, Pamela R.; Westmark, Cara J.; Wang, SuQing; Levenson, Jonathan; O’Riordan, Kenneth J.; Burger, Corinna; Malter, James S.

    2010-01-01

    Some forms of learning and memory, and their electrophysiologic correlate, long-term potentiation (LTP), require dendritic translation. We demonstrate that Pin1, a peptidyl-prolyl isomerase, is present in dendritic spines and shafts and inhibits protein synthesis induced by glutamatergic signaling. Pin1 suppression increased dendritic translation, possibly through eIF4E binding proteins 1 and 2 (4E-BP1/2) and eukaryotic translation initiation factor 4E (eIF4E). Consistent with increased protein synthesis, hippocampal slices from Pin−/− mice had normal early LTP (E-LTP) but significantly enhanced late LTP (L-LTP) compared to wild-type controls. Protein kinase C ζ (PKCζ) and protein kinase M ζ (PKMζ) were increased in Pin1−/− mouse brain and their activity was required to maintain dendritic translation. PKMζ interacted with and inhibited Pin1 by phosphorylating Ser16. Therefore, glutamate-induced, dendritic protein synthesis is sequentially regulated by Pin1 and PKMζ signaling. PMID:20215645

  17. DNA Methyltransferase protein synthesis is reduced in CXXC finger protein 1-deficient embryonic stem cells.

    PubMed

    Butler, Jill S; Palam, Lakshmi R; Tate, Courtney M; Sanford, Jeremy R; Wek, Ronald C; Skalnik, David G

    2009-05-01

    CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life.

  18. Semi-synthesis of thioamide containing proteins.

    PubMed

    Wang, Yanxin J; Szantai-Kis, D Miklos; Petersson, E James

    2015-05-14

    Our laboratory has shown that the thioamide, a single atom O-to-S substitution, can be a versatile fluorescence quenching probe that is minimally-perturbing when placed at many locations in a protein sequence. In order to make these and other thioamide experiments applicable to full-sized proteins, we have developed methods for incorporating thioamides by generating thiopeptide fragments through solid phase synthesis and ligating them to protein fragments expressed in E. coli. To install donor fluorophores, we have adapted unnatural amino acid mutagenesis methods, including the generation of new tRNA synthetases for the incorporation of small, intrinsically fluorescent amino acids. We have used a combination of these two methods, as well as chemoenzymatic protein modification, to efficiently install sidechain and backbone modifications to generate proteins labeled with fluorophore/thioamide pairs.

  19. Antibiotics that target protein synthesis.

    PubMed

    McCoy, Lisa S; Xie, Yun; Tor, Yitzhak

    2011-01-01

    The key role of the bacterial ribosome makes it an important target for antibacterial agents. Indeed, a large number of clinically useful antibiotics target this complex translational ribonucleoprotein machinery. The majority of these compounds, mostly of natural origin, bind to one of the three key ribosomal sites: the decoding (or A-site) on the 30S, the peptidyl transferase center (PTC) on the 50S, and the peptide exit tunnel on the 50S. Antibiotics that bind the A-site, such as the aminoglycosides, interfere with codon recognition and translocation. Peptide bond formation is inhibited when small molecules like oxazolidinones bind at the PTC. Finally, macrolides tend to block the growth of the amino acid chain at the peptide exit tunnel. In this article, the major classes of antibiotics that target the bacterial ribosome are discussed and classified according to their respective target. Notably, most antibiotics solely interact with the RNA components of the bacterial ribosome. The surge seen in the appearance of resistant bacteria has not been met by a parallel development of effective and broad-spectrum new antibiotics, as evident by the introduction of only two novel classes of antibiotics, the oxazolidinones and lipopeptides, in the past decades. Nevertheless, this significant health threat has revitalized the search for new antibacterial agents and novel targets. High resolution structural data of many ribosome-bound antibiotics provide unprecedented insight into their molecular contacts and mode of action and inspire the design and synthesis of new candidate drugs that target this fascinating molecular machine.

  20. N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

    PubMed

    Kamita, Masahiro; Kimura, Yayoi; Ino, Yoko; Kamp, Roza M; Polevoda, Bogdan; Sherman, Fred; Hirano, Hisashi

    2011-04-01

    N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.

  1. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  2. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upreg...

  3. Elongation factors in protein synthesis.

    PubMed

    Kraal, B; Bosch, L; Mesters, J R; de Graaf, J M; Woudt, L P; Vijgenboom, E; Heinstra, P W; Zeef, L A; Boon, C

    1993-01-01

    Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

  4. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  5. Effects of quebracho tannin extract (Schinopsis balansae Engl.) and activated charcoal on nitrogen balance, rumen microbial protein synthesis and faecal composition of growing Boer goats.

    PubMed

    Al-Kindi, Amal; Dickhoefer, Uta; Schlecht, Eva; Sundrum, Albert; Schiborra, Anne

    2016-08-01

    Under irrigated arid conditions, organic fertiliser rich in slowly decomposable nitrogen (N) and carbon (C) is needed for soil fertility maintenance. Feeding ruminants with condensed tannins will lower ruminal protein degradation, reduce urinary N excretion and might increase the faecal fraction of slowly decomposable N. Supplementation with activated charcoal (AC) might enrich manure with slowly degrading C. Therefore, we investigated the effects of feeding quebracho tannin extract (QTE) and AC on the N balance of goats, the efficiency of microbial protein synthesis in the rumen (EMPS) and the composition of faeces. The feeding trial comprised three periods; in each period, 12 male Boer goats (28 ± 3.9 kg live weight) were assigned to six treatments: a Control diet (per kg diet 500 g grass hay and 500 g concentrate) and to further five treatments the Control diet was supplemented with QTE (20 g and 40 g/kg; diets QTE2 and QTE4, respectively), with AC (15 g and 30 g/kg, diets AC1.5 and AC3.0, respectively) and a mixture of QTE (20 g/kg) plus AC (15 g/kg) (diet QTEAC). In addition to the N balance, EMPS was calculated from daily excretions of purine derivatives, and the composition of faecal N was determined. There was no effect of QTE and AC supplementation on the intake of organic matter (OM), N and fibre, but apparent total tract digestibility of OM was reduced (p = 0.035). Feeding QTE induced a shift in N excretion from urine to faeces (p ≤ 0.001) without altering N retention. Total N excretion tended to decrease with QTE treatments (p = 0.053), but EMPS was not different between treatments. Faecal C excretion was higher in QTE and AC treatments (p = 0.001) compared with the Control, while the composition of faecal N differed only in concentration of undigested dietary N (p = 0.001). The results demonstrate that QTE can be included into diets of goats up to 40 g/kg, without affecting N utilisation, but simultaneously increasing the

  6. The lipid peroxidation product 4-hydroxy-trans-2-nonenal causes protein synthesis in cardiac myocytes via activated mTORC1-p70S6K-RPS6 signaling.

    PubMed

    Calamaras, Timothy D; Lee, Charlie; Lan, Fan; Ido, Yasuo; Siwik, Deborah A; Colucci, Wilson S

    2015-05-01

    Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

  7. Chronic leucine supplementation of a low protein diet increases protein synthesis in skeletal muscle and visceral tissues of neonatal pigs through mTOR signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine acutely stimulates protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that leucine supplementation of a low protein diet will enhance protein synthesis and mTOR signaling in the neonate for prolonged periods. Fasted 5-d-old pigs (n=6–8...

  8. Design, Synthesis, Acaricidal/Insecticidal Activity, and Structure-Activity Relationship Studies of Novel Oxazolines Containing Sulfone/Sulfoxide Groups Based on the Sulfonylurea Receptor Protein-Binding Site.

    PubMed

    Yu, Xiuling; Liu, Yuxiu; Li, Yongqiang; Wang, Qingmin

    2016-04-20

    Enormous compounds containing sulfone/sulfoxide groups have been used in a variety of fields, especially in drug and pesticide design. To search for novel environmentally benign and ecologically safe pesticides with unique modes of action, a series of 2,4-diphenyl-1,3-oxazolines containing sulfone/sulfoxide groups as chitin synthesis inhibitors (CSIs) were designed and synthesized on the basis of the sulfonylurea receptor protein-binding site for CSIs. Their structures were characterized by (1)H and (13)C nuclear magnetic resonance and high-resolution mass spectrometry. The acaricidal and insecticidal activities of the new compounds were evaluated. It was found that most of the target compounds displayed wonderful acaricidal activities against spider mite (Tetranychus cinnabarinus) larvae and eggs. Especially compounds I-4, II-3, and II-4 displayed higher activities than commercial etoxazole at a concentration of 2.5 mg L(-1). Some target compounds exhibited insecticidal activities against lepidopteran pests. The present work demonstrated that these compounds containing sulfone/sulfoxide groups could be considered as potential candidates for the development of novel acaricides in the future.

  9. Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors*

    PubMed Central

    Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

    2015-01-01

    Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. PMID:26134564

  10. Computational model of the fathead minnow hypothalamic-pituitary-gonadal axis: Incorporating protein synthesis in improving predictability of responses to endocrine active chemicals.

    PubMed

    Breen, Miyuki; Villeneuve, Daniel L; Ankley, Gerald T; Bencic, David; Breen, Michael S; Watanabe, Karen H; Lloyd, Alun L; Conolly, Rory B

    2016-01-01

    There is international concern about chemicals that alter endocrine system function in humans and/or wildlife and subsequently cause adverse effects. We previously developed a mechanistic computational model of the hypothalamic-pituitary-gonadal (HPG) axis in female fathead minnows exposed to a model aromatase inhibitor, fadrozole (FAD), to predict dose-response and time-course behaviors for apical reproductive endpoints. Initial efforts to develop a computational model describing adaptive responses to endocrine stress providing good fits to empirical plasma 17β-estradiol (E2) data in exposed fish were only partially successful, which suggests that additional regulatory biology processes need to be considered. In this study, we addressed short-comings of the previous model by incorporating additional details concerning CYP19A (aromatase) protein synthesis. Predictions based on the revised model were evaluated using plasma E2 concentrations and ovarian cytochrome P450 (CYP) 19A aromatase mRNA data from two fathead minnow time-course experiments with FAD, as well as from a third 4-day study. The extended model provides better fits to measured E2 time-course concentrations, and the model accurately predicts CYP19A mRNA fold changes and plasma E2 dose-response from the 4-d concentration-response study. This study suggests that aromatase protein synthesis is an important process in the biological system to model the effects of FAD exposure.

  11. Synthesis in Escherichia coli of two smaller enzymically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene.

    PubMed Central

    Mo, Y Y; Seshu, J; Wang, D; Mallavia, L P

    1998-01-01

    FK506-binding proteins (FKBPs) have been identified in a variety of eukaryotic and prokaryotic organisms. Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Coxiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (PPIase) activity. Further characterization of the cbmip gene has identified two additional proteins with molecular masses of 15.5 and 15.0 kDa that are synthesized, in addition to the 23.5 kDa CbMip, when expressed in Escherichia coli. Amino acid sequencing at the N-terminus combined with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation. When the internal methionines serving as start sites were replaced with lysine by site-directed mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was abolished even though the synthesis of 23.5 kDa CbMip was intact. This confirmed that the 15.5 and 15.0 kDa proteins are indeed generated by translational reinitiation and are not degradation products of the 23.5 kDa protein. Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit PPIase activity. Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15. 0 kDa proteins are designated as C. burnetii FKBP (Cb-FKBP) analogues I and II, respectively. TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indicating that structural variations could allow for different subcellular compartmentalization of similar proteins. Western-blot analysis of lysates of purified C. burnetii using a CbMip-specific monoclonal antibody revealed the presence of a protein migrating at approximately 15 kDa, indicating the presence of smaller

  12. ALTERATION IN MICROSOMAL PROTEIN SYNTHESIS DURING EARLY DEVELOPMENT OF MOUSE BRAIN*

    PubMed Central

    Johnson, Terry C.; Belytschko, Gail

    1969-01-01

    The loss of protein synthesis during early mouse-brain development was shown to be the result, at least in part, of the inability of microsomes obtained from more mature neural tissue to participate in rapid polypeptide synthesis. The loss of brain microsomal activity was observed shortly after birth and continued until the animals were approximately ten days old. Despite the difference in synthetic activity, sucrose gradient profiles of microsomes and polyribosomes from young and more mature brain tissue were quite similar. The loss in protein synthesis was shown to be independent of available mRNA and not attributable to aminoacyl-RNA synthetases and tRNA binding activity. PMID:5257009

  13. Optimizing the measurement of mitochondrial protein synthesis in human skeletal muscle.

    PubMed

    Burd, Nicholas A; Tardif, Nicolas; Rooyackers, Olav; van Loon, Luc J C

    2015-01-01

    The measurement of mitochondrial protein synthesis after food ingestion, contractile activity, and/or disease is often used to provide insight into skeletal muscle adaptations that occur in the longer term. Studies have shown that protein ingestion stimulates mitochondrial protein synthesis in human skeletal muscle. Minor differences in the stimulation of mitochondrial protein synthesis occur after a single bout of resistance or endurance exercise. There appear to be no measurable differences in mitochondrial protein synthesis between critically ill patients and aged-matched controls. However, the mitochondrial protein synthetic response is reduced at a more advanced age. In this paper, we discuss the challenges involved in the measurement of human skeletal muscle mitochondrial protein synthesis rates based on stable isotope amino acid tracer methods. Practical guidelines are discussed to improve the reliability of the measurement of mitochondrial protein synthesis rates. The value of the measurement of mitochondrial protein synthesis after a single meal or exercise bout on the prediction of the longer term skeletal muscle mass and performance outcomes in both the healthy and disease populations requires more work, but we emphasize that the measurements need to be reliable to be of any value to the field.

  14. Effect of a protein synthetic inhibitor on in vivo estimates of protein synthesis in dogs

    SciTech Connect

    Schwenk, W.F.; Rubanyi, E.; Haymond, M.W.

    1987-05-01

    In vivo estimates of nonoxidative leucine disappearance have frequently been used as estimates of leucine incorporation into protein. To attempt to assess this extrapolation to protein synthesis, seven overnight fasted dogs received primed 4-h infusions of emetine, an alkaloid known to inhibit protein synthesis at the translational level. Protein metabolism was studied using infusions of (1-/sup 14/C)leucine and ..cap alpha..-(4,5-/sup 3/H)ketoisocaproate (KIC) and the steady-state specific activities of the leucine moiety (e.g., (/sup 14/C)KIC and (/sup 3/H)leucine) reciprocal to the infused isotopes as estimates of intracellular leucine specific activities. Plasma leucine and KIC concentrations increased, as did leucine oxidation. Estimates of nonoxidative leucine disappearance decreased by approx. 70%, and estimates of the endogenous leucine rate of appearance decreased by approx. 40% using either the /sup 14/C or /sup 3/H data. They conclude that, although in vivo estimates of leucine metabolism are not quantitative, rapid changes in whole-body estimates of protein synthesis can be predicted during infusion of labeled leucine.

  15. The Sensitivity of Memory Consolidation and Reconsolidation to Inhibitors of Protein Synthesis and Kinases: Computational Analysis

    ERIC Educational Resources Information Center

    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…

  16. Possible involvement of poly(A) in protein synthesis.

    PubMed Central

    Jacobson, A; Favreau, M

    1983-01-01

    The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect. Images PMID:6137807

  17. Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

    PubMed

    Wilson, Gabriel J; Moulton, Christopher J; Garlick, Peter J; Anthony, Tracy G; Layman, Donald K

    2012-11-13

    Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced

  18. Age-related changes in the synthesis and phosphorylation of proteins

    SciTech Connect

    Butler, J.A.; Heydari, A.; Richardson, A.

    1986-03-01

    It is well documented that the protein synthetic activity of liver tissue decreases significantly with age. However, very little information is available on the effect of age on the synthesis or phosphorylation of individual proteins. Hepatocytes were isolated from 5- to 30-month-old male Fischer F344 rats, and proteins were labeled with either (/sup 3/H)-valine or (/sup 32/P)-phosphate. Two-dimensional polyacrylamide gel electrophoresis was used to monitor the synthesis and phosphorylation of a wide variety of proteins. A dramatic increase or decrease in the synthesis of approximately 2 to 3% of the proteins was observed. Most of the proteins whose synthesis increased with age were found to be plasma proteins, e.g., acute phase proteins, synthesized by the liver. In general, the synthesis of most proteins decreased 20 to 40% with age. The phosphorylation of most proteins (over 200) did not appear to change with age. However the phosphorylation of two acidic proteins (molecular weights of 148 Kd and 130 Kd and pIs of 5.4 and 5.36, respectively) decreased with age while the phosphorylation of a basic protein (molecular weight of 57 Kd and pI of 8.09) increased with age.

  19. Understanding Protein Synthesis: An Interactive Card Game Discussion

    ERIC Educational Resources Information Center

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a…

  20. Protein synthesis during sleep consolidates cortical plasticity in vivo

    PubMed Central

    Seibt, Julie; Dumoulin, Michelle C.; Aton, Sara J.; Coleman, Tammi; Watson, Adam; Naidoo, Nirinjini; Frank, Marcos G.

    2012-01-01

    SUMMARY Sleep consolidates experience-dependent brain plasticity, but the precise cellular mechanisms mediating this process are unknown [1]. De novo cortical protein synthesis is one possible mechanism. In support of this hypothesis, sleep is associated with increased brain protein synthesis [2, 3] and transcription of mRNAs involved in protein synthesis regulation [4, 5]. Protein synthesis in turn is critical for memory consolidation and persistent forms of plasticity in vitro and in vivo [6, 7]. However, it is unknown if cortical protein synthesis in sleep serves similar functions. We investigated the role of protein synthesis in the sleep-dependent consolidation of a classic form of cortical plasticity in vivo (ocular dominance plasticity: ODP [8, 9]) in the cat visual cortex. We show that intracortical inhibition of mammalian target of rapamycin (mTOR)-dependent protein synthesis during sleep abolishes consolidation, but has no effect on plasticity induced during wakefulness. Sleep also promotes phosphorylation of protein synthesis regulators (i.e. 4E-BP1 and eEF2) and the translation (but not transcription) of key plasticity-related mRNAs (ARC and BDNF). These findings show that sleep promotes cortical mRNA translation. Interruption of this process has functional consequences, as it abolishes the consolidation of experience in the cortex. PMID:22386312

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

    SciTech Connect

    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.

  2. Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

    PubMed Central

    Kourtis, Nikos; Tavernarakis, Nektarios

    2017-01-01

    Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals. PMID:28286807

  3. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    SciTech Connect

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M.; Wolf-Yadlin, Alejandro; Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C.; Jahan, Tahmina A.; Krasnoselsky, Alexei L.; Palermo, Robert E.; Katze, Michael G.

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

  4. mTORC1-independent reduction of retinal protein synthesis in type 1 diabetes.

    PubMed

    Fort, Patrice E; Losiewicz, Mandy K; Pennathur, Subramaniam; Jefferson, Leonard S; Kimball, Scot R; Abcouwer, Steven F; Gardner, Thomas W

    2014-09-01

    Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy.

  5. Effect of acute smoke exposure on hepatic protein synthesis.

    PubMed

    Garrett, R J; Jackson, M A

    1979-05-01

    In vivo hepatic protein synthesis was monitored in female rats under control and smoke-exposed conditions. During the 15 min period after i.v. administration of [3H]proline protein synthesis was 206 +/- 35 nmol of proline per mg of DNA for sham-control animals. When animals were subjected to acute exposure to cigarette smoke, protein synthesis was inhibited and the extent of inhibition was positively correlated with the dosage of smoke (32%, 15 puffs; 66%, 60 puffs). The inhibitory effect of whole smoke on protein synthesis was unaltered by passing the smoke through either charcoal or cambridge filters. Carbon monoxide in smoke is not removed by either type of filter. At a level comparable to that in cigarette smoke carbon monoxide depressed hepatic protein synthesis to the same extent as did whole or filtered smoke.

  6. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men.

    PubMed

    Mitchell, Cameron J; McGregor, Robin A; D'Souza, Randall F; Thorstensen, Eric B; Markworth, James F; Fanning, Aaron C; Poppitt, Sally D; Cameron-Smith, David

    2015-10-21

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h(-1) in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

  7. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

    PubMed Central

    Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

    2015-01-01

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

  8. Role of RNA and Protein Synthesis in Abscission

    PubMed Central

    Abeles, F. B.

    1968-01-01

    The cell separation aspect of abscission is thought to involve the action of specific cell wall degrading enzymes. Enzymes represent synthesis which in turn is preceded by the synthesis of specific RNA molecules, and it follows that inhibition of either of these processes would also block abscission. Since abscission is a localized phenomenon usually involving 2 or 3 cell layers, RNA and protein synthesis should also be localized. Manipulations of plant material which either accelerate or retard abscission may be due to the regulation of RNA and protein synthesis. This paper is a review of literature concerned with these and related questions. Images PMID:16657020

  9. Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

    PubMed

    Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

    2014-11-01

    Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis.

  10. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  11. Muscle and liver protein synthesis in growing rats fed diets containing raw legumes as the main source of protein

    SciTech Connect

    Goena, M.; Santidrian, S.; Cuevillas, F.; Larralde, J.

    1986-03-01

    Although legumes are widely used as protein sources, their effects on protein metabolism remain quite unexplored. The authors have measured the rates of gastrocnemius muscle and liver protein synthesis in growing rats fed ad libitum over periods of 12 days on diets containing raw field bean (Vicia faba L.), raw kidney bean (Phaseolus vulgaris L.), and raw bitter vetch (Vicia ervilia L.) as the major sources of protein. Diets were isocaloric and contained about 12% protein. Protein synthesis was evaluated by the constant-intravenous-infusion method, using L-//sup 14/C/-tyrosine, as well as by the determination of the RNA-activity (g of newly synthesized protein/day/g RNA). Results showed that, as compared to well-fed control animals, those fed the raw legume diets exhibited a marked reduction in the rate of growth with no changes in the amount of food intake (per 100 g b.wt.). These changes were accompanied by a significant reduction in the rate of muscle protein synthesis in all legume-treated rats, being this reduction greater in the animals fed the Ph. vulgaris and V. ervilia diets. Liver protein synthesis was slightly higher in the rats fed the V. faba and V. ervilia diets, and smaller in the Ph. vulgaris-fed rats. It is suggested that both sulfur amino acid deficiency and the presence of different anti-nutritive factors in raw legumes may account for these effects.

  12. Erythropoietin Does Not Enhance Skeletal Muscle Protein Synthesis Following Exercise in Young and Older Adults

    PubMed Central

    Lamon, Séverine; Zacharewicz, Evelyn; Arentson-Lantz, Emily; Gatta, Paul A. Della; Ghobrial, Lobna; Gerlinger-Romero, Frederico; Garnham, Andrew; Paddon-Jones, Douglas; Russell, Aaron P.

    2016-01-01

    Purpose: Erythropoietin (EPO) is a renal cytokine that is primarily involved in hematopoiesis while also playing a role in non-hematopoietic tissues expressing the EPO-receptor (EPOR). The EPOR is present in human skeletal muscle. In mouse skeletal muscle, EPO stimulation can activate the AKT serine/threonine kinase 1 (AKT) signaling pathway, the main positive regulator of muscle protein synthesis. We hypothesized that a single intravenous EPO injection combined with acute resistance exercise would have a synergistic effect on skeletal muscle protein synthesis via activation of the AKT pathway. Methods: Ten young (24.2 ± 0.9 years) and 10 older (66.6 ± 1.1 years) healthy subjects received a primed, constant infusion of [ring-13C6] L-phenylalanine and a single injection of 10,000 IU epoetin-beta or placebo in a double-blind randomized, cross-over design. 2 h after the injection, the subjects completed an acute bout of leg extension resistance exercise to stimulate skeletal muscle protein synthesis. Results: Significant interaction effects in the phosphorylation levels of the members of the AKT signaling pathway indicated a differential activation of protein synthesis signaling in older subjects when compared to young subjects. However, EPO offered no synergistic effect on vastus lateralis mixed muscle protein synthesis rate in young or older subjects. Conclusions: Despite its ability to activate the AKT pathway in skeletal muscle, an acute EPO injection had no additive or synergistic effect on the exercise-induced activation of muscle protein synthesis or muscle protein synthesis signaling pathways. PMID:27458387

  13. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon.

  14. Coordinated regulation of protein synthesis and degradation by mTORC1.

    PubMed

    Zhang, Yinan; Nicholatos, Justin; Dreier, John R; Ricoult, Stéphane J H; Widenmaier, Scott B; Hotamisligil, Gökhan S; Kwiatkowski, David J; Manning, Brendan D

    2014-09-18

    Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.

  15. A resource dependent protein synthesis model for evaluating synthetic circuits.

    PubMed

    Halter, Wolfgang; Montenbruck, Jan Maximilian; Tuza, Zoltan A; Allgöwer, Frank

    2017-03-09

    Reliable in silico design of synthetic gene networks necessitates novel approaches to model the process of protein synthesis under the influence of limited resources. We present such a novel protein synthesis model which originates from the Ribosome Flow Model and among other things describes the movement of RNA-polymerase and ribosomes on mRNA and DNA templates, respectively. By analyzing the convergence properties of this model based upon geometric considerations, we present additional insights into the dynamic mechanisms of the process of protein synthesis. Further, we demonstrate how this model can be used to evaluate the performance of synthetic gene circuits under different loading scenarios.

  16. Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

    PubMed

    Liu, Tao; Nair, Somarajan J; Lescarbeau, André; Belani, Jitendra; Peluso, Stéphane; Conley, James; Tillotson, Bonnie; O'Hearn, Patrick; Smith, Sherri; Slocum, Kelly; West, Kip; Helble, Joseph; Douglas, Mark; Bahadoor, Adilah; Ali, Janid; McGovern, Karen; Fritz, Christian; Palombella, Vito J; Wylie, Andrew; Castro, Alfredo C; Tremblay, Martin R

    2012-10-25

    Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

  17. Ribosomal analysis of rapid rates of protein synthesis in the Antarctic sea urchin Sterechinus neumayeri.

    PubMed

    Pace, Douglas A; Maxson, Robert; Manahan, Donal T

    2010-02-01

    Previous research has shown that developing stages of the Antarctic sea urchin Sterechinus neumayeri have high rates of protein synthesis that are comparable to those of similar species living in much warmer waters. Direct measurements of the biosynthetic capacities of isolated ribosomes have not been reported for marine organisms living in the extreme-cold environment of Antarctica. Such measurements are required for a mechanistic understanding of how the critical and highly complex processes involved in protein synthesis are regulated in animals living in the coldest marine environment on Earth (< -1 degrees C). We tested the hypothesis that high rates of protein synthesis in the cold are a direct result of high biosynthetic capacities of ribosomes engaged in protein synthesis. Our results show that the rate at which ribosomes manufacture proteins (i.e., the peptide elongation rate) at -1 degrees C is surprisingly similar to rates measured in other sea urchin species at temperatures that are over 15 degrees C warmer. Average peptide elongation rates for a range of developmental stages of the Antarctic sea urchin were 0.36 codons s(-1) (+/- 0.05, SE). On the basis of subcellular rate determinations of ribosomal activity, we calculated stage-specific rates of protein synthesis for blastulae and gastrulae to be 3.7 and 6.5 ng protein h(-1), respectively. These findings support the conclusion that the high rates of biosynthesis previously reported for the Antarctic sea urchin are an outcome of high ribosomal activities.

  18. DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

    PubMed Central

    Zarucki-Schulz, T; Jerez, C; Goldberg, G; Kung, H F; Huang, K H; Brot, N; Weissbach, H

    1979-01-01

    The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results. Images PMID:160561

  19. Presynaptic Protein Synthesis Is Required for Long-Term Plasticity of GABA Release.

    PubMed

    Younts, Thomas J; Monday, Hannah R; Dudok, Barna; Klein, Matthew E; Jordan, Bryen A; Katona, István; Castillo, Pablo E

    2016-10-19

    Long-term changes of neurotransmitter release are critical for proper brain function. However, the molecular mechanisms underlying these changes are poorly understood. While protein synthesis is crucial for the consolidation of postsynaptic plasticity, whether and how protein synthesis regulates presynaptic plasticity in the mature mammalian brain remain unclear. Here, using paired whole-cell recordings in rodent hippocampal slices, we report that presynaptic protein synthesis is required for long-term, but not short-term, plasticity of GABA release from type 1 cannabinoid receptor (CB1)-expressing axons. This long-term depression of inhibitory transmission (iLTD) involves cap-dependent protein synthesis in presynaptic interneuron axons, but not somata. Translation is required during the induction, but not maintenance, of iLTD. Mechanistically, CB1 activation enhances protein synthesis via the mTOR pathway. Furthermore, using super-resolution STORM microscopy, we revealed eukaryotic ribosomes in CB1-expressing axon terminals. These findings suggest that presynaptic local protein synthesis controls neurotransmitter release during long-term plasticity in the mature mammalian brain.

  20. Protein synthesis as an integral quality control mechanism during ageing.

    PubMed

    Charmpilas, Nikolaos; Daskalaki, Ioanna; Papandreou, Margarita Elena; Tavernarakis, Nektarios

    2015-09-01

    Ageing is manifested as functional and structural deterioration that affects cell and tissue physiology. mRNA translation is a central cellular process, supplying cells with newly synthesized proteins. Accumulating evidence suggests that alterations in protein synthesis are not merely a corollary but rather a critical factor for the progression of ageing. Here, we survey protein synthesis regulatory mechanisms and focus on the pre-translational regulation of the process exerted by non-coding RNA species, RNA binding proteins and alterations of intrinsic RNA properties. In addition, we discuss the tight relationship between mRNA translation and two central pathways that modulate ageing, namely the insulin/IGF-1 and TOR signalling cascades. A thorough understanding of the complex interplay between protein synthesis regulation and ageing will provide critical insights into the pathogenesis of age-related disorders, associated with impaired proteostasis and protein quality control.

  1. Application of a nonradioactive method of measuring protein synthesis in industrially relevant Chinese hamster ovary cells.

    PubMed

    Dadehbeigi, Nazanin; Dickson, Alan James

    2013-01-01

    Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a "snapshot" to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells.

  2. Predictors of Muscle Protein Synthesis after Severe Pediatric Burns

    PubMed Central

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

    2015-01-01

    Background 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 post-injury, and identify predictors that influence this response. Study design 87 children with ≥40% total body surface area (TBSA) burn were included. Patients participated in stable isotope infusion studies at 1, 2 and ~ 4 weeks post-burn, and at 6, 12 and 24 months post-injury to determine skeletal muscle fractional synthesis rate. Generalized estimating equations with log link normal distribution were applied to account for clustering of patients and control for patient characteristics. Results Patients (8±6 years) had large (62, 51–72% TBSA) and deep (47±21% TBSA third degree) burns. Muscle fractional synthesis rate was elevated throughout the first 12 months post-burn compared to established values from healthy young adults. Muscle fractional synthesis rate was lower in boys, children >3 years old, and when burns were >80% TBSA. Conclusions Muscle protein synthesis is elevated for at least one year after injury, suggesting that greater muscle protein turnover is a component of the long-term pathophysiological response to burn trauma. Muscle protein synthesis is highly affected by gender, 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 victims. PMID:25807408

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

    PubMed

    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.

  4. Molecular insights into protein synthesis with proline residues.

    PubMed

    Melnikov, Sergey; Mailliot, Justine; Rigger, Lukas; Neuner, Sandro; Shin, Byung-Sik; Yusupova, Gulnara; Dever, Thomas E; Micura, Ronald; Yusupov, Marat

    2016-12-01

    Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.

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

    PubMed Central

    Twiss, Jeffery L.; Kalinski, Ashley L.; Sachdeva, Rahul; Houle, John D.

    2016-01-01

    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. PMID:27857722

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

    PubMed

    Twiss, Jeffery L; Kalinski, Ashley L; Sachdeva, Rahul; Houle, John D

    2016-09-01

    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.

  7. Selective inhibition of virus protein synthesis by prostaglandin A1: a translational block associated with HSP70 synthesis.

    PubMed Central

    Amici, C; Giorgi, C; Rossi, A; Santoro, M G

    1994-01-01

    Cyclopentenone prostaglandins are potent inhibitors of virus replication. The antiviral activity has been associated with the induction of 70-kDa heat shock protein (HSP70) synthesis. In this report, we describe that in African green monkey kidney cells infected with Sendai virus (SV) and treated with prostaglandin A1 (PGA1), SV protein synthesis was selectively blocked as long as HSP70 was being synthesized by the host cell. The block appeared to be at the translational level, as indicated by the following (i) PGA1 had no effect on SV primary transcription, and a dramatic decrease in the abundance of SV mRNA occurred only at later stages of infection; and (ii) treatment with PGA1 started at 6 h postinfection, at which time SV mRNA had already accumulated in infected cells, did not suppress the levels of NP mRNA, but it reduced the amount of ribosome-bound NP mRNA and caused a dramatic decrease in the level of genomic RNA. The PGA1-induced block of SV protein synthesis appeared to be cell mediated, since it was prevented by actinomycin D, while PGA1 had no effect on SV mRNA translation in vitro. The possibility that HSP70 could be a mediator of the antiviral effect is suggested by the fact that treatment with other classical inducers of HSP70, including sodium arsenite, cadmium, and heat shock at 42 degrees C for 5 h, also selectively prevented SV protein synthesis as long as heat shock protein synthesis occurred. Moreover, SV protein synthesis was not inhibited by PGA1 in murine Friend erythroleukemic cells, which lack the ability to induce HSP70 expression in response to PGA1. Images PMID:7933069

  8. The origin of polynucleotide-directed protein synthesis

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1989-01-01

    If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

  9. Semisynthetic tRNA complement mediates in vitro protein synthesis.

    PubMed

    Cui, Zhenling; Stein, Viktor; Tnimov, Zakir; Mureev, Sergey; Alexandrov, Kirill

    2015-04-08

    Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

  10. Cryopreservation of Plant Mitochondria as a Tool for Protein Import or in Organello Protein Synthesis Studies.

    PubMed Central

    Schieber, O.; Dietrich, A.; Marechal-Drouard, L.

    1994-01-01

    Cryopreserved chloroplasts and thylakoids have recently been proven to be suitable for protein import and integration assays. The possibility of recovering intact plant mitochondria after storage would also facilitate a wide range of investigations that are currently underway on the molecular biology of these organelles, e.g. mitochondrial transcription, RNA editing, in organello protein synthesis, and protein or transfer RNA import. Therefore, we addressed the question whether cryopreservation of isolated plant mitochondria was also possible. Tobacco (Nicotiana tabacum) or broad bean (Vicia faba) mitochondria were quick frozen and stored in liquid nitrogen in the presence of various concentrations of ethylene glycol as a cryoprotectant. After thawing, up to 90% of the mitochondria stored in 5 to 10% ethylene glycol appeared to retain an intact outer membrane and normal oxidative phosphorylation activity. Their ultrastructural aspect, observed by electron microscopy, was similar to that of freshly prepared mitochondria. Furthermore, efficient in organello protein synthesis was carried out with mitochondria stored in the presence of 7.5% ethylene glycol. Finally, the precursor of the [beta] subunit of the mitochondrial F1-ATPase from Nicotiana plumbaginifolia was successfully translocated into V. faba cryopreserved mitochondria and processed. These data demonstrate that plant mitochondria cryopreserved under the conditions described here remain functional and can be used for a variety of physiological and biochemical studies. PMID:12232314

  11. Bacterial Obg proteins: GTPases at the nexus of protein and DNA synthesis.

    PubMed

    Kint, Cyrielle; Verstraeten, Natalie; Hofkens, Johan; Fauvart, Maarten; Michiels, Jan

    2014-08-01

    Obg proteins (also known as ObgE, YhbZ and CgtA) are conserved P-loop GTPases, essential for growth in bacteria. Like other GTPases, Obg proteins cycle between a GTP-bound ON and a GDP-bound OFF state, thereby controlling cellular processes. Interestingly, the in vitro biochemical properties of Obg proteins suggest that they act as sensors for the cellular GDP/GTP pools and adjust their activity according to the cellular energy status. Obg proteins have been attributed a host of cellular functions, including roles in essential cellular processes (DNA replication, ribosome maturation) and roles in different stress adaptation pathways (stringent response, sporulation, general stress response). This review summarizes the current knowledge on Obg activity and function. Furthermore, we present a model that integrates the different functions of Obg by assigning it a fundamental role in cellular physiology, at the hub of protein and DNA synthesis. In particular, we believe that Obg proteins might provide a connection between different global pathways in order to fine-tune cellular processes in response to a given energy status.

  12. Cell-free protein synthesis: applications in proteomics and biotechnology.

    PubMed

    He, Mingyue

    2008-01-01

    Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

  13. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-04-21

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise.

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

    SciTech Connect

    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.

  15. Amino acid metabolism and protein synthesis in lactating rats fed on a liquid diet.

    PubMed Central

    Barber, T; García de la Asunción, J; Puertes, I R; Viña, J R

    1990-01-01

    1. Amino acid metabolism was studied in control virgin rats, lactating rats and virgin rats protein-pair-fed with the lactating rats (high-protein virgin rats). 2. Urinary excretion of nitrogen and urea was higher in lactating than in control virgin rats, and in high-protein virgin rats it was higher than in lactating rats. 3. The activities of urea-cycle enzymes (units/g) were higher in high-protein virgin than in lactating rats, except for arginase. In lactating rats the activities of carbamoyl-phosphate synthase, ornithine carbamoyltransferase and argininosuccinate synthase were lower than in control virgin rats. When the liver size is considered, the activities in lactating rats were similar to those in high-protein virgin rats, except for arginase. 4. N-Acetylglutamate content was higher in high-protein virgin rats than in the other two groups. 5. The rate of urea synthesis from precursors by isolated hepatocytes was higher in high-protein virgin rats than in the other two groups. 6. The flooding-dose method (L-[4-3H]phenylalanine) for measuring protein synthesis was used. The absolute synthesis rates of mammary gland, liver and small-intestinal mucosa were higher in lactating rats than in the other two groups, and in high-protein virgin rats than in control virgin rats 7. These results show that the increased needs for amino acids during lactation are met by hyperphagia and by a nitrogen-sparing mechanism. PMID:2396994

  16. Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization.

    PubMed

    Costache, Vlad; Bilotto, Stefania; Laguerre, Laurent; Bellé, Robert; Cosson, Bertrand; Cormier, Patrick; Morales, Julia

    2012-05-01

    The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.

  17. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    PubMed

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress.

  18. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    SciTech Connect

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-06-15

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [{sup 3}H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [{sup 3}H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-{kappa}B, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  19. β-Adrenergic receptor blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in humans

    PubMed Central

    Robinson, Matthew M.; Bell, Christopher; Peelor, Frederick F.

    2011-01-01

    β-Adrenergic receptor (AR) signaling is a regulator of skeletal muscle protein synthesis and mitochondrial biogenesis in mice. We hypothesized that β-AR blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in adult humans. Six healthy men (mean ± SD: 26 ± 6 yr old, 39.9 ± 4.9 ml·kg−1·min−1 peak O2 uptake, 26.7 ± 2.0 kg/m2 body mass index) performed 1 h of stationary cycle ergometer exercise (60% peak O2 uptake) during 1) β-AR blockade (intravenous propranolol) and 2) administration of saline (control). Skeletal muscle mitochondrial, myofibrillar, and sarcoplasmic protein synthesis rates were assessed using [2H5]phenylalanine incorporation into skeletal muscle proteins after exercise. The mRNA content of signals for mitochondrial biogenesis was determined using real-time PCR. β-AR blockade decreased mitochondrial (from 0.217 ± 0.076 to 0.135 ± 0.031%/h, P < 0.05), but not myofibrillar or sarcoplasmic, protein synthesis rates. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA was increased ∼2.5-fold (P < 0.05) at 5 h compared with 1 h postexercise but was not influenced by β-AR blockade. We conclude that decreased β-AR signaling during cycling can blunt the postexercise increase in mitochondrial protein synthesis rates without affecting mRNA content. PMID:21613574

  20. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    PubMed

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  1. Predictors of muscle protein synthesis after severe pediatric burns

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. 9-Fluorenylmethyloxycarbonyl/ tbutyl-based convergent protein synthesis.

    PubMed

    Barlos, K; Gatos, D

    1999-01-01

    Besides linear solid phase peptide synthesis, segment condensation in solution and chemical ligation, convergent peptide synthesis (CPS) was developed in order to enable the efficient preparation of complex peptides and small proteins. According to this synthetic strategy, solid phase synthesized and suitably protected peptide fragments corresponding to the entire peptide/protein-sequence are condensed on a solid support or in solution, to the target protein. This review summarizes CPS performed utilizing the mild 9-fluorenylmethyloxycarbonyl/tbutyloxycarbonyl-based protecting scheme for the amino acids.

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

    PubMed

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

    2013-03-06

    Memory consolidation has been suggested to be protein synthesis dependent. Previous 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 phosphatase and tensin homolog deleted on chromosome 10 (PTEN), 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, knockdown of calpain-2, but not calpain-1, by small interfering RNA 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.

  4. Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae.

    PubMed

    Dever, Thomas E; Kinzy, Terri Goss; Pavitt, Graham D

    2016-05-01

    In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.

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

    PubMed

    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.

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

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    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. PMID:27367725

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

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-06-28

    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.

  8. Synthesis and Evaluation of In Vitro DNA/Protein Binding Affinity, Antimicrobial, Antioxidant and Antitumor Activity of Mononuclear Ru(II) Mixed Polypyridyl Complexes.

    PubMed

    Putta, Venkat Reddy; Chintakuntla, Nagamani; Mallepally, Rajender Reddy; Avudoddi, Srishailam; K, Nagasuryaprasad; Nancherla, Deepika; V V N, Yaswanth; R S, Prakasham; Surya, Satyanarayana Singh; Sirasani, Satyanarayana

    2016-01-01

    The four novel Ru(II) complexes [Ru(phen)2MAFIP](2+) (1) [MAFIP = 2-(5-(methylacetate)furan-2-yl)-1 H-imidazo[4,5-f] [1, 10]phenanthroline, phen = 1,10-Phenanthroline], [Ru(bpy)2MAFIP](2+) (2) (bpy = 2,2'-bipyridine) and [Ru(dmb)2MAFIP](2+) (3) (dmb = 4,4'-dimethyl-2,2'-bipyridine) and [Ru(hdpa)2MAFIP](2+) (4) (hdpa = 2,2-dipyridylamine) have been synthesized and fully characterized via elemental analysis, NMR spectroscopy, EI-MS and FT-IR spectroscopy. In addition, the DNA-binding behaviors of the complexes 1-4 with calf thymus DNA were investigated by UV-Vis absorption, fluorescence studies and viscosity measurement. The DNA-binding experiments showed that the complexes 1-4 interact with CT-DNA through an intercalative mode. BSA protein binding affinity of synthesized complexes was determined by UV/Vis absorption and fluorescence emission titrations. The binding affinity of ruthenium complexes was supported by molecular docking. The photoactivated cleavage of plasmid pBR322 DNA by ruthenium complexes 1-4 was investigated. All the synthesized compounds were tested for antimicrobial activity by using three Gram-negative (Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa) and three Gram-positive (Micrococcus luteus, Bacillus subtilis and Bacillus megaterium) organisms, these results indicated that complex 3 was more activity compared to other complexes against all tested microbial strains while moderate antimicrobial activity profile was noticed for complex 4. The antioxidant activity experiments show that the complexes exhibit moderate antioxidant activity. The cytotoxicity of synthesized complexes on HeLa cell lines has been examined by MTT assay. The apoptosis assay was carried out with Acridine Orange (AO) staining methods and the results indicate that complexes can induce the apoptosis of HeLa cells. The cell cycle arrest investigated by flow cytometry and these results indicate that complexes 1-4 induce the cell cycle arrest at G0/G1

  9. Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor.

    PubMed

    Mikulík, Karel; Bobek, Jan; Bezousková, Silvia; Benada, Oldrich; Kofronová, Olga

    2002-11-29

    Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.

  10. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    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.

  11. His6 tag-assisted chemical protein synthesis

    NASA Astrophysics Data System (ADS)

    Bang, Duhee; Kent, Stephen B. H.

    2005-04-01

    To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. affinity purification | native chemical ligation

  12. His6 tag-assisted chemical protein synthesis.

    PubMed

    Bang, Duhee; Kent, Stephen B H

    2005-04-05

    To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses.

  13. His6 tag-assisted chemical protein synthesis

    PubMed Central

    Bang, Duhee; Kent, Stephen B. H.

    2005-01-01

    To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. PMID:15784744

  14. A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles. Involvement of a protein kinase C mediated MAP kinase 3/1 self- activation loop

    PubMed Central

    Yang, Peixin; Roy, Shyamal K.

    2006-01-01

    Summary FSH- or EGF-induced granulosa cell proliferation in intact preantral follicles depends on a novel PKC-mediated MAPK3/1 self-activation loop. The objective was to reveal whether a PKC-mediated self-sustaining MAPK3/1 activation loop was necessary for FSH- or EGF-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors. FSH or EGF phosphorylated RAF1, MAP2K1 and MAPK3/1. However, relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for CDK4 activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3h was independent of EGFR kinase activity, but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1 and PLA2G4. Inhibition of PKC activity as late as 4h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase. PMID:16525034

  15. Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells.

    PubMed

    Yao, Kang; Yin, Yulong; Li, Xilong; Xi, Pengbin; Wang, Junjun; Lei, Jian; Hou, Yongqing; Wu, Guoyao

    2012-06-01

    α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.

  16. Nucleic acid and protein synthesis during lateral root initiation in Marsilea quadrifolia (Marsileaceae)

    NASA Technical Reports Server (NTRS)

    Lin, B. L.; Raghavan, V.

    1991-01-01

    The pattern of DNA, RNA, and protein synthesis during lateral root initiation in Marsilea quadrifolia L. was monitored by autoradiography of incorporated of 3H-thymidine, 3H-uridine, and 3H-leucine, respectively. DNA synthesis was associated with the enlargement of the lateral root initial prior to its division. Consistent with histological studies, derivatives of the lateral root initial as well as the cells of the adjacent inner cortex and pericycle of the parent root also continued to synthesize DNA. RNA and protein synthetic activities were found to be higher in the lateral root initials than in the endodermal initials of the same longitudinal layer. The data suggest a role for nucleic acid and protein synthesis during cytodifferentiation of a potential endodermal cell into a lateral root initial.

  17. Prolonged stimulation of protein synthesis by leucine is dependent on amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine is unique among the amino acids in its ability to enhance protein synthesis by activating translation initiation (Kimball and Jefferson, 2005). Our laboratory has shown that raising leucine to postprandial levels, whilst keeping all other amino acids at the post absorptive, level acutely st...

  18. Stimulation of muscle protein synthesis by leucine is dependent on plasma amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported that a physiological increase in plasma leucine increased translation initiation factor activity during 60- and 120-min leucine infusion. Muscle protein synthesis was stimulated at 60 min but not at 120 min, perhaps due to the decrease (-50%) in plasma essential amino acids (AA). ...

  19. The chemical basis for the origin of the genetic code and the process of protein synthesis

    NASA Technical Reports Server (NTRS)

    1982-01-01

    The major thrust is to understand just how the process of protein synthesis, including that very important aspect, genetic coding, came to be. Two aspects of the problem: the chemistry of active aminoacyl species; and affinities between amino acids and nucleotides, and specifically, how these affinities might affect the chemistry between the two are stressed.

  20. The cell-free protein synthesis system from wheat germ.

    PubMed

    Takai, Kazuyuki; Endo, Yaeta

    2010-01-01

    The wheat-germ cell-free protein synthesis system had been one of the most efficient eukaryotic cell-free systems since it was first developed in 1964. However, radio-labeled amino acids had long been essential for detection of the products. Since the discovery of a method for prevention of the contamination by a protein synthesis inhibitor originated from endosperm, the wheat cell-free system has found a wide variety of applications in postgenomic high-throughput screening, structural biology, medicine, and so on. In this chapter, we describe a method for preparation of the cell-free extract and a standard protein synthesis method, as the methods for the applications are found in later chapters.

  1. Synthesis of peptide sequences derived from fibril-forming proteins.

    PubMed

    Scanlon, Denis B; Karas, John A

    2011-01-01

    The pathogenesis of a large number of diseases, including Alzheimer's Disease, Parkinson's Disease, and Creutzfeldt-Jakob Disease (CJD), is associated with protein aggregation and the formation of amyloid, fibrillar deposits. Peptide fragments of amyloid-forming proteins have been found to form fibrils in their own right and have become important tools for unlocking the mechanism of amyloid fibril formation and the pathogenesis of amyloid diseases. The synthesis and purification of peptide sequences derived from amyloid fibril-forming proteins can be extremely challenging. The synthesis may not proceed well, generating a very low quality crude product which can be difficult to purify. Even clean crude peptides can be difficult to purify, as they are often insoluble or form fibrils rapidly in solution. This chapter presents methods to recognise and to overcome the difficulties associated with the synthesis, and purification of fibril-forming peptides, illustrating the points with three synthetic examples.

  2. Prolonged cell-free protein synthesis in a batch system using wheat germ extract.

    PubMed

    Kawarasaki, Y; Nakano, H; Yamane, T

    1994-10-01

    Reaction conditions of cell-free protein synthesis using wheat germ extract were examined to prolong the period of protein synthesis in a batch reaction. By optimizing conditions for ATP regeneration system involved in the cell-free system, protein synthesis continued about 4 hours, so that about 17 micrograms dihydrofolate reductase protein was obtained in 1 ml of a reaction mixture. It suggests that maintaining ATP concentration is the primary requirement for long-life cell-free protein synthesis.

  3. The relationship between protein synthesis and protein degradation in object recognition memory.

    PubMed

    Furini, Cristiane R G; Myskiw, Jociane de C; Schmidt, Bianca E; Zinn, Carolina G; Peixoto, Patricia B; Pereira, Luiza D; Izquierdo, Ivan

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory.

  4. Pharmacological induction of heat shock protein 68 synthesis in cultured rat astrocytes.

    PubMed

    Nishimura, R N; Dwyer, B E

    1995-12-15

    The induction of the highly inducible 70-kDa heat shock protein (HSP 70) is associated with thermotolerance and survival from many other types of stress. This investigation studied the pharmacological induction of HSP 68 (HSP 68 is the rat homolog of human HSP 70) by 1,10-phenanthroline in cultured rat astrocytes under conditions that activated heat shock transcription factor-1 without inducing HSP 68 synthesis. Two conditions that activate heat shock transcription factor-1 and promote its binding to the heat shock element without subsequent transcription of HSP 68 mRNA, intracellular acidosis and exposure to salicylate, showed synthesis of HSP 68 when 1,10-phenanthroline was added to culture medium after the activation of heat shock transcription factor-1. 1,10-phenanthroline mimicked heat shock by inducing HSP 68 mRNA and protein under both conditions. 1,10-phenanthroline added alone to culture medium did not induce the synthesis of HSP 68 or activate heat shock transcription factor-1. These findings strongly suggest a multistep activation for HSP 68 synthesis and also demonstrate that the synthesis of HSP 68 can be pharmacologically regulated.

  5. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

    PubMed

    Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

    2015-09-15

    Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1.

  6. A Recombinant Collagen-mRNA Platform for Controllable Protein Synthesis.

    PubMed

    Sun, Liping; Xiong, Yunjing; Bashan, Anat; Zimmerman, Ella; Shulman Daube, Shirley; Peleg, Yoav; Albeck, Shira; Unger, Tamar; Yonath, Hagith; Krupkin, Miri; Matzov, Donna; Yonath, Ada

    2015-07-06

    We have developed a collagen-mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin-healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell-free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase-mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.

  7. Effect of hypothalamic electrical stimulation on protein synthesis in organs of adult and old rats

    SciTech Connect

    Frol'kis, V.V.; Muradyan, K.K.; Rushkevich, Yu.E.; Mozzhukhina, T.G.; Khilobok, I.Yu.; Gol'dshtein, N.B.

    1986-12-01

    Age differences in hypothalamic regulation of total protein synthesis in different organs and also of liver chromatin proteins were compared in this investigation. Rats were used in the experiments and the intensity of protein synthesis was judged from the relative specific radioactivity which was determined as the ratio of the specific radioactivities of acid-insoluble and acid-soluble materials, separated by means of nitrocellulose membrane filters. Protein was determined by two-wave spectrophotometry and the radioactivity of all samples was measured on a Mark III radio spectrometer. The investigations showed that hypothalmic electrical stimulation causes a marked increase in /sup 3/H-leucine incorporation into protein of active and inactive liver chromatin.

  8. Effects of Whey, Caseinate, or Milk Protein Ingestion on Muscle Protein Synthesis after Exercise.

    PubMed

    Kanda, Atsushi; Nakayama, Kyosuke; Sanbongi, Chiaki; Nagata, Masashi; Ikegami, Shuji; Itoh, Hiroyuki

    2016-06-03

    Whey protein (WP) is characterized as a "fast" protein and caseinate (CA) as a "slow" protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p < 0.05) in FSR compared with SP at different times (WP, 60 min; MP, 90 and 120 min; CA, 120 min). Although statistical analysis could not be performed, the calculated the area under the curve (AUC) values for FSR following this trend were: MP, 534.61; CA, 498.22; WP, 473.46; and SP, 406.18. We conclude that ingestion of MP, CA or WP causes the initial peak time in muscle protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP.

  9. Effects of Whey, Caseinate, or Milk Protein Ingestion on Muscle Protein Synthesis after Exercise

    PubMed Central

    Kanda, Atsushi; Nakayama, Kyosuke; Sanbongi, Chiaki; Nagata, Masashi; Ikegami, Shuji; Itoh, Hiroyuki

    2016-01-01

    Whey protein (WP) is characterized as a “fast” protein and caseinate (CA) as a “slow” protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p < 0.05) in FSR compared with SP at different times (WP, 60 min; MP, 90 and 120 min; CA, 120 min). Although statistical analysis could not be performed, the calculated the area under the curve (AUC) values for FSR following this trend were: MP, 534.61; CA, 498.22; WP, 473.46; and SP, 406.18. We conclude that ingestion of MP, CA or WP causes the initial peak time in muscle protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP. PMID:27271661

  10. Control of 6-(D-threo-1',2'-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity.

    PubMed Central

    Gütlich, M; Witter, K; Bourdais, J; Veron, M; Rödl, W; Ziegler, I

    1996-01-01

    6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum. PMID:8660315

  11. Control of 6-(D-threo-1',2'-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity.

    PubMed

    Gütlich, M; Witter, K; Bourdais, J; Veron, M; Rödl, W; Ziegler, I

    1996-02-15

    6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.

  12. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  13. Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine.

    PubMed

    Esteghamat-Panah, Roya; Hadadzadeh, Hassan; Farrokhpour, Hossein; Simpson, Jim; Abdolmaleki, Amir; Abyar, Fatemeh

    2017-02-15

    A new mononuclear rhodium(III) complex, [Rh(bzimpy)Cl3] (bzimpy = 2,6-bis(2-benzimidazolyl)pyridine), was synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with fish sperm DNA (FS-DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement in order to evaluate the possible DNA-binding mode and to calculate the corresponding DNA-binding constant. The results reveal that the Rh(III) complex interacts with DNA through groove binding mode with a binding affinity on the order of 10(4). In addition, the binding of the Rh(III) complex to bovine serum albumin (BSA) was monitored by UV-Vis and fluorescence emission spectroscopy at different temperatures. The mechanism of the complex interaction was found to be static quenching. The thermodynamic parameters (ΔH, ΔS, and ΔG) obtained from the fluorescence spectroscopy data show that van der Waals interactions and hydrogen bonds play a major role in the binding of the Rh(III) complex to BSA. For the comparison of the DNA- and BSA-binding affinities of the free bzimpy ligand with its Rh(III) complex, the absorbance titration and fluorescence quenching experiments of the free bzimpy ligand with DNA and BSA were carried out. Competitive experiments using eosin Y and ibuprofen as site markers indicated that the complex was mainly located in the hydrophobic cavity of site I of the protein. These experimental results were confirmed by the results of molecular docking. Finally, the in vitro cytotoxicity properties of the Rh(III) complex against the MCF-7, K562, and HT-29 cell lines were evaluated and compared with those of the free ligand (bzimpy). It was found that the complexation process improved the anticancer activity significantly.

  14. Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content.

    PubMed

    Kanda, Atsushi; Nakayama, Kyosuke; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2013-09-28

    It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague–Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.

  15. Enhanced in vivo protein synthesis in circulating immune cells of ICU patients.

    PubMed

    Januszkiewicz, Anna; Klaude, Maria; Loré, Karin; Andersson, Jan; Ringdén, Olle; Rooyackers, Olav; Wernerman, Jan

    2007-11-01

    Insufficient function of the immune system contributes to a poor prognosis in intensive care unit (ICU) patients. However, the immune system function is not easily monitored and evaluated. In vivo protein synthesis determination in immune competent cells offers a possibility to quantify immunological activation. The aim of this descriptive study was to determine the in vivo fractional protein synthesis rate (FSR) in immune cells of ICU patients during the initial phase of the critical illness. Patients (n = 20) on ventilator treatment in the general ICU were studied during their first week of ICU stay. FSR was determined in circulating T lymphocytes, mononuclear cells, the whole population of blood leukocytes, and in stationary immune cells of palatine tonsils during a 90-min period by a flooding technique. Healthy, adult subjects (n = 11), scheduled for elective ear, nose, and throat surgery served as a control group. The FSR in leukocytes and mononuclear cells of ICU patients was higher compared with the control group. In contrast, the FSR of circulating T lymphocytes and of tonsillar cells was not different from that in the healthy subjects. In summary, the ICU patients showed a distinct polarization of metabolic responses during the initial phase of the critical illness. The in vivo rate of protein synthesis was high in the circulating mononuclear cells and leukocytes, reflecting enhanced metabolic activity in these cell populations. Determination of the in vivo protein synthesis rate may be used as a tool to obtain additional information on activation of the immune system.

  16. An efficient synthesis of an exo-enone analogue of LL-Z1640-2 and evaluation of its protein kinase inhibitory activities.

    PubMed

    Wang, Stephanie Q; Goh, Shermin S; Chai, Christina L L; Chen, Anqi

    2016-01-14

    An efficient synthesis of an exo-enone analogue (5) of resorcylic acid lactone (RAL), natural product LL-Z1640-2 (1), has been achieved using a Ni-catalysed regioselective reductive coupling macrocyclisation of an alkyne-aldehyde as a key step. The synthetic route is significantly shorter than those for the natural product and avoids the isomerisation problem of the cis-double bond in the molecule. The preliminary biological evaluation showed that the exo-enone analogue is a potent inhibitor of several important kinases relevant to cancer drug development.

  17. Discovery and Analysis of 4H-Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors▿

    PubMed Central

    Ribble, Wendy; Hill, Walter E.; Ochsner, Urs A.; Jarvis, Thale C.; Guiles, Joseph W.; Janjic, Nebojsa; Bullard, James M.

    2010-01-01

    Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyl-tRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC50s) ranging from 3 to 14 μM. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five- or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 μg/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition. PMID:20696870

  18. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Gilbertson, V.

    1999-01-01

    The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

  19. The unphosphorylated EIIA(Ntr) protein represses the synthesis of alkylresorcinols in Azotobacter vinelandii.

    PubMed

    Muriel-Millán, Luis Felipe; Moreno, Soledad; Romero, Yanet; Bedoya-Pérez, Leidy Patricia; Castañeda, Miguel; Segura, Daniel; Espín, Guadalupe

    2015-01-01

    Upon encystment induction, Azotobacter vinelandii produces the phenolic lipids alkylresorcinols (ARs) that are structural components of the cysts. The enzymes responsible for the ARs synthesis are encoded in the arsABCD operon, whose expression is activated by ArpR. The transcription of arpR is initiated from an RpoS dependent promoter. The nitrogen-related phosphotransferase system (PTS(Ntr)) is a global regulatory system present in Gram negative bacteria. It comprises the EI(Ntr), NPr and EIIA(Ntr) proteins encoded by ptsP, ptsO and ptsN genes respectively. These proteins participate in a phosphoryl-group transfer from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. In A. vinelandii, the non-phosphorylated form of EIIA(Ntr) was previously shown to repress the synthesis of poly-ß-hydroxybutyrate. In this work, we show that PTS(Ntr) also regulates the synthesis of ARs. In a strain that carries unphosphorylated EIIA(Ntr), the expression of arpR was reduced, while synthesis of ARs and transcription of arsA were almost abrogated. The expression of arpR from an RpoS-independent promoter in this strain restored the ARs synthesis. Taken together these results indicate that unphosphorylated EIIA(Ntr) negatively affects activation of arpR transcription by RpoS.

  20. Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Shenton, Daniel; Grant, Chris M

    2003-01-01

    The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione. We report here the target proteins which are modified in yeast cells in response to H(2)O(2). In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified. The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities. The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed. In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway. Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH. Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis. Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation. In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation. PMID:12755685

  1. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    PubMed

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Synthesis of a cyanopeptide-analogue with trypsin activating properties.

    PubMed

    Radau, G; Rauh, D

    2000-04-17

    An efficient synthesis of a peptidic analogue of cyanobacterial metabolites with proposed serine protease inhibitory activity has been developed. Surprisingly, one trypsin activating compound was obtained.

  4. Translational Control of Specific Uterine Protein Synthesis After Estrogen Induction

    PubMed Central

    DeAngelo, Anthony B.; Fujimoto, George I.

    1973-01-01

    The rate of leucine incorporation into a specific estrogen-induced protein of immature rat isolated by gel electrophoresis declines rapidly between 2-4 hr after estrogen stimulation in vivo followed by incubation in vitro. Actinomycin D present during the in vitro phase prevents this decline, and elicits a “superinduction” effect at 2 hr. Labeled induced protein remains stable during this time period, indicating that its decline is due to a reduction in synthesis capacity for the inducible protein, rather than to its degradation. A second injection of hormone at 3 hr has no effect on the reduced level of synthesis capacity for induced protein noted at 4 hr in the rat uterus. PMID:4509650

  5. The spatial and temporal regulation of the hormonal signal. Role of mitochondria in the formation of a protein complex required for the activation of cholesterol transport and steroids synthesis.

    PubMed

    Poderoso, Cecilia; Duarte, Alejandra; Cooke, Mariana; Orlando, Ulises; Gottifredi, Vanesa; Solano, Angela R; Lemos, Jose R; Podestá, Ernesto J

    2013-05-22

    The mitochondria are critical for steroidogenesis since the ability of cholesterol to move into mitochondria to be available for cytochrome P450, CYP11A1, determines the efficacy of steroid production. Several proteins kinases, such as PKA, MEK and ERK which are essential to complete steroidogenesis, form a mitochondria-associated complex. The protein-protein interactions between kinases and key factors during the transport of cholesterol takes place in the contact sites between the two mitochondrial membranes; however, no mitochondrial targeting sequence has been described for these kinases. Here we discuss the possibility that mitochondrial reorganization may be mediating a compartmentalized cellular response. This reorganization could allow the physical interaction between the hormone-receptor complex and the enzymatic and lipidic machinery necessary for the complete steroid synthesis and release. The movement of organelles in specialized cells could impact on biological processes that include, but are not limited to, steroid synthesis.

  6. Leucine acts as a nutrient signal to stimulate protein synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial rise in amino acids and insulin independently stimulates protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to amino acids. We have shown that the postprandial rise in leucine, but not isoleucine or valine, acutely stimulates muscle pro...

  7. The Teaching of Protein Synthesis--A Microcomputer Based Method.

    ERIC Educational Resources Information Center

    Goodridge, Frank

    1983-01-01

    Describes two computer programs (BASIC for 32K Commodore PET) for teaching protein synthesis. The first is an interactive test of base-pairing knowledge, and the second generates random DNA nucleotide sequences, with instructions for substitution, insertion, and deletion printed out for each student. (JN)

  8. Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

    ERIC Educational Resources Information Center

    Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

    2004-01-01

    To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

  9. Problem-Solving Test: The Mechanism of Protein Synthesis

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: protein synthesis, ribosomes, amino acids, peptides, peptide bond, polypeptide chain, N- and C-terminus, hemoglobin, [alpha]- and [beta]-globin chains, radioactive labeling, [[to the third power]H] and [[to the fourteenth power]C]leucine, cytosol, differential centrifugation, density…

  10. Frog Foam Nest Protein Diversity and Synthesis.

    PubMed

    Hissa, Denise Cavalcante; Bezerra, Walderly Melgaço; Freitas, Cléverson Diniz Teixeira De; Ramos, Márcio Viana; Lopes, José Luiz De Souza; Beltramini, Leila Maria; Roberto, Igor Joventino; Cascon, Paulo; Melo, Vânia Maria Maciel

    2016-08-01

    Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest.

  11. Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

    PubMed

    Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

    2016-01-01

    Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

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

    PubMed Central

    Liu, Yiyong; Sellegounder, Durai; Sun, Jingru

    2016-01-01

    Upon pathogen infection, microbial killing pathways and cellular stress pathways are rapidly activated by the host innate immune system. These pathways must be tightly regulated because insufficient or excessive immune responses have deleterious consequences. Increasing evidence indicates that the nervous system regulates the immune system to confer coordinated protection to the host. However, the precise mechanisms of neural-immune communication remain unclear. Previously we have demonstrated that OCTR-1, a neuronal G protein-coupled receptor, functions in the sensory neurons ASH and ASI to suppress innate immune responses in non-neural tissues against Pseudomonas aeruginosa in Caenorhabditis elegans. In the current study, by using a mass spectrometry-based quantitative proteomics approach, we discovered that OCTR-1 regulates innate immunity by suppressing translation and the unfolded protein response (UPR) pathways at the protein level. Functional assays revealed that OCTR-1 inhibits specific protein synthesis factors such as ribosomal protein RPS-1 and translation initiation factor EIF-3.J to reduce infection-triggered protein synthesis and UPR. Translational inhibition by chemicals abolishes the OCTR-1-controlled innate immune responses, indicating that activation of the OCTR-1 pathway is dependent on translation upregulation such as that induced by pathogen infection. Because OCTR-1 downregulates protein translation activities, the OCTR-1 pathway could function to suppress excessive responses to infection or to restore protein homeostasis after infection. PMID:27833098

  13. BDNF Facilitates L-LTP Maintenance in the Absence of Protein Synthesis through PKMζ

    PubMed Central

    Mei, Fan; Nagappan, Guhan; Ke, Yang; Sacktor, Todd C.; Lu, Bai

    2011-01-01

    Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase ζ (PKMζ) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMζ reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMζ, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMζ level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMζ at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMζ in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMζ. PMID:21747912

  14. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    PubMed

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

  15. Sulfate resupply accentuates protein synthesis in coordination with nitrogen metabolism in sulfur deprived Brassica napus.

    PubMed

    Zhang, Qian; Lee, Bok-Rye; Park, Sang-Hyun; Zaman, Rashed; Avice, Jean-Christophe; Ourry, Alain; Kim, Tae-Hwan

    2015-02-01

    To investigate the regulatory interactions between S assimilation and N metabolism in Brassica napus, de novo synthesis of amino acids and proteins was quantified by (15)N and (34)S tracing, and the responses of transporter genes, assimilatory enzymes and metabolites pool involving in nitrate and sulfate metabolism were assessed under continuous sulfur supply, sulfur deprivation and sulfate resupply after 3 days of sulfur (S) deprivation. S-deprived plants were characterized by a strong induction of sulfate transporter genes, ATP sulfurylase (ATPS) and adenosine 5'-phosphosulfate reductase (APR), and by a repressed activity of nitrate reductase (NR) and glutamine synthetase (GS). Sulfate resupply to the S-deprived plants strongly increased cysteine, amino acids and proteins concentration. The increase in sulfate and cysteine concentration caused by sulfate resupply was not matched with the expression of sulfate transporters and the activity of ATPS and APR which were rapidly decreased by sulfate resupply. A strong induction of O-acetylserine(thiol)lyase (OASTL), NR and GS upon sulfate resupply was accompanied with the increase in cysteine, amino acids and proteins pool. Sulfate resupply resulted in a strong increase in de novo synthesis of amino acids and proteins, as evidenced by the increases in N and S incorporation into amino acids (1.8- and 2.4-fold increase) and proteins (2.2-and 6.3-fold increase) when compared to S-deprived plants. The results thus indicate that sulfate resupply followed by S-deprivation accelerates nitrate assimilation for protein synthesis.

  16. Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

    PubMed Central

    Choung-Hee Lee, Elaine; Deonarine, Andrew; Strange, Kevin

    2012-01-01

    Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage

  17. The effect of elevated plasma phenylalanine levels on protein synthesis rates in adult rat brain.

    PubMed Central

    Dunlop, D S; Yang, X R; Lajtha, A

    1994-01-01

    Increasing the plasma phenylalanine concentration to levels as high as 0.560-0.870 mM (over ten times normal levels) had no detectable effect on the rate of brain protein synthesis in adult rats. The average rates for 7-week-old rats were: valine, 0.58 +/- 0.05%/h, phenylalanine, 0.59 +/- 0.06%/h, and tyrosine, 0.60 +/- 0.09%/h, or 0.59 +/- 0.06%/h overall. Synthesis rates calculated on the basis of the specific activity of the tRNA-bound amino acid were slightly lower (4% lower for phenylalanine) than those based on the brain free amino acid pool. Similarly, the specific activities of valine and phenylalanine in microdialysis fluid from striatum were practically the same as those in the brain free amino acid pool. Thus the specific activities of the valine and phenylalanine brain free pools are good measures of the precursor specific activity for protein synthesis. In any event, synthesis rates, whether based on the specific activities of the amino acids in the brain free pool or those bound to tRNA, were unaffected by elevated levels of plasma phenylalanine. Brain protein synthesis rates measured after the administration of quite large doses of phenylalanine (> 1.5 mumol/g) or valine (15 mumol/g) were in agreement (0.62 +/- 0.01 and 0.65 +/- 0.01%/h respectively) with the rates determined with infusions of trace amounts of amino acids. Thus the technique of stabilizing precursor-specific activity, and pushing values in the brain close to those of the plasma, by the administration of large quantities of precursor, appears to be valid. PMID:8093014

  18. Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

    NASA Technical Reports Server (NTRS)

    Howard, G.; Steffen, J. M.; Geoghegan, T. E.

    1989-01-01

    The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

  19. Response of in vivo protein synthesis in T lymphocytes and leucocytes to an endotoxin challenge in healthy volunteers

    PubMed Central

    Januszkiewicz, A; Loré, K; EsséN, P; Andersson, B; Mcnurlan, M A; Garlick, P J; RingdéN, O; Andersson, J; Wernerman, J

    2002-01-01

    In vivo determination of protein synthesis in immune cells reflects metabolic activity and immunological activation. An intravenous injection of endotoxin to healthy volunteers was used as a human sepsis model, and in vivo protein synthesis of T lymphocytes and leucocytes was measured. The results were related to plasma concentrations of selected cytokines, peripheral cell counts and subpopulations of immune cells. The subjects (n = 8 + 8) were randomized to an endotoxin (4 ng/kg) or a saline group. In vivo protein synthesis was determined twice: before and 1–2·5 h after the endotoxin/saline injection. Protein synthesis decreased in isolated T lymphocytes, but increased in leucocytes. Plasma levels of TNF-α, IL-8, IL-6, IL-1 ra and IL-10 were elevated, whereas IL-2 and IFN-γ, produced predominantly by T lymphocytes, did not change in response to endotoxin. Neutrophils increased, whereas lymphocytes and monocytes decreased 2·5 h after the endotoxin injection. Flow cytometry revealed a drop in total CD3+ T lymphocytes and CD56+ natural killer cells, accompanied by an increase in CD15+ granulocytes. In summary, in vivo protein synthesis decreased in T lymphocytes, while the total leucocyte population showed a concomitant increase immediately after the endotoxin challenge. The changes in protein synthesis were accompanied by alterations in immune cell subpopulations and in plasma cytokine levels. PMID:12390314

  20. Response of in vivo protein synthesis in T lymphocytes and leucocytes to an endotoxin challenge in healthy volunteers.

    PubMed

    Januszkiewicz, A; Loré, K; Essén, P; Andersson, B; McNurlan, M A; Garlick, P J; Ringdén, O; Andersson, J; Wernerman, J

    2002-11-01

    In vivo determination of protein synthesis in immune cells reflects metabolic activity and immunological activation. An intravenous injection of endotoxin to healthy volunteers was used as a human sepsis model, and in vivo protein synthesis of T lymphocytes and leucocytes was measured. The results were related to plasma concentrations of selected cytokines, peripheral cell counts and subpopulations of immune cells. The subjects (n = 8 + 8) were randomized to an endotoxin (4 ng/kg) or a saline group. In vivo protein synthesis was determined twice: before and 1-2.5 h after the endotoxin/saline injection. Protein synthesis decreased in isolated T lymphocytes, but increased in leucocytes. Plasma levels of TNF-alpha, IL-8, IL-6, IL-1 ra and IL-10 were elevated, whereas IL-2 and IFN-gamma, produced predominantly by T lymphocytes, did not change in response to endotoxin. Neutrophils increased, whereas lymphocytes and monocytes decreased 2.5 h after the endotoxin injection. Flow cytometry revealed a drop in total CD3+ T lymphocytes and CD56+ natural killer cells, accompanied by an increase in CD15+ granulocytes. In summary, in vivo protein synthesis decreased in T lymphocytes, while the total leucocyte population showed a concomitant increase immediately after the endotoxin challenge. The changes in protein synthesis were accompanied by alterations in immune cell subpopulations and in plasma cytokine levels.

  1. Design, Synthesis, and Antibacterial Activities of Novel Heterocyclic Arylsulphonamide Derivatives.

    PubMed

    Singh, Anuradha; Srivastava, Ritika; Singh, Ramendra K

    2017-02-13

    Design, synthesis, and antibacterial activities of a series of arylsulphonamide derivatives as probable peptide deformylase (PDF) inhibitors have been discussed. Compounds have been designed following Lipinski's rule and after docking into the active site of PDF protein (PDB code: 1G2A) synthesized later on. Furthermore, to assess their antibacterial activity, screening of the compound was done in vitro conditions against Gram-positive and Gram-negative bacterial strains. In silico, studies revealed these compounds as potential antibacterial agents and this fact was also supported by their prominent scoring functions. Antibacterial results indicated that these molecules possessed a significant activity against Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Escherichia coli with MIC values ranging from 0.06 to 0.29 μM. TOPKAT results showed that high LD50 values and the compounds were assumed non-carcinogenic when various animal models were studied computationally.

  2. HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation.

    PubMed

    Della Fazia, Maria Agnese; Castelli, Marilena; Bartoli, Daniela; Pieroni, Stefania; Pettirossi, Valentina; Piobbico, Danilo; Viola-Magni, Mariapia; Servillo, Giuseppe

    2005-07-15

    The liver has the ability to autonomously regulate growth and mass. Following partial hepatectomy, hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in hepatocyte proliferation. To understand the mechanisms responsible for the proliferative response, we studied liver regeneration by characterization of novel genes that are activated in residual hepatocytes. A regenerating liver cDNA library screening was performed with cDNA-subtracted probes derived from regenerating and normal liver. Here, we describe the biology of Hops (for hepatocyte odd protein shuttling). HOPS is a novel shuttling protein that contains an ubiquitin-like domain, a putative NES and a proline-rich region. HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. Evidence shows that cAMP governs HOPS export in hepatocytes of normal and regenerating liver and is mediated via CRM-1. We demonstrate that HOPS binds to elongation factor eEF-1A and interferes in protein synthesis. HOPS overexpression in H-35-hepatoma and 3T3-NIH cells strongly reduces proliferation.

  3. Fixed metabolic costs for highly variable rates of protein synthesis in sea urchin embryos and larvae.

    PubMed

    Pace, Douglas A; Manahan, Donal T

    2006-01-01

    Defining the physiological mechanisms that set metabolic rates and the 'cost of living' is important for understanding the energy costs of development. Embryos and larvae of the sea urchin Lytechinus pictus (Verrill) were used to test hypotheses regarding differential costs of protein synthesis in animals differing in size, rates of protein synthesis, and physiological feeding states. For embryos, the rate of protein synthesis was 0.22+/-0.014 ng protein embryo(-1) h(-1) (mean +/- s.e.m.) and decreased in unfed larvae to an average rate of 0.05+/-0.001 ng protein larva(-1) h(-1). Fed larvae had rates of synthesis that were up to 194 times faster than unfed larvae (9.7+/-0.81 ng protein larva(-1) h(-1)). There was no significant difference, however, in the cost of protein synthesis between these larvae with very different physiological states. Furthermore, the cost of synthesis in the larval stages was also similar to costs measured for blastula and gastrula embryos of 8.4+/-0.99 J mg(-1) protein synthesized. The cost of protein synthesis was obtained using both direct ('inhibitor') and indirect ('correlative') measurements; both methods gave essentially identical results. Protein synthesis accounted for up to 54+/-8% of metabolic rate in embryos. Percent of metabolism accounted for by protein synthesis in larvae was dependent on their physiological feeding state, with protein synthesis accounting for 16+/-4% in unfed larvae and 75+/-11% in fed larvae. This regulation of metabolic rate was due to differential rates of synthesis for a fixed energy cost per unit mass of protein synthesized. The cost of synthesizing a unit of protein did not change with increasing rates of protein synthesis. We conclude that the cost of protein synthesis is independent of the rate of synthesis, developmental stage, size and physiological feeding state during sea urchin development.

  4. The PURE system for the cell-free synthesis of membrane proteins.

    PubMed

    Kuruma, Yutetsu; Ueda, Takuya

    2015-09-01

    Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large-molecular-weight complexes, such as the SecYEG translocon or ATP synthase. Here we describe a protocol for the cell-free synthesis of membrane proteins using the protein synthesis using recombinant elements (PURE) system, and for subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane and complex formation in the membrane. In addition, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within 1 d. In contrast to alternative methods using living cells, this protocol can also help to prevent the difficulties in membrane protein purification and the risks of protein aggregation during reconstitution into lipid membranes.

  5. Synthesis and Anticancer Activity of Epipolythiodiketopiperazine Alkaloids

    PubMed Central

    Boyer, Nicolas; Morrison, Karen C.; Kim, Justin; Hergenrother, Paul J.; Movassaghi, Mohammad

    2013-01-01

    The epipolythiodiketopiperazine (ETP) alkaloids are a highly complex class of natural products with potent anticancer activity. Herein, we report the application of a flexible and scalable synthesis, allowing the construction of dozens of ETP derivatives. The evaluation of these compounds against cancer cell lines in culture allows for the first expansive structure–activity relationship (SAR) to be defined for monomeric and dimeric ETP-containing natural products and their synthetic cognates. Many ETP derivatives demonstrate potent anticancer activity across a broad range of cancer cell lines, and kill cancer cellsviainduction of apoptosis. Several traits thatbode well for the translational potential of the ETP class of natural products includeconcise and efficient synthetic access, potent induction of apoptotic cell death, activity against a wide range of cancer types, and a broad tolerance for modifications at multiple sitesthat should facilitate small-molecule drug development, mechanistic studies, and evaluation in vivo. PMID:23914293

  6. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    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. PMID:22427882

  7. Action of phenylephrine on protein synthesis in liver cells.

    PubMed Central

    Menaya, J; Parrilla, R; Ayuso, M S

    1987-01-01

    The alpha-adrenergic agonist phenylephrine was found to inhibit protein labelling from [3H]valine in isolated liver cells. This effect is only observable under conditions of partial Ca2+ depletion and in cells displaying maximal rates of protein labelling, i.e. cells isolated from fed animals or from starved animals when incubated in the presence of alanine. The ability of phenylephrine to inhibit protein labelling at near-saturating concentrations of the amino acid precursor indicates that this alpha-agonist actually decreases the rate of protein synthesis. The possibility that phenylephrine acts by making cellular Ca2+ availability further limiting can be ruled out, since alanine stimulates protein labelling under conditions of severe Ca2+ depletion obtained by pretreatment of the cells with EGTA. The following observations indicate that the phenylephrine action may be mediated by an increase in cellular cyclic AMP content: (1) a close relationship was found between the abilities of phenylephrine to inhibit protein labelling and to increase cyclic AMP content; (2) cyclic AMP mimics the phenylephrine action only in cells partially depleted of Ca2+; (3) the alpha 1-antagonist prazosin, which inhibited the phenylephrine-mediated increase in cyclic AMP, also abolished the effect on protein synthesis. PMID:2829846

  8. Effects of growth rate on cell extract performance in cell-free protein synthesis.

    PubMed

    Zawada, James; Swartz, James

    2006-07-05

    Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.

  9. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy.

  10. Leucine pulses enhance skeletal muscle protein synthesis during continuous feeding in neonatal pigs

    PubMed Central

    Boutry, Claire; El-Kadi, Samer W.; Suryawan, Agus; Wheatley, Scott M.; Orellana, Renán A.; Kimball, Scot R.; Nguyen, Hanh V.

    2013-01-01

    Infants unable to maintain oral feeding can be nourished by orogastric tube. We have shown that orogastric continuous feeding restricts muscle protein synthesis compared with intermittent bolus feeding in neonatal pigs. To determine whether leucine infusion can be used to enhance protein synthesis during continuous feeding, neonatal piglets received the same amount of formula enterally by orogastric tube for 25.25 h continuously (CON) with or without LEU or intermittently by bolus every 4 h (BOL). For the CON+LEU group, leucine pulses were administered parenterally (800 μmol·kg−1·h−1) every 4 h. Insulin and glucose concentrations increased after the BOL meal and were unchanged in groups fed continuously. LEU infusion during CON feeding increased plasma leucine after the leucine pulse and decreased essential amino acids compared with CON feeding. Protein synthesis in longissimus dorsi (LD), gastrocnemius, and soleus muscles, but not liver or heart, were greater in CON+LEU and BOL than in the CON group. BOL feeding increased protein synthesis in the small intestine. Muscle S6K1 and 4E-BP1 phosphorylation and active eIF4E·eIF4G complex formation were higher in CON+LEU and BOL than in CON but AMPKα, eIF2α, and eEF2 phosphorylation were unchanged. LC3-II-to-total LC3 ratio was lower in CON+LEU and BOL than in CON, but there were no differences in atrogin-1 and MuRF-1 abundance and FoxO3 phosphorylation. In conclusion, administration of leucine pulses during continuous orogastric feeding in neonates increases muscle protein synthesis by stimulating translation initiation and may reduce protein degradation via the autophagy-lysosome, but not the ubiquitin-proteasome pathway. PMID:23839523

  11. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  12. Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

    PubMed Central

    Huang, Yanmei; Ainsley, Joshua A.; Reijmers, Leon G.; Jackson, F. Rob

    2013-01-01

    Abstract Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity. PMID:24348200

  13. Transcriptional regulation of storage protein synthesis during dicotyledon seed filling.

    PubMed

    Verdier, Jérôme; Thompson, Richard D

    2008-09-01

    Seeds represent a major source of nutrients for human and animal livestock diets. The nutritive value of seeds is largely due to storage products which accumulate during a key phase of seed development, seed filling. In recent years, our understanding of the mechanisms regulating seed filling has advanced significantly due to the diversity of experimental approaches used. This review summarizes recent findings related to transcription factors that regulate seed storage protein accumulation. A framework for the regulation of storage protein synthesis is established which incorporates the events before, during and after seed storage protein synthesis. The transcriptional control of storage protein synthesis is accompanied by physiological and environmental controls, notably through the action of plant hormones and other intermediary metabolites. Finally, recent post-genomics analyses on different model plants have established the existence of a conserved seed filling process involving the master regulators (LEC1, LEC2, ABI3 and FUS3) but also revealed certain differences in fine regulation between plant families.

  14. Activated platelets signal chemokine synthesis by human monocytes.

    PubMed Central

    Weyrich, A S; Elstad, M R; McEver, R P; McIntyre, T M; Moore, K L; Morrissey, J H; Prescott, S M; Zimmerman, G A

    1996-01-01

    Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions. PMID:8617886

  15. Potency and Spectrum of Activity of AN3365, a Novel Boron-Containing Protein Synthesis Inhibitor, Tested against Clinical Isolates of Enterobacteriaceae and Nonfermentative Gram-Negative Bacilli

    PubMed Central

    Alley, M. R. K.; Sader, Helio S.; Biedenbach, Douglas J.; Jones, Ronald N.

    2013-01-01

    AN3365 (MIC50/90, 0.5/1 μg/ml) was active against Enterobacteriaceae, including a subset of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains (MIC50/90, 1/2 μg/ml). AN3365 inhibited 98.0 and 92.2% of wild-type (MIC50/90, 2/8 μg/ml) and carbapenem-resistant (MIC50/90, 4/8 μg/ml) Pseudomonas aeruginosa strains, respectively, at ≤8 μg/ml. AN3365 also demonstrated activity against wild-type Acinetobacter baumannii (MIC50/90, 2/8 μg/ml) and Stenotrophomonas maltophilia (MIC50/90, 2/4 μg/ml), while it was less active against multidrug-resistant A. baumannii (MIC50/90, 8/16 μg/ml) and Burkholderia cepacia (MIC50/90, 8/32 μg/ml). PMID:23507283

  16. Stem cell function and stress response are controlled by protein synthesis

    PubMed Central

    Blanco, Sandra; Bandiera, Roberto; Popis, Martyna; Hussain, Shobbir; Lombard, Patrick; Aleksic, Jelena; Sajini, Abdulrahim; Tanna, Hinal; Cortés-Garrido, Rosana; Gkatza, Nikoletta; Dietmann, Sabine; Frye, Michaela

    2016-01-01

    Summary Whether protein synthesis and cellular stress response pathways interact to control stem cell function is currently unknown. Here, we show that skin stem cells synthesize less protein than their immediate progenitors in vivo, even when forced to proliferate. Our analyses reveal that activation of stress response pathways drives both a global reduction of protein synthesis and altered translational programmes that together promote stem cell functions and tumourigenesis. Mechanistically we show that inhibition of post-transcriptional cytosine-5 methylation locks stem cells in this distinct translational inhibition programme. Paradoxically, this inhibition renders stem cells hypersensitive to cytotoxic stress, as tumour regeneration after treatment with 5-fluorouracil is blocked. Thus, stem cells must revoke translation inhibition pathways to regenerate a tissue or tumour. PMID:27306184

  17. Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor.

    PubMed

    Pereira, Sandro F F; Gonzalez, Ruben L; Dworkin, Jonathan

    2015-06-23

    In nature, most organisms experience conditions that are suboptimal for growth. To survive, cells must fine-tune energy-demanding metabolic processes in response to nutrient availability. Here, we describe a novel mechanism by which protein synthesis in starved cells is down-regulated by phosphorylation of the universally conserved elongation factor Tu (EF-Tu). Phosphorylation impairs the essential GTPase activity of EF-Tu, thereby preventing its release from the ribosome. As a consequence, phosphorylated EF-Tu has a dominant-negative effect in elongation, resulting in the overall inhibition of protein synthesis. Importantly, this mechanism allows a quick and robust regulation of one of the most abundant cellular proteins. Given that the threonine that serves as the primary site of phosphorylation is conserved in all translational GTPases from bacteria to humans, this mechanism may have important implications for growth-rate control in phylogenetically diverse organisms.

  18. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    PubMed

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  19. Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

    PubMed Central

    Weinstock, Matthew T.; Jacobsen, Michael T.; Kay, Michael S.

    2014-01-01

    Mirror-image proteins (composed of d-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger d-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein. The ability of chaperones to fold d-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic d-protein. We report the total chemical synthesis of a 312-residue GroEL/ES-dependent protein, DapA, in both l- and d-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both l- and d-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold d-proteins for drug development and mirror-image synthetic biology applications. PMID:25071217

  20. Polyaromatic compounds alter placental protein synthesis in pregnant rats

    SciTech Connect

    Shiverick, K.T.; Ogilvie, S.; Medrano, T. )

    1991-03-15

    The administration of the polyaromatic compounds {beta}-naphthoflavone ({beta}NF) and 3-methylcholanthrene (3MC) to pregnant rats during mid-gestation has been shown to produce marked feto-placental growth retardation. This study examined secretory protein synthesis in placental tissue from rats following administration of {beta}NF on gestation days (gd) 11-14 or 3MC on gd 12-14. Explants of placental basal zone tissue were cultured for 24 hours in serum-free medium in the presence of ({sup 3}H)leucine. Secreted proteins were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis followed by either fluorography or immunostaining. Total incorporation of ({sup 3}H)leucine into secreted proteins was not altered in BZ explants from {beta}NF or 3MC-treated animals. However a selective decrease was observed in ({sup 3}H)leucine incorporation into a major complex of proteins with apparent molecular weight of 25-30,000 and isoelectric point between 5.3 to 5.7. This group of proteins has been further identified as being related to rat pituitary growth hormone (GH) using N-terminal amino acid microsequencing of individual spots from 2-D SDS-PA gels. This is the first report that synthesis of GH-related proteins by rat placenta is decreased following {beta}NF and 3MC administration, a change which may underlie the feto-placental growth retardation associated with these polyaromatic compounds.

  1. Protein Lysine Methyltransferase G9a Inhibitors: Design, Synthesis, and Structure Activity Relationships of 2,4-Diamino-7-aminoalkoxy-quinazolines.†

    PubMed Central

    Liu, Feng; Chen, Xin; Allali-Hassani, Abdellah; Quinn, Amy M.; Wigle, Tim J.; Wasney, Gregory A.; Dong, Aiping; Senisterra, Guillermo; Chau, Irene; Siarheyeva, Alena; Norris, Jacqueline L.; Kireev, Dmitri B.; Jadhav, Ajit; Herold, J. Martin; Janzen, William P.; Arrowsmith, Cheryl H.; Frye, Stephen V.; Brown, Peter J.; Simeonov, Anton; Vedadi, Masoud; Jin, Jian

    2010-01-01

    Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is over expressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth and the di-methylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. Based on the structural insights revealed by this co-crystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison Ki = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date. PMID:20614940

  2. Protein lysine methyltransferase G9a inhibitors: design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines.

    PubMed

    Liu, Feng; Chen, Xin; Allali-Hassani, Abdellah; Quinn, Amy M; Wigle, Tim J; Wasney, Gregory A; Dong, Aiping; Senisterra, Guillermo; Chau, Irene; Siarheyeva, Alena; Norris, Jacqueline L; Kireev, Dmitri B; Jadhav, Ajit; Herold, J Martin; Janzen, William P; Arrowsmith, Cheryl H; Frye, Stephen V; Brown, Peter J; Simeonov, Anton; Vedadi, Masoud; Jin, Jian

    2010-08-12

    Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is overexpressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth, and the dimethylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. On the basis of the structural insights revealed by this cocrystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison K(i) = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date.

  3. Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts 1

    PubMed Central

    Vasconcelos, Aurea C.

    1976-01-01

    Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [35S]methionine or [3H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea. PMID:16659752

  4. Question 7: Optimized Energy Consumption for Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Szaflarski, Witold; Nierhaus, Knud H.

    2007-10-01

    In our previous contribution (Nierhaus, Orig Life Evol Biosph, this volume, 2007) we mentioned that life had solved the problem of energy supply in three major steps, and that these steps also mark major stages during the development of life. We further outlined a possible scenario concerning a minimal translational apparatus focusing on the essential components necessary for protein synthesis. Here we continue that consideration by addressing on one of the main problems of early life, namely avoiding wasteful energy loss. With regard to the limiting energy supply of early living systems, i.e. those of say more than 3,000 Ma, a carefully controlled and product oriented energy consumption was in demand. In recent years we learned how a bacterial cell avoids energy drain, thus being able to pump most of the energy into protein synthesis. These lessons must be followed by the design of a minimal living system, which is surveyed in this short article.

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

    SciTech Connect

    Horst, M.N. )

    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.

  6. Modifications to the translational apparatus which affect the regulation of protein synthesis in sea urchin embryos

    SciTech Connect

    Scalise, F.W.

    1988-01-01

    Protein synthesis can be regulated at a number of cellular levels. I have examined how modifications to specific components of the protein synthetic machinery are involved in regulating the efficiency of initiation of translation during early sea urchin embryogenesis. It is demonstrated that Ca{sup 2+} concentrations exceeding 500 uM cause the inhibition of protein synthesis in cell-free translation lysates prepared from sea urchin embryos. Specific changes in the state of phosphorylation of at least 8 proteins occur during this Ca{sup 2+}-mediated repression of translation. Analysis of these proteins has indicated that, unlike mammalian systems, there is no detectable level of Ca{sup 2+}-dependent phosphorylation of the {alpha}subunit eIF-2. Two of the proteins which do become phosphorylated in response to Ca{sup 2+} are calmodulin and an isoelectric form of sea urchin eIF-4D. In addition, 2 proteins which share similarities with kinases involved in the regulation of protein synthesis in mammalian cells, also become phosphorylated. I have investigated the consequences of changes in eIF-4D during sea urchin embryogenesis because it has been proposed that a polyamine-mediated conversion of lysine to hypusine in this factor may enhance translational activity. It is demonstrated that ({sup 3}H) spermidine-derived radioactivity is incorporated into a number of proteins when sea urchin embryos are labeled in vivo, and that the pattern of individual proteins that become labeled changes over the course of the first 30 hr of development.

  7. Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

    SciTech Connect

    Kopecky, J.; Baudysova, M.; Zanotti, F.; Janikova, D.; Pavelka, S.; Houstek, J. )

    1990-12-25

    In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-(35S)methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.

  8. The Roles of RNA in the Synthesis of Protein

    PubMed Central

    Moore, Peter B.; Steitz, Thomas A.

    2011-01-01

    The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding. PMID:21068149

  9. SYNTHESIS OF PROTEINS BY NATIVE CHEMICAL LIGATION USING FMOC-BASED CHEMISTRY

    SciTech Connect

    Camarero, J A; Mitchell, A R

    2005-01-20

    C-terminal peptide {alpha}-thioesters are valuable intermediates in the synthesis/semisynthesis of proteins by native chemical ligation. They are prepared either by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. The present paper reviews the different methods available for the chemical synthesis of peptide {alpha}-thioesters using Fmoc-based SPPS.

  10. Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa.

    PubMed

    Hu, Yanmei; Keniry, Megan; Palmer, Stephanie O; Bullard, James M

    2016-08-01

    Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 μM. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 μg/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines.

  11. Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

    PubMed Central

    Hu, Yanmei; Keniry, Megan; Palmer, Stephanie O.

    2016-01-01

    Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa. This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 μM. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 μg/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines. PMID:27246774

  12. Identification of lipid synthesis and secretion proteins in bovine milk.

    PubMed

    Lu, Jing; van Hooijdonk, Toon; Boeren, Sjef; Vervoort, Jacques; Hettinga, Kasper

    2014-02-01

    Lactation physiology is a process that is only partly understood. Proteomics techniques have shown to be useful to help advance the knowledge on lactation physiology in human and rodent species but have not been used as major tools for dairy cows, except for mastitis. In this paper, advanced non-targeted proteomics techniques (Filter aided sample preparation and NanoLC-Orbitrap-MS/MS) were applied to study the milk fat globule membrane and milk serum fraction, resulting in the identification of 246 proteins. Of these, 23 transporters and enzymes were related to lipid synthesis and secretion in mammary gland and their functions are discussed in detail. The identification of these intracellular transporters and enzymes in milk provides a possibility of using milk itself to study lipid synthesis and secretion pathways. This full-scale scan of milk proteins by using non-targeted proteomic analysis helps to reveal the important proteins involved in lipid synthesis and secretion for further examination in targeted studies.

  13. [Effect of precursors and cofactors of nucleic acid and protein synthesis on the response of cortical neurons induced by polarization].

    PubMed

    Kruglikov, R I; Maĭzelis, M Ia; Zabludovskiĭ, A L

    1977-01-01

    Injection of K-orotate and folic acid in different proportions and of vitamine B12 produces changes in the S35-methionine inclusion in the proteins of the sensorimotor cortex, basal ganglia, hypothalamus and hippocampus depending on the proportions of the injected agents. In animals with activation of the synthesis in the brain, surface anode polarization increased the mean frequency of spike activity of the neurones in the sensorimotor cortex and reduced the relative number of units, which responded to polarization by inhibition, as compared with the control animals and those in which no activation of protein synthesis was observed. The characteristics of cortical unit responses to surface anode polarization in experimental rats are apparently due to changes in the chemoreactive properties of their membranes, which set in under the influence of changes in the nucleic acid and protein synthesis in these neurones.

  14. Effect of hyperbaric oxygenation on carbohydrate metabolism protein synthesis in the myocardium during sustained hypodynamia

    NASA Technical Reports Server (NTRS)

    Makarov, G. A.

    1980-01-01

    Glycolysis and the intensity of protein synthesis were studied in 140 white male rats in subcellular fractions of the myocardium during 45 day hypodynamia and hyperbaric oxygenation. Hypodynamia increased: (1) the amount of lactic acids; (2) the amount of pyruvic acid; (3) the lactate/pyruvate coefficient; and (4) the activities of aldolase and lactate dehydrogenase. Hyperbaric oxygenation was found to have a favorable metabolic effect on the animals with hypodynamia.

  15. High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.

    PubMed

    Takemori, Nobuaki; Takemori, Ayako; Matsuoka, Kazuhiro; Morishita, Ryo; Matsushita, Natsuki; Aoshima, Masato; Takeda, Hiroyuki; Sawasaki, Tatsuya; Endo, Yaeta; Higashiyama, Shigeki

    2015-02-01

    Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry.

  16. Betulin Phosphonates; Synthesis, Structure, and Cytotoxic Activity.

    PubMed

    Chrobak, Elwira; Bębenek, Ewa; Kadela-Tomanek, Monika; Latocha, Małgorzata; Jelsch, Christian; Wenger, Emmanuel; Boryczka, Stanisław

    2016-08-26

    Betulin derivatives are a widely studied group of compounds of natural origin due to their wide spectrum of biological activities. This paper describes new betulin derivatives, containing a phosphonate group. The allyl-vinyl isomerization and synthesis of acetylenic derivatives have been reported. Structural identification of products as E and Z isomers has been carried out using ¹H-, (13)C-, (31)P-NMR, and crystallographic analysis. The crystal structure in the orthorhombic space group and analysis of crystal packing contacts for 29-diethoxyphosphoryl-28-cyclopropylpropynoyloxy-lup-20E(29)-en-3β-ol 8a are reported. All new compounds were tested in vitro for their antiproliferative activity against human T47D (breast cancer), SNB-19 (glioblastoma), and C32 (melanoma) cell lines.

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

    PubMed

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J F; Vollmer, Waldemar; Breukink, Eefjan; Terrak, Mohammed

    2017-02-24

    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 with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.

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

    PubMed Central

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J. F.; Vollmer, Waldemar; Breukink, Eefjan; 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 with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell’s need to ensure appropriate use of the limited pool of lipid II. PMID:28233869

  19. Identification of Chemical Compounds That Inhibit Protein Synthesis in Pseudomonas aeruginosa.

    PubMed

    Palmer, Stephanie O; Hu, Yanmei; Keniry, Megan; Bullard, James M

    2016-11-21

    Four inhibitory compounds were identified using a poly-uridylic acid (polyU) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa in an in vitro screen of a synthetic compound library. The compounds were specific for inhibition of bacterial protein synthesis. In enzymatic assays, the compounds inhibited protein synthesis with IC50 values ranging from 20 to 60 μM. Minimum inhibitory concentrations (MICs) were determined in cultures for a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae All the compounds were observed to have broad-spectrum activity and inhibited an efflux pump mutant strain of P. aeruginosa with MICs of 0.5-16 μg/mL. The molecular target of two compounds was determined to be PheRS. These two compounds were bacteriostatic against both Gram-positive and Gram-negative pathogens. In competition assays, they were not observed to compete with the natural substrates ATP or phenylalanine for active site binding. The other two compounds directly inhibited the ribosome and were bactericidal against both Gram-positive and Gram-negative pathogens. In cytotoxicity MTT testing in human cell lines, the compounds were shown to be from 2500- to 30,000-fold less active than the control staurosporine.

  20. Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells

    PubMed Central

    Marczin, Nándor; Go, Carolyn Y; Papapetropoulos, Andreas; Catravas, John D

    1998-01-01

    The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC).As determined by cyclic GMP accumulation, substrate (L-arginine)- and inhibitor (NG-monomethyl-L-arginine, NMMA)-sensitive iNOS activity was significantly elevated in CH- or ANI-treated RASMC after 24 h.Lipopolysaccharide (LPS) produced a time-dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS-induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration-dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 μM and inhibition at higher concentrations.Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS- and CH-treated cells.CH-induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor.RT-PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS- or CH stimulation alone.These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine-treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins. PMID:9535031

  1. Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis

    PubMed Central

    Liu, Han-Hsuan

    2016-01-01

    Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. SIGNIFICANCE STATEMENT Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual

  2. Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

    PubMed Central

    Siddell, S G; Ellis, R J

    1975-01-01

    The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts. Images PLATE 1 PMID:1147911

  3. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  4. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  5. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

  6. Sexually dimorphic effect of aging on skeletal muscle protein synthesis

    PubMed Central

    2012-01-01

    Background Although there appear to be no differences in muscle protein turnover in young and middle aged men and women, we have reported significant differences in the rate of muscle protein synthesis between older adult men and women. This suggests that aging may affect muscle protein turnover differently in men and women. Methods We measured the skeletal muscle protein fractional synthesis rate (FSR) by using stable isotope-labeled tracer methods during basal postabsorptive conditions and during a hyperaminoacidemic-hyperinsulinemic-euglycemic clamp in eight young men (25–45 y), ten young women (25–45 y), ten old men (65–85 y) and ten old women (65–85 y). Results The basal muscle protein FSR was not different in young and old men (0.040 ± 0.004 and 0.043 ± 0.005%·h-1, respectively) and combined insulin, glucose and amino acid infusion significantly increased the muscle protein FSR both in young (to 0.063 ± 0.006%·h-1) and old (to 0.051 ± 0.008%·h-1) men but the increase (0.023 ± 0.004 vs. 0.009 ± 0.004%·h-1, respectively) was ~60% less in the old men (P = 0.03). In contrast, the basal muscle protein FSR was ~30% greater in old than young women (0.060 ± 0.003 vs. 0.046 ± 0.004%·h-1, respectively; P < 0.05) and combined insulin, glucose and amino acid infusion significantly increased the muscle protein FSR in young (P < 0.01) but not in old women (P = 0.10) so that the FSR was not different between young and old women during the clamp (0.074 ± 0.006%·h-1 vs. 0.072 ± 0.006%·h-1, respectively). Conclusions There is sexual dimorphism in the age-related changes in muscle protein synthesis and thus the metabolic processes responsible for the age-related decline in muscle mass. PMID:22620287

  7. A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells.

    PubMed

    Castorena, Kathryn M; Weeks, Spencer A; Stapleford, Kenneth A; Cadwallader, Amy M; Miller, David J

    2007-08-01

    The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.

  8. Effect of insulin on the compartmentation of glutamate for protein synthesis

    SciTech Connect

    Brown, A.B.; Mohan, C.; Bessman, S.P.

    1986-03-05

    The effect of insulin on the formation of CO/sub 2/ and incorporation of 1-/sup 14/C glutamine and U-/sup 14/C acetate into protein was studied in isolated rat hepatocytes. Insulin caused an 18% increase in /sup 14/CO/sub 2/ production from U-/sup 14/C acetate in comparison to a 10% increase from 1-/sup 14/C glutamate. Insulin caused a greater increase in the incorporation of tracer acetate carbons into hepatocyte protein. Hydrolysis of labeled protein and subsequent determination of glutamate specific activity revealed that incorporation of acetate carbons into protein as glutamate was about 52% greater in the presence of insulin. These results demonstrate the existence of two compartments of glutamate for protein synthesis: (i) glutamate generated in the Krebs cycle through transamination of a-ketoglutarate; (ii) cytosolic glutamate. Insulin had a greater stimulatory effect on the incorporation of glutamate generated in the Krebs cycle.

  9. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley.

    PubMed

    Mork-Jansson, Astrid; Bue, Ann Kristin; Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

  10. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

    PubMed Central

    Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.). PMID:26172838

  11. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  12. Protein-synthesis-dependent induction of annexin I by glucocorticoid.

    PubMed Central

    Wong, W T; Frost, S C; Nick, H S

    1991-01-01

    We demonstrate that annexin I/lipocortin I (lipo I) gene expression is regulated by dexamethasone (DEX) in mouse 3T3-L1 fibroblasts and LA-4 lung epithelial cells. We have characterized this induction further in 3T3-L1 fibroblasts. At 24 h after addition of DEX, the levels of lipo I mRNA and protein increased 5-fold and 1.5-fold respectively. Time-course experiments revealed that the induction was delayed by 2-4 h after DEX addition. Half-maximal induction of both lipo I mRNA and protein was achieved with 10 nM-DEX. Both actinomycin D and cycloheximide blocked the DEX effect on lipo I mRNA expression. These results indicate that the induction of lipo I by DEX has a transcriptional component and requires protein synthesis de novo. Images Fig. 1. Fig. 2. Fig. 4. Fig. 6. Fig. 7. Fig. 8. PMID:1827255

  13. Design, Synthesis, and Anti-inflammatory Properties of Orally Active 4-(Phenylamino)-pyrrolo[2,1-f][1,2,4]triazine p38[alpha] Mitogen-Activated Protein Kinase Inhibitors

    SciTech Connect

    Hynes, Jr., John; Dyckman, Alaric J.; Lin, Shuqun; Wrobleski, Stephen T.; Wu, Hong; Gillooly, Kathleen M.; Kanner, Steven B.; Lonial, Herinder; Loo, Derek; McIntyre, Kim W.; Pitt, Sidney; Shen, Ding Ren; Shuster, David J.; Yang, XiaoXia; Zhang, Rosemary; Behnia, Kamelia; Zhang, Hongjian; Marathe, Punit H.; Doweyko, Arthur M.; Tokarski, John S.; Sack, John S.; Pokross, Matthew; Kiefer, Susan E.; Newitt, John A.; Barrish, Joel C.; Dodd, John; Schieven, Gary L.; Leftheris, Katerina

    2008-06-30

    A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.

  14. Alphavirus RNA synthesis and non-structural protein functions.

    PubMed

    Rupp, Jonathan C; Sokoloski, Kevin J; Gebhart, Natasha N; Hardy, Richard W

    2015-09-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

  15. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    PubMed Central

    Thoring, Lena; Wüstenhagen, Doreen A.; Borowiak, Maria; Stech, Marlitt; Sonnabend, Andrei; Kubick, Stefan

    2016-01-01

    Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA. PMID:27684475

  16. Genetic studies of an Escherichia coli K-12 temperature-sensitive mutant defective in membrane protein synthesis.

    PubMed Central

    Sato, T; Ohki, M; Yura, T; Ito, K

    1979-01-01

    The mutant divE42(Ts) of Escherichia coli K-12, defective in the synthesis of membrane proteins and in the transcription of the lac operon at high temperature, has been further characterized. It was found that a mutation (divE42) located at about min 22 on the E. coli chromosome map is responsible for the Lac- phenotype and temperature-sensitive growth. The mutation could be contransduced with serC, pyrD, or pyrC by phage P1 at a frequency of 4, 16, or 0.5%, respectively, the gene order being serC-pyrD-ompA-sulA-divE-pyrC. Examination of temperature-independent revertants and Pyr+ transductants revealed that all the mutant phenotypes examined (deficiencies in the increase of activities of some membrane enzymes, expression of the lac operon, and synthesis of several other proteins) are due to a single mutation (divE42) which is recessive to the wild-type (divE+) allele. Protein synthesis in the mutant was also analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Synthesis of a number of proteins, including membrane proteins, was found to decrease significantly, whereas that of an elongation factor, EF-Tu, increased upon transfer of a log-phase culture to high temperature (42 degrees C). These effects of temperature shift-up on protein synthesis were evident within 5 min under the conditions used. Images PMID:374381

  17. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  18. Protein synthesis in tomato-fruit locule tissue

    PubMed Central

    Davies, J. W.; Cocking, E. C.

    1967-01-01

    1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating 14C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20–45μμmoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10–20μμmoles of amino acid/mg. of protein; 40–150μμg. of carbon/mg. of protein was incorporated in 10min. from 14C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10–20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants. PMID:5340735

  19. Installing hydrolytic activity into a completely de novo protein framework

    NASA Astrophysics Data System (ADS)

    Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

  20. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

  1. Synthesis of the blood circulating C-terminal fragment of insulin-like growth factor (IGF)-binding protein-4 in its native conformation. Crystallization, heparin and IGF binding, and osteogenic activity.

    PubMed

    Fernández-Tornero, Carlos; Lozano, Rosa M; Rivas, Germán; Jiménez, M Angeles; Ständker, Ludger; Díaz-Gonzalez, Diana; Forssmann, Wolf-Georg; Cuevas, Pedro; Romero, Antonio; Giménez-Gallego, Guillermo

    2005-05-13

    Insulin-like growth factor-binding proteins play a critical role in a wide variety of important physiological processes. It has been demonstrated that both an N-terminal and a C-terminal fragment of insulin-like growth factor-binding protein-4 exist and accumulate in the circulatory system, these fragments accounting for virtually the whole amino acid sequence of the protein. The circulating C-terminal fragment establishes three disulfide bridges, and the binding pattern of these has recently been defined. Here we show that the monodimensional 1H NMR spectrum of the C-terminal fragment is typical of a protein with a relatively close packed tertiary structure. This fragment can be produced in its native conformation in Escherichia coli, without the requirement of further refolding procedures, when synthesis is coupled to its secretion from the cell. The recombinant protein crystallizes with the unit cell parameters of a hexagonal system. Furthermore, it binds strongly to heparin, acquiring a well defined oligomeric structure that interacts with insulin-like growth factors, and promotes bone formation in cultures of murine calvariae.

  2. Synthesis, SAR, and series evolution of novel oxadiazole-containing 5-lipoxygenase activating protein inhibitors: discovery of 2-[4-(3-{(r)-1-[4-(2-amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915).

    PubMed

    Takahashi, Hidenori; Riether, Doris; Bartolozzi, Alessandra; Bosanac, Todd; Berger, Valentina; Binetti, Ralph; Broadwater, John; Chen, Zhidong; Crux, Rebecca; De Lombaert, Stéphane; Dave, Rajvee; Dines, Jonathon A; Fadra-Khan, Tazmeen; Flegg, Adam; Garrigou, Michael; Hao, Ming-Hong; Huber, John; Hutzler, J Matthew; Kerr, Steven; Kotey, Adrian; Liu, Weimin; Lo, Ho Yin; Loke, Pui Leng; Mahaney, Paige E; Morwick, Tina M; Napier, Spencer; Olague, Alan; Pack, Edward; Padyana, Anil K; Thomson, David S; Tye, Heather; Wu, Lifen; Zindell, Renee M; Abeywardane, Asitha; Simpson, Thomas

    2015-02-26

    The synthesis, structure-activity relationship (SAR), and evolution of a novel series of oxadiazole-containing 5-lipoxygenase-activating protein (FLAP) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (IC50 < 100 nM). Optimization of binding and functional potencies, as well as physicochemical properties resulted in the identification of compound 69 (BI 665915) that demonstrated an excellent cross-species drug metabolism and pharmacokinetics (DMPK) profile and was predicted to have low human clearance. In addition, 69 was predicted to have a low risk for potential drug-drug interactions due to its cytochrome P450 3A4 profile. In a murine ex vivo whole blood study, 69 demonstrated a linear dose-exposure relationship and a dose-dependent inhibition of LTB4 production.

  3. Bringing the science of proteins into the realm of organic chemistry: total chemical synthesis of SEP (synthetic erythropoiesis protein).

    PubMed

    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.

  4. Biologically active proteins from natural product extracts.

    PubMed

    O'Keefe, B R

    2001-10-01

    The term "biologically active proteins" is almost redundant. All proteins produced by living creatures are, by their very nature, biologically active to some extent in their homologous species. In this review, a subset of these proteins will be discussed that are biologically active in heterologous systems. The isolation and characterization of novel proteins from natural product extracts including those derived from microorganisms, plants, insects, terrestrial vertebrates, and marine organisms will be reviewed and grouped into several distinct classes based on their biological activity and their structure.

  5. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  6. Leucine supplementation of a low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with amino acids. Leucine appears to be the most effective single amino acid to trigger these effects. To examine the response to enteral leucine supplementation...

  7. NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10*

    PubMed Central

    Yang, Yongjie; Cimen, Huseyin; Han, Min-Joon; Shi, Tong; Deng, Jian-Hong; Koc, Hasan; Palacios, Orsolya M.; Montier, Laura; Bai, Yidong; Tong, Qiang; Koc, Emine C.

    2010-01-01

    A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3. PMID:20042612

  8. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii.

    PubMed

    Sato, Atsushi; Matsumura, Rie; Hoshino, Naomi; Tsuzuki, Mikio; Sato, Norihiro

    2014-01-01

    Triacylglycerol (TG) synthesis is induced for energy and carbon storage in algal cells under nitrogen(N)-starved conditions, and helps prevent reactive oxygen species (ROS) 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 (DGAT) 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 DGAT 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.

  9. Ribosome recycling: An essential process of protein synthesis.

    PubMed

    Kiel, Michael C; Kaji, Hideko; Kaji, Akira

    2007-01-01

    A preponderance of textbooks outlines cellular protein synthesis (translation) in three basic steps: initiation, elongation, and termination. However, researchers in the field of translation accept that a vital fourth step exists; this fourth step is called ribosome recycling. Ribosome recycling occurs after the nascent polypeptide has been released during the termination step. Despite the release of the polypeptide, ribosomes remain bound to the mRNA and tRNA. It is only during the fourth step of translation that ribosomes are ultimately released from the mRNA, split into subunits, and are free to bind new mRNA, thus the term "ribosome recycling." This step is essential to the viability of cells. In bacteria, it is catalyzed by two proteins, elongation factor G and ribosome recycling factor, a near perfect structural mimic of tRNA. Eukaryotic organelles such as mitochondria and chloroplasts possess ribosome recycling factor and elongation factor G homologues, but the nature of ribosome recycling in eukaryotic cytoplasm is still under investigation. In this review, the discovery of ribosome recycling and the basic mechanisms involved are discussed so that textbook writers and teachers can include this vital step, which is just as important as the three conventional steps, in sections dealing with protein synthesis.

  10. A Simple Protein Synthesis Model for the PURE System Operation.

    PubMed

    Mavelli, Fabio; Marangoni, Roberto; Stano, Pasquale

    2015-06-01

    The encapsulation of transcription-translation (TX-TL) cell-free machinery inside lipid vesicles (liposomes) is a key element in synthetic cell technology. The PURE system is a TX-TL kit composed of well-characterized parts, whose concentrations are fine tunable, which works according to a modular architecture. For these reasons, the PURE system perfectly fulfils the requirements of synthetic biology and is widely used for constructing synthetic cells. In this work, we present a simplified mathematical model to simulate the PURE system operations. Based on Michaelis-Menten kinetics and differential equations, the model describes protein synthesis dynamics by using 9 chemical species, 6 reactions and 16 kinetic parameters. The model correctly predicts the time course for messenger RNA and protein production and allows quantitative predictions. By means of this model, it is possible to foresee how the PURE system species affect the mechanism of proteins synthesis and therefore help in understanding scenarios where the concentration of the PURE system components has been modified purposely or as a result of stochastic fluctuations (for example after random encapsulation inside vesicles). The model also makes the determination of response coefficients for all species involved in the TX-TL mechanism possible and allows for scrutiny on how chemical energy is consumed by the three PURE system modules (transcription, translation and aminoacylation).

  11. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    PubMed

    Krinsky, Nitzan; Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    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.

  12. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    PubMed Central

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    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. PMID:27768741

  13. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    PubMed

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

  14. Excessive energy intake does not modify fed-state tissue protein synthesis rates in adult rats.

    PubMed

    Adéchian, Solange; Giardina, Silvana; Rémond, Didier; Papet, Isabelle; Buonocore, Daniela; Gaudichon, Claire; Dardevet, Dominique; Marzatico, Fulvio; Mosoni, Laurent

    2009-07-01

    The impact of chronic excessive energy intake on protein metabolism is still controversial. Male Wistar rats were fed ad libitum during 5 weeks with either a high-fat high-sucrose diet (HF: n = 9) containing 45% of total energy as lipids (protein 14%; carbohydrate 40% with 83.5% sucrose) or a standard diet (controls: n = 10). Energy intake and body weight were recorded. At the end of the experiment, we measured body composition, metabolic parameters (plasma amino acid, lipid, insulin, and glucose levels), inflammatory parameter (plasma alpha2-macroglobulin), oxidative stress parameters (antioxidant enzyme activities, lipoperoxidation (LPO), protein carbonyl content in liver and muscle), and in vivo fed-state fractional protein synthesis rates (FSRs) in muscle and liver. Energy intake was significantly higher in HF compared with control rats (+28%). There were significant increases in body weight (+8%), body fat (+21%), renal (+41%), and epidydimal (+28%) fat pads in HF compared with control rats. No effect was observed in other tissue weights (liver, muscle, spleen, kidneys, intestine). Liver and muscle FSRs, plasma levels of lipids, glucose, insulin and alpha2-macroglobulin, soleus and liver glutathione reductase and peroxidase activities, MnSOD activity, LPO, and protein carbonyl content were not altered by the HF diet. Only soleus muscle and liver Cu/ZnSOD activity and soleus muscle catalase activities were reduced in HF rats compared with control rats. Thus, chronic excessive energy intake and increased adiposity, in the absence of other metabolic alterations, do not stimulate fed-state tissue protein synthesis rates.

  15. mTOR's role in ageing: protein synthesis or autophagy?

    PubMed

    Hands, Sarah L; Proud, Christopher G; Wyttenbach, Andreas

    2009-07-20

    The molecular and cellular mechanisms that regulate ageing are currently under scrutiny because ageing is linked to many human diseases. The nutrient sensing TOR pathway is emerging as a key regulator of ageing. TOR signaling is complex affecting several crucial cellular functions and two such functions, which show clear effects on ageing, are protein synthesis and autophagy. In this article we discuss the relative importance of both these processes in ageing, identify how TOR regulates translation and autophagy and speculate on links between the TOR signaling network and ageing pathways.

  16. Turnover of whole body proteins and myofibrillar proteins in middle-aged active men

    SciTech Connect

    Zackin, M.; Meredith, C.; Frontera, W.; Evans, W.

    1986-03-05

    Endurance-trained older men have a higher proportion of lean tissue and greater muscle cell oxidative capacity, reversing age-related trends and suggesting major changes in protein metabolism. In this study, protein turnover was determined in 6 middle-aged (52+/-1 yr) men who were well trained (VO/sub 2/ max 55.2+/-5.0 ml O/sub 2//kg.min) and lean (body fat 18.9+/-2.8%, muscle mass 36.6+/-0.6%). The maintained habitual exercise while consuming 0.6, 0.9 or 1.2 g protein/kg.day for 10-day periods. N flux was measured from /sup 15/N in urea after oral /sup 15/N-glycine administration. Myofibrillar protein breakdown was estimated from urinary 3-methyl-histidine. Dietary protein had no effect on turnover rates, even when N balance was negative. Whole body protein synthesis was 3.60+/-0.12 g/kg.day and breakdown was 3.40+/-0.14 g/kg.day for all N intakes. Whole body protein flux, synthesis and breakdown were similar to values reported for sedentary young (SY) or sedentary old (SO) men on comparable diets. 3-me-his (3.67+/-0.14 ..mu..mol/kg.day) was similar to values reported for SY but higher (p<0.01) than for SO. Myofibrillar protein breakdown per unit muscle mass (185+/-7 ..mu..mol 3-me-his/g creatinine) was higher (p<0.01) than for SY or SO. In active middle-aged men, myofibrillar proteins may account for a greater proportion of whole body protein turnover, despite an age-related reduction in muscle mass.

  17. Nitrogen Assimilation and Protein Synthesis in Wheat Seedlings As Affected by Mineral Nutrition. I. Macronutrients 1

    PubMed Central

    Harper, James E.; Paulsen, Gary M.

    1969-01-01

    Deficiencies of each macronutrient (N, P, K, Ca. Mg, S, and Fe) decreased the specific activity of nitrate reductase from Triticum aestivum L. seedlings. Nitrate content was decreased by N, P, K, Ca, and Mg deficiencies and unaffected by S and Fe deficiencies. Glutamic acid dehydrogenase activity was decreased by N, P, and S deficiencies, unchanged by K deficiency, and increased by Ca, Mg, and Fe deficiencies. Glutamine synthetase activity closely paralleled nitrate reductase activity and was decreased by deficiencies of N, P, K, Ca, Mg, and S. Glutamic-oxaloacetic transaminase was not sensitive to macronutrient deficiencies. High 14C-leucine incorporation into tissue sections of N-, P-, K-, Ca-, and S-deficient seedlings did not appear indicative of protein synthesis rates in intact seedlings. Nutritional deficiencies apparently depleted endogenous amino acid pools and caused less inhibition of exogenous 14C-leucine incorporation into protein. PMID:16657034

  18. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    PubMed

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

  19. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  20. Rational Design of Protein C Activators

    PubMed Central

    Barranco-Medina, Sergio; Murphy, Mary; Pelc, Leslie; Chen, Zhiwei; Di Cera, Enrico; Pozzi, Nicola

    2017-01-01

    In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance. PMID:28294177

  1. Cost of protein synthesis and energy allocation during development of antarctic sea urchin embryos and larvae.

    PubMed

    Pace, Douglas A; Manahan, Donal T

    2007-04-01

    Cold environments represent a substantial volume of the biosphere. To study developmental physiology in subzero seawater temperatures typically found in the Southern Ocean, rates and costs of protein synthesis were measured in embryos and larvae of Sterechinus neumayeri, the Antarctic sea urchin. Our analysis of the "cost of living" in extreme cold for this species shows (1) that cost of protein synthesis is strikingly low during development, at 0.41 +/- 0.05 J (mg protein synthesized)(-1) (n = 16); (2) that synthesis cost is fixed and independent of synthesis rate; and (3) that a low synthesis cost permits high rates of protein turnover at -1 degrees C, at rates comparable to those of temperate species of sea urchin embryos developing at 15 degrees C. With a low synthesis cost, even at the highest synthesis rates measured (gastrulae), the proportion of total metabolism accounted for by protein synthesis in the Antarctic sea urchin was 54%-a value similar to that of temperate sea urchin embryos. In the Antarctic sea urchin, up to 87% of metabolic rate can be accounted for by the combined energy costs of protein synthesis and the sodium pump. We conclude that, in Antarctic sea urchin embryos, high rates of protein synthesis can be supported in extreme-cold environments while still maintaining low rates of respiration.

  2. Nicotine promotes Streptococcus mutans extracellular polysaccharide synthesis, cell aggregation and overall lactate dehydrogenase activity.

    PubMed

    Huang, R; Li, M; Gregory, R L

    2015-08-01

    Several epidemiology studies have reported a positive relationship between smoking and dental caries. Nicotine, an alkaloid component of tobacco, has been demonstrated to stimulate biofilm formation and metabolic activity of Streptococcus mutans, one of the most important pathogens of dental caries. The first aim of the present study was to explore the possible mechanisms leading to increased biofilm by nicotine treatment from three aspects, extracellular polysaccharides (EPS) synthesis, glucosyltransferase (Gtf) synthesis and glucan-binding protein (Gbp) synthesis at the mRNA and protein levels. The second aim was to investigate how nicotine affects S. mutans virulence, particular in lactate dehydrogenase (LDH) activity. Confocal laser scanning microscopy results demonstrated that both biofilm bacterial cell numbers and EPS were increased by nicotine. Gtf and GbpA protein expression of S. mutans planktonic cells were upregulated while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression trends of those genes were mostly consistent with results on protein level but not statistically significant, and gtfD and gbpD of biofilm cells were inhibited. Nicotine was not directly involved in S. mutans LDH activity. However, since it increases the total number of bacterial cells in biofilm, the overall LDH activity of S. mutans biofilm is increased. In conclusion, nicotine stimulates S. mutans planktonic cell Gtf and Gbp expression. This leads to more planktonic cells attaching to the dental biofilm. Increased cell numbers within biofilm results in higher overall LDH activity. This contributes to caries development in smokers.

  3. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis.

  4. Fast and efficient MCR-based synthesis of clickable rhodamine tags for protein profiling.

    PubMed

    Brauch, Sebastian; Henze, Michael; Osswald, Bianca; Naumann, Kai; Wessjohann, Ludger A; van Berkel, Sander S; Westermann, Bernhard

    2012-02-07

    Protein profiling probes are important tools for studying the composition of the proteome and as such have contributed greatly to the understanding of various complex biological processes in higher organisms. For this purpose the application of fluorescently labeled activity or affinity probes is highly desirable. Especially for in vivo detection of low abundant target proteins, otherwise difficult to analyse by standard blotting techniques, fluorescently labeled profiling probes are of high value. Here, a one-pot protocol for the synthesis of activated fluorescent labels (i.e. azide, alkynyl or NHS), based on the Ugi-4-component reaction (Ugi-4CR), is presented. As a result of the peptoidic structure formed, the fluorescent properties of the products are pH insensitive. Moreover, the applicability of these probes, as exemplified by the labeling of model protein BSA, will be discussed.

  5. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis.

    PubMed

    Venteicher, Andrew S; Abreu, Eladio B; Meng, Zhaojing; McCann, Kelly E; Terns, Rebecca M; Veenstra, Timothy D; Terns, Michael P; Artandi, Steven E

    2009-01-30

    Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase, the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for telomerase function. TCAB1 associates with active telomerase enzyme, established telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association, and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.

  6. Reversible Inhibition of Mitochondrial Protein Synthesis during Linezolid-Related Hyperlactatemia▿

    PubMed Central

    Garrabou, Glòria; Soriano, Alejandro; López, Sònia; Guallar, Jordi P.; Giralt, Marta; Villarroya, Francesc; Martínez, Jose A.; Casademont, Jordi; Cardellach, Francesc; Mensa, Josep; Miró, Òscar

    2007-01-01

    The objective of the present study was to determine the mitochondrial toxicity mechanisms of linezolid-related hyperlactatemia. Five patients on a long-term schedule of linezolid treatment were studied during the acute phase of hyperlactatemia and after clinical recovery and lactate normalization following linezolid withdrawal. Mitochondrial studies were performed with peripheral blood mononuclear cells and consisted of measurement of mitochondrial mass, mitochondrial protein synthesis homeostasis (cytochrome c oxidase [COX] activity, COX-II subunit expression, COX-II mRNA abundance, and mitochondrial DNA [mtDNA] content), and overall mitochondrial function (mitochondrial membrane potential and intact-cell oxidative capacity). During linezolid-induced hyperlactatemia, we found extremely reduced protein expression (16% of the remaining content compared to control values [100%], P < 0.001) for the mitochondrially coded, transcribed, and translated COX-II subunit. Accordingly, COX activity was also found to be decreased (51% of the remaining activity, P < 0.05). These reductions were observed despite the numbers of COX-II mitochondrial RNA transcripts being abnormally increased (297%, P = 0.10 [not significant]) and the mitochondrial DNA content remaining stable. These abnormalities persisted even after the correction for mitochondrial mass, which was mildly decreased during the hyperlactatemic phase. Most of the mitochondrial abnormalities returned to control ranges after linezolid withdrawal, lactate normalization, and clinical recovery. Linezolid inhibits mitochondrial protein synthesis, leading to decreased mitochondrial enzymatic activity, which causes linezolid-related hyperlactatemia, which resolves upon discontinuation of linezolid treatment. PMID:17194826

  7. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

    NASA Astrophysics Data System (ADS)

    Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

    2016-05-01

    Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

  8. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials: Model Comparison and Predictions.

    PubMed

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

    2015-07-29

    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 mechanistic model were compared with those of two other models, DVE1994 and NRC-2001, that are frequently used in common international feeding practice. DVE1994 predictions for intestinally digestible rumen undegradable protein (ARUP) for starchy concentrates were higher (27 vs 18 g/kg DM, p < 0.05, SEM = 1.2) than predictions by the NRC-2001, whereas there was no difference in predictions for ARUP from protein concentrates among the three models. DVE2010 and NRC-2001 had highest estimations of intestinally digestible microbial protein for starchy (92 g/kg DM in DVE2010 vs 46 g/kg DM in NRC-2001 and 67 g/kg DM in DVE1994, p < 0.05 SEM = 4) and protein concentrates (69 g/kg DM in NRC-2001 vs 31 g/kg DM in DVE1994 and 49 g/kg DM in DVE2010, p < 0.05 SEM = 4), respectively. Potential protein supplies predicted by tested models from starchy and protein concentrates are widely different, and comparable direct measurements are needed to evaluate the actual ability of different models to predict the potential protein supply to dairy cows from different feedstuffs.

  9. Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle.

    PubMed

    McDaneld, T G; Hannon, K; Moody, D E

    2006-06-01

    Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of beta-adrenergic receptor agonists. Because beta-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of approximately 34% (P<0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area (P<0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation (P<0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area (P<0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.

  10. Codon-specific and general inhibition of protein synthesis by the tRNA-sequestering minigenes.

    PubMed

    Delgado-Olivares, Luis; Zamora-Romo, Efraín; Guarneros, Gabriel; Hernandez-Sanchez, Javier

    2006-07-01

    The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.

  11. Evidence of VTA and LC control of protein synthesis required for the behavioral tagging process.

    PubMed

    Moncada, Diego

    2017-02-01

    Several works have shown that the formation of different long-term memories relies on a behavioral tagging process. In other words, to establish a lasting memory, at least two parallel processes must occur: the setting of a learning tag (triggered during learning) that defines where a memory could be stored, and the synthesis of proteins, that once captured at tagged sites will effectively allow the consolidation process to occur. This work focused in studying which brain structures are responsible of controlling the synthesis of those proteins at the brain areas where memory is being stored. It combines electrical activation of the ventral tegmental area (VTA) and/or the locus coeruleus (LC), with local pharmacological interventions and weak and strong behavioral trainings in the inhibitory avoidance and spatial object recognition tasks in rats. The results presented here strongly support the idea that the VTA is a brain structure responsible for regulating the consolidation of memories acting through the D1/D5 dopaminergic receptors of the hippocampus to control the synthesis of new proteins required for this process. Moreover, they provide evidence that the LC may be a second structure with a similar role, acting independently and complementary to the VTA, through the β-adrenergic receptors of the hippocampus.

  12. Evolution of Protein Synthesis from an RNA World

    PubMed Central

    Noller, Harry F.

    2012-01-01

    SUMMARY Because of the molecular complexity of the ribosome and protein synthesis, it is a challenge to imagine how translation could have evolved from a primitive RNA World. Two specific suggestions are made here to help to address this, involving separate evolution of the peptidyl transferase and decoding functions. First, it is proposed that translation originally arose not to synthesize functional proteins, but to provide simple (perhaps random) peptides that bound to RNA, increasing its available structure space, and therefore its functional capabilities. Second, it is proposed that the decoding site of the ribosome evolved from a mechanism for duplication of RNA. This process involved homodimeric “duplicator RNAs,” resembling the anticodon arms of tRNAs, which directed ligation of trinucleotides in response to an RNA template. PMID:20610545

  13. Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors.

    PubMed

    Salehi, Amin S M; Shakalli Tang, Miriam J; Smith, Mark T; Hunt, Jeremy M; Law, Robert A; Wood, David W; Bundy, Bradley C

    2017-03-21

    Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor β in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

  14. Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells.

    PubMed

    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.

  15. Effect of Anoxia on Energy Charge and Protein Synthesis in Rice Embryo

    PubMed Central

    Mocquot, Bernard; Prat, Christiane; Mouches, Claude; Pradet, Alain

    1981-01-01

    Energy charge, adenine nucleotide levels, and protein synthesis were studied during the transfer of rice seedlings from air to anoxia. Within minutes, the energy charge value dropped from 0.90 in air to 0.50 in the seed and 0.60 in the coleoptile after the transfer to a nitrogen atmosphere, and then increased to a value of 0.80 during the subsequent hours. The sum of nucleotides also dropped to 60% of the value in air in the seeds and to 30% in the coleoptiles. However, during the anaerobic growth of coleoptiles, a considerable increase in the nucleotide pool occurred. The incorporation of amino acids into proteins was measured at different stages in anoxic treatment. In rice embryo, we observed a considerable protein synthesis correlated with a high value of energy charge under anoxia. The analysis of labeled proteins by two-dimensional polyacrylamide gel electrophoresis showed a modified pattern of polypeptides synthesized during anoxic treatment. Some of these proteins were intensively labeled and appeared to be induced by anaerobic treatment. Our data indicate that high metabolic activity occurs in rice embryo under anoxia, which can be correlated with a high energy charge value. These phenomena may be part of the mechanisms which permit the adaptation of rice embryos to anaerobiosis. Images PMID:16661971

  16. Cbl proteins in platelet activation.

    PubMed

    Buitrago, Lorena; Tsygankov, Alexander; Sanjay, Archana; Kunapuli, Satya P

    2013-01-01

    Platelets play a fundamental role in hemostasis. Their functional responses have to be tightly controlled as any disturbance may lead to bleeding disorders or thrombosis. It is thus important to clearly identify and understand the signaling mechanisms involved in platelet function. An important role of c-Cbl and Cbl-b ubiquitin ligases in platelet functional responses and in hematological malignancies has been recently described. Cbl proteins perform negative and positive regulation of several signaling pathways in platelets. In this review, we explore the role of Cbl proteins in platelet functional responses.

  17. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  18. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  19. Further studies on the stimulation of protein synthesis in androgen-dependent tissues by testosterone

    PubMed Central

    Mainwaring, W. I. P.; Wilce, P. A.

    1972-01-01

    1. By using centrifugation through a discontinuous sucrose gradient, four microsomal fractions are obtained from the prostate gland. 2. Administration of androgens to castrated rats stimulates protein synthesis in all fractions, particularly in the heavy rough fraction. 3. Androgens also increase the content of protein, RNA and phospholipid in the heavy rough fraction. 4. Time-course experiments in vivo show that androgens induce a rapid increase in the synthesis of ribosomal precursor RNA preceding the synthesis of new microsomal fraction and the increase in protein synthesis. PMID:4655422

  20. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  1. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.

    PubMed

    Blackburn, Matthew C; Petrova, Ekaterina; Correia, Bruno E; Maerkl, Sebastian J

    2016-04-20

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

  2. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

    PubMed Central

    Failmezger, Jurek; Nitschel, Robert; Sánchez-Kopper, Andrés; Kraml, Michael; Siemann-Herzberg, Martin

    2016-01-01

    Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems. PMID:27992588

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

    PubMed Central

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael C.

    2014-01-01

    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 (OTSs) 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. PMID:24959531

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

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    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. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    SciTech Connect

    Sampson, D.A.; Jansen, G.R.

    1985-04-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

  6. Solution phase parallel synthesis and evaluation of MAPK inhibitory activities of close structural analogues of a Ras pathway modulator.

    PubMed

    Lu, Yingchun; Sakamuri, Sukumar; Chen, Quin-Zene; Keng, Yen-Fang; Khazak, Vladimir; Illgen, Katrin; Schabbert, Silke; Weber, Lutz; Menon, Sanjay R

    2004-08-02

    A solution phase parallel synthesis approach was undertaken to rapidly explore the structure-activity relationship of an inhibitor of the Ras/Raf protein interaction identified from a small molecule compound library. Evaluation of the MAPK pathway signaling inhibitory activity of the synthesized analogues as well as their antiproliferative activity and ability to inhibit soft agar growth were performed.

  7. Effect of Acyclovir on Viral Protein Synthesis in Cells Infected with Herpes Simplex Virus Type 1

    PubMed Central

    Furman, Phillip A.; McGuirt, Paul V.

    1983-01-01

    The effect of the antiviral agent 9-(2-hydroxyethoxymethyl)guanine (acyclovir) on herpes simplex virus type 1 protein synthesis during virus replication was examined. Treatment of infected cells with acyclovir markedly affected the amounts of the four major glycosylated and certain non-glycosylated viral polypeptides synthesized; other viral polypeptides were made in normal amounts. The reduced amount of late protein synthesis was most likely due to the inhibition of progeny viral DNA synthesis by acyclovir. Images PMID:6301368

  8. Long-term leucine induced stimulation of muscle protein synthesis is amino acid dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infusing leucine for 1 h increases skeletal muscle protein synthesis in the neonate, but this is not sustained for 2 h unless the corresponding fall in amino acids is prevented. This study aimed to determine whether a continuous leucine infusion can stimulate protein synthesis for a prolonged period...

  9. Post-prandial changes in protein synthesis in red drum (Sciaenops ocellatus) larvae.

    PubMed

    McCarthy, Ian D; Fuiman, Lee A

    2011-06-01

    Protein synthesis is one of the major energy-consuming processes in all living organisms. Post-prandial changes in protein synthesis have been studied in a range of animal taxa but have been little studied in fish larvae. Using the flooding-dose method, we measured post-prandial changes in whole-body rates of protein synthesis in regularly fed red drum Sciaenops ocellatus (Linnaeus) larvae for 24-28 h following their daily meal. Fractional rates of protein synthesis increased from a baseline (pre-feeding) rate of 16% day(-1) to a post-prandial peak of 48% day(-1) ca. 8 h after feeding before declining to 12% day(-1) after 24-28 h. The overall mean daily rate of protein synthesis was calculated as 27% day(-1). Although suggested as energetically impossible in larval poikilotherms, our results show that rates in excess of 30% day(-1) can be attained by larval fishes for a few hours but are not sustained. The average daily energetic cost of protein synthesis was estimated as 34% of daily total oxygen consumption, ranging from 19% immediately before feeding to 61% during the post-prandial peak in protein synthesis. This suggests that during the post-prandial peak, protein synthesis will require a large proportion of the hourly energy production, which, given the limited metabolic scope in fish larvae, may limit the energy that could otherwise be allocated to other energy-costly functions, such as foraging and escape responses.

  10. On the Role of Hippocampal Protein Synthesis in the Consolidation and Reconsolidation of Object Recognition Memory

    ERIC Educational Resources Information Center

    Rossato, Janine I.; Bevilaqua, Lia R. M.; Myskiw, Jociane C.; Medina, Jorge H.; Izquierdo, Ivan; Cammarota, Martin

    2007-01-01

    Upon retrieval, consolidated memories are again rendered vulnerable to the action of metabolic blockers, notably protein synthesis inhibitors. This has led to the hypothesis that memories are reconsolidated at the time of retrieval, and that this depends on protein synthesis. Ample evidence indicates that the hippocampus plays a key role both in…

  11. Recalling an Aversive Experience by Day-Old Chicks Is Not Dependent on Somatic Protein Synthesis

    ERIC Educational Resources Information Center

    Mileusnic, Radmila; Lancashire, Christine L.; Rose, Steven P. R.

    2005-01-01

    Long-term memory is dependent on protein synthesis and inhibiting such synthesis following training results in amnesia for the task. Proteins synthesized during training must be transported to the synapse and disrupting microtubules with Colchicines, and hence, blocking transport, results in transient amnesia. Reactivating memory for a previously…

  12. Translate to divide: сontrol of the cell cycle by protein synthesis

    PubMed Central

    Polymenis, Michael; Aramayo, Rodolfo

    2015-01-01

    Protein synthesis underpins much of cell growth and, consequently, cell multiplication. Understanding how proliferating cells commit and progress into the cell cycle requires knowing not only which proteins need to be synthesized, but also what determines their rate of synthesis during cell division. PMID:28357283

  13. Effects of aging and life-prolonging diet on thyroid regulation of protein synthesis.

    PubMed

    Gromakova, I A; Konovalenko, O A

    2004-03-01

    The effect of thyroxin on the intensity of protein synthesis in rats of different age was studied during natural aging and in rats maintained on a low-caloric diet inhibiting aging. The intensity of protein synthesis decreased and the reaction to hormonal stimulus was absent in animals fed life-prolonging diet.

  14. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

  15. Protein-directed synthesis of highly monodispersed, spherical gold nanoparticles and their applications in multidimensional sensing

    PubMed Central

    Leng, Yumin; Fu, Ling; Ye, Liqun; Li, Bo; Xu, Xiumei; Xing, Xiaojing; He, Junbao; Song, Yuling; Leng, Chaoliang; Guo, Yongming; Ji, Xiaoxu; Lu, Zhiwen

    2016-01-01

    An in-situ reduction method has been reported to prepare gold nanoparticles (GNPs) of 40–110 nm by using the green reducing agents of proteins, which are activated by H2O2 and the superoxide anion (). The protein of collagen turns HAuCl4 to the aqueous Au(I) ainions, which are further reduced by other proteins to be highly monodispersed and spherical GNPs of different sizes. The GNPs reduced by different proteins are found to be with the exposed {100} facets, the distinctive UV-vis absorption spectra and various colors (See Fig. 1). By means of extracting the color responses, such as red, green and blue (RGB) alterations, an in-situ reduction method-based multidimensional sensing platform is fabricated in the process of GNPs synthesis. Without further modification of GNPs, nine common proteins are found to be well detected and discriminated at different concentrations. Moreover, this sensing platform also demonstrates great potentials in qualitative and semiquantitative analysis on the individuals of these proteins with high sensitivity. Furthermore, the validation of this multidimensional sensing platform has been carried out by analysis on the spiked proteins in human urine and the target proteins in complex matrix (e.g. lysozyme in human tear). PMID:27353703

  16. Synthesis of (-)-arctigenin derivatives and their anticancer activity.

    PubMed

    Gui-Rong, Chen; Li-Ping, Cai; De-Qiang, Dou; Ting-Guo, Kang; Hong-Fu, Li; Fu-Rui, Li; Ning, Jiang

    2012-01-01

    The natural dibenzylbutyrolactone type lignanolide (-)-arctigenin, which was prepared from fructus arctii, showed obvious anticancer activity. The synthesis of four new (-)-arctigenin derivatives and their anticancer bioactivities were examined. The structures of the four new synthetic derivatives were elucidated.

  17. Synthesis and in vitro antiproliferative activities of quinoline derivatives.

    PubMed

    Broch, Sidonie; Aboab, Bettina; Anizon, Fabrice; Moreau, Pascale

    2010-04-01

    The synthesis of new di- and trimeric quinoline derivatives is described as well as their in vitro antiproliferative activities toward a human fibroblast primary culture and two human solid cancer cell lines (MCF-7 and PA 1).

  18. N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

    PubMed

    Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

    2010-07-01

    Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

  19. Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis.

    PubMed

    Nagano, Hikaru; Sugihara, Shouhei; Takagi, Hisanori; Ogasawara, Tomio; Endo, Yaeta; Takai, Kazuyuki

    2008-01-01

    The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution of protein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryotic elongation factors (eEFs) 1A, 1B, and 2. The eEF1A and eEF2 fractions supported polyphenylalanine synthesis.

  20. In vivo downregulation of protein synthesis in the snail Helix apersa during estivation.

    PubMed

    Pakay, Julian L; Withers, Philip C; Hobbs, Andrew A; Guppy, Michael

    2002-07-01

    Protein synthesis is downregulated during metabolic depression in a number of systems where the metabolic depression is effected by obvious extrinsic cues. The metabolic depression of the estivating land snail Helix apersa occurs in the absence of any obvious physiological stress and has an intrinsic component independent of temperature, pH, O(2) status, or osmolality. We show that this metabolic depression is accompanied by a downregulation of protein synthesis in vivo. The rate of protein synthesis decreases in two major tissues during estivation: to 23% and 53% of the awake rate in hepatopancreas and foot muscle, respectively. We show from calculations of the theoretical contribution of protein synthesis to total O(2) consumption that the depression of protein synthesis must be a significant, obligate, in vivo component of metabolic depression in H. aspersa.

  1. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

    PubMed

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md; Gregory, Brian; Samsel, Leigh; Zengel, Janice M; Lindahl, Lasse

    2013-12-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

  2. Pokeweed Antiviral Protein, a Ribosome Inactivating Protein: Activity, Inhibition and Prospects

    PubMed Central

    Domashevskiy, Artem V.; Goss, Dixie J.

    2015-01-01

    Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant’s defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction—a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics. PMID:25635465

  3. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

    PubMed Central

    You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

    2015-01-01

    ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

  4. Synthesis of fluorescent dipeptidomimetics and their ribosomal incorporation into green fluorescent protein.

    PubMed

    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.

  5. ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.

    PubMed

    Alissandratos, Apostolos; Caron, Karine; Loan, Thomas D; Hennessy, James E; Easton, Christopher J

    2016-12-16

    E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

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

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

    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.

  7. Pokeweed antiviral protein increases HIV-1 particle infectivity by activating the cellular mitogen activated protein kinase pathway.

    PubMed

    Mansouri, Sheila; Kutky, Meherzad; Hudak, Katalin A

    2012-01-01

    Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase that exhibits antiviral activity against several viruses. The enzyme removes purine bases from the messenger RNAs of the retroviruses Human immunodeficiency virus-1 and Human T-cell leukemia virus-1. This depurination reduces viral protein synthesis by stalling elongating ribosomes at nucleotides with a missing base. Here, we transiently expressed PAP in cells with a proviral clone of HIV-1 to examine the effect of the protein on virus production and quality. PAP reduced virus production by approximately 450-fold, as measured by p24 ELISA of media containing virions, which correlated with a substantial decline in virus protein synthesis in cells. However, particles released from PAP-expressing cells were approximately 7-fold more infectious, as determined by single-cycle infection of 1G5 cells and productive infection of MT2 cells. This increase in infectivity was not likely due to changes in the processing of HIV-1 polyproteins, RNA packaging efficiency or maturation of virus. Rather, expression of PAP activated the ERK1/2 MAPK pathway to a limited extent, resulting in increased phosphorylation of viral p17 matrix protein. The increase in infectivity of HIV-1 particles produced from PAP-expressing cells was compensated by the reduction in virus number; that is, virus production decreased upon de novo infection of cells over time. However, our findings emphasize the importance of investigating the influence of heterologous protein expression upon host cells when assessing their potential for antiviral applications.

  8. Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis.

    PubMed

    Schoborg, Jennifer A; Hodgman, C Eric; Anderson, Mark J; Jewett, Michael C

    2014-05-01

    Cell-free protein synthesis (CFPS) platforms are now considered a powerful tool for synthesizing a variety of proteins at scales from pL to 100 L with accelerated process development pipelines. We previously reported the advancement of a novel yeast-based CFPS platform. Here, we studied factors that cause termination of yeast CFPS batch reactions. Specifically, we characterized the substrate and byproduct concentrations in batch, fed-batch, and semi-continuous reaction formats through high-performance liquid chromatography (HPLC) and chemical assays. We discovered that creatine phosphate, the secondary energy substrate, and nucleoside triphosphates were rapidly degraded during batch CFPS, causing a significant drop in the reaction's energy charge (E.C.) and eventual termination of protein synthesis. As a consequence of consuming creatine phosphate, inorganic phosphate accumulated as a toxic byproduct. Additionally, we measured amino acid concentrations and found that aspartic acid was rapidly consumed. By adopting a semi-continuous reaction format, where passive diffusion enables substrate replenishment and byproduct removal, we achieved over a 70% increase in active superfolder green fluorescent protein (sfGFP) as compared with the batch system. This study identifies targets for the future improvement of the batch yeast CFPS reaction. Moreover, it outlines a detailed, generalized method to characterize and improve other CFPS platforms.

  9. Altered Protein Synthesis is a Trigger for Long-term Memory Formation

    PubMed Central

    Klann, Eric; Sweatt, J. David

    2008-01-01

    Summary There is ongoing debate concerning whether new protein synthesis is necessary for, or even contributes to, memory formation and storage. This review summarizes a contemporary model proposing a role for altered protein synthesis in memory formation and its subsequent stabilization. One defining aspect of the model is that altered protein synthesis serves as a trigger for memory consolidation. Thus, we propose that specific alterations in the pattern of neuronal protein translation serve as an initial event in long-term memory formation. These specific alterations in protein read-out result in the formation of a protein complex that then serves as a nidus for subsequent perpetuating reinforcement by a positive feedback mechanism. The model proposes this scenario as a minimal but requisite component for long-term memory formation. Our description specifies three aspects of prevailing scenarios for the role of altered protein synthesis in memory that we feel will help clarify what, precisely, is typically proposed as the role for protein translation in memory formation. First, that a relatively short initial time window exists wherein specific alterations in the pattern of proteins translated (not overall protein synthesis) is involved in initializing the engram. Second, that a self-perpetuating positive feedback mechanism maintains the altered pattern of protein expression (synthesis or recruitment) locally. Third, that other than the formation and subsequent perpetuation of the unique initializing proteins, ongoing constitutive protein synthesis is all that is minimally necessary for formation and maintenance of the engram. We feel that a clear delineation of these three principles will assist in interpreting the available experimental data, and propose that the available data are consistent with a role for protein synthesis in memory. PMID:17919940

  10. Functional effects of a pathogenic mutation in Cereblon (CRBN) on the regulation of protein synthesis via the AMPK-mTOR cascade.

    PubMed

    Lee, Kwang Min; Yang, Seung-Joo; Choi, Ja-Hyun; Park, Chul-Seung

    2014-08-22

    Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate-activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK-mTOR cascade in Crbn-knock-out mice, ectopic expression of the wild-type CRBN increased protein synthesis by inhibiting endogenous AMPK. Unlike the wild-type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR-dependent repression of protein synthesis in Crbn-deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.

  11. Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

    PubMed

    Joshi, Manali; Kulkarni, Abhijeet; Pal, Jayanta K

    2013-11-01

    Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

  12. Design, synthesis, and evaluation of an alpha-helix mimetic library targeting protein-protein interactions.

    PubMed

    Shaginian, Alex; Whitby, Landon R; Hong, Sukwon; Hwang, Inkyu; Farooqi, Bilal; Searcey, Mark; Chen, Jiandong; Vogt, Peter K; Boger, Dale L

    2009-04-22

    The design and solution-phase synthesis of an alpha-helix mimetic library as an integral component of a small-molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate alpha-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 x 20 x 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i + 4, i + 7 amino acid side chains of an alpha-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long-term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead alpha-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an alpha-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

  13. Alphavirus RNA synthesis and non-structural protein functions

    PubMed Central

    Rupp, Jonathan C.; Sokoloski, Kevin J.; Gebhart, Natasha N.

    2015-01-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field. PMID:26219641

  14. Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains.

    PubMed

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-06-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.

  15. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    PubMed Central

    Poortmans, J.R.; Carpentier, A.; Pereira-Lancha, L.O.; Lancha, A.

    2012-01-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, 2H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg−1·day−1 compared to 0.8 g·kg−1·day−1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h. PMID:22666780

  16. Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

    PubMed Central

    Tetlow, Ian J.; Wait, Robin; Lu, Zhenxiao; Akkasaeng, Rut; Bowsher, Caroline G.; Esposito, Sergio; Kosar-Hashemi, Behjat; Morell, Matthew K.; Emes, Michael J.

    2004-01-01

    Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with γ-32P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with γ-32P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein–protein

  17. Kinetics and mechanism of the synthesis of a novel protein-based plastic using subcritical water.

    PubMed

    Abdelmoez, Wael; Yoshida, Hiroyuki

    2008-01-01

    We investigated the intermolecular mechanism and kinetics of the synthesis of a novel biodegradable protein-based plastic from bovine serum albumin under subcritical water conditions using batch reactors. The reaction mechanism could be viewed as a chain reaction stabilized by the formation of intermolecular disulfide bonds. The kinetic analysis was based on non-steady-state kinetics using a theoretical model developed in one of our previous works. The activation energy and pre-exponential factor were found to be 7.2 kJ/mol and 0.9 s-1, respectively. These low values signify that the reaction is relatively temperature-insensitive with some diffusion limitation.

  18. The endocannabinoid 2-arachidonoylglycerol dysregulates the synthesis of proteins by the human syncytiotrophoblast.

    PubMed

    Costa, M A; Fonseca, B M; Mendes, A; Braga, J; Teixeira, N A; Correia-da-Silva, G

    2016-03-01

    In recent years, endocannabinoids emerged as new players in various reproductive events. Recently, we demonstrated the involvement of 2-arachidonoylglycerol (2-AG) in human cytotrophoblast apoptosis and syncytialization. However, 2-AG impact in hormone production by the syncytiotrophoblast (hST) was never studied. In this work, we demonstrate that 2-AG activates cannabinoid (CB) receptors, exerting an inhibitory action on cyclic AMP/protein kinase A (cAMP/PKA) and mitogen-activated protein kinase (MAPK) p38 pathways, and enhancing ERK 1/2 phosphorylation. Furthermore, 2-AG affects the synthesis of human chorionic gonadotropin (hCG), leptin, aromatase, 3-β-hydroxysteroid dehydrogenase (3-β-HSD), and placental protein 13 (PP13). These 2-AG effects are mediated by the activation of CB receptors, in a mechanism that may involve p38, ERK 1/2 and cAMP/PKA pathways, which participate in the regulation of placental proteins expression. To our knowledge, this is the first study that associates the endocannabinoid signalling and endocrine placental function, shedding light on a role for 2-AG in the complex network of molecules that orchestrate the production of placental proteins essential for the gestational success.

  19. A unified view of the initiation of protein synthesis.

    PubMed

    Nakamoto, Tokumasa

    2006-03-17

    The mechanism of the initiation of protein synthesis is discussed in terms of two different hypotheses in which each emphasized a different possible element of the process: the Shine-Dalgarno (SD) hypothesis ascribed an essential role to recognition of the SD segment by the ribosomal RNA; it is supported by a variety of experiments but conflicting evidence negates its obligatory nature. In contrast, our hypothesis highlighted the role of the structure of the mRNA and proposes that the initiation codon is selected by virtue of its unique accessibility. The rationale for the importance of accessibility in the selection of the initiation site is discussed. An analysis and a recapitulation of the initiation process and ribosomal specificity are presented. The apparent conflicts with the SD hypothesis are resolved in a unified mechanism where accessibility is the dominant factor.

  20. Competition For Resources in a Model for Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Cook, Larry; Zia, Royce

    2009-03-01

    The Totally Asymmetric Simple Exclusion Process (TASEP) is often used to explore translation during protein synthesis. The particles represent ribosomes that move along mRNA, which is represented by the one-dimensional lattice. Unlike ordinary TASEP where the supply of particles is unlimited, there is a finite number of ribosome in a cell. In addition, there are many genes which compete for this pool of ribosomes. Thus, we are motivated to consider the effects of multiple TASEPs (of varying lengths) coupled to a single, finite reservoir of particles. In particular, the total occupation numbers, the density profiles and the particle currents of individual TASEPs are studied, as the overall reservoir of particles is varied. Both Monte Carlo simulation results and analytic considerations will be presented.

  1. Design and synthesis of a protein. beta. -turn mimetic

    SciTech Connect

    Olson, G.L.; Voss, M.E.; Hill, D.E.; Kahn, M.; Madison, V.S.; Cook, C.M. )

    1990-01-03

    A nine-membered-ring lactam system (1) has been chosen as a framework for the development of non-peptide molecules to mimic structural features of peptide and protein {beta}-turns. The synthesis of model di- and tetrapeptide mimetics starting from 1,5-cyclooctadiene derivatives is reported. In the model dipeptide mimetic (9), the amide linkages is trans (NMR, X-ray) and functional groups at positions adjacent to the lactam amide bond correspond closely to the side-chain positions of residues i + 1 and i + 2 of classical type II{prime} {beta}-turns. In the model tetrapeptide mimetic (30), all four side chains of low-energy trans amide conformers of the mimetic are well matched to their peptide counterparts.

  2. SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

    PubMed Central

    Raff, Rudolf A.; Greenhouse, Gerald; Gross, Kenneth W.; Gross, Paul R.

    1971-01-01

    Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified. PMID:5165266

  3. Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis

    SciTech Connect

    Ortiz, W. )

    1990-05-01

    Growing cultures of photoheterotrophic Euglena gracilis experience an increase in chlorophyll accumulation during the initial phase of the temperature-induced bleaching response suggesting an increase in the synthesis of plastid components at the bleaching temperature of 33{degree}C. A primary goal of this work was to establish whether an increase in the synthesis of plastid proteins accompanies the observed increase in chlorophyll accumulation. In vivo pulse-labeling experiments with ({sup 35}S)sodium sulfate were carried out with cells grown at room temperature or at 33{degree}C. The synthesis of a number of plastid polypeptides of nucleocytoplasmic origin, including some presumably novel polypeptides, increased in cultures treated for 15 hours at 33{degree}C. In contrast, while synthesis of thylakoid proteins by the plastid protein synthesis machinery decreased modestly, synthesis of the large subunit of the enzyme ribulosebisphosphate carboxylase was strongly affected at the elevated temperature. Synthesis of novel plastid-encoded polypeptides was not induced at the bleaching temperature. It is concluded that protein synthesis in plastids declines during the initial phase of the temperature response in Euglena despite an overall increase in cellular protein synthesis and an increase in chlorophyll accumulation per cell.

  4. In vivo effects of the Epstein-Barr virus small RNA EBER-1 on protein synthesis and cell growth regulation.

    PubMed

    Laing, Kenneth G; Elia, Androulla; Jeffrey, Ian; Matys, Volker; Tilleray, Vivienne J; Souberbielle, Bernard; Clemens, Michael J

    2002-06-05

    Recent studies have suggested a role for the Epstein-Barr virus-encoded RNA EBER-1 in malignant transformation. EBER-1 inhibits the activity of the protein kinase PKR, an inhibitor of protein synthesis with tumour suppressor properties. In human 293 cells and murine embryonic fibroblasts, transient expression of EBER-1 promoted total protein synthesis and enhanced the expression of cotransfected reporter genes. However reporter gene expression was stimulated equally well in cells from control and PKR knockout mice. NIH 3T3 cells stably expressing EBER-1 exhibited a greatly increased frequency of colony formation in soft agar, and protein synthesis in these cells was relatively resistant to inhibition by the calcium ionophore A23187. Nevertheless clones containing a high concentration of EBER-1 were not invariably tumourigenic. We conclude that EBER-1 can enhance protein synthesis by a PKR-independent mechanism and that, although this RNA may contribute to the oncogenic potential of Epstein-Barr virus, its expression is not always sufficient for malignant transformation.

  5. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    PubMed

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  6. Stimulation of tentoxin synthesis by aged-culture filtrates and continued synthesis in the presence of protein inhibitors.

    PubMed

    Sheu, J T; Talburt, D E

    1986-02-01

    Tentoxin, a cyclic tetrapeptide produced by Alternaria alternata (Fries) Keissler, induces chlorosis in certain seedling plants. It can be extracted from culture filtrates of the fungus. Tentoxin production is stimulated and increased by using a mixture of aged culture filtrates and modified Richards solution. Aged culture filtrates can be obtained from 3-week-old or older cultures of A. alternata in modified Richards solution or Pratts solution. A mixture of aged culture filtrate and fresh medium in the ratio 2:3 gives the maximal enhancement of tentoxin production. This growth system provided us with a model for studying the effects of protein synthesis inhibitors on tentoxin production. Two antibiotics which inhibit protein synthesis at the ribosomal level were tested on growth, protein synthesis, and tentoxin production in A. alternata cultures. Cycloheximide at concentrations of 500 mug/ml or emetine at concentrations of 250 mug/ml did not inhibit tentoxin synthesis, although they stopped mycelial growth and protein synthesis of the fungus at the logarithmic growth stage in the enhancement medium. These results led us to conclude that tentoxin, like certain other bioactive cyclic peptides, is synthesized by a nonribosomal peptide synthesis mechanism.

  7. The effect of synthetic homopolymer poly I:C on the synthesis of nucleic acids, protein and interferon in spleen cells normally and with radiation

    NASA Technical Reports Server (NTRS)

    Antropova, Y. N.; Konstantinova, I. V.; Fuks, B. B.; Talosh, M. Y.; Veysfeyler, Y. K.

    1974-01-01

    A comparative study is reported of the effect of the synthetic homopolymer poly I:C and Newcastle Disease virus on the synthesis of RNA, DNA, total protein and interferon in the spleen of nonradiated and radiated mice. In radiated animals, poly I:C and NDV had no stimulating effect on the synthesis of RNA; administration of both inducers to radiated mice did not significantly affect the content of lymphoid cellular elements in the spleen. However, while reduction of RNA synthesis, caused by radiation, also increases slightly under the effect of poly I:C and the virus, the synthesis of interferon in spleen cells and in the entire body is activated.

  8. Intracellular synthesis of silver nanoparticle by actinobacteria and its antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Otari, S. V.; Patil, R. M.; Ghosh, S. J.; Thorat, N. D.; Pawar, S. H.

    2015-02-01

    Intracellular synthesis of silver nanoparticles (AgNPs) using Rhodococcus spp. is demonstrated. The synthesized nanoparticles were characterized by UV-Vis spectroscopy, X-ray diffraction, energy dispersive spectroscopy, Fourier trans-form infrared spectroscopy, and transmission electron microscopy. Transmission electron microscopy study of microorganisms' revealed synthesis of nanoparticle was occurring inside the cell, in the cytoplasm. AgNPs ranged from 5 to 50 nm. Formed nanoparticles were stable in the colloidal solution due to presence of proteins on the surface. AgNPs showed excellent bactericidal and bacteriostatic activity against pathogenic microorganisms.

  9. Intermittent bolus feeding has a greater stimulatory effect on protein synthesis in skeletal muscle than continuous feeding in neonatal pigs.

    PubMed

    Gazzaneo, María C; Suryawan, Agus; Orellana, Renán A; Torrazza, Roberto Murgas; El-Kadi, Samer W; Wilson, Fiona A; Kimball, Scot R; Srivastava, Neeraj; Nguyen, Hanh V; Fiorotto, Marta L; Davis, Teresa A

    2011-12-01

    Orogastric tube feeding, using either continuous or intermittent bolus delivery, is common in infants for whom normal feeding is contraindicated. To compare the impact of different feeding strategies on muscle protein synthesis, after withholding food overnight, neonatal pigs received a complete formula orally as a bolus feed every 4 h or were continuously fed. Protein synthesis rate and translational mechanisms in skeletal muscle were examined after 0, 24, and 25.5 h. Plasma amino acid and insulin concentrations increased minimally and remained constant in continuously fed compared to feed-deprived pigs; however, the pulsatile meal feeding pattern was mimicked in bolus-fed pigs. Muscle protein synthesis was stimulated by feeding and the greatest response occurred after a bolus meal. Bolus but not continuous feeds increased polysome aggregation, the phosphorylation of protein kinase B, tuberous sclerosis complex 2, proline-rich Akt substrate of 40 kDa, eukaryotic initiation factor (eIF) 4E binding protein (4EBP1), and rp S6 kinase and enhanced dissociation of the 4EBP1 ·eIF4E complex and formation of the eIF4E ·eIF4G complex compared to feed deprivation (P < 0.05). Activation of insulin receptor substrate-1, regulatory associated protein of mammalian target of rapamycin, AMP-activated protein kinase, eukaryotic elongation factor 2, and eIF2α phosphorylation were unaffected by either feeding modality. These results suggest that in neonates, intermittent bolus feeding enhances muscle protein synthesis to a greater extent than continuous feeding by eliciting a pulsatile pattern of amino acid- and insulin-induced translation initiation.

  10. Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

    PubMed

    Xie, Xiaolei; Le, Li; Fan, Yanxin; Lv, Lin; Zhang, Junjie

    2012-07-01

    Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

  11. Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein.

    PubMed Central

    Imani, F; Jacobs, B L

    1988-01-01

    In this report we demonstrate that reovirus serotype 1-infected cells contain an inhibitor of the interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase. We provide evidence that suggests that the virus-encoded sigma 3 protein is likely responsible for this kinase inhibitory activity. We could not detect activation of the dsRNA-dependent protein kinase in extracts prepared from either interferon-treated or untreated reovirus serotype 1-infected mouse L cells under conditions that led to activation of the kinase in extracts prepared from either interferon-treated or untreated, uninfected cells. Extracts from reovirus-infected cells blocked activation of kinase in extracts from interferon-treated cells when the two were mixed prior to assay. The kinase inhibitory activity in extracts of reovirus-infected cells could be overcome by adding approximately 100-fold excess of dsRNA over the amount required to activate kinase in extracts of uninfected cells. Kinase inhibitory activity in extracts of interferon-treated, virus-infected cells could be overcome with somewhat less dsRNA (approximately 10-fold excess). Most of the inhibitory activity in the extracts could be removed by adsorption with immobilized anti-reovirus sigma 3 serum or immobilized dsRNA, suggesting that the dsRNA-binding sigma 3 protein is necessary for kinase inhibitory activity. Purified sigma 3 protein, when added to reaction mixtures containing partially purified kinase, inhibited enzyme activation. Control of activation of this kinase, which can modify eukaryotic protein synthesis initiation factor 2, may be relevant to the sensitivity of reovirus replication to treatment of cells with interferon and to the shutoff of host protein synthesis in reovirus-infected cells. Images PMID:2460857

  12. The role of protein synthesis in memory consolidation: Progress amid decades of debate

    PubMed Central

    Hernandez, Pepe J.; Abel, Ted

    2009-01-01

    A major component of consolidation theory holds that protein synthesis is required to produce the synaptic modification needed for long-term memory storage. Protein synthesis inhibitors have played a pivotal role in the development of this theory. However, these commonly used drugs have unintended effects that have prompted some to reevaluate the role of protein synthesis in memory consolidation. Here we review the role of protein synthesis in memory formation as proposed by consolidation theory calling special attention to the controversy involving the non-specific effects of a group of protein synthesis inhibitors commonly used to study memory formation in vivo. We argue that molecular and genetic approaches that were subsequently applied to the problem of memory formation confirm the results of less selective pharmacological studies. Thus, to a certain extent, the debate over the role of protein synthesis in memory based on interpretational difficulties inherent to the use of protein synthesis inhibitors may be somewhat moot. We conclude by presenting avenues of research we believe will best provide answers to both long-standing and more recent questions facing field of learning and memory. PMID:18053752

  13. Leucine alleviates dexamethasone-induced suppression of muscle protein synthesis via synergy involvement of mTOR and AMPK pathways

    PubMed Central

    Wang, Xiao J.; Yang, Xin; Wang, Ru X.; Jiao, Hong C.; Zhao, Jing P.; Song, Zhi G.; Lin, Hai

    2016-01-01

    Glucocorticoids (GCs) are negative muscle protein regulators that contribute to the whole-body catabolic state during stress. Mammalian target of rapamycin (mTOR)-signalling pathway, which acts as a central regulator of protein metabolism, can be activated by branched-chain amino acids (BCAA). In the present study, the effect of leucine on the suppression of protein synthesis induced by GCs and the pathway involved were investigated. In vitro experiments were conducted using cultured C2C12 myoblasts to study the effect of GCs on protein synthesis, and the involvement of mTOR pathway was investigated as well. After exposure to dexamethasone (DEX, 100 μmol/l) for 24 h, protein synthesis in muscle cells was significantly suppressed (P<0.05), the phosphorylations of mTOR, ribosomal protein S6 protein kinase 1 (p70s6k1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) were significantly reduced (P<0.05). Leucine supplementation (5 mmol/l, 10 mmol/l and 15 mmol/l) for 1 h alleviated the suppression of protein synthesis induced by DEX (P<0.05) and was accompanied with the increased phosphorylation of mTOR and decreased phosphorylation of AMPK (P<0.05). Branched-chain amino transferase 2 (BCAT2) mRNA level was not influenced by DEX (P>0.05) but was increased by leucine supplementation at a dose of 5 mmol/l (P<0.05). PMID:27129299

  14. Design, synthesis, and characterization of protein-based nanostructures

    NASA Astrophysics Data System (ADS)

    Modica, Justin Alan

    This thesis outlines a modular method for the bottom-up fabrication of molecular architectures and functional molecular assemblies too large for common organic synthesis and too small for current lithographic techniques. The construction of these assemblies relies on the reactions of bifunctional recombinant proteins with small molecule linkers that exploit the rapid kinetics and superior selectivity of enzymes. The selectivity of the bond forming reactions yield precisely-defined covalent assemblies with dimensions on the order of 10 - 100 nm. Preequilibration of reactants ensure rapid kinetics at nano- to low micromolar concentrations even as the molecules approach MDa molecular weights. Consequently, this 'dock-and-lock' strategy offers a superior alternative to standard bioconjugation chemistries available to fabricate biomolecular assemblies. Using this strategy, we are able to prepare and isolate discrete, monodisperse molecules that have linear, cyclic and star geometries. Our ability to isolate these discreet molecular objects offers a distinct advantage over non-covalent biomolecular self-assembly systems as the sizes, and therefore structures, of our assemblies are not concentration dependant. Furthermore, the modularity of our approach allows the incorporation of functional protein domains into architectures that retain their native functionality even in the context of a very complex molecular environment. We show that oligomers of the functional domains and their contraction upon treatment with a calcium stimulus is linearly proportional to the degree of oligomerization thus providing a means toward rational functional nanomaterials design.

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

    PubMed

    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

  16. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  17. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  18. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.

  19. Differential regulation of protein synthesis and mTOR signaling in skeletal muscle and visceral tissues of neonatal pigs after a meal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis (PS) increases after a meal in neonates, but the time course of the changes in PS in different tissues after a meal is unknown. We aimed to evaluate the changes in tissue PS, mammalian target of rapamycin complex 1 (mTORC1) activation, and proportion of ribosomal protein (rp) mRNAs...

  20. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  1. Threonine Affects Intestinal Function, Protein Synthesis and Gene Expression of TOR in Jian Carp (Cyprinus carpio var. Jian)

    PubMed Central

    Feng, Lin; Peng, Yan; Wu, Pei; Hu, Kai; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Li, Shu-Hong; Zhou, Xiao-Qiu

    2013-01-01

    This study aimed to investigate the effects of threonine (Thr) on the digestive and absorptive ability, proliferation and differentiation of enterocytes, and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian). First, seven isonitrogenous diets containing graded levels of Thr (7.4–25.2 g/kg diet) were fed to the fishes for 60 days. Second, enterocyte proliferation and differentiation were assayed by culturing enterocytes with graded levels of Thr (0–275 mg/l) in vitro. Finally, enterocytes were cultured with 0 and 205 mg/l Thr to determine protein synthesis. The percent weight gain (PWG), specific growth rate, feed intake, feed efficiency, protein retention value, activities of trypsin, lipase and amylase, weights and protein contents of hepatopancreas and intestine, folds heights, activities of alkaline phosphatase (AKP), γ- glutamyl transpeptidase and Na+/K+-ATPase in all intestinal segments, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities in hepatopancreas, and 4E-BP2 gene expression in muscle, hepatopancreas and intestinal segments were significantly enhanced by Thr (p<0.05). However, the plasma ammonia concentration and TOR gene expression decreased (p<0.05). In vitro, Thr supplement significantly increased cell numbers, protein content, the activities of GOT, GPT, AKP and Na+/K+-ATPase, and protein synthesis rate of enterocytes, and decreased LDH activity and ammonia content in cell medium (p<0.05). In conclusion, Thr improved growth, digestive and absorptive capacity, enterocyte proliferation and differentiation, and protein synthesis and regulated TOR and 4E-BP2 gene expression in juvenile Jian carp. The dietary Thr requirement of juvenile Jian carp was 16.25 g/kg diet (51.3 g/kg protein) based on quadratic regression analysis of PWG. PMID:23922879

  2. Effect of weight loss on the rate of muscle protein synthesis during fasted and fed conditions in obese older adults.

    PubMed

    Villareal, Dennis T; Smith, Gordon I; Shah, Krupa; Mittendorfer, Bettina

    2012-09-01

    Although weight loss ameliorates many of the metabolic abnormalities associated with obesity, there has been reluctance to prescribe weight loss in obese, older individuals because of the fear that it will cause debilitating loss of muscle mass and impair physical function. To gain insight into the mechanisms responsible for the weight loss-induced changes in muscle mass, we measured the rate of muscle protein synthesis (by using stable isotope labeled tracer methodology) during basal, postabsorptive conditions and during mixed meal ingestion in eight obese, older adults: (i) before weight loss therapy, (ii) ~3 months after starting the weight loss intervention (i.e., during the active weight loss phase), when subjects had lost ~7% of their initial body weight, and (iii) after they had lost ~10% of their body weight and maintained this new body weight for ~6 months (~12 months after starting the weight loss intervention). The basal muscle protein fractional synthesis rate (FSR) was not affected by weight loss. Mixed meal ingestion stimulated the rate of muscle protein synthesis, and the anabolic response (i.e., increase in the protein synthesis rate above basal values) was greater (P < 0.05) during negative energy balance and active weight loss at 3 months (0.033 ± 0.012%·per hour, mean ± s.e.m.) than during weight maintenance before and at 12 months of weight loss therapy (0.003 ± 0.003 and 0.008 ± 0.012%·per hour, respectively). We conclude that during dietary calorie restriction and weight loss in older adults, the rate of muscle protein synthesis is not impaired. Thus, the loss of muscle mass must be mediated predominately by adverse effects of dietary calorie restriction on muscle proteolysis.

  3. Synthesis of benzopolycyclic cage amines: NMDA receptor antagonist, trypanocidal and antiviral activities

    PubMed Central

    Torres, Eva; Duque, María D.; López-Querol, Marta; Taylor, Martin C.; Naesens, Lieve; Ma, Chunlong; Pinto, Lawrence H.; Sureda, Francesc X.; Kelly, John M.; Vázquez, Santiago

    2012-01-01

    The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano-5H-benzocyclononen-7-amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypanocidal activities. Two compounds are endowed with micromolar anti vesicular stomatitis virus activity, while only one compound shows micromolar anti-influenza activity. The anti-influenza activity of this compound does not seem to be mediated by blocking of the M2 protein. PMID:22178660

  4. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

  5. Altered response of protein synthesis to nutritional state and endurance training in old rats.

    PubMed

    Mosoni, L; Valluy, M C; Serrurier, B; Prugnaud, J; Obled, C; Guezennec, C Y; Mirand, P P

    1995-02-01

    This study was undertaken to determine whether the loss of muscle protein mass during aging could be explained by a reduced sensitivity of muscle protein synthesis to feeding and exercise. Male Wistar rats aged 12 and 24 mo were exercised by treadmill running for 4 mo. Protein synthesis was measured by the flooding dose method in tibialis anterior, soleus, and liver of conscious rested, trained rats and age-matched controls in the postprandial or in the postabsorptive state. No marked change with age could be detected in basal muscle protein synthesis. In contrast, protein synthesis was stimulated in adult but not in old rats by feeding in tibialis anterior and by exercise in soleus. In liver, protein synthesis was not modified by age but was stimulated by feeding and by exercise, which improved the response to feeding. We conclude that the impact of nutrition on muscle protein synthesis is blunted in old age, which could contribute to the age-related loss of nutrition-sensitive muscle proteins.

  6. Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics.

    PubMed

    Xiao, Lin; Xian, Hongxu; Lee, Kit Yee; Xiao, Bin; Wang, Hongyan; Yu, Fengwei; Shen, Han-Ming; Liou, Yih-Cherng

    2015-10-09

    Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.

  7. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

    PubMed Central

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P.; Wang, Shanshan; Krug, Robert M.

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  8. Establishment and optimization of a wheat germ cell-free protein synthesis system and its application in venom kallikrein.

    PubMed

    Wang, Yunpeng; Xu, Wentao; Kou, Xiaohong; Luo, Yunbo; Zhang, Yanan; Ma, Biao; Wang, Mengsha; Huang, Kunlun

    2012-08-01

    Wheat germ cell-free protein synthesis systems have the potential to synthesize functional proteins safely and with high accuracy, but the poor energy supply and the instability of mRNA templates reduce the productivity of this system, which restricts its applications. In this report, phosphocreatine and pyruvate were added to the system to supply ATP as a secondary energy source. After comparing the protein yield, we found that phosphocreatine is more suitable for use in the wheat germ cell-free protein synthesis system. To stabilize the mRNA template, the plasmid vector, SP6 RNA polymerase, and Cu(2+) were optimized, and a wheat germ cell-free protein synthesis system with high yield and speed was established. When plasmid vector (30 ng/μl), SP6 RNA polymerase (15 U), phosphocreatine (25 mM), and Cu(2+) (5 mM) were added to the system and incubated at 26°C for 16 h, the yield of venom kallikrein increased from 0.13 to 0.74 mg/ml. The specific activity of the recombinant protein was 1.3 U/mg, which is only slightly lower than the crude venom kallikrein (1.74 U/mg) due to the lack of the sugar chain. In this study, the yield of venom kallikrein was improved by optimizing the system, and a good foundation has been laid for industrial applications and for further studies.

  9. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ.

    PubMed

    Nepomuceno, Gabriella M; Chan, Katie M; Huynh, Valerie; Martin, Kevin S; Moore, Jared T; O'Brien, Terrence E; Pollo, Luiz A E; Sarabia, Francisco J; Tadeus, Clarissa; Yao, Zi; Anderson, David E; Ames, James B; Shaw, Jared T

    2015-03-12

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ's GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3.

  10. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ

    PubMed Central

    2015-01-01

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ’s GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3. PMID:25815151

  11. Induction of heat-shock protein synthesis in chondrocytes at physiological temperatures.

    PubMed

    Madreperla, S A; Louwerenburg, B; Mann, R W; Towle, C A; Mankin, H J; Treadwell, B V

    1985-01-01

    Induction of heat-shock protein (HSP) synthesis is demonstrated in cultured calf-chondrocytes at temperatures shown to occur in normal human cartilage during experiments subjecting intact cadaverous hip joints to the parameters of level walking. A 70,000 MW heat-shock protein (HSP-70) is synthesized by chondrocytes at temperatures above 39 degrees C, while induction of synthesis of a 110,000 MW HSP only occurs at temperatures of 45 degrees C or greater. These differences in critical temperatures for induction, and data showing differences in kinetics of induction and repression of synthesis, suggest that there are differences in the mechanism of induction of the two HSPs. The duration of HSP synthesis and inhibition of synthesis of normal cellular proteins is directly proportional to the duration and magnitude of the temperature rise. Possible relationships between these new findings and the initiation and progression of degenerative joint disease are discussed.

  12. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Garg, S.; Schmidt, D. K.

    1986-01-01

    The utility of augmenting displays to aid the human operator in controlling high order complex systems is well known. Analytical evaluation of various display designs for a simple k/s sup 2 plant in a compensatory tracking task using an optimal Control Model (OCM) of human behavior is carried out. This analysis reveals that significant improvement in performance should be obtained by skillful integration of key information into the display dynamics. The cooperative control synthesis technique previously developed to design pilot-optimal control augmentation is extended to incorporate the simultaneous design of performance enhancing augmented displays. The application of the cooperative control synthesis technique to the design of augmented displays is discussed for the simple k/s sup 2 plant. This technique is intended to provide a systematic approach to design optimally augmented displays tailored for specific tasks.

  13. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Garg, S.; Schmidt, D. K.

    1985-01-01

    The utility of augmenting displays to aid the human operator in controlling high order complex systems is well known. Analytical evaluations of various display designs for a simple k/s-squared plant in a compensatory tracking task using an Optimal Control Model (OCM) of human behavior is carried out. This analysis reveals that significant improvement in performance should be obtained by skillful integration of key information into the display dynamics. The cooperative control synthesis technique previously developed to design pilot-optimal control augmentation is extended to incorporate the simultaneous design of performance enhancing augmented displays. The application of the cooperative control synthesis technique to the design of augmented displays is discussed for the simple k/s-squared plant. This technique is intended to provide a systematic approach to design optimally augmented displays tailored for specific tasks.

  14. Activation of catalysts for synthesizing methanol from synthesis gas

    DOEpatents

    Blum, David B.; Gelbein, Abraham P.

    1985-01-01

    A method for activating a methanol synthesis catalyst is disclosed. In this method, the catalyst is slurried in an inert liquid and is activated by a reducing gas stream. The activation step occurs in-situ. That is, it is conducted in the same reactor as is the subsequent step of synthesizing methanol from a methanol gas stream catalyzed by the activated catalyst still dispersed in a slurry.

  15. Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein

    PubMed Central

    Casperson, Shanon L.; Sheffield-Moore, Melinda; Hewlings, Susan J.; Paddon-Jones, Douglas

    2013-01-01

    SUMMARY Background & aim Protein-energy supplementation is routinely employed to combat muscle loss. However, success is often compromised by increased satiety, poor palatability, high costs and low compliance. Methods For 2-weeks we supplemented meals of older individuals with leucine (4 g/meal; 3 meals/day; days 2–14). Metabolic studies were performed prior to (Day 1) and following (Day 15) supplementation. Leucine was not provided on metabolic study days. Venous blood and vastus lateralis muscle biopsies were obtained during a primed constant infusion of L-[ring-13C6] phenylalanine. Mixed muscle fractional synthesis rate (FSR), body composition and markers of nutrient signaling (mTOR, 4E-BP1 and p70S6K1 phosphorylation) were measured before and after a low protein/carbohydrate simulated meal. Results The meal modestly increased FSR on Day 1 (postabsorptive: 0.063 ± 0.004 vs. postprandial: 0.075 ± 0.006%/h; p = 0.03), however, two weeks of leucine supplementation increased postabsorptive FSR (p = 0.004) and the response to the meal (p = 0.01) (postabsorptive: 0.074 ± 0.007 vs. postprandial: 0.10 ± 0.007%/h). Changes in FSR were mirrored by increased phosphorylation of mTOR, 4E-BP1 and p70S6K1 (p ≤ 0.1). No change in fat free mass was observed (p > 0.05). Conclusions In older adults, leucine supplementation may improve muscle protein synthesis in response to lower protein meals. PMID:22357161

  16. Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

    SciTech Connect

    Onali, P.; Olianas, M.C.

    1987-03-23

    In rat striatal synaptosomes, 4..beta..-phorbol 12-myristate 13-acetate (PMA) and 4 ..beta..-phorbol 12,13-dibutyrate (PDBu), two activators of Ca/sup 2 +/-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C) tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 ..mu..M PMA and 1 ..mu..M PDBu. 4 ..beta..-Phorbol and 4 ..beta..-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 ..mu..M. PMA did not change the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C)DOPA. Addition of 1 mM EGTA to a Ca/sup 2 +/-free incubation medium failed to affect PMA stimulation. KCl (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KCl addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis. 37 references, 3 figures, 3 tables.

  17. Cell-free synthesis system suitable for disulfide-containing proteins

    SciTech Connect

    Matsuda, Takayoshi; Watanabe, Satoru; Kigawa, Takanori

    2013-02-08

    Highlights: ► Cell-free synthesis system suitable for disulfide-containing proteins is proposed. ► Disulfide bond formation was facilitated by the use of glutathione buffer. ► DsbC catalyzed the efficient shuffling of incorrectly formed disulfide bonds. ► Milligram quantities of functional {sup 15}N-labeled BPTI and lysozyme C were obtained. ► Synthesized proteins were both catalytically functional and properly folded. -- Abstract: Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional {sup 15}N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.

  18. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates – A Substudy

    PubMed Central

    Hursel, Rick; Martens, Eveline A. P.; Gonnissen, Hanne K. J.; Hamer, Henrike M.; Senden, Joan M. G.; van Loon, Luc J. C.; Westerterp-Plantenga, Margriet S.

    2015-01-01

    Background Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. Objective To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. Methods A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. Results After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low

  19. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  20. Effect of oxidative stress, produced by cumene hydroperoxide, on the various steps of protein synthesis. Modifications of elongation factor-2.

    PubMed

    Ayala, A; Parrado, J; Bougria, M; Machado, A

    1996-09-20

    We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.

  1. The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.

    PubMed

    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.

  2. Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis, and ATP production in human skeletal muscle without amino acid replacement.

    PubMed

    Barazzoni, Rocco; Short, Kevin R; Asmann, Yan; Coenen-Schimke, Jill M; Robinson, Matthew M; Nair, K Sreekumaran

    2012-11-01

    Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-¹³C]leucine as a tracer, we measured the fractional synthetic rate of mitochondrial as well as sarcoplasmic and mixed muscle proteins in 18 participants during sustained (7-h) insulin or saline infusion (n = 9 each). We also measured muscle ATP production, mitochondrial enzyme activities, mRNA levels of mitochondrial genes, and phosphorylation of signaling proteins regulating protein synthesis. The concentration of circulating essential AA decreased during insulin infusion. Mitochondrial, sarcoplasmic, and mixed muscle PS rates were also lower during insulin (2-7 h) than during saline infusions despite increased mRNA levels of selected mitochondrial genes. Under these conditions, insulin did not alter mitochondrial enzyme activities and ATP production. These effects were associated with enhanced phosphorylation of Akt but not of protein synthesis activators mTOR, p70(S6K), and 4EBP1. In conclusion, sustained physiological hyperinsulinemia without AA replacement did not stimulate PS of mixed muscle or protein subfractions and did not alter muscle mitochondrial ATP production in healthy humans. These results support that insulin and AA act in conjunction to stimulate muscle mitochondrial function and mitochondrial protein synthesis.

  3. All about that Amide Bond: The Sixth Chemical Protein Synthesis (CPS) Meeting.

    PubMed

    Weller, Caroline E; Chatterjee, Champak

    2015-11-01

    Endless potential: The sixth Chemical Protein Synthesis Meeting, held recently in St. Augustine, Florida, showed the potential of peptide and protein chemistry when applied toward understanding and controlling complex biological processes. This report highlights the diverse and cutting-edge protein chemistry presented at the meeting.

  4. Response of rat brain protein synthesis to ethanol and sodium barbital

    SciTech Connect

    Tewari, S.; Greenberg, S.A.; Do, K.; Grey, P.A.

    1987-01-01

    Central nervous system (CNS) depressants such as ethanol and barbiturates under acute or chronic conditions can induce changes in rat brain protein synthesis. While these data demonstrate the individual effects of drugs on protein synthesis, the response of brain protein synthesis to alcohol-drug interactions is not known. The goal of the present study was to determine the individual and combined effects of ethanol and sodium barbital on brain protein synthesis and gain an understanding of the mechanisms by which these alterations in protein synthesis are produced. Specifically, the in vivo and in vitro effects of sodium barbital (one class of barbiturates which is not metabolized by the hepatic tissue) were examined on brain protein synthesis in rats made physically dependent upon ethanol. Using cell free brain polysomal systems isolated from Control, Ethanol and 24 h Ethanol Withdrawn rats, data show that sodium barbital, when intubated intragastrically, inhibited the time dependent incorporation of /sup 14/C) leucine into protein by all three groups of ribosomes. Under these conditions, the Ethanol Withdrawn group displayed the largest inhibition of the /sup 14/C) leucine incorporation into protein when compared to the Control and Ethanol groups. In addition, sodium barbital when added at various concentrations in vitro to the incubation medium inhibited the incorporation of /sup 14/C) leucine into protein by Control and Ethanol polysomes. The inhibitory effects were also obtained following preincubation of ribosomes in the presence of barbital but not cycloheximide. Data suggest that brain protein synthesis, specifically brain polysomes, through interaction with ethanol or barbital are involved in the functional development of tolerance. These interactions may occur through proteins or polypeptide chains or alterations in messenger RNA components associated with the ribosomal units.

  5. Liposome chaperon in cell-free membrane protein synthesis: one-step preparation of KcsA-integrated liposomes and electrophysiological analysis by the planar bilayer method.

    PubMed

    Ando, M; Akiyama, M; Okuno, D; Hirano, M; Ide, T; Sawada, S; Sasaki, Y; Akiyoshi, K

    2016-02-01

    Chaperoning functions of liposomes were investigated using cell-free membrane protein synthesis. KcsA potassium channel-reconstituted liposomes were prepared directly using cell-free protein synthesis. In the absence of liposomes, all synthesized KcsA protein aggregated. In the presence of liposomes, however, synthesized KcsA spontaneously integrated into the liposome membrane. The KscA-reconstituted liposomes were transferred to the planar bilayer across a small hole in a thin plastic sheet and the channel function of KcsA was examined. The original electrophysiological activities, such as voltage- and pH-dependence, were observed. These results suggested that in cell-free membrane protein synthesis, liposomes act as chaperones, preventing aggregation and assisting in folding and tetrameric formation, thereby allowing full channel activity.

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

    PubMed Central

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-01-01

    Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway. PMID:27007909

  7. Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

    SciTech Connect

    Hodge, B.O.; Kream, B.E.

    1988-05-01

    We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of (/sup 3/H)proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis.

  8. Synthesis and antitumour activity of 4-aminoquinazoline derivatives

    NASA Astrophysics Data System (ADS)

    Lipunova, G. N.; Nosova, E. V.; Charushin, V. N.; Chupakhin, O. N.

    2016-07-01

    Pieces of data on the synthesis and antitumour activity of 4-aminoquinazolines are summarized and analyzed. Key methods for the synthesis of these compounds are considered, primarily cyclocondensation of carboxylic acid derivatives, as well as the oxidation of quinazolines and the cyclization of disubstituted thioureas. Improvements of synthetic schemes for erlotinib, gefitinib and lapatinib, which are the best-known pharmaceuticals based on compounds of the title class, are also considered. Synthetic strategies and biological activities for new 4-aminoquinazoline derivatives that are EGFR-tyrosine kinase inhibitors, multiactive compounds, and labelled compounds for use as positron emission tomography (PET) imaging agents are discussed. The bibliography includes 263 references.

  9. Catalytic Activation of Nitrogen Dioxide for Selective Synthesis of Nitroorganics

    DTIC Science & Technology

    2015-01-15

    AFRL-OSR-VA-TR-2015-0035 Catalytic activation of nitrogen dioxide for selective synthesis SETH BROWN UNIVERSITY OF NOTRE DAME DU LAC Final Report 01...8-98) v Prescribed by ANSI Std. Z39.18 12-01-2015 Final 15 Aug 2011 - 14 Aug 2014 Catalytic activation of nitrogen dioxide for selective synthesis...reductive elimination of the nitroarene has not. Nitrogen dioxide can be used as a source of the nitro group in reactions with arylboronic acids or their

  10. Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

    PubMed Central

    Miller, Jennifer L.; Cimen, Huseyin; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially-encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCδ, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications. PMID:19702336

  11. [Changes in heat shock protein synthesis and thermotolerance of Arabidopsis thaliana seedlings as a result of inhibition of Hsp90 by geldanamycin].

    PubMed

    Kozeko, L G

    2014-01-01

    The influence of geldanamycin (GA), which is a specific inhibitor of heat shock protein Hsp90 activities, on synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA was shown to induce synthesis of these stress proteins under normal conditions. Treatment of seeds with the Hsp90 inhibitor resulted in the elevated constitutive levels of Hsp70 and Hsp90 in seedlings as well as increased induction of their synthesis under heat shock, at that the effect of GA increased with its concentration. These up-regulation of Hsp promoted thermotolerance of seedlings. The obtained results are considered as evidence for autoregulation of heat shock protein synthesis and regulation of plant tolerance by Hsp90.

  12. Role of RNA Synthesis in the Estrogen Induction of a Specific Uterine Protein*

    PubMed Central

    DeAngelo, Anthony B.; Gorski, Jack

    1970-01-01

    The rate of amino acid incorporation into a specific uterine protein (induced protein band) isolated by gel electrophoresis has been shown to be markedly stimulated within an hour after estrogen administration. Injection of actinomycin D (8 mg/kg) prior to estrogen blocks the synthesis of induced protein. The accumulation of the product of the actinomycin D-sensitive step (induced protein band RNA) is significant 15 minutes after estrogen, and its synthesis would appear to be initiated as soon as the estrogen-receptor complex reaches the nucleus. Blocking protein synthesis with puromycin or cycloheximide did not affect the accumulation of induced protein band RNA, indicating that this is one of the earliest macromolecular synthetic events to occur after estrogen administration. PMID:5269235

  13. Muscle protein synthesis in response to nutrition and exercise.

    PubMed

    Atherton, P J; Smith, K

    2012-03-01

    Muscle protein synthesis (MPS) is the driving force behind adaptive responses to exercise and represents a widely adopted proxy for gauging chronic efficacy of acute interventions, (i.e. exercise/nutrition). Recent findings in this arena have been progressive. Nutrient-driven increases in MPS are of finite duration (∼1.5 h), switching off thereafter despite sustained amino acid availability and intramuscular anabolic signalling. Intriguingly, this 'muscle-full set-point' is delayed by resistance exercise (RE) (i.e. the feeding × exercise combination is 'more anabolic' than nutrition alone) even 24 h beyond a single exercise bout, casting doubt on the importance of nutrient timing vs. sufficiency per se. Studies manipulating exercise intensity/workload have shown that increases in MPS are negligible with RE at 20-40% but maximal at 70-90% of one-repetition maximum when workload is matched (according to load × repetition number). However, low-intensity exercise performed to failure equalises this response. Analysing distinct subcellular fractions (e.g. myofibrillar, sarcoplasmic, mitochondrial) may provide a readout of chronic exercise efficacy in addition to effect size in MPS per se, i.e. while 'mixed' MPS increases similarly with endurance and RE, increases in myofibrillar MPS are specific to RE, prophetic of adaptation (i.e. hypertrophy). Finally, the molecular regulation of MPS by exercise and its regulation via 'anabolic' hormones (e.g. IGF-1) has been questioned, leading to discovery of alternative mechanosensing-signalling to MPS.

  14. Relief memory consolidation requires protein synthesis within the nucleus accumbens.

    PubMed

    Bruning, Johann E A; Breitfeld, Tino; Kahl, Evelyn; Bergado-Acosta, Jorge R; Fendt, Markus

    2016-06-01

    Relief learning refers to the association of a stimulus with the relief from an aversive event. The thus-learned relief stimulus then can induce, e.g., an attenuation of the startle response or approach behavior, indicating positive valence. Previous studies revealed that the nucleus accumbens is essential for the acquisition and retrieval of relief memory. Here, we ask whether the nucleus accumbens is also the brain site for consolidation of relief memory into a long-term form. In rats, we blocked local protein synthesis within the nucleus accumbens by local infusions of anisomycin at different time points during a relief conditioning experiment. Accumbal anisomycin injections immediately after the relief conditioning session, but not 4 h later, prevented the consolidation into long-term relief memory. The retention of already consolidated relief memory was not affected by anisomycin injections. This identifies a time window and site for relief memory consolidation. These findings should complement our understanding of the full range of effects of adverse experiences, including cases of their distortion in humans such as post-traumatic stress disorder and/or phobias.

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

    PubMed Central

    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

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

    PubMed

    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.

  17. Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Zhang, Ming; Zhu, Gengrui; Lv, Dongwen; Wang, Yaping; Zhu, Dong; Yan, Yueming

    2017-01-01

    Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation. PMID:28194157

  18. Protein synthesis of muscle fractions from the small intestine in alcohol fed rats.

    PubMed Central

    Preedy, V R; Peters, T J

    1990-01-01

    The effects of chronic ethanol feeding on the amounts and synthesis rates of cytoplasmic, contractile, and stromal protein fractions were investigated in the small intestine of eight pairs of immature and seven pairs of mature rats. Treated rats were fed ethanol as 36% of total energy in a nutritionally adequate liquid diet. Paired controls were fed isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose. After six weeks the total cytoplasmic and contractile protein content in immature rats was reduced by 18% and 31%, respectively (p less than or equal to 0.007). The decline in the stromal protein content (26%) was not statistically significant (p = 0.130). In mature rats the protein contents were also reduced in the cytoplasmic (25%, p = 0.035) and contractile (27%, p = 0.005) protein fractions, though the stromal protein fraction was unaltered (p = 0.913). In immature rats fractional rates of protein synthesis in cytoplasmic and contractile protein fractions of the small intestine were unaltered by chronic ethanol feeding (p less than or equal to 0.853). In mature rats, the synthesis rates of corresponding fractions declined, by 18% and 31%, respectively, but were also not statistically significant (p less than or equal to 0.369). Absolute rates of protein synthesis in immature rats fell by 6% (p = 0.549) in the cytoplasmic and 31% in the contractile protein fraction (p = 0.045). In mature rats, the corresponding reductions were 38% (p = 0.106) and 48% (p = 0.033), respectively. Virtually no radioactivity could be detected in the stromal fraction, signifying very low synthesis rates. Chronic ethanol feeding reduces the amount of protein in the small intestine of the immature and mature rat with the contractile protein fraction showing the greatest decrease. In the absence of statistically significant reductions in fractional synthesis rates a partial adaptation in turnover rates may have occurred. PMID:2323594

  19. Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts.

    PubMed

    Abraham, A K; Kolseth, S; Pihl, A

    1982-05-17

    Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.

  20. Pyrophosphate-condensing activity linked to nucleic acid synthesis.

    PubMed Central

    Volloch, V Z; Rits, S; Tumerman, L

    1979-01-01

    In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation. Images PMID:88040

  1. Enteral leucine supplementation increases protein synthesis in skeletal and cardiac muscles and visceral tissues of neonatal pigs through mTORC1-dependent pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine activates mammalian target of rapamycin (mTOR) to upregulate protein synthesis (PS). To examine enteral Leu effects on PS and signaling activation, 5-d-old piglets were fed for 24 h diets containing: (i) LP, (ii) LP+L, or (iii) HP. PS in skeletal muscles, heart, liver, pancreas, and jejunum...

  2. Activation of formylmethanofurna synthesis in cell extracts of Methanobacterium thermoautotrophicum

    SciTech Connect

    Bobik, T.A.; Wolfe, R.S. )

    1989-03-01

    In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO{sub 2} fixation reaction that is an early step in CO{sub 2} reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O{sup 3}-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO{sub 2} reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier.

  3. A Better Understanding of Protein Structure and Function by the Synthesis and Incorporation of Selenium- and Tellurium Containing Tryptophan Analogs

    SciTech Connect

    Helmey, Sherif Samir; Rice, Ambrose Eugene; Hatch, Duane Michael; Silks, Louis A.; Marti-Arbona, Ricardo

    2016-08-17

    Unnatural heavy metal-containing amino acid analogs have shown to be very important in the analysis of protein structure, using methods such as X-ray crystallography, mass spectroscopy, and NMR spectroscopy. Synthesis and incorporation of selenium-containing methionine analogs has already been shown in the literature however with some drawbacks due to toxicity to host organisms. Thus synthesis of heavy metal tryptophan analogs should prove to be more effective since the amino acid tryptophan is naturally less abundant in many proteins. For example, bioincorporation of β-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]SeTrp) and β-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]SeTrp) has been shown in the following proteins without structural or catalytic perturbations: human annexin V, barstar, and dihydrofolate reductase. The reported synthesis of these Se-containing analogs is currently not efficient for commercial purposes. Thus a more efficient, concise, high-yield synthesis of selenotryptophan, as well as the corresponding, tellurotryptophan, will be necessary for wide spread use of these unnatural amino acid analogs. This research will highlight our progress towards a synthetic route of both [6,7]SeTrp and [6,7]TeTrp, which ultimately will be used to study the effect on the catalytic activity of Lignin Peroxidase (LiP).

  4. Comparative cell signalling activity of ultrapure recombinant chaperonin 60 proteins from prokaryotes and eukaryotes.

    PubMed

    Maguire, Maria; Poole, Stephen; Coates, Anthony R M; Tormay, Peter; Wheeler-Jones, Caroline; Henderson, Brian

    2005-06-01

    Heat-shock protein (hsp)60/chaperonin 60 is a potent immunogen which has recently been claimed to have cell-signalling actions upon myeloid and vascular endothelial cells. The literature is controversial with different chaperonin 60 proteins producing different patterns of cellular activation and the ever-present criticism that activity is the result of bacterial contaminants. To clarify the situation we have cloned, expressed and purified to homogeneity the chaperonin 60 proteins from Chlamydia pneumoniae, Helicobacter pylori and the human mitochondrion. These highly purified proteins were compared for their ability to stimulate human peripheral blood mononuclear cell (PBMC) cytokine synthesis and vascular endothelial cell adhesion protein expression. In spite of their significant sequence homology, the H. pylori protein was the most potent PBMC activator with the human protein the least potent. PBMC activation by C. pneumoniae and human, but not H. pylori, chaperonin 60 was blocked by antibody neutralization of Toll-like receptor-4. The C. pneumoniae chaperonin 60 was the most potent endothelial cell activator, with the human protein being significantly less active than bacterial chaperonin 60 proteins. These results have implications for the role of chaperonin 60 proteins as pathological factors in autoimmune and cardiovascular disease, and raise the possibility that each of these proteins may result in different pathological effects in such diseases.

  5. Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange Mass Spectrometry

    PubMed Central

    Landgraf, Rachelle R.; Goswami, Devrishi; Rajamohan, Francis; Harris, Melissa S.; Calabrese, Matthew; Hoth, Lise R.; Magyar, Rachelle; Pascal, Bruce D.; Chalmers, Michael J.; Busby, Scott A.; Kurumbail, Ravi; Griffin, Patrick R.

    2013-01-01

    Summary AMP-Activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme by hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit suggesting that the molecular binding site of latter resides between the α and β subunits. The distinct short and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands. PMID:24076403

  6. Converting a Natural Protein Compartment into a Nanofactory for the Size-Constrained Synthesis of Antimicrobial Silver Nanoparticles.

    PubMed

    Giessen, Tobias W; Silver, Pamela A

    2016-12-16

    Engineered biological systems are used extensively for the production of high value and commodity organics. On the other hand, most inorganic nanomaterials are still synthesized via chemical routes. By engineering cellular compartments, functional nanoarchitectures can be produced under environmentally sustainable conditions. Encapsulins are a new class of microbial nanocompartments with promising applications in nanobiotechnology. Here, we engineer the Thermotoga maritima encapsulin EncTm to yield a designed compartment for the size-constrained synthesis of silver nanoparticles (Ag NPs). These Ag NPs exhibit uniform shape and size distributions as well as long-term stability. Ambient aqueous conditions can be used for Ag NP synthesis, while no reducing agents or solvents need to be added. The antimicrobial activity of the synthesized protein-coated or shell-free Ag NPs is superior to that of silver nitrate and citrate-capped Ag NPs. This study establishes encapsulins as an engineerable platform for the synthesis of biogenic functional nanomaterials.

  7. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Garg, S.; Schmidt, D. K.

    1985-01-01

    A technique is developed that is intended to provide a systematic approach to synthesizing display augmentation for optimal manual control in complex, closed-loop tasks. A cooperative control synthesis technique, previously developed to design pilot-optimal control augmentation for the plant, is extended to incorporate the simultaneous design of performance enhancing displays. The technique utilizes an optimal control model of the man in the loop. It is applied to the design of a quickening control law for a display and a simple K/s(2) plant, and then to an F-15 type aircraft in a multi-channel task. Utilizing the closed loop modeling and analysis procedures, the results from the display design algorithm are evaluated and an analytical validation is performed. Experimental validation is recommended for future efforts.

  8. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Gary, Sanjay; Schmidt, David K.

    1987-01-01

    A technique is developed that is intended to provide a systematic approach to synthesizing display augmentation for optimal manual control in complex, closed-loop tasks. A cooperative control synthesis technique, previously developed to design pilot-optimal control augmentation for the plant, is extended to incorporate the simultaneous design of performance enhancing displays. The technique utilizes an optimal control model of the man in the loop. It is applied to the design of a quickening control law for a display and a simple K/(s squared) plant, and then to an F-15 type aircraft in a multichannel task. Utilizing the closed-loop modeling and analysis procedures, the results from the display design algorithm are evaluated and an analytical validation is performed. Experimental validation is recommended for future efforts.

  9. The substrate for long-lasting memory: if not protein synthesis, then what?

    PubMed

    Routtenberg, Aryeh

    2008-03-01

    The prevailing textbook view that de novo protein synthesis is required for memory (e.g., [Bear, M. F., Connors, B., & Paradiso, M. 2006. Neuroscience. Lippincott, New York]) is seriously flawed and an alternative hypothesis has been proposed in which post-translational modification (PTM) of proteins already synthesized and already present within the synapse is 'the' substrate for long-lasting memory. Protein synthesis serves a replenishment role. The first part of this review discusses how long-lasting memory can be achieved with 'only' PTM of existing synaptic proteins. The second part critically reviews a recent report published in Neuron 2007 that exemplifies the current view of protein synthesis and memory while also illustrating how these results can be understood within this new PTM framework. A necessary yet unexpected conclusion to emerge from consideration of the consequences of a PTM mechanism as the necessary, sufficient and exclusive substrate for long-lasting memory, is that the central Hebbian dogma that cells that 'fire together, wire together' is an unlikely mechanism for long-lasting memory. Thus, a unique feature of the PTM model is that longevity of information storage is achieved not by stability of the synaptic mechanism, but by impermanent pseudoredundant circuits. This is so because PTM is a reversible process and thus any permanent connection, any 'lasting effect' cannot be in the form of stable synapse formation. We have therefore proposed a solution in which network level processes regulate cellular mechanisms, even as such mechanisms regulate the network. Thus, synapses are 'meta-stabilized' by regulated feedback mediated by the circuit in which the synapse is embedded. For example, spontaneous activity is proposed to be a substrate feedback mechanism we term 'cryptic rehearsal' to sustain for some period of time after learning an approximation to the state initially created by input. Additionally, because the duplication of these traces

  10. Motualevic Acids and Analogs: Synthesis and Antimicrobial Structure Activity Relationships

    PubMed Central

    Cheruku, Pradeep; Keffer, Jessica L.; Dogo-Isonagie, Cajetan; Bewley, Carole A.

    2010-01-01

    Synthesis of the marine natural products motualevic acids A, E, and analogs in which modifications have been made to the ω-brominated lipid (E)-14,14-dibromotetra-deca-2,13-dienoic acid or amino acid unit are reported, together with antimicrobial activities against Staphylococcus aureus, methicillin-resistant S. aureus, Enterococcus faecium, and vancomycin-resistant Enterococcus. PMID:20538459

  11. Synthesis and antimicrobial activity of some novel dicationic sulphonophanes.

    PubMed

    Rajakumar, Perumal; Satheeshkumar, Chinnadurai; Mohanraj, Gunasekaran; Mathivanan, Narayanasamy

    2011-07-01

    The synthesis of some novel imidazole-based dicationic sulfonophanes incorporating various spacer units is described. All the sulphonophanes exhibit good antibacterial and antifungal activity against five bacterial strains Bacillus subtilis, Staphylococcus aureus, Vibrio cholera, Escherichia coli, Proteus vulgaris and human pathogenic fungus Candida albicans.

  12. Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland.

    PubMed

    Ho, A K; Price, D M; Terriff, D; Chik, C L

    2006-06-27

    Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.

  13. Lack of dependance of transcription-induced cytosine deaminations on protein synthesis.

    PubMed

    Mokkapati, Sanath Kumar; Bhagwat, Ashok S

    2002-10-31

    Transcription-induced mutations (TIM) is a phenomenon in Escherichia coli in which transcription promotes C to T and other mutations in a strand-specific manner. Because the processes of transcription and translation are coupled in prokaryotes and some models regarding creating a hypermutagenic state in E. coli require new protein synthesis, we tested the possibility that TIM was dependent on efficient synthesis of proteins. We used puromycin to reversibly inhibit protein synthesis and found that it had little effect on mRNA synthesis, plasmid copy-number or TIM. Our results show that TIM is not dependent on efficient translation of mRNA and this helps eliminate certain models concerning the mechanism underlying TIM.

  14. Signaling of angiotensin II-induced vascular protein synthesis in conduit and resistance arteries in vivo

    PubMed Central

    Daigle, Christine; Martens, Fabrice MAC; Girardot, Daphné; Dao, Huy Hao; Touyz, Rhian M; Moreau, Pierre

    2004-01-01

    Background From in vitro studies, it has become clear that several signaling cascades are involved in angiotensin II-induced cellular hypertrophy. The aim of the present study was to determine some of the signaling pathways mediating angiotensin II (Ang II)-induced protein synthesis in vivo in large and small arteries. Methods Newly synthesized proteins were labeled during 4 hours with tritiated leucine in conscious control animals, or animals infused for 24 hours with angiotensin II (400 ng/kg/min). Hemodynamic parameters were measure simultaneously. Pharmacological agents affecting signaling cascades were injected 5 hours before the end of Ang II infusion. Results Angiotensin II nearly doubled the protein synthesis rate in the aorta and small mesenteric arteries, without affecting arterial pressure. The AT1 receptor antagonist Irbesartan antagonized the actions of Ang II. The Ang II-induced protein synthesis was associated with increased extracellular signal-regulated kinases (ERK)1/2 phosphorylation in aortic, but not in mesenteric vessels. Systemic administration of PD98059, an inhibitor of the ERK-1/2 pathway, produced a significant reduction of protein synthesis rate in the aorta, and only a modest decrease in mesenteric arteries. Rapamycin, which influences protein synthesis by alternative signaling, had a significant effect in both vessel types. Rapamycin and PD98059 did not alter basal protein synthesis and had minimal effects on arterial pressure. Conclusion ERK1/2 and rapamycin-sensitive pathways are involved in pressure-independent angiotensin II-induced vascular protein synthesis in vivo. However, their relative contribution may vary depending on the nature of the artery under investigation. PMID:15134586

  15. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    SciTech Connect

    Nosek, T.M.; Adams, R.J.

    1986-03-05

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7..mu..M compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7..mu..M ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100..mu..M CYC or LEU did not affect NKA activity or /sup 3/H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA.

  16. Photo-synthesis of protein-based nanoparticles and the application in drug delivery

    SciTech Connect

    Xie, Jinbing; Wang, Hongyang; Cao, Yi; Qin, Meng Wang, Wei

    2015-07-15

    Recently, protein-based nanoparticles as drug delivery systems have attracted great interests due to the excellent behavior of high biocompatibility and biodegradability, and low toxicity. However, the synthesis techniques are generally costly, chemical reagents introduced, and especially present difficulties in producing homogeneous monodispersed nanoparticles. Here, we introduce a novel physical method to synthesize protein nanoparticles which can be accomplished under physiological condition only through ultraviolet (UV) illumination. By accurately adjusting the intensity and illumination time of UV light, disulfide bonds in proteins can be selectively reduced and the subsequent self-assembly process can be well controlled. Importantly, the co-assembly can also be dominated when the proteins mixed with either anti-cancer drugs, siRNA, or active targeting molecules. Both in vitro and in vivo experiments indicate that our synthesized protein–drug nanoparticles (drug-loading content and encapsulation efficiency being ca. 8.2% and 70%, respectively) not only possess the capability of traditional drug delivery systems (DDS), but also have a greater drug delivery efficiency to the tumor sites and a better inhibition of tumor growth (only 35% of volume comparing to the natural growing state), indicating it being a novel drug delivery system in tumor therapy.

  17. Fractional synthesis rates of DNA and protein in rabbit skin are not correlated.

    PubMed

    Zhang, Xiao-jun; Chinkes, David L; Wu, Zhanpin; Martini, Wenjun Z; Wolfe, Robert R

    2004-09-01

    We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-(13)C(6)]glucose and L-[(15)N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-(2)H(5)]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 +/- 0.59%/d in whole skin and 3.08 +/- 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 +/- 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 +/- 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis.

  18. Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12).

    PubMed Central

    Biocca, S; Cattaneo, A; Calissano, P

    1984-01-01

    Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity. Images PMID:6585787

  19. Effects of oral meal feeding on whole body protein breakdown and protein synthesis in cachectic pancreatic cancer patients

    PubMed Central

    van Dijk, David PJ; van de Poll, Marcel CG; Moses, Alastair GW; Preston, Thomas; Olde Damink, Steven WM; Rensen, Sander S; Deutz, Nicolaas EP; Soeters, Peter B; Ross, James A; Fearon, Kenneth CH; Dejong, Cornelis HC

    2015-01-01

    Background Pancreatic cancer is often accompanied by cachexia, a syndrome of severe weight loss and muscle wasting. A suboptimal response to nutritional support may further aggravate cachexia, yet the influence of nutrition on protein kinetics in cachectic patients is poorly understood. Methods Eight cachectic pancreatic cancer patients and seven control patients received a primed continuous intravenous infusion of l-[ring-2H5]phenylalanine and l-[3,3-2H2]tyrosine for 8 h and ingested sips of water with l-[1-13C]phenylalanine every 30 min. After 4 h, oral feeding was started. Whole body protein breakdown, protein synthesis, and net protein balance were calculated. Results are given as median with interquartile range. Results Baseline protein breakdown and protein synthesis were higher in cachectic patients compared with the controls (breakdown: 67.1 (48.1–79.6) vs. 45.8 (42.6–46.3) µmol/kg lean body mass/h, P = 0.049; and synthesis: 63.0 (44.3–75.6) vs. 41.8 (37.6–42.5) µmol/kg lean body mass/h, P = 0.021). During feeding, protein breakdown decreased significantly to 45.5 (26.9–51.1) µmol/kg lean body mass/h (P = 0.012) in the cachexia group and to 33.7 (17.4–37.1) µmol/kg lean body mass/h (P = 0.018) in the control group. Protein synthesis was not affected by feeding in cachectic patients: 58.4 (46.5–76.1) µmol/kg lean body mass/h, but was stimulated in controls: 47.9 (41.8–56.7) µmol/kg lean body mass/h (P = 0.018). Both groups showed a comparable positive net protein balance during feeding: cachexia: 19.7 (13.1–23.7) and control: 16.3 (13.6–25.4) µmol/kg lean body mass/h (P = 0.908). Conclusion Cachectic pancreatic cancer patients have a higher basal protein turnover. Both cachectic patients and controls show a comparable protein anabolism during feeding, albeit through a different pattern of protein kinetics. In cachectic patients, this is primarily related to reduced protein breakdown, whereas in controls, both protein breakdown and

  20. Specific activation of human interleukin-5 depends on de novo synthesis of an AP-1 complex.

    PubMed

    Schwenger, Gretchen T F; Kok, Chee Choy; Arthaningtyas, Estri; Thomas, Marc A; Sanderson, Colin J; Mordvinov, Viatcheslav A

    2002-12-06

    It is clear from the biology of eosinophilia that a specific regulatory mechanism must exist. Because interleukin-5 (IL5) is the key regulatory cytokine, it follows that a gene-specific control of IL5 expression must exist that differs even from closely related cytokines such as IL4. Two features of IL5 induction make it unique compared with other cytokines; first, induction by cyclic adenosine monophosphate (cAMP), which inhibits other T-cell-derived cytokines, and second, sensitivity to protein synthesis inhibitors, which have no effect on other cytokines. This study has utilized the activation of different transcription factors by different stimuli in a human T-cell line to study the role of conserved lymphokine element 0 (CLE0) in the specific induction of IL5. In unstimulated cells the ubiquitous Oct-1 binds to CLE0. Stimulation induces de novo synthesis of the AP-1 members JunD and Fra-2, which bind to CLE0. The amount of IL5 produced correlates with the production of the AP-1 complex, suggesting a key role in IL5 expression. The formation of the AP-1 complex is essential, but the rate-limiting step is the synthesis of AP-1, especially Fra-2. This provides an explanation for the sensitivity of IL5 to protein synthesis inhibitors and a mechanism for the specific induction of IL5 compared with other cytokines.

  1. Protein synthesis elongation factor EF-1 alpha expression and longevity in Drosophila melanogaster.

    PubMed Central

    Shikama, N; Ackermann, R; Brack, C

    1994-01-01

    It has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in elongation factor EF-1 alpha. Transgenic Drosophila melanogaster flies carrying an additional copy of the EF-1 alpha gene under control of a heat-inducible promoter have an extended lifespan, further indicating that the EF-1 alpha gene may play an important role in determining longevity. To test this hypothesis, we have quantitated EF-1 alpha mRNA, EF-1 alpha protein, and the EF-1 alpha complex-formation activity in these transgenic flies. Furthermore, we have tested whether the transgene construct is functional--i.e., whether transgenic mRNA is induced when flies are grown at higher temperature. The results show that although there is a clear difference in mean lifespan between the EF-1 alpha transgenic (E) flies and the control transgenic (C) flies, E flies do not express more EF-1 alpha protein or mRNA than C flies kept at the same experimental conditions. Although the transgene can be induced when E flies are heat-shocked at 37 degrees C, transgenic mRNA is not detectable in E flies aged at 29 degrees C. In both lines, the loss in catalytic activity with age is the same. We conclude that the E flies examined here do not live longer because of overexpressing the EF-1 alpha gene. Images PMID:8183891

  2. Robust production of recombinant phosphoproteins using cell-free protein synthesis

    PubMed Central

    Oza, Javin P.; Aerni, Hans R.; Pirman, Natasha L.; Barber, Karl W.; ter Haar, Charlotte M.; Rogulina, Svetlana; Amrofell, Matthew B.; Isaacs, Farren J.; Rinehart, Jesse; Jewett, Michael C.

    2015-01-01

    Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins.