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Sample records for active protein synthesis

  1. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  2. Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression.

    PubMed

    Andruska, Neal D; Zheng, Xiaobin; Yang, Xujuan; Mao, Chengjian; Cherian, Mathew M; Mahapatra, Lily; Helferich, William G; Shapiro, David J

    2015-04-14

    Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration. PMID:25825714

  3. Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase.

    PubMed

    Li, Jian; Lawton, Thomas J; Kostecki, Jan S; Nisthal, Alex; Fang, Jia; Mayo, Stephen L; Rosenzweig, Amy C; Jewett, Michael C

    2016-02-01

    Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL(-1) were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4 . Scale-up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs. PMID:26356243

  4. Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

    PubMed

    Yagasaki, Kazumi; Morisaki, Naoko; Kitahara, Yoshiro; Miura, Atsuhito; Funabiki, Ryuhei

    2003-11-01

    Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C. PMID:19003213

  5. 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.

  6. Modular Total Synthesis of Protein Kinase C Activator (-)-Indolactam V.

    PubMed

    Haynes-Smith, Jeremy; Diaz, Italia; Billingsley, Kelvin L

    2016-05-01

    A concise, eight-step total synthesis of (-)-indolactam V, a nanomolar agonist of protein kinase C, is reported. The synthesis relies upon an efficient copper-catalyzed amino acid arylation to establish the indole C4-nitrogen bond. This cross-coupling method is applicable to a range of hydrophobic amino acids, providing a platform for further diversification of indolactam alkaloid scaffolds and studies on their potent biological activity. PMID:27074538

  7. Phrenic long-term facilitation requires spinal serotonin receptor activation and protein synthesis.

    PubMed

    Baker-Herman, Tracy L; Mitchell, Gordon S

    2002-07-15

    Respiratory long-term facilitation (LTF) is a form of serotonin-dependent plasticity induced by intermittent hypoxia. LTF is manifested as a long-lasting increase in respiratory amplitude (and frequency) after the hypoxic episodes have ended. We tested the hypotheses that LTF of phrenic amplitude requires spinal serotonin receptor activation and spinal protein synthesis. A broad-spectrum serotonin receptor antagonist (methysergide) or protein synthesis inhibitors (emetine or cycloheximide) were injected intrathecally in the cervical spinal cord of anesthetized rats. Control rats, injected with vehicle (artificial CSF), exhibited an augmented phrenic burst amplitude after three 5 min episodes of hypoxia (78 +/- 15% above baseline, 60 min after hypoxia; p < 0.05), indicating LTF. Pretreatment with methysergide, emetine, or cycloheximide attenuated or abolished phrenic LTF (20 +/- 4, 0.2 +/- 11, and 20 +/- 2%, respectively; all p > 0.05). With protein synthesis inhibitors, phrenic LTF differed from control by 15 min after intermittent hypoxia. As an internal control against unintended drug distribution, we measured respiratory LTF in hypoglossal (XII) motor output. At 60 min after intermittent hypoxia, all treatment groups exhibited similar XII LTF (artificial CSF, 44 +/- 10%; methysergide, 40 +/- 5%; emetine, 35 +/- 9%; and cycloheximide, 57 +/- 29%; all p < 0.05), suggesting that drugs were restricted at effective doses to the spinal cord. We conclude that phrenic LTF requires spinal serotonin receptor activation and protein synthesis. Serotonin receptors on phrenic motoneuron dendrites may induce new protein synthesis, thereby giving rise to phrenic LTF. PMID:12122082

  8. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

    PubMed Central

    Zinck, R; Cahill, M A; Kracht, M; Sachsenmaier, C; Hipskind, R A; Nordheim, A

    1995-01-01

    Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction. PMID:7651411

  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. PMID:18789668

  10. Long Lasting Protein Synthesis- and Activity-Dependent Spine Shrinkage and Elimination after Synaptic Depression

    PubMed Central

    Ramiro-Cortés, Yazmín; Israely, Inbal

    2013-01-01

    Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD) mediated by metabotropic glutamate receptors (mGluRs) through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation. PMID:23951097

  11. An inhibitory factor for cell-free protein synthesis from Salmonella enteritidis exhibits cytopathic activity against Chinese hamster ovary cells.

    PubMed

    Iwamaru, Y; Miyake, M; Arii, J; Tanabe, Y; Noda, M

    2001-12-01

    A factor inhibiting cell-free protein synthesis was purified from Salmonella enteritidis cell lysate by sequential ammonium sulfate precipitation, chromatography on anion exchange and hydrophobic interaction columns, and polyacrylamide disc gel electrophoresis. The purified factor, which was named SIPS (Salmonella inhibitor of protein synthesis), inhibited in vitro protein synthesis in rabbit reticulocyte lysate and had a molecular mass of 38 kDa, estimated by PAGE under denaturing conditions. SIPS was also cytopathic for Chinese hamster ovary cells. The N-terminal amino acid sequence (20 residues) of SIPS was found to be identical to that of mature L-asparaginase II of Escherichia coli. Indeed, the purified SIPS exhibited asparaginase activity, E. coli L-asparaginase II had cytopathic activity and inhibited in vitro protein synthesis. The results suggest that at least a part of cytotoxicity and inhibition of cell-free protein synthesis caused by S. enteritidis is a property of the bacterial L-asparaginase. PMID:11747376

  12. 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. PMID:25837301

  13. Gateway synthesis of daphnane congeners and their protein kinase C affinities and cell-growth activities

    NASA Astrophysics Data System (ADS)

    Wender, Paul A.; Buschmann, Nicole; Cardin, Nathan B.; Jones, Lisa R.; Kan, Cindy; Kee, Jung-Min; Kowalski, John A.; Longcore, Kate E.

    2011-08-01

    The daphnane diterpene orthoesters constitute a structurally fascinating family of natural products that exhibit a remarkable range of potent biological activities. Although partial activity information is available for some natural daphnanes, little information exists for non-natural congeners or on how changes in structure affect mode of action, function, potency or selectivity. A gateway strategy designed to provide general synthetic access to natural and non-natural daphnanes is described and utilized in the synthesis of two novel members of this class. In this study, a commercially available tartrate derivative was elaborated through a key late-stage diversification intermediate into B-ring yuanhuapin analogues to initiate exploration of the structure-function relationships of this class. Protein kinase C was identified as a cellular target for these agents, and their activity against human lung and leukaemia cell lines was evaluated. The natural product and a novel non-natural analogue exhibited significant potency, but the epimeric epoxide was essentially inactive.

  14. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    PubMed

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules. PMID:27319340

  15. Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

    SciTech Connect

    Fujibayashi, S.; Wenger, D.A.

    1986-11-15

    Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When (/sup 35/S)methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the (/sup 35/S)methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.

  16. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  17. Steroid-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders. Correlation with natriferic activity.

    PubMed Central

    Geheb, M; Alvis, R; Owen, A; Hercker, E; Cox, M

    1984-01-01

    We have identified a group of proteins (Mr approximately 70 000-80 000; pI approximately 5.5-6.0) in giant-toad (Bufo marinus) urinary bladders whose synthesis appears to be related to aldosterone-stimulated Na+ transport. Spironolactone, a specific mineralocorticoid antagonist in renal epithelia, inhibits the synthesis of these proteins as well as the natriferic effect of the hormone. Since a variety of other steroids (some of which are traditionally considered to be glucocorticoids) also stimulate Na+ transport in toad urinary bladders, we examined whether their natriferic activity was expressed in a fashion similar to that of aldosterone. Short-circuit current was used to measure Na+ transport, and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. At a concentration of approximately 100 nM, dexamethasone, corticosterone and aldosterone were equinatriferic. Dexamethasone and aldosterone had identical dose-response curves, maximal and half-maximal activity being evident at concentrations of approximately 100 nM and 10 nM respectively. In contrast, at a concentration of approximately 10 nM, corticosterone had no effect on Na+ transport. The natriferic activities of these three steroids correlate with their known affinities for the putative mineralocorticoid receptor in toad urinary bladders. Natriferic concentrations of dexamethasone and corticosterone (140 nM) induced the synthesis of proteins with characteristics identical with those induced by aldosterone. Spironolactone, at an antagonist/agonist ratio of 2000:1, inhibited steroid-induced Na+ transport and the synthesis of these proteins. Thus it appears that all natriferic steroids share a common mechanism of action in toad urinary bladders. Natriferic activity can be correlated not only with relative steroid-receptor affinity but also with the induction of a specific group of epithelial-cell proteins. Images Fig. 1. Fig

  18. Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs.

    PubMed

    Rahman, M A; Summerton, J; Foster, E; Cunningham, K; Stirchak, E; Weller, D; Schaup, H W

    1991-01-01

    Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics. PMID:1821653

  19. MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas.

    PubMed

    Grzmil, Michal; Huber, Roland M; Hess, Daniel; Frank, Stephan; Hynx, Debby; Moncayo, Gerald; Klein, Dominique; Merlo, Adrian; Hemmings, Brian A

    2014-02-01

    High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically. PMID:24401275

  20. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis

    PubMed Central

    Jeanblanc, Jerome; Logrip, Marian L.; Janak, Patricia H.; Ron, Dorit

    2013-01-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  1. 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. PMID:26334346

  2. Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation.

    PubMed

    Rawls, J A; Pusztai, R; Green, M

    1990-12-01

    The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F. PMID:2173783

  3. 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.

  4. Synthesis of Lipidated Proteins.

    PubMed

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented. PMID:27444727

  5. 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. PMID:17101792

  6. Possible involvement of AMP-activated protein kinase in PGE1-induced synthesis of osteoprotegerin in osteoblasts

    PubMed Central

    KAINUMA, SHINGO; OTSUKA, TAKANOBU; KUROYANAGI, GEN; YAMAMOTO, NAOHIRO; MATSUSHIMA-NISHIWAKI, RIE; KOZAWA, OSAMU; TOKUDA, HARUHIKO

    2016-01-01

    AMP-activated protein kinase (AMPK) is firmly established as a central regulator of cellular energy homeostasis. We have previously reported that prostaglandin E1 (PGE1) stimulates the synthesis of osteoprotegerin through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. The present study investigated the involvement of AMPK in PGE1-induced osteoprotegerin synthesis in MC3T3-E1 cells. The levels of osteoprotegerin were measured using an enzyme-linked immunosorbent assay, while the phosphorylation of AMPK, acetyl-CoA carboxylase, p38 MAP kinase and SAPK/JNK were analyzed by western blotting. In addition, the mRNA expression levels of osteoprotegerin were determined by a reverse transcription-quantitative polymerase chain reaction. It was revealed that PGE1 significantly induced the phosphorylation of the α and β subunits of AMPK in a time-dependent manner (P<0.05). In addition, acetyl-CoA carboxylase, a direct substrate of AMPK, was significantly phosphorylated by PGE1 (P<0.05). Compound C, an AMPK inhibitor, was revealed to suppress the phosphorylation of acetyl-CoA carboxylase, which significantly reduced the release and mRNA expression levels of PGE1-stimulated osteoprotegerin (P<0.05). However, the PGE1-induced phosphorylation of p38 MAP kinase and SAPK/JNK were not affected by compound C. The results of the present study indicated that AMPK may positively regulate PGE1-stimulated osteoprotegerin synthesis in osteoblasts; thus providing novel insight into the regulatory mechanisms underlying bone metabolism. PMID:27168848

  7. Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

    PubMed Central

    Malarkey, K.; Chilvers, E. R.; Lawson, M. F.; Plevin, R.

    1995-01-01

    1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF. Images Figure 1 Figure 2 Figure 5 Figure 6 Figure 7 PMID:8564258

  8. The LMP1 oncogene of EBV activates PERK and the unfolded protein response to drive its own synthesis

    PubMed Central

    Lee, Dong Yun

    2008-01-01

    The oncogene latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) without a ligand drives proliferation of EBV-infected B cells. Its levels vary in cells of clonal populations by more than 100-fold, which leads to multiple distinct activities of the oncogene. At intermediate levels it drives proliferation, and at high levels it inhibits general protein synthesis by inducing phosphorylation of eukaryotic initiation factor 2α (eIF2α). We have found that LMP1 activates PERK to induce phosphorylation of eIF2α, which upregulates activating transcription factor 4 (ATF4) expression. ATF4, in turn, transactivates LMP1's own promoter. LMP1 activates not only PERK but also inositol requiring kinase 1 (IRE1) and ATF6, 3 pathways of the unfolded protein response (UPR). Increasing expression levels of LMP1 induced a dose-dependent increase in IRE1 activity, as measured by its “splicing” of XBP-1. These infected B cells secrete immunoglobins independent of the levels of LMP1, indicating that only a threshold level of XBP-1 is required for the secretion. These findings indicate that LMP1's activation of the UPR is a normal event in a continuum of LMP1's expression that leads both to stimulatory and inhibitory functions and regulates the physiology of EBV-infected B cells in multiple, unexpected modes. PMID:18042799

  9. 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

  10. Design and synthesis of an activity-based protein profiling probe derived from cinnamic hydroxamic acid.

    PubMed

    Ai, Teng; Qiu, Li; Xie, Jiashu; Geraghty, Robert J; Chen, Liqiang

    2016-02-15

    In our continued effort to discover new anti-hepatitis C virus (HCV) agents, we validated the anti-replicon activity of compound 1, a potent and selective anti-HCV hydroxamic acid recently reported by us. Generally favorable physicochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties exhibited by 1 made it an ideal parent compound from which activity-based protein profiling (ABPP) probe 3 was designed and synthesized. Evaluation of probe 3 revealed that it possessed necessary anti-HCV activity and selectivity. Therefore, we have successfully obtained compound 3 as a suitable ABPP probe to identify potential molecular targets of compound 1. Probe 3 and its improved analogs are expected to join a growing list of ABPP probes that have made important contributions to not only the studies of biochemical and cellular functions but also discovery of selective inhibitors of protein targets. PMID:26753813

  11. Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

    PubMed

    Giraud, Francis; Alves, Georges; Debiton, Eric; Nauton, Lionel; Théry, Vincent; Durieu, Emilie; Ferandin, Yoan; Lozach, Olivier; Meijer, Laurent; Anizon, Fabrice; Pereira, Elisabeth; Moreau, Pascale

    2011-07-14

    The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents. PMID:21623630

  12. Water Stress and Protein Synthesis

    PubMed Central

    Dhindsa, R. S.; Cleland, R. E.

    1975-01-01

    Water stress causes a reduction in hydrostatic pressure and can cause an increase in abscisic acid in plant tissues. To assess the possible role of abscisic acid and hydrostatic pressure in water stress effects, we have compared the effects of water stress, abscisic acid, and an imposed hydrostatic pressure on the rate and pattern of protein synthesis in Avena coleoptiles. Water stress reduces the rate and changes the pattern of protein synthesis as judged by a double labeling ratio technique, Abscisic acid reduces the rate but does not alter the pattern of protein synthesis. Gibberellic acid reverses the abscisic acid-induced but not the stress-induced inhibition of protein synthesis. The effect of hydrostatic pressure depends on the gas used. With a 19: 1 N2-air mixture, the rate of protein synthesis is increased in stressed but not in turgid tissues. An imposed hydrostatic pressure alters the pattern of synthesis in stressed tissues, but does not restore the pattern to that found in turgid tissues. Because of the differences in response, we conclude that water stress does not affect protein synthesis via abscisic acid or reduced hydrostatic pressure. PMID:16659167

  13. Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins.

    PubMed

    Luna, Milene S A; Hortencio, Thiago M A; Ferreira, Zulma S; Yamanouye, Norma

    2009-05-01

    The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NFkappaB and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NFkappaB and AP-1. Activation of NFkappaB and AP-1 depended on phospholipase C and protein kinase A. Activation of NFkappaB also depended on protein kinase C. Isoprenaline activated both NFkappaB and AP-1, and phenylephrine activated NFkappaB and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production. PMID:19411547

  14. 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

  15. Evidence of two mechanisms for the activation of the glucose transporter GLUT1 by anisomycin: p38(MAP kinase) activation and protein synthesis inhibition in mammalian cells.

    PubMed Central

    Barros, L F; Young, M; Saklatvala, J; Baldwin, S A

    1997-01-01

    1. Inhibitors of protein synthesis stimulate sugar transport in mammalian cells through activation of plasma membrane GLUT1, the housekeeping isoform of the glucose transporter. However, it has been reported that some of these compounds, in addition to their effect on protein synthesis, also activate protein kinases. 2. In the present study we have explored the role of these two effects on GLUT1 activation. In 3T3-L1 adipocytes and Clone 9 cells, stimulation of sugar transport by puromycin, a translational inhibitor that does not activate kinases, was not detectable until 90 min after exposure. In contrast, stimulation by anisomycin, a potent Jun-NH2-terminal kinase (JNK) agonist, exhibited no lag phase. An intermediate response was observed to emetine and cycloheximide, weak activators of JNK. 3. The potency of anisomycin to stimulate transport acutely (30 min of exposure) was 5- to 10-fold greater than for its chronic stimulation of transport, measured after 4 h of exposure. The stimulation of transport by a low concentration of anisomycin (0.3 microM) was transient, peaked at 30-60 min and it was inhibited (IC50 < 1 microM) by SB203580, which indicates that its mediator is not JNK, but the homologous p38(MAP kinase) (p38(MAPK)). In contrast, the responses to 4 h exposure to 300 microM anisomycin or puromycin were refractory to SB203580. 4. Exposure to anisomycin resulted in rapid activation of p38(MAPK). Activation of both p38(MAPK) and GLUT1 by 0.3 microM anisomycin was cancelled by puromycin. 5. We conclude that the activation of GLUT1 in response to anisomycin includes two components: a delayed component involving translational inhibition and a fast, puromycin-inhibitable component that is secondary to activation of p38(MAPK). Images Figure 2 Figure 7 PMID:9401960

  16. Plasma reciprocal pool specific activity predicts that of intracellular free leucine for protein synthesis

    SciTech Connect

    Horber, F.F.; Horber-Feyder, C.M.; Krayer, S.; Schwenk, W.F.; Haymond, M.W. )

    1989-09-01

    We previously proposed that, during the infusion of either labeled leucine or its alpha-ketoacid, alpha-ketoisocaproate (KIC), the plasma specific activity (SA) of the transaminated product of the infused tracer (reciprocal pool SA) may better reflect the intracellular leucine SA than the plasma SA of either infused tracer (primary pool SA). To test this hypothesis, 14 dogs were simultaneously infused intravenously with (3H)leucine and (14C)KIC, and blood and tissue compartments were sampled. The ratios of (3H)-leucine to (14C)leucine (3H)/(14C)leucine in mixed tissue proteins and in the intracellular space of striated muscle were the same as the ratio of the isotope infusion rates and similar, although slightly lower (P less than 0.01), than (3H)KIC/(14C)leucine SA (ratio of reciprocal pool SA) in plasma. Plasma (3H)KIC/(14C)leucine SA were essentially identical to the (3H)/(14C) of leucine in (1) mixed liver proteins, (2) intrahepatic free leucine, and (3) fibrin. The (3H)/(14C)leucine in mixed renal proteins and in the intracellular space of kidney and erythrocytes were similar to those of the venous plasma (3H)/(14C)leucine SA. The plasma (3H)KIC and (14C)leucine SA (the reciprocal pool SA) were similar to the SA of (3H)- and (14C)leucine in the intracellular space of all organs investigated with the exception of kidney. Therefore, in postabsorptive dogs, the plasma SA of the transaminated product of the infused labeled KIC or leucine is an excellent predictor of the intracellular leucine SA in all tissues investigated with the exception of kidney.

  17. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

    PubMed Central

    Uozumi, N; Sakurai, K; Sasaki, T; Takekawa, S; Yamagata, H; Tsukagoshi, N; Udaka, S

    1989-01-01

    The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes. Images PMID:2464578

  18. Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy, isolation, physico-chemical characterization and biological activity.

    PubMed

    Patel, P S; Huang, S; Fisher, S; Pirnik, D; Aklonis, C; Dean, L; Meyers, E; Fernandes, P; Mayerl, F

    1995-09-01

    Bacillaene, a novel polyene antibiotic, was discovered and isolated from fermentation broths of a strain of Bacillus subtilis. The novel antibiotic has a nominal molecular weight of 580 and an empirical formula of C35H48O7. Bacillaene is active against a broad spectrum of bacteria in agar-plate diffusion assays. Studies in vitro indicate that the antibiotic inhibits prokaryotic protein synthesis but not eukaryotic protein synthesis. Cell survival studies performed with strains of Escherichia coli indicate that the antibiotic is a bacteriostatic agent. PMID:7592068

  19. Protein Synthesis in Relation to Ripening of Pome Fruits 1

    PubMed Central

    Frenkel, Chaim; Klein, Isaac; Dilley, D. R.

    1968-01-01

    Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. 14C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases during the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment with cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis. PMID:16656897

  20. Sodium Phenylbutyrate Enhances Astrocytic Neurotrophin Synthesis via Protein Kinase C (PKC)-mediated Activation of cAMP-response Element-binding Protein (CREB)

    PubMed Central

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

    2013-01-01

    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 (Ser133) 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. PMID:23404502

  1. Chlorolissoclimides: New inhibitors of eukaryotic protein synthesis

    PubMed Central

    Robert, Francis; Gao, Hong Qing; Donia, Marwa; Merrick, William C.; Hamann, Mark T.; Pelletier, Jerry

    2006-01-01

    Lissoclimides are cytotoxic compounds produced by shell-less molluscs through chemical secretions to deter predators. Chlorinated lissoclimides were identified as the active component of a marine extract from Pleurobranchus forskalii found during a high-throughput screening campaign to characterize new protein synthesis inhibitors. It was demonstrated that these compounds inhibit protein synthesis in vitro, in extracts prepared from mammalian and plant cells, as well as in vivo against mammalian cells. Our results suggest that they block translation elongation by inhibiting translocation, leading to an accumulation of ribosomes on mRNA. These data provide a rationale for the cytotoxic nature of this class of small molecule natural products. PMID:16540697

  2. Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.

    PubMed Central

    Bulleid, N J; Bassel-Duby, R S; Freedman, R B; Sambrook, J F; Gething, M J

    1992-01-01

    Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1520279

  3. 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...

  4. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

    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. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were tr...

  5. "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…

  6. 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

  7. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

    PubMed

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

    2016-03-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278. PMID:26981393

  8. Design, synthesis, and protein methyltransferase activity of a unique set of constrained amine containing compounds.

    PubMed

    Zhou, Hao; Che, Xin; Bao, Guochen; Wang, Na; Peng, Li; Barnash, Kimberly D; Frye, Stephen V; James, Lindsey I; Bai, Xu

    2016-09-15

    Epigenetic alterations relate to various human diseases, and developing inhibitors of Kme regulatory proteins is considered to be a new frontier for drug discovery. We were inspired by the known multicyclic ligands, UNC669 and UNC926, which are the first reported small molecule ligands for a methyl-lysine binding domain. We hypothesized that reducing the conformational flexibility of the key amine moiety of UNC669 would result in a unique set of ligands. Twenty-five novel compounds containing a fused bi- or tricyclic amine or a spirocyclic amine were designed and synthesized. To gauge the potential of these amine-containing compounds to interact with Kme regulatory proteins, the compounds were screened against a panel of 24 protein methyltransferases. Compound 13 was discovered as a novel scaffold that interacts with SETD8 and could serve as a starting point for the future development of PKMT inhibitors. PMID:27528434

  9. Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK.

    PubMed

    Garcia, Bibian; Martinez-de-Mena, Raquel; Obregon, Maria-Jesus

    2012-10-01

    Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat. PMID:22766489

  10. Enhancement of RNA Synthesis, Protein Synthesis, and Abscission by Ethylene

    PubMed Central

    Abeles, F. B.; Holm, R. E.

    1966-01-01

    Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed. PMID:16656405

  11. 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.

  12. 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.

  13. Facile synthesis of borofragments and their evaluation in activity-based protein profiling

    PubMed Central

    Adachi, Shinya; Cognetta, Armand B.; Niphakis, Micah J.; He, Zhi; Zajdlik, Adam; St Denis, Jeffrey D.; Scully, Conor C. G.; Cravatt, Benjamin F.

    2015-01-01

    The discovery of enzyme inhibitors relies on synthetic methods that enable rapid and modular construction of small molecules. Heterocyclic fragments designed to maximize enthalpic interactions with their protein targets represent a particularly desirable class of molecules. Here we describe a reagent that enables straightforward construction of “borofragments”, in which a heterocycle is separated from the boron center by two or three rotatable bonds. The stability of these molecules depends on the MIDA group which likely acts as a slow-release element under biological conditions. Borofragments can be used to discover inhibitors of enzymes that use catalytic oxygen nucleophiles. We have employed this method to identify inhibitors of ABHD10 and the predicted carboxypeptidase CPVL. This technique should be applicable to other classes of targets. PMID:25633248

  14. Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation.

    PubMed

    Jacobs, René L; Lingrell, Susanne; Dyck, Jason R B; Vance, Dennis E

    2007-02-16

    5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK. PMID:17179149

  15. Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

    SciTech Connect

    Ogura, Hirotsugu; Tsukumo, Yoshinori; Sugimoto, Hikaru; Igarashi, Masayuki; Nagai, Kazuo; Kataoka, Takao

    2008-04-01

    The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in a dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.

  16. 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...

  17. 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)

  18. The solid phase synthesis of a protein activator for lecithin-cholesterol acyltransferase corresponding to human plasma apoC-I.

    PubMed Central

    Sigler, G F; Soutar, A K; Smith, L C; Gotto, A M; Sparrow, J T

    1976-01-01

    Apolipoprotein C-I, a protein constituent of the very low density lipoproteins of human plasma, consists of a single chain of 57 amino acids. The total synthesis of a protein corresponding to apolipoprotein C-I in physical properties and compositions was accomplished by solid phase techniques employing a modified polystrene incorporating spacer groups between the point of attachment of the first residue and the polymer matrix. The synthetic apoprotein was shown to activate lecithin:cholesterol acyltransferase to the same extent as the native protein. Comparative lipid-binding studies with dimyristoyl phosphatidylcholine gave complexes for native and synthetic apoprotein which floated at the same density after ultracentrifugation in KBr gradients and had virtually the same lipid:protein ratios. Images PMID:179085

  19. The solid phase synthesis of a protein activator for lecithin-cholesterol acyltransferase corresponding to human plasma apoC-I.

    PubMed

    Sigler, G F; Soutar, A K; Smith, L C; Gotto, A M; Sparrow, J T

    1976-05-01

    Apolipoprotein C-I, a protein constituent of the very low density lipoproteins of human plasma, consists of a single chain of 57 amino acids. The total synthesis of a protein corresponding to apolipoprotein C-I in physical properties and compositions was accomplished by solid phase techniques employing a modified polystrene incorporating spacer groups between the point of attachment of the first residue and the polymer matrix. The synthetic apoprotein was shown to activate lecithin:cholesterol acyltransferase to the same extent as the native protein. Comparative lipid-binding studies with dimyristoyl phosphatidylcholine gave complexes for native and synthetic apoprotein which floated at the same density after ultracentrifugation in KBr gradients and had virtually the same lipid:protein ratios. PMID:179085

  20. Noradrenaline synthesis after sympathetic nerve activation in rat atria and its dependence on calcium but not CAM kinase II and protein kinases A or C.

    PubMed Central

    Kotsonis, P.; Binko, J.; Majewski, H.

    1996-01-01

    1. The biosynthesis of noradrenaline following sympathetic nerve activation was investigated in rat atria. In particular the time course of noradrenaline synthesis changes, the relationship of changes in synthesis to transmitter release and the possible roles of second messengers and protein kinases were examined. 2. Rat atria incubated with the precursor [3H]-tyrosine synthesized [3H]-noradrenaline. Synthesis was enhanced following pulsatile electrical field stimulation (3 Hz for 5 min) with the bulk of the increase occurring in the first 45 min after the commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [3H]-noradrenaline and the radioactive outflow in response to electrical field stimulation (3 Hz for 5 min) was taken as an index of noradrenaline release. 3. Stimulation-induced (S-I) noradrenaline synthesis was significantly correlated to S-I noradrenaline release for a variety of procedures which modulate noradrenaline release by mechanisms altering Ca2+ entry into the neurone (r2 = 0.99): those which decreased release: tetrodotoxin (0.3 microM), Ca(2+)-free medium, lowering the frequency of nerve activation to 1 Hz, and those which increased release, tetraethylammonium (0.3 mM), phentolamine (1 microM) and the combination of phentolamine (1 microM) and adenosine (10 microM). On the strength of this relationship we suggest that Ca2+ entry is a determining factor in S-I synthesis changes rather than the amount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibitor KN-62 (10 microM) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4. The cell permeable cyclic AMP analogue, 8-bromoadenosine 3',5'-monophosphate (BrcAMP, 90 and 270 microM), dose-dependently increased basal [3H]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) antagonist, Rp-8

  1. Activation of macrophage peroxisome proliferator-activated receptor-gamma by diclofenac results in the induction of cyclooxygenase-2 protein and the synthesis of anti-inflammatory cytokines.

    PubMed

    Ayoub, Samir S; Botting, Regina M; Joshi, Amrish N; Seed, Michael P; Colville-Nash, Paul R

    2009-07-01

    Cyclooxygenase-2 (COX-2) is an inducible isoform of the COX family of enzymes central to the synthesis of pro-inflammatory prostaglandins. Induction of COX-2 is mediated by many endogenous and exogenous molecules that include pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS). It has been demonstrated that COX-2 can also be induced by diclofenac in cultured J774.2 macrophages. This induction was delayed compared to COX-2 induced by LPS and paracetamol selectively inhibited activity of this protein. The aim of the present study was to determine the transcription factor involved in the production of COX-2 after treatment of J774.2 cells with 500 microM diclofenac. Pre-treatment of cells with the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) antagonists GW9662 (0.1-1 microM) or biphenol A Diglycidyl Ether (100-200 microM) resulted in reduction of the induction of COX-2 by diclofenac, but not by LPS. Induction of COX-2 by the PPAR-gamma agonist 15deoxyDelta(12,14)prostaglandin J(2) was also reduced when the cells were pre-treated with the PPAR-gamma antagonists BADGE or GW9662. On the other hand, pre-treatment of cells with the nuclear factor-kappa-B (NF-kappaB) Super-repressor IkappaBalpha (150-600 nM) reduced the induction of COX-2 by LPS, but not by diclofenac. We, therefore, have identified that PPAR-gamma activation is a requirement for COX-2 induction after diclofenac stimulation of J774.2 cells. These results along with the finding that treatment of J774.2 macrophages with diclofenac resulted in the release of the anti-inflammatory cytokines, interleukin-10 and transforming growth factor-beta suggest that the diclofenac-induced COX-2 protein may possess anti-inflammatory actions. PMID:19219624

  2. EFFECT OF THE LEUCINE ANALOGS, ALPHA-KETOISOCAPROIC ACID (KIC) AND NORLEUCINE, ON MUSCLE PROTEIN SYNTHESIS AND TRANSLATION INITIATION FACTOR ACTIVATION IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonatal pigs; however, the chemical structure responsible for this effect has not been identified. We have shown that isoleucine and valine are not able to stimulate protein synthesis when raised in plasma withi...

  3. 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,…

  4. Interactions between the Fusarium toxin deoxynivalenol and lipopolysaccharides on the in vivo protein synthesis of acute phase proteins, cytokines and metabolic activity of peripheral blood mononuclear cells in pigs.

    PubMed

    Kullik, K; Brosig, B; Kersten, S; Valenta, H; Diesing, A-K; Panther, P; Reinhardt, N; Kluess, J; Rothkötter, H-J; Breves, G; Dänicke, S

    2013-07-01

    The in vivo effects of the Fusarium toxin deoxynivalenol (DON) on albumin and fibrinogen synthesis in pigs and metabolic activity of porcine peripheral blood mononuclear cells (PBMCs) were studied alone or in combination with lipopolysaccharides (LPSs) in order to examine proposed synergistic effects of both substances. A total of 36 male castrated pigs (initial weight of 26 kg) were used. Uncontaminated (Control) and naturally DON-contaminated (chronic oral DON, 3.1mg/kg diet) wheat was fed for 37 days. On the day of protein synthesis measurement, pigs recruited from the Control group were treated once intravenously with (iv) DON (100 μg/kg live weight (LW)/h), iv LPS (7.5 μg/kgLW/h) or a combination of both substances, and six pigs from the chronic oral group were treated once with iv LPS. A treatment with DON alone exhibited no alterations of acute phase protein synthesis and metabolic activity of PBMC. There was no evidence that the chosen dosing regimen of DON had influences on the induced sub-acute stage of sepsis, as the LPS challenge, irrespective of DON co-exposure, mediated an acute phase reaction with a typical decrease of albumin synthesis, as well as changes in cytokine concentration and a loss of metabolic activity in PBMC. PMID:23500770

  5. 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)

  6. 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. PMID:27114253

  7. Storage Protein Synthesis in Maize

    PubMed Central

    Larkins, Brian A.; Bracker, Charles E.; Tsai, C. Y.

    1976-01-01

    Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg2+ contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels. Images PMID:16659563

  8. T-2 mycotoxin inhibits mitochondrial protein synthesis

    SciTech Connect

    Pace, J.G.; Watts, M.R.; Canterbury, W.J.

    1988-01-01

    The authors investigated the effect of T-2 toxin on rat liver mitochondrial protein synthesis. Isolated rat liver mitochondria were supplemented with an S-100 supernatant from rat liver and an external ATP-generating system. An in-vitro assay employing cycloheximide, and inhibitor of cytoplasmic protein synthesis, and chloramphenicol, and inhibitor of mitochondrial protein synthesis, to distinguish mitochondrial protein synthesis from the cytoplasmic process. Amino acid incorporation into mitochondria was dependent on the concentration of mitochondria and was inhibited by chloramphenicol. The rate of uptake of tritium leucine into mitochondrial protein was unaffected by the addition of T-2 toxin and was not a rate-limiting step in incorporation. However, 0.02 micrograms/ml of T-2 toxin decreased the rate of protein synthesis inhibition correlated with the amount of T-2 toxin taken up by the mitochondria. While T-2 toxin is known to inhibit eukaryotic protein synthesis, this is the first time T-2 was shown to inhibit mitochondrial protein synthesis.

  9. Synthesis, Protein Levels, Activity and Phosphorylation State of Tyrosine Hydroxylase in Mesoaccumbens and Nigrostriatal Dopamine Pathways of Chronically Food-restricted Rats

    PubMed Central

    Pan, Yan; Berman, Yemiliya; Haberny, Sandra; Meller, Emanuel; Carr, Kenneth D.

    2006-01-01

    Chronic food restriction (FR) enhances the rewarding and motor-activating effects of abused drugs, and is accompanied by changes in dopamine (DA) dynamics and increased D-1 DA receptor-mediated cell signaling and transcriptional responses in nucleus accumbens (NAc). However, little is known about effects of FR on DA synthetic activity in the mesoaccumbens and nigrostriatal pathways. In Experiment 1 of the present study, tyrosine hydroxylase (TH) gene expression was measured in ventral tegmental area and substantia nigra, using real time RT-PCR and in situ hybridization; no differences were observed between FR and ad libitum fed (AL) rats. In Experiment 2, TH protein levels, determined by Western blot, were found to be elevated in NAc and caudate-putamen (CPu) of FR relative to AL rats. In the absence of increased transcription, this may reflect a slowing of TH degradation. In Experiments 3 and 4, DA synthetic activity was assessed by Western blot measurement of TH phosphorylation at Ser-40, and HPLC measurement of in vivo tyrosine hydroxylation rate, as reflected by DOPA accumulation following administration of a decarboxylase inhibitor (NSD-1015; 100 mg/kg, i.p.). Basal phospho-Ser(40)-TH levels did not differ between groups but DOPA accumulation was decreased by FR. Decreased DOPA synthesis, despite increased levels of TH protein, may reflect the inhibitory effect of increased DA binding to TH protein or decreased concentrations of cofactor tetrahydrobiopterin. Finally, in response to d-amphetamine (0.5 and 5.0 mg/kg, i.p.), phospho-Ser(40)-TH was selectively decreased in NAc of FR rats. This suggests increased feedback inhibition of DA synthesis - a possible consequence of postsynaptic receptor hypersensitivity, or increased extracellular DA concentration. These results indicate that FR increases TH protein levels, but may decrease the capacity for DA synthesis by decreasing TH activity. According to this scheme, the previously observed upregulation of striatal

  10. Cell-free translation of Drosophila C virus RNA: identification of a virus protease activity involved in capsid protein synthesis and further studies on in vitro processing of cricket paralysis virus specified proteins.

    PubMed

    Reavy, B; Moore, N F

    1983-01-01

    Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsid precursors of either of the viruses. PMID:6307220

  11. Lipoxygenase inhibitors suppress IL-2 synthesis: relationship with rise of [Ca++]i and the events dependent on protein kinase C activation.

    PubMed

    Dornand, J; Sekkat, C; Mani, J C; Gerber, M

    1987-11-01

    The present study was performed in an attempt to understand the mechanism involved in the inhibition of interleukin 2 (IL-2) synthesis by lipoxygenase (LO) pathway inhibitors. Using the two IL-2-producing lymphoid cell lines, (Jurkat and EL4 cells), we showed first that the inhibitory effect of the phenolic compounds tested (NDGA, BHA and caffeic acid) acted on lymphoid cells themselves and not on eventual monocytic or granulocytic contaminant cells. Secondly, these inhibitors were demonstrated as exerting their effect on two levels: they affected the events controlled by both second messengers implicated in T cell activation, namely rise of intracellular free calcium concentration [( Ca++]i) and protein kinase C (PKC) activation. For this purpose, LO inhibitor effects have been compared: (a) on IL-2 production by the two different lines: Jurkat cells, which need both signals, and EL4 cells, which require only PKC activation for the induction of this production; and (b) on the events induced by the different ways of Jurkat cell activation: PHA (or anti-CD3 monoclonal antibody) versus calcium ionophore. These results are discussed with respect to an eventual involvement of arachidonic acid [AA] derivatives in IL-2 synthesis. PMID:3123378

  12. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  13. 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.

  14. Monitoring protein synthesis in single live cancer cells.

    PubMed

    Tu, Chengyi; Santo, Loredana; Mishima, Yuko; Raje, Noopur; Smilansky, Zeev; Zoldan, Janet

    2016-05-16

    Protein synthesis is generally under sophisticated and dynamic regulation to meet the ever-changing demands of a cell. Global up or down-regulation of protein synthesis and the shift of protein synthesis location (as shown, for example, during cellular stress or viral infection) are recognized as cellular responses to environmental changes such as nutrient/oxygen deprivation or to alterations such as pathological mutations in cancer cells. Monitoring protein synthesis in single live cells can be a powerful tool for cancer research. Here we employed a microfluidic platform to perform high throughput delivery of fluorescent labeled tRNAs into multiple myeloma cells with high transfection efficiency (∼45%) and high viability (>80%). We show that the delivered tRNAs were actively recruited to the ER for protein synthesis and that treatment with puromycin effectively disrupted this process. Interestingly, we observed the scattered distribution of tRNAs in cells undergoing mitosis, which has not been previously reported. Fluorescence lifetime analysis detected extensive FRET signals generated from tRNAs labeled as FRET pairs, further confirming that the delivered tRNAs were used by active ribosomes for protein translation. Our work demonstrates that the microfluidic delivery of FRET labeled tRNAs into living cancer cells can provide new insights into basic cancer metabolism and has the potential to serve as a platform for drug screening, diagnostics, or personalized medication. PMID:26956582

  15. Reduction of liver function delays resumption of postpartum ovarian activity and alters the synthesis of acute phase proteins in dairy cows.

    PubMed

    Montagner, Paula; Krause, Ana Rita Tavares; Schwegler, Elizabeth; Weschenfelder, Marina Menoncin; Rabassa, Viviane Rohrig; Schneider, Augusto; Pereira, Rubens Alves; Brauner, Cássio Cassal; Del Pino, Francisco Augusto Burkert; Gonçalves, Fernanda Medeiros; Corrêa, Marcio Nunes

    2016-06-01

    The aim of this study was to evaluate the concentration of acute phase proteins, milk production, and resumption of postpartum ovarian activity of clinically healthy dairy cows in a semi-extensive system with different Liver Functionality Index (LFI) values. The animals were divided into two groups: Low LFI (LLFI: -7 to -12; n: 10) and High LFI (HLFI: -7 to -4; n: 10). Animals with LLFI had lower paraoxonase activity and lower albumin concentration in the pre- and postpartum periods (P<0.05), higher non-esterified fatty acids prepartum (P<0.005), and higher haptoglobin concentration postpartum (P<0.01). The LLFI group showed lower resumption of ovarian activity until 44days postpartum (29%; P<0.05) than HLFI (86%). Milk production did not differ between groups. Therefore, this study suggests that the LFI is an important biomarker of synthesis of acute phase proteins and the first ovulation interval, and it can be used to improve the production and reproductive performance. PMID:27234541

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

    PubMed

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

    2015-07-01

    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. PMID:26054976

  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. Synthesis and structure-activity relationships of 2-amino-3-carboxy-4-phenylthiophenes as novel atypical protein kinase C inhibitors

    PubMed Central

    Titchenell, Paul M.; Hollis Showalter, H. D.; Pons, Jean-François; Barber, Alistair J.; Jin, Yafei

    2013-01-01

    Recent evidence suggests atypical protein kinase C (aPKC) isoforms are required for both TNF- and VEGF-induced breakdown of the blood-retinal barrier (BRB) and endothelial permeability to 70kDa dextran or albumin. A chemical library screen revealed a series of novel small molecule phenylthiophene based inhibitors of aPKC isoforms that effectively block permeability in cell culture and in vivo. In an effort to further elucidate the structural requirements of this series of inhibitors, we detail in this study a structure-activity relationship (SAR) built on screening hit 1, which expands on our initial pharmacophore model. The biological activity of our analogues was evaluated in models of bona fide aPKC-dependent signaling including NFκB driven-gene transcription as a marker for an inflammatory response and VEGF/TNF-induced vascular endothelial permeability. The EC50 for the most efficacious inhibitors (6, 32) was in the low nanomolar range in these two cellular assays. Our study demonstrates the key structural elements that confer inhibitory activity and highlights the requirement for electron-donating moieties off the C-4 aryl moiety of the 2-amino-3-carboxy-4-phenylthiophene backbone. These studies suggest that this class has potential for further development into small molecule aPKC inhibitors with therapeutic efficacy in a host of diseases involving increased vascular permeability and inflammation. PMID:23566515

  19. Nucleolar localization of Small G protein RhoA is associated with active RNA synthesis in human carcinoma HEp-2 cells

    PubMed Central

    LI, YUEYING; HU, YONG; CHE, LILONG; JIA, JUNHAI; CHEN, MIN

    2016-01-01

    Previous studies have demonstrated that the nuclear localization of ras homolog family member A (RhoA), with prominent concentration in the nucleolus, is a common feature in human cancer tissues and cancer cell lines. Although a previous study has demonstrated that the nuclear translocation of RhoA occurs via active transport, a process that occurs through importin α in a nuclear factor-κB-dependent manner, the mechanism, biological function and pathological meaning of the nucleolar residency of RhoA remain to be elucidated. As the cell nucleolus is the site of ribosome biosynthesis, the aim of the present study was to investigate the association between RNA synthesis and the nucleolar localization of RhoA, as well as the molecular mechanisms underlying the residency of RhoA in the nucleolus of HEp-2 (human larynx epithelial carcinoma) cells. Indirect immunofluorescence microscopy was used to evaluate the subcellular distribution of nuclear RhoA, and immunoblotting analysis was used to determine the total cellular protein level of RhoA. Consistent with the results of previous studies, untreated HEp-2 cells exhibited bright nucleolar staining, indicating an increased concentration of RhoA in the nucleoli. Treatment with actinomycin D for the inhibition of RNA synthesis caused a redistribution of RhoA from the nucleoli to the nucleoplasm with a speckled staining pattern. Immunoblotting revealed that neither the total cellular amount of RhoA nor the integrity of RhoA was affected by treatment with actinomycin D. In cells that were treated at a decreased concentration (0.05 mg/l) of actinomycin D, the redistribution of RhoA was reversible following the removal of the drug from the culture medium. However, this reversal was not observed at an increased drug concentration (1 mg/l). Overall, to the best of our knowledge, the results of the present study provide the first in situ evidence that the inhibition of RNA synthesis induces a redistribution of nucleolar RhoA to

  20. 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. PMID:25151089

  1. 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.

  2. 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. PMID:26491045

  3. The Role of Protein Synthesis in the Senescence of Leaves

    PubMed Central

    Martin, Colin; Thimann, Kenneth V.

    1972-01-01

    The senescence of oat leaves has been studied by following the loss of chlorophyll and protein and the increase of α-amino nitrogen, after detachment and darkening. Protein synthesis and the amounts of proteolytic enzymes in the leaves have been determined directly. The process of senescence is shown to be a sequential one in which protein synthesis,most probably the formation of a proteolytic enzyme with l-serine in its active center, is of prime importance. The evidence is as follows. Firstly, l-serine specifically enhances senescence, especially in presence of kinetin. Secondly, cycloheximide, which inhibits protein synthesis in other systems, delays senescence and prevents the serine enhancement. Although requiring higher concentrations, cycloheximide can be as effective as kinetin in inhibiting senescence. It is shown directly that cycloheximide prevents protein synthesis in oat leaves under the same conditions as when it prevents senescence. Thirdly, leaves have been shown to contain two proteinases, with pH optima at 3 and 7.5, whose activity increases during senescence, even though the total leaf protein is decreasing. The amounts of both these enzymes present after 3 days are clearly increased by serine, and are greatly decreased by cycloheximide or by kinetin. The role of kinetin in delaying senescence thus may rest on its ability to suppress protease formation. PMID:16657898

  4. 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.

  5. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

    PubMed Central

    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.

    2012-01-01

    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. PMID:22453275

  6. 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.

  7. Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors with anti-HIV Activity.

    PubMed

    Sancineto, Luca; Mariotti, Alice; Bagnoli, Luana; Marini, Francesca; Desantis, Jenny; Iraci, Nunzio; Santi, Claudio; Pannecouque, Christophe; Tabarrini, Oriana

    2015-12-24

    The interest in the synthesis of Se-containing compounds is growing with the discovery of derivatives exhibiting various biological activities. In this manuscript, we have identified a series of 2,2'-diselenobisbenzamides (DISeBAs) as novel HIV retroviral nucleocapsid protein 7 (NCp7) inhibitors. Because of its pleiotropic functions in the whole viral life cycle and its mutation intolerant nature, NCp7 represents a target of great interest which is not reached by any anti-HIV agent in clinical use. Using the diselenobisbenzoic scaffold, amino acid, and benzenesulfonamide derivatives were prepared and biologically profiled against different models of HIV infection. The incorporation of amino acids such as glycine and glutamate into DISeBAs 7 and 8 resulted in selective anti-HIV activity against both acutely and chronically infected cells as well as an interesting virucidal effect. DISeBAs demonstrated broad antiretroviral activity, encompassing HIV-1 drug-resistant strains including clinical isolates, as well as simian immunodeficiency virus (SIV). Time of addition experiments, along with the observed dose dependent inhibition of the Gag precursor proper processing, confirmed that their mechanism of action is based on NCp7 inhibition. PMID:26613134

  8. Synthesis and structure-activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein.

    PubMed

    Jain, Hiteshkumar D; Zhang, Chunchun; Zhou, Shuo; Zhou, Hao; Ma, Jun; Liu, Xiaoxiang; Liao, Xuebin; Deveau, Amy M; Dieckhaus, Christine M; Johnson, Michael A; Smith, Kirsten S; Macdonald, Timothy L; Kakeya, Hideaki; Osada, Hiroyuki; Cook, James M

    2008-04-15

    Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These analogues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole N(a)-H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the substituents present on the indole N(a)-H or the indole C-2 position influenced the mechanism of action of these analogues. Analogues 68 (IC(50)=10 microM) and 67 (IC(50)=19 microM) were 7-fold and 3.5-fold more potent, respectively, than 1 (IC(50)=68 microM) in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three human carcinoma cell lines: H520 (IC(50)=11.9 microM), MCF-7 (IC(50)=17.0 microM) and PC-3 (IC(50)=11.1 microM) and was equipotent with etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210 cell proliferation and may be useful tools in labeling BCRP. PMID:18321710

  9. Selective memory generalization by spatial patterning of protein synthesis

    PubMed Central

    O’Donnell, Cian; Sejnowski, Terrence J.

    2014-01-01

    Summary Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings we proposed a novel two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. PMID:24742462

  10. 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.

  11. 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

  12. EFFECT OF ANTIBIOTICS AND INHIBITORS ON M PROTEIN SYNTHESIS

    PubMed Central

    Brock, Thomas D.

    1963-01-01

    Brock, Thomas D. (Western Reserve University, Cleveland, Ohio). Effect of antibiotics and inhibitors on M protein synthesis. J. Bacteriol. 85:527–531. 1963.—This work extends the observations of Fox and Krampitz on M protein synthesis in nongrowing cells of streptococci. A survey of a large number of antibiotics and other potential inhibitors was made. Some substances bring about inhibition of fermentation and inhibit M protein synthesis because they deprive the cell of the energy needed for this process. A second group of substances inhibit growth at concentrations tenfold or more lower than they inhibit M protein synthesis. These are the antibiotics which inhibit synthesis of cell wall or other structures in growing cells, but do not affect protein synthesis. A third group of substances inhibit growth and M protein synthesis at the same concentration. These substances probably inhibit growth because they inhibit general protein synthesis, and are therefore specific inhibitors of protein synthesis. In this class are chloramphenicol, erythromycin, and the tetracyclines. Several other antibiotics of previously unknown mode of action are in this class. A fourth group of substances had no effect on M protein synthesis. No substances were found which inhibited M protein synthesis at a lower concentration than that which inhibited growth. M protein synthesis in nongrowing cells may be a useful model system for obtaining a detailed understanding of protein synthesis. PMID:14042928

  13. Synthesis and Structure of a Ternary Copper(II) Complex with Mixed Ligands of Diethylenetriamine and Picrate: DNA/Protein-Binding Property and In Vitro Anticancer Activity Studies.

    PubMed

    Shi, Ya-Ning; Zheng, Kang; Zhu, Ling; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2015-05-01

    Based on the importance of the design and synthesis of transition metal complexes with noncovalent DNA/protein-binding abilities in the field of metallo pharmaceuticals, a new mononuclear ternary copper(II) complex with mixed ligands of diethylenetriamine (dien) and picrate anion (pic), identified as [Cu(dien)(pic)](pic), was synthesized and characterized by elemental analysis, molar conductivity measurement, infrared spectrum, electronic spectral studies, and single-crystal X-ray diffractometry. The structure analysis reveals that the copper(II) complex crystallizes in the monoclinic space group P21 /c, and the copper(II) ion has a distorted square pyramidal coordination geometry. A two-dimensional supramolecular structure is formed through hydrogen bonds. The DNA/bovine serum albumin (BSA)-binding properties of the complex are explored, indicating that the complex can interact with herring sperm DNA via intercalation mode and bind to BSA responsible for quenching of tryptophan fluorescence by static quenching mechanism. The in vitro anticancer activity shows that the copper(II) complex is active against the selected tumor cell lines. PMID:25652782

  14. Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.

    PubMed

    Hong, Seok Hoon; Ntai, Ioanna; Haimovich, Adrian D; Kelleher, Neil L; Isaacs, Farren J; Jewett, Michael C

    2014-06-20

    Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins enables the creation of biopolymers, proteins, and enzymes with new chemical properties, new structures, and new functions. To achieve this, amber (TAG codon) suppression has been widely applied. However, the suppression efficiency is limited due to the competition with translation termination by release factor 1 (RF1), which leads to truncated products. Recently, we constructed a genomically recoded Escherichia coli strain lacking RF1 where 13 occurrences of the amber stop codon have been reassigned to the synonymous TAA codon (rEc.E13.ΔprfA). Here, we assessed and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from this strain. We observed the synthesis of 190±20 μg/mL of modified soluble superfolder green fluorescent protein (sfGFP) containing a single p-propargyloxy-L-phenylalanine (pPaF) or p-acetyl-L-phenylalanine. As compared to the parent rEc.E13 strain with RF1, this results in a modified sfGFP synthesis improvement of more than 250%. Beyond introducing a single NSAA, we further demonstrated benefits of CFPS from the RF1-deficient strains for incorporating pPaF at two- and five-sites per sfGFP protein. Finally, we compared our crude S30 extract system to the PURE translation system lacking RF1. We observed that our S30 extract based approach is more cost-effective and high yielding than the PURE translation system lacking RF1, ∼1000 times on a milligram protein produced/$ basis. Looking forward, using RF1-deficient strains for extract-based CFPS will aid in the synthesis of proteins and biopolymers with site-specifically incorporated NSAAs. PMID:24328168

  15. 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.

  16. 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.

  17. Synthesis and SAR study of 4-hydroxy-3-(2-hydroxynapthalene-1-yl)phenyl)-arylsulfonamides: Heat shock protein 90 (Hsp90) inhibitors with submicromolar activity in an in vitro assay

    PubMed Central

    Ganesh, Thota; Thepchatri, Pahk; Li, Lian; Du, Yuhong; Fu, Haian; Snyder, James P.; Sun, Aiming

    2008-01-01

    Heat shock protein 90 is emerging as an important target in cancer chemotherapy. In a program directed towards identifying novel chemical probes for Hsp90, we found 4-hydroxy-3-(2-hydroxynapthalene-1-yl)phenyl)benzene sulfonamide as an Hsp90 inhibitor with very weak activity. In this report we present a new and general method for the synthesis of variety of analogs around this scaffold and discuss their structure activity relationships. PMID:18762423

  18. Postnatal ontogeny of skeletal muscle protein synthesis in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The neonatal period is characterized by rapid growth and elevated rates of synthesis and accretion of skeletal muscle proteins. The fractional rate of muscle protein synthesis is very high at birth and declines rapidly with age. The elevated capacity for muscle protein synthesis in the neonatal pig ...

  19. Postnatal ontogeny of skeletal muscle protein synthesis in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The neonatal period is characterized by rapid growth and elevated rates of synthesis and accretion of skeletal muscle proteins. The fractional rate of muscle protein synthesis is very high at birth and declines rapidly with development. The elevated capacity for muscle protein synthesis in the neo...

  20. Thyroid hormone stimulation of plasma protein synthesis in cultured hepatocytes.

    PubMed

    Hertzberg, K M; Pindyck, J; Mosesson, M W; Grieninger, G

    1981-01-25

    The direct effect of thyroid hormones on hepatocellular plasma protein synthesis has been studied in primary monolayer cultures derived from chick embryo liver. The chemically defined medium used for plating and maintaining the cultures contained no other hormones, protein, or serum supplement. Addition of physiological concentrations (10 nM) of triiodothyronine or thyroxine produced 3-fold or greater increases in the rates of synthesis of fibrinogen and three other major secreted proteins. By comparison albumin, transferrin, and total protein synthesis were not substantially increased. The enhanced synthesis of selected plasma proteins could be detected 6 h after initial addition of triiodothyronine. Exposure of the cells to the hormone for only 30 min was nearly as effective as continuous exposure in eliciting the ultimate response. Triiodothyronine exerted its half-maximal effect at a concentration of 1 nM. Diminished potency was associated with less iodination of the hormone; a marked reduction was noted with di-iodinated thyronine and no stimulatory activity at all with either mono- or non-iodinated thyronine. PMID:7451459

  1. Feeding-induced time course of changes in protein synthesis in neonatal pig skeletal muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feeding increases protein synthesis by promoting translation initiation, and in the neonate, this increase is greatest in skeletal muscle. This study aimed to identify the feeding-induced time course of the changes in protein synthesis and translation factor activation in muscle. Piglets (n=36; 5-7 ...

  2. 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 ...

  3. 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...

  4. 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…

  5. 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 ...

  6. 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

  7. 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.

  8. 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. PMID:24944023

  9. Cumulative Muscle Protein Synthesis and Protein Intake Requirements.

    PubMed

    Simmons, Erin; Fluckey, James D; Riechman, Steven E

    2016-07-17

    Muscle protein synthesis (MPS) fluctuates widely over the course of a day and is influenced by many factors. The time course of MPS responses to exercise and the influence of training and nutrition can only be pieced together from several different investigations and methods, many of which create unnatural experimental conditions. Measurements of cumulative MPS, the sum synthesis over an extended period, using deuterium oxide have been shown to accurately reflect muscle responses and may allow investigations of the response to exercise, total protein intake requirements, and interaction with protein timing in free-living experimental conditions; these factors have yet to be carefully integrated. Such studies could include clinical and athletic populations to integrate nutritional and exercise recommendations and help guide their revisions to optimize the skeletal muscle function that is so important to overall health. PMID:27215586

  10. 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.

  11. Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes

    PubMed Central

    Nishikawa, Takehiro; Sunami, Takeshi; Matsuura, Tomoaki; Yomo, Tetsuya

    2012-01-01

    Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.” In vitro compartmentalization (IVC) is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment, in vitro protein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized by in vitro protein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP and β-glucuronidase are described. We discuss the future directions for this method and its applications. PMID:22957209

  12. 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...

  13. An oxygen-regulated switch in the protein synthesis machinery

    PubMed Central

    Uniacke, James; Holterman, Chet E.; Lachance, Gabriel; Franovic, Aleksandra; Jacob, Mathieu D.; Fabian, Marc R.; Payette, Josianne; Holcik, Martin; Pause, Arnim; Lee, Stephen

    2016-01-01

    SUMMARY Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eukaryotic translation initiation factor 4E (eIF4E) binding to the 7-methylguanosine (m7-GpppG) 5′cap of mRNAs1,2. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms3–6. While the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells7,8. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition9. Here, we uncover an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. PAR-CLIP10 analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array mRNAs, including the epidermal growth factor receptor (EGFR). Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5′cap and targets mRNAs to polysomes for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen tension switches the basic translation initiation machinery. PMID:22678294

  14. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis

    PubMed Central

    Nilsson, Tobias; Lundin, Camilla Rydström; Nordlund, Gustav; Ädelroth, Pia; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain. PMID:27063297

  15. Synthesis of a novel chitosan-based Ce(IV) complex with proteolytic activity in vitro toward edible biological proteins.

    PubMed

    Li, Yan; Liu, Bingjie; Liu, Zihui; Meng, Xianghong; Wang, Dongfeng

    2016-04-20

    The occurrence of enzymatic activities is attributed to proper spatial organization of functional groups from first principles. A novel chitosan-based Ce(IV) complex (CC[Ce(IV)]), an artificial metalloproteinase, was synthesized by attaching cyclen, Ce(IV), and chlorophyll-Cu(II) to a chitosan-based matrix. The enzymatic hydrolytic efficiency (HE) and the procedure of catalyzing myoglobin (Mb) by CC[Ce(IV)] in vitro were investigated using spectrophotometry, electrophoresis, and liquid chromatography. The results showed that the HE of Mb was up to 60% at 60°C within 24h, displaying a catalytic proficiency. The pseudo-first-order kinetic constant (kobs) for CC[Ce(IV)] treatment within 24h was 3.85×10(-2)h(-1), higher than that for α-chymotrypsin treatment, which was 2.63×10(-2)h(-1). Moreover, the peptide bond derived from Asp-Phe/Phe-Asp in Mb could be specifically cleaved by CC[Ce(IV)], which could simulate the functionality of α-chymotrypsin. This work provides an experimental basis for potential utilization of the chitosan-based Ce(IV) complexes in the food industry. PMID:26876839

  16. Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis.

    PubMed

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2016-05-01

    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation. PMID:26822063

  17. Pyridalyl inhibits cellular protein synthesis in insect, but not mammalian, cell lines.

    PubMed

    Moriya, Koko; Hirakura, Setsuko; Kobayashi, Jun; Ozoe, Yoshihisa; Saito, Shigeru; Utsumi, Toshihiko

    2008-09-01

    To gain insight into the mechanism of action and selectivity of the insecticidal activity of pyridalyl, the cytotoxicity of pyridalyl against various insect and mammalian cell lines was characterized by measuring the inhibition of cellular protein synthesis. When the effect of pyridalyl on the cellular protein synthesis in Sf9 cells was evaluated by measuring the incorporation of [(3)H]leucine, rapid and significant inhibition of protein synthesis was observed. However, pyridalyl did not inhibit protein synthesis in a cell-free protein synthesis system, indicating that pyridalyl does not directly inhibit protein synthesis. No obvious cytotoxicity was observed against any of the mammalian cell lines tested. In the case of insect cell lines, remarkable differences in the cytotoxicity of pyridalyl were observed: the highest cytotoxicity (IC50 mM) was found against Sf9 cells derived from Spodoptera frugiperda, whereas no obvious cytotoxicity was observed against BmN4 cells derived from Bombyx mori. Measurements of the insecticidal activity of pyridalyl against Spodoptera litura and B. mori revealed a correlation between the cytotoxicity against cultured cell lines and the insecticidal activity. From these observations, it was concluded that the selective inhibition of cellular protein synthesis by pyridalyl might contribute significantly to the insecticidal activity and the selectivity of this compound. PMID:18454491

  18. 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...

  19. Peroxidase-like Catalytic Activity of Copper-Mediated Protein-Inorganic Hybrid Nanoflowers and Nanofibers of β-Lactoglobulin and α-Lactalbumin: Synthesis, Spectral Characterization, Microscopic Features, and Catalytic Activity.

    PubMed

    Thawari, Atul Gajanan; Rao, Chebrolu Pulla

    2016-04-27

    A free Cys-SH-containing protein, β-lactoglobulin (β-LG), and another protein not possessing the same, viz., apo-α-lactoglobulin (apo-α-LA), were used in studies to demonstrate the role of this amino acid, along with its secondary structure, in the formation of a protein dimer and a protein-inorganic hybrid nanoflower and in the creation of the peroxidase-like activity of the nanomaterials produced when the proteins were treated with varying Cu(2+) concentration under different pH conditions. An increase in the pH as well as the Cu(2+) mole ratio results in increasing dimer formation in case of β-LG due to the presence of free Cys121-SH, while the dimer is not formed in case of apo-α-LA under the same conditions. The role of Cys in the dimer formation has been demonstrated both by MALDI and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies. Both of the proteins exhibited changes in their secondary structures to different extents as a function of pH, and the structures were stabilized by Cu(2+) interactions, as studied by CD and fluorescence spectroscopy. The small and spherical nanoparticles formed at pH 7 with lower equivalents of Cu(2+) join together to form larger aggregates at higher equivalents of Cu(2+). For the same concentration at pH 9, both the aggregates and the nanoflowers were noticed. However, at pH 12, the Cu(2+) binding induces the formation of fibers along with the flowers. Both the nanoflowers and nanofibers exhibited peroxidase-like activity in a catalytic manner. Nanoflowers were also shown to detect phenol in the concentration range from 10 to 200 μM. The copper-induced nanobiomaterial obtained in the case of apo-α-LA also exhibited peroxidase-like activity. Thus, this paper deals with the green synthesis of copper-induced protein (β-LG/apo-α-LA)-inorganic hybrid nanomaterials that are important due to their applications as nanobiomaterials. PMID:27049752

  20. 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

  1. Accelerated chemical synthesis of peptides and small proteins

    PubMed Central

    Miranda, Les P.; Alewood, Paul F.

    1999-01-01

    The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10–15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the “difficult” C-terminal sequence of HIV-1 proteinase (residues 81–99); fragment 65–74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize “difficult couplings,” rapid access to peptides for biological and structure–activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods. PMID:9989998

  2. Robust Chemical Synthesis of Membrane Proteins through a General Method of Removable Backbone Modification.

    PubMed

    Zheng, Ji-Shen; He, Yao; Zuo, Chao; Cai, Xiao-Ying; Tang, Shan; Wang, Zhipeng A; Zhang, Long-Hua; Tian, Chang-Lin; Liu, Lei

    2016-03-16

    Chemical protein synthesis can provide access to proteins with post-translational modifications or site-specific labelings. Although this technology is finding increasing applications in the studies of water-soluble globular proteins, chemical synthesis of membrane proteins remains elusive. In this report, a general and robust removable backbone modification (RBM) method is developed for the chemical synthesis of membrane proteins. This method uses an activated O-to-N acyl transfer auxiliary to install in the Fmoc solid-phase peptide synthesis process a RBM group with switchable reactivity toward trifluoroacetic acid. The method can be applied to versatile membrane proteins because the RBM group can be placed at any primary amino acid. With RBM, the membrane proteins and their segments behave almost as if they were water-soluble peptides and can be easily handled in the process of ligation, purification, and mass characterizations. After the full-length protein is assembled, the RBM group can be readily removed by trifluoroacetic acid. The efficiency and usefulness of the new method has been demonstrated by the successful synthesis of a two-transmembrane-domain protein (HCV p7 ion channel) with site-specific isotopic labeling and a four-transmembrane-domain protein (multidrug resistance transporter EmrE). This method enables practical synthesis of small- to medium-sized membrane proteins or membrane protein domains for biochemical and biophysical studies. PMID:26943264

  3. Ultrafast sonochemical synthesis of protein-inorganic nanoflowers

    PubMed Central

    Batule, Bhagwan S; Park, Ki Soo; Kim, Moon Il; Park, Hyun Gyu

    2015-01-01

    We developed a simple but efficient method to synthesize protein-inorganic hybrid nanostructures with a flower-like shape (nanoflowers), which relies on sonication to facilitate the synthesis of the nanoflowers. With this technique, we synthesized nanoflowers containing laccase as a model protein and copper phosphate within 5 minutes at room temperature. The resulting laccase nanoflowers yielded greatly enhanced activity, stability, and reusability, and their usefulness was successfully demonstrated by applying them in the colorimetric detection of epinephrine. The strategy developed could be used to rapidly synthesize nanoflowers for various applications in biosensor and enzyme catalysis and would expand the utilization of nanoflowers in diverse fields of biotechnology. PMID:26346235

  4. Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise.

    PubMed

    Beelen, Milou; Zorenc, Antoine; Pennings, Bart; Senden, Joan M; Kuipers, Harm; van Loon, Luc J C

    2011-06-01

    This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise. PMID:21364122

  5. 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. PMID:27046020

  6. Mitochondrial Protein Synthesis, Import, and Assembly

    PubMed Central

    Fox, Thomas D.

    2012-01-01

    The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

  7. 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

  8. 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. PMID:26875912

  9. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    PubMed

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

  10. 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…

  11. Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles

    PubMed Central

    CHAN, JENNY; KHAN, SHAKILA N.; HARVEY, ISABELLE; MERRICK, WILLIAM; PELLETIER, JERRY

    2004-01-01

    The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation. PMID:14970397

  12. Organization and Regulation of Mitochondrial Protein Synthesis.

    PubMed

    Ott, Martin; Amunts, Alexey; Brown, Alan

    2016-06-01

    Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria. PMID:26789594

  13. Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis.

    PubMed

    Wang, Jia-Xing; Fang, Ge-Min; He, Yao; Qu, Da-Liang; Yu, Min; Hong, Zhang-Yong; Liu, Lei

    2015-02-01

    Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO2 in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q. PMID:25475965

  14. Acute effects of enteral leucine supplementation of a low protein diet on muscle protein synthesis in neonatal pigs

    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 insulin and amino acids (AA), particularly leucine. We hypothesized that enteral Leu supplementation of a low protein diets in neonatal pigs will acutely in...

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

    PubMed

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

    2012-01-01

    Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications. PMID:22008973

  16. Synthesis and antimicrobial activity of squalamine analogue.

    PubMed

    Kim, H S; Choi, B S; Kwon, K C; Lee, S O; Kwak, H J; Lee, C H

    2000-08-01

    Synthesis and antimicrobial activity of squalamine analogue 2 are reported. The synthesis of 2 was accomplished from bisnoralcohol 3. The spermidine moiety was introduced via reductive amination of an appropriately functionalized 3beta-aminosterol with spermidinyl aldehyde 17 utilizing sodium triacetoxyborohydride as the reducing agent. Compound 2 shows weaker antimicrobial activity than squalamine. PMID:11003150

  17. Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells.

    PubMed

    Alcantara, Ethel H; Shin, Mee-Young; Sohn, Ho-Yong; Park, Youn-Moon; Kim, Taewan; Lim, Jae-Hwan; Jeong, Hyung-Jin; Kwon, Soon-Tae; Kwun, In-Sook

    2011-11-01

    Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0-10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 μM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 μM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 μM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation. PMID:21292464

  18. 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.

  19. 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.

  20. 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 practical…

  1. mTORC1-Independent Reduction of Retinal Protein Synthesis in Type 1 Diabetes

    PubMed Central

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

    2014-01-01

    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. Ins2Akita 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. PMID:24740573

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

    PubMed

    Marsh, E Neil G

    2014-10-21

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

  3. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones.

    PubMed

    McKee, E E; Ferguson, M; Bentley, A T; Marks, T A

    2006-06-01

    The effects of a variety of oxazolidinones, with different antibacterial potencies, including linezolid, on mitochondrial protein synthesis were determined in intact mitochondria isolated from rat heart and liver and rabbit heart and bone marrow. The results demonstrate that a general feature of the oxazolidinone class of antibiotics is the inhibition of mammalian mitochondrial protein synthesis. Inhibition was similar in mitochondria from all tissues studied. Further, oxazolidinones that were very potent as antibiotics were uniformly potent in inhibiting mitochondrial protein synthesis. These results were compared to the inhibitory profiles of other antibiotics that function by inhibiting bacterial protein synthesis. Of these, chloramphenicol and tetracycline were significant inhibitors of mammalian mitochondrial protein synthesis while the macrolides, lincosamides, and aminoglycosides were not. Development of future antibiotics from the oxazolidinone class will have to evaluate potential mitochondrial toxicity. PMID:16723564

  4. L Protein Requirement for In Vitro RNA Synthesis by Vesicular Stomatitis Virus

    PubMed Central

    Emerson, Suzanne U.; Wagner, Robert R.

    1973-01-01

    The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis. PMID:4357510

  5. 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

  6. Renal protein synthesis in diabetes mellitus: effects of insulin and insulin-like growth factor I

    SciTech Connect

    Barac-Nieto, M.; Lui, S.M.; Spitzer, A. )

    1991-06-01

    Is increased synthesis of proteins responsible for the hypertrophy of kidney cells in diabetes mellitus Does the lack of insulin, and/or the effect of insulin-like growth factor I (IGFI) on renal tubule protein synthesis play a role in diabetic renal hypertrophy To answer these questions, we determined the rates of 3H-valine incorporation into tubule proteins and the valine-tRNA specific activity, in the presence or absence of insulin and/or IGFI, in proximal tubule suspension isolated from kidneys of streptozotocin diabetic and control rats. The rate of protein synthesis increased, while the stimulatory effects of insulin and IGFI on tubule protein synthesis were reduced, early (96 hours) after induction of experimental diabetes. Thus, hypertrophy of the kidneys in experimental diabetes mellitus is associated with increases in protein synthesis, rather than with decreases in protein degradation. Factor(s) other than the lack of insulin, or the effects of IGFI, must be responsible for the high rate of protein synthesis present in the hypertrophying tubules of diabetic rats.

  7. Modeling protein synthesis from a physicist's perspective: A toy model

    NASA Astrophysics Data System (ADS)

    Basu, Aakash; Chowdhury, Debashish

    2007-10-01

    Proteins are polymers of amino acids. These macromolecules are synthesized by intracellular machines called ribosomes. Although the experimental investigation of protein synthesis has been a traditional area of research in molecular cell biology, important quantitative models of protein synthesis have been reported in research journals devoted to statistical physics and related interdisciplinary topics. From the perspective of a physicist, protein synthesis is the classical transport of interacting ribosomes on a messenger RNA (mRNA) template that dictates the sequence of the amino acids on the protein. We discuss appropriate simplification of the models and methods. In particular, we develop and analyze a simple toy model using some elementary techniques of nonequilibrium statistical mechanics and predict the average rate of protein synthesis and the spatial organization of the ribosomes in the steady state.

  8. Straightforward thiol-mediated protein labelling with DTPA: Synthesis of a highly active 111In-annexin A5-DTPA tracer

    PubMed Central

    2012-01-01

    Background Annexin A5 (anxA5) has been found useful for molecular imaging of apoptosis and other biological processes. Methods Here, we report an optimised two-step synthesis of annexin A5-diethylene triamine pentaacetic acid (DTPA) (anxA5-DTPA) for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a single purification step. The use of a recombinant annexin A5 (cys-anxA5) with a single thiol group allowed regionally specific coupling, without affecting the binding domain of cys-anxA5. Results The metal complexing capacity of anxA5-DTPA was investigated by labelling with 111In3+ and Eu3+. Binding of modified anxA5-DTPA to apoptotic cells was tested in competition experiments with a fluorescent anxA5 derivative (anxA5-FITC) using flow cytometry and compared with that of wildtype anxA5 or non-binding anxA5-DTPA (M1234-anxA5-DTPA). The binding affinity to apoptotic cells of the anxA5-DTPA conjugate does not differ from that of wildtype anxA5. Conclusions This two-step synthesis of annexin A5-DTPA resulted in biologically active anxA5-DTPA, which can be labelled with radionuclides for use in SPECT and PET imaging. PMID:22541756

  9. 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

  10. 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.

  11. Interrelation between protein synthesis, proteostasis and life span.

    PubMed

    Arnsburg, Kristin; Kirstein-Miles, Janine

    2014-02-01

    The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity. PMID:24653664

  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. PMID:24898563

  14. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  15. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.

    PubMed Central

    Semenza, G L; Wang, G L

    1992-01-01

    We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis. Images PMID:1448077

  16. Synthesis of fully active biotinylated analogues of parathyroid hormone and parathyroid hormone-related protein as tools for the characterization of parathyroid hormone receptors.

    PubMed

    Roubini, E; Duong, L T; Gibbons, S W; Leu, C T; Caulfield, M P; Chorev, M; Rosenblatt, M

    1992-04-28

    The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys13 or Lys26 residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)NH2, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1314656

  17. Synthesis, characterization and crystal structure of cobalt(III) complexes containing 2-acetylpyridine thiosemicarbazones: DNA/protein interaction, radical scavenging and cytotoxic activities.

    PubMed

    Manikandan, Rajendran; Viswanathamurthi, Periasamy; Velmurugan, Krishnaswamy; Nandhakumar, Raju; Hashimoto, Takeshi; Endo, Akira

    2014-01-01

    The synthesis, structure and biological studies of cobalt(III) complexes supported by NNS-tridentate ligands are reported. Reactions of 2-acetylpyridine N-substituted thiosemicarbazone (HL(1-3)) with [CoCl2(PPh3)2] resulted [Co(L(1-3))2]Cl (1-3) which were characterized by elemental analysis and various spectral studies. The molecular structure of the complex 1 has been determined by single crystal X-ray diffraction studies. In vitro DNA binding studies of complexes 1-3 carried out by fluorescence studies and the results revealed the binding of complexes to DNA via intercalation. The binding constant (Kb) values of complexes 1-3 from fluorescence experiments showed that the complex 3 has greater binding propensity for DNA. The DNA cleavage activity of the complexes 1 and 3 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using fluorescence spectroscopic method, which showed that the complexes 1-3 could bind strongly with BSA. The antioxidant property of the complexes was evaluated to test their free-radical scavenging ability. Furthermore, in vitro cytotoxicity of the complexes against MCF-7 and A431 cell lines was assayed which showed higher activity and efficiently vanished the cancer cells even at low concentrations. PMID:24342132

  18. MATERNAL PROTEIN HOMEOSTASIS AND MILK PROTEIN SYNTHESIS DURING FEEDING AND FASTING IN HUMANS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about amino acid (aa) and protein metabolism in lactating women. We hypothesized: 1) aa sources other than the plasma acid pool provide substrate for milk protein synthesis in humans; and 2) if albumin was one such source, then albumin fractional synthesis rate (FSR) is higher in th...

  19. 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

  20. Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

    PubMed

    Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W

    2016-06-01

    Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale. PMID:27227514

  1. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

    PubMed

    Shankaran, Mahalakshmi; King, Chelsea L; Angel, Thomas E; Holmes, William E; Li, Kelvin W; Colangelo, Marc; Price, John C; Turner, Scott M; Bell, Christopher; Hamilton, Karyn L; Miller, Benjamin F; Hellerstein, Marc K

    2016-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  2. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  3. Presynaptic protein synthesis required for NT-3-induced long-term synaptic modulation

    PubMed Central

    2011-01-01

    Background Neurotrophins elicit both acute and long-term modulation of synaptic transmission and plasticity. Previously, we demonstrated that the long-term synaptic modulation requires the endocytosis of neurotrophin-receptor complex, the activation of PI3K and Akt, and mTOR mediated protein synthesis. However, it is unclear whether the long-term synaptic modulation by neurotrophins depends on protein synthesis in pre- or post-synaptic cells. Results Here we have developed an inducible protein translation blocker, in which the kinase domain of protein kinase R (PKR) is fused with bacterial gyrase B domain (GyrB-PKR), which could be dimerized upon treatment with a cell permeable drug, coumermycin. By genetically targeting GyrB-PKR to specific cell types, we show that NT-3 induced long-term synaptic modulation requires presynaptic, but not postsynaptic protein synthesis. Conclusions Our results provide mechanistic insights into the cell-specific requirement for protein synthesis in the long-term synaptic modulation by neurotrophins. The GyrB-PKR system may be useful tool to study protein synthesis in a cell-specific manner. PMID:21211057

  4. Temperature-Regulated Protein Synthesis by Leptospira interrogans

    PubMed Central

    Nally, Jarlath E.; Timoney, John F.; Stevenson, Brian

    2001-01-01

    Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection. PMID:11119530

  5. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27098003

  7. Rational Design, Synthesis and Evaluation of Coumarin Derivatives as Protein-protein Interaction Inhibitors.

    PubMed

    De Luca, Laura; Agharbaoui, Fatima E; Gitto, Rosaria; Buemi, Maria Rosa; Christ, Frauke; Debyser, Zeger; Ferro, Stefania

    2016-09-01

    Herein we describe the design and synthesis of a new series of coumarin derivatives searching for novel HIV-1 integrase (IN) allosteric inhibitors. All new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 IN enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75. A combined approach of docking and molecular dynamic simulations has been applied to clarify the activity of the new compounds. Specifically, the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA) was calculated, whereas hydrogen bond occupancies were monitored throughout simulations methods. PMID:27546050

  8. 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.

  9. Xanthane sesquiterpenoids: structure, synthesis and biological activity.

    PubMed

    Vasas, Andrea; Hohmann, Judit

    2011-04-01

    The aim of this review is to survey the naturally occurring xanthanes and xanthanolides, their structures, biological activities, structure–activity relationships and synthesis. There has been no comprehensive review of this topic previously. On the basis of 126 references, 112 compounds are summarized. PMID:21321751

  10. Cell-free protein synthesis in microfluidic array devices.

    PubMed

    Mei, Qian; Fredrickson, Carl K; Simon, Andrew; Khnouf, Ruba; Fan, Z Hugh

    2007-01-01

    We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. PMID:17924644

  11. Multiple Post-translational Modifications Affect Heterologous Protein Synthesis*

    PubMed Central

    Tokmakov, Alexander A.; Kurotani, Atsushi; Takagi, Tetsuo; Toyama, Mitsutoshi; Shirouzu, Mikako; Fukami, Yasuo; Yokoyama, Shigeyuki

    2012-01-01

    Post-translational modifications (PTMs) are required for proper folding of many proteins. The low capacity for PTMs hinders the production of heterologous proteins in the widely used prokaryotic systems of protein synthesis. Until now, a systematic and comprehensive study concerning the specific effects of individual PTMs on heterologous protein synthesis has not been presented. To address this issue, we expressed 1488 human proteins and their domains in a bacterial cell-free system, and we examined the correlation of the expression yields with the presence of multiple PTM sites bioinformatically predicted in these proteins. This approach revealed a number of previously unknown statistically significant correlations. Prediction of some PTMs, such as myristoylation, glycosylation, palmitoylation, and disulfide bond formation, was found to significantly worsen protein amenability to soluble expression. The presence of other PTMs, such as aspartyl hydroxylation, C-terminal amidation, and Tyr sulfation, did not correlate with the yield of heterologous protein expression. Surprisingly, the predicted presence of several PTMs, such as phosphorylation, ubiquitination, SUMOylation, and prenylation, was associated with the increased production of properly folded soluble proteins. The plausible rationales for the existence of the observed correlations are presented. Our findings suggest that identification of potential PTMs in polypeptide sequences can be of practical use for predicting expression success and optimizing heterologous protein synthesis. In sum, this study provides the most compelling evidence so far for the role of multiple PTMs in the stability and solubility of heterologously expressed recombinant proteins. PMID:22674579

  12. 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.

  13. Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

    PubMed Central

    Swedes, J S; Dial, M E; McLaughlin, C S

    1979-01-01

    Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited. PMID:374362

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

    PubMed Central

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

    2013-01-01

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

  15. Bacterial Heat Shock Protein Activity

    PubMed Central

    Maleki, Farajollah; Khosravi, Afra; Nasser, Ahmad; Taghinejad, Hamid

    2016-01-01

    Bacteria are exposed to different types of stress in their growth conditions. They have developed appropriate responses, modulated by the re-modeling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive in a variety range of nature. Proteins are essential components for biologic activity in the eukaryotic and prokaryotic cell. Heat Shock Proteins (HSP) have been identified from various organisms and have critical role in cell hemostasis. Chaperone can sense environment and have different potential role in the organism evolution. PMID:27134861

  16. The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs

    PubMed Central

    Signer, Robert A.J.; Qi, Le; Zhao, Zhiyu; Thompson, David; Sigova, Alla A.; Fan, Zi Peng; DeMartino, George N.; Young, Richard A.; Sonenberg, Nahum; Morrison, Sean J.

    2016-01-01

    Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis. PMID:27492367

  17. 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

    2016-01-01

    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

  18. 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

  19. 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. PMID:26170084

  20. Acetaldehyde inhibition of protein synthesis in isolated rat pancreatic acini

    SciTech Connect

    Majumdar, A.P.; Haiman, M.J.; Zylbert, B.A.; Billy, H.T.; Vesenka, G.D.; Geokas, M.C.

    1986-03-30

    Exposure of isolated dispersed pancreatic acini to increasing concentrations of ethanol (5 to 500 mM) or acetaldehyde (0.5 to 100 mM) produced a progressive inhibition of (3H)leucine incorporation into both cellular (those remaining in the cell) and secretory (those released into the medium) proteins. Whereas 500 mM ethanol caused 90-95% inhibition in the synthesis of cellular and secretory proteins, the concentration of acetaldehyde needed to produce a similar inhibition was found to be 50 mM. All subsequent experiments were performed with 12.5 mM acetaldehyde, a concentration that consistently inhibited acinar protein synthesis by about 50%. The acetaldehyde-mediated inhibition of acinar protein synthesis was partially normalized when this metabolite was removed after 30 min during a 90-min incubation period. In the presence of acetaldehyde, the secretion of 3H-pulse-labeled proteins, but not amylase, trypsinogen, or chymotrypsinogen, was greatly depressed. Acetaldehyde also caused a marked reduction in (3H)uridine incorporation into acinar RNA. The entry of (3H)uridine, (3H)leucine, and (3H)aminoisobutyric acid into isolated acini was found to be slightly (15-25%) decreased by acetaldehyde. It is concluded that acetaldehyde exerts a direct toxic effect on isolated dispersed pancreatic acini as evidenced by diminution of both protein and RNA synthesis and decreased secretion of the newly synthesized proteins. This inhibitory effect of acetaldehyde could be partially reversed.

  1. 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

  2. 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.

  3. 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

  4. Andes Virus Nucleocapsid Protein Interrupts Protein Kinase R Dimerization To Counteract Host Interference in Viral Protein Synthesis

    PubMed Central

    Wang, Zekun

    2014-01-01

    ABSTRACT Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. IMPORTANCE Protein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the

  5. Cell-free protein synthesis systems derived from cultured mammalian cells.

    PubMed

    Brödel, Andreas K; Wüstenhagen, Doreen A; Kubick, Stefan

    2015-01-01

    We present a technology for the production of target proteins using novel cell-free systems derived from cultured human K562 cells and Chinese hamster ovary (CHO) cells. The protocol includes the cultivation of cells, the preparation of translationally active lysates, and the cell-free synthesis of desired proteins. An efficient expression vector based on the internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV) was constructed for both systems. The coupled batch-based platforms enable the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. The glycosylation of erythropoietin demonstrates the successful performance of posttranslational modifications in the novel cell-free systems. Protein yields of approximately 20 μg/ml (K562-based cell-free system) and 50 μg/ml (CHO-based cell-free system) of active firefly luciferase are obtained in the coupled transcription-translation systems within 3 h. As a result, both cell-free protein synthesis systems serve as powerful tools for high-throughput proteomics. PMID:25502197

  6. 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.

  7. 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). ...

  8. Prolonged stimulation of muscle protein synthesis by leucine in neonates is dependent on amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rise in amino acids and insulin after a meal independently stimulate protein synthesis in skeletal muscle of neonates by activating the intracellular signalling pathways that regulate mRNA translation. Leucine, in particular, is important in mediating the response to amino acids. Previously, w...

  9. 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.

  10. Proteins of the rat prostate. II. Synthesis of new proteins in the ventral lobe during castration-induced regression

    SciTech Connect

    Lee, C.; Sensibar, J.A.

    1987-10-01

    Ventral prostates from adult Sprague-Dawley rats at different days postcastration were cut into one to two mm.3 pieces and incubated in medium containing S-35-methionine (100 uCi/ml.) at 37 C under 95% oxygen and 5% carbon dioxide for four hours. The incubated tissues were subjected to two-dimensional electrophoresis and radiofluorography. Over 100 spots were developed in the fluorograms. Three groups of spots, representing cytoskeletal proteins, androgen-dependent proteins and castration-induced proteins, were further evaluated by a computer-based densitometer. The level of densitometry absorption is proportional to the amount of radioactivity in each spot. The synthesis of cytoskeletal proteins, such as actin and tropomyosin, were relatively constant throughout the course of prostatic regression. The rate of synthesis of androgen-dependent proteins declined rapidly from a high level of synthesis before castration to a non-detectable level by Day 3 postcastration. However, three proteins, which were either not synthesized (spot G and spot H) or synthesized at a very low level (spot I) before castration, were the major proteins synthesized by the prostate during early stages of its regression. The rate of synthesis of these proteins reached a peak by Day 4 postcastration, declined rapidly and remained at a low level thereafter. The respective molecular weights and isoelectric points for these three proteins were 33 Kd and 7.2 for spot G, 38 Kd and 5.3 for spot H and 64 Kd and 6.0 for spot I. Previous findings showed that prostatic regression in rats was associated with a surge of activities in proteolytic enzymes which peaked five to six days postcastration.

  11. DNA Nanoparticles for Improved Protein Synthesis In Vitro

    PubMed Central

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

    2016-01-01

    Abstract The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro. PMID:26821778

  12. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    PubMed Central

    MacBeth, K J; Lee, C A

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells. In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis. PMID:8454361

  13. Bacterial Protein Synthesis as a Target for Antibiotic Inhibition.

    PubMed

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

    Protein synthesis occurs on macromolecular machines, called ribosomes. Bacterial ribosomes and the translational machinery represent one of the major targets for antibiotics in the cell. Therefore, structural and biochemical investigations into ribosome-targeting antibiotics provide not only insight into the mechanism of action and resistance of antibiotics, but also insight into the fundamental process of protein synthesis. This review summarizes the recent advances in our understanding of protein synthesis, particularly with respect to X-ray and cryoelectron microscopy (cryo-EM) structures of ribosome complexes, and highlights the different steps of translation that are targeted by the diverse array of known antibiotics. Such findings will be important for the ongoing development of novel and improved antimicrobial agents to combat the rapid emergence of multidrug resistant pathogenic bacteria. PMID:27481773

  14. The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis.

    PubMed

    Pan, Meihui; Liang Js, Jun-shan; Fisher, Edward A; Ginsberg, Henry N

    2002-02-01

    Although microsomal triglyceride transfer protein (MTP) and newly synthesized triglyceride (TG) are critical for co-translational targeting of apolipoprotein B (apoB100) to lipoprotein assembly in hepatoma cell lines, their roles in the later stages of lipoprotein assembly remain unclear. Using N-acetyl-Leu-Leu-norleucinal to prevent proteasomal degradation, HepG2 cells were radiolabeled and chased for 0-90 min (chase I). The medium was changed and cells chased for another 150 min (chase II) in the absence (control) or presence of Pfizer MTP inhibitor CP-10447 (CP). As chase I was extended, inhibition of apoB100 secretion by CP during chase II decreased from 75.9% to only 15% of control (no CP during chase II). Additional studies were conducted in which chase I was either 0 or 90 min, and chase II was in the presence of [(3)H]glycerol and either BSA (control), CP (inhibits both MTP activity and TG synthesis),BMS-1976360-1) (BMS) (inhibits only MTP activity), or triacsin C (TC) (inhibits only TG synthesis). When chase I was 0 min, CP, BMS, and TC reduced apoB100 secretion during chase II by 75.3, 73.9, and 53.9%. However, when chase I was 90 min, those agents reduced apoB100 secretion during chase II by only 16.0, 19.2, and 13.9%. Of note, all three inhibited secretion of newly synthesized TG during chase II by 80, 80, and 40%, whether chase I was 0 or 90 min. In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions. Oleic acid increased secretion of TG-enriched apoB100-lipoproteins similarly in the absence or presence of either of CP, BMS, or TC. We conclude that neither MTP nor newly synthesized TG is necessary for the later stages of apoB100-lipoprotein assembly and secretion in either HepG2 or McA-RH7777 cells. PMID:11704664

  15. Quantifying elongation rhythm during full-length protein synthesis.

    PubMed

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

    2013-07-31

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

  16. Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

    PubMed

    Lapasset, Laure; Pradet-Balade, Bérengère; Vergé, Valérie; Lozano, Jean-Claude; Oulhen, Nathalie; Cormier, Patrick; Peaucellier, Gérard

    2008-11-01

    Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E. PMID:18361417

  17. 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

  18. 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

    2016-01-01

    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

  19. Antiviral activities of whey proteins.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  20. Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

    The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma×rat glioma hybrid cells. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5′-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A2 adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA). In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells. PMID:11959806

  1. Redundant pathways for Cdc2 activation in Xenopus oocyte: either cyclin B or Mos synthesis

    PubMed Central

    Haccard, Olivier; Jessus, Catherine

    2006-01-01

    Xenopus oocytes are arrested in meiotic prophase I. Progesterone induces the resumption of meiotic maturation, which requires continuous protein synthesis to bring about Cdc2 activation. The identification of the newly synthesized proteins has long been a goal. Two plausible candidates have received extensive study. The synthesis of cyclin B and of c-Mos, a kinase that activates the mitogen-activated protein kinase pathway in oocytes, is clearly upregulated by translational control in response to progesterone. Recent studies suggest that ablation of either c-Mos or cyclin B synthesis by antisense oligonucleotides does not block meiotic maturation. Here, however, we show that when both pathways are simultaneously inhibited, progesterone no longer triggers maturation; adding back either c-Mos or cyclin B restores meiotic maturation. We conclude that the specific synthesis of either B-type cyclins or c-Mos, induced by progesterone, is required to induce meiotic maturation. The two pathways seem to be functionally redundant. PMID:16374506

  2. 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. PMID:23388415

  3. Action of Inhibitors of RNA and Protein Synthesis on Cell Enlargement 1

    PubMed Central

    Noodén, Larry D.; Thimann, Kenneth V.

    1966-01-01

    Further studies with inhibitors of protein synthesis are presented to support the conclusion, drawn from work with chloramphenicol, that protein synthesis is a critical limiting factor in auxin-induced cell expansion. The indoleacetic acid-induced elongation of oat coleoptile sections was strongly inhibited by dl-p-fluorophenylalanine, and the inhibition is antagonized by phenylalanine. Puromycin at 10−4 m very strongly inhibited the indoleacetic acid-induced growth of oat coleoptile and artichoke tuber sections and exerted a less powerful effect on pea stem sections. As found earlier with chloramphenicol, concentrations of puromycin effective in inhibiting the growth of coleoptile sections had quantitatively similar effects on protein synthesis, as measured by the incorporation of C14-leucine into protein of the coleoptile tissue. Several analogues of RNA bases were also tested, but while 8-azaguanine very strongly inhibited growth of artichoke tuber disks, 6-azauracil was the only one of this group clearly inhibitory to growth in coleoptile or pea stem sections. Actinomycin D actively inhibited both elongation and the incorporation of C14-leucine into protein in oat coleoptile sections. Inhibition of the 2 processes went closely parallel. Actinomycin D also powerfully inhibited growth of artichoke tuber disks. All the compounds effective in inhibiting growth generally inhibited the uptake of leucine as well. The possibility that auxin causes cell enlargement in plants by inducing the synthesis of a messenger RNA and of one or more new but unstable enzymes, is discussed. Possible but less favored alternative explanations are: A) that auxin induces synthesis of a wall protein, or B) that the continued synthesis of some other unstable protein (by a process independent of auxin) may be a prerequisite for cell enlargement. PMID:5904588

  4. 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...

  5. Semi-synthesis of labeled proteins for spectroscopic applications.

    PubMed

    De Rosa, Lucia; Russomanno, Anna; Romanelli, Alessandra; D'Andrea, Luca Domenico

    2013-01-01

    Since the introduction of SPPS by Merrifield in the 60s, peptide chemists have considered the possibility of preparing large proteins. The introduction of native chemical ligation in the 90s and then of expressed protein ligation have opened the way to the preparation of synthetic proteins without size limitations. This review focuses on semi-synthetic strategies useful to prepare proteins decorated with spectroscopic probes, like fluorescent labels and stable isotopes, and their biophysical applications. We show that expressed protein ligation, combining the advantages of organic chemistry with the easy and size limitless recombinant protein expression, is an excellent strategy for the chemical synthesis of labeled proteins, enabling a single protein to be functionalized at one or even more distinct positions with different probes. PMID:23282535

  6. 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

  7. Activation and de novo synthesis of hydrogenase in chlamydomonas.

    PubMed

    Roessler, P G; Lien, S

    1984-12-01

    Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase. PMID:16663954

  8. Activation and de novo synthesis of hydrogenase in Chlamydomonas

    SciTech Connect

    Roessler, P.G.; Lien, S.

    1984-12-01

    Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogenase in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process. Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg/sup 2 +/, Ca/sup 2 +/, and iron does not lead to active hydrogenase formation. Furthermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place. The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.

  9. Discovery and analysis of 4H-pyridopyrimidines, a class of selective bacterial protein synthesis inhibitors.

    PubMed

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

    2010-11-01

    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 (IC(50)s) 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

  10. 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.

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

    PubMed Central

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

    2007-01-01

    Recent findings suggest that functional plasticity phenomena such as long-term potentiation (LTP) and long-term depression (LTD) – cellular processes underlying memory – are restricted to functional dendritic compartments. It was also shown, however, that a relatively strong activation of a synaptic input can abolish compartment restrictions. Our data support these findings and we present one cellular pathway responsible for uncompartmentalization of the normally localized plasticity processes by the action of rolipram, an inhibitor of type 4 phosphodiesterases. In contrast with compartment-restricted information processing, uncompartmentalization requires transcription. In the search for system relevance of compartmentalization versus uncompartmentalization we describe firstly data which show that more cognitive information processing in rats' behaviour may follow rules of compartmentalization, whereas stressful, more life-threatening, inputs abolish compartment-restricted information processing involving transcription. Our findings allow us to suggest that consolidation of processes which take place during the cognitive event most probably depend on local protein synthesis, whereas stress immediately induces gene expression in addition, resulting in a compartment-unspecific up-regulation of plasticity-related proteins (PRPs), providing the entire neuron with a higher level of ‘reactiveness’. These data would provide a specific functional cellular mechanism to respond differentially and effectively to behaviourally weighted inputs. PMID:17702813

  12. 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...

  13. 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. PMID:26541872

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

    PubMed Central

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

    2013-01-01

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

  15. In vitro protein synthesis capacities in a cold stenothermal and a temperate eurythermal pectinid.

    PubMed

    Storch, D; Heilmayer, O; Hardewig, I; Pörtner, H-O

    2003-09-01

    The translational system was isolated from the gills of the Antarctic scallop Adamussium colbecki (Smith) and the European scallop Aequipecten opercularis (Linnaeus) for in vitro protein synthesis capacities microg protein mg FW(-1) day(-1)) and the translational capacities of RNA (k(RNA in vitro) mg protein mg RNA(-1) day(-1)). In vitro protein synthesis capacity in the cold-adapted pectinid at 0 degrees C was similar to the one found in the temperate scallop at 25 degrees C. These findings might reflect cold compensated rates in Adamussium colbecki, partly explainable by high tissue levels of RNA. Cold-compensated in vitro protein synthesis capacities may further result from increments in the translational capacity of RNA. The thermal sensitivity of the translation machinery was slightly different in the two species, with significantly lower levels of Arrhenius activation energies E(a) and Q(10) in Adamussium colbecki in the temperature range 0-15 degrees C. Reduced protein synthesis and translational capacities were found in vitro in gills of long-term aquarium-maintained Adamussium colbecki and were accounted for by a loss of protein synthesis machinery, i.e. a reduction in RNA levels, as well as a decrease in the amount of protein synthesized per milligram of RNA (RNA translational capacity, k(RNA in vitro)). Such changes may involve food uptake or mirror metabolic depression strategies, like those occurring during winter. Consequences of high in vitro RNA translational capacities found in the permanently cold-adapted species are discussed in the context of seasonal food availability and growth rates at high latitudes. PMID:12905006

  16. Characterization of the proteostasis roles of glycerol accumulation, protein degradation and protein synthesis during osmotic stress in C. elegans.

    PubMed

    Burkewitz, Kristopher; Choe, Keith P; Lee, Elaine Choung-Hee; 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 brought

  17. 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...

  18. 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)

  19. 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…

  20. The Development of an Interactive Videodisc Program on Protein Synthesis.

    ERIC Educational Resources Information Center

    Hazan, Charlene Corey

    An interactive videodisk (IVD) program was developed to reinforce learning of the biological concept of protein synthesis for high school students. The laser videodisc "The Living Textbook Life Science" was the source of frames, and the authoring system of G. Smith was used to create the disc. The interactive program was designed to make the…

  1. 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…

  2. Enhanced skeletal muscle protein synthesis rates in pigs treated with somatotropin requires fed amino acids levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic somatotropin (pST) treatment in pigs increases skeletal muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin alone could not account for the pST-induced increase in protein synthesis. This study...

  3. AMINO ACIDS AUGMENT MUSCLE PROTEIN SYNTHESIS IN NEONATAL PIGS DURING ENDOTOXEMIA BY MODULATING TRANSLATION INITIATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In adults, sepsis reduces protein synthesis in skeletal muscle by restraining translation. The effect of sepsis on amino acid-stimulated muscle protein synthesis has not been determined in neonates, a population who is highly anabolic and whose muscle protein synthesis rates are uniquely sensitive ...

  4. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  5. Role of N-acetylglutamate turnover in urea synthesis of rats given proteins of different quality.

    PubMed

    Tujioka, Kazuyo; Fukaya, Yuka; Sano, Atushi; Hayase, Kazutoshi; Yokogoshi, Hidehiko

    2005-04-01

    The purpose of this study was to find whether the synthesis and degradation of N-acetylglutamate would affect urea synthesis when the dietary protein quality was manipulated. Experiments were done on three groups of rats given diets containing 10 g gluten, 10 g casein or 10 g whole egg protein/100 g for 10 d. The urinary excretion of urea, the liver concentrations of N-acetylglutamate and free glutamate, the liver activity of N-acetylglutamate synthetase increased with the decline in quality of dietary protein. A reverse correlation was observed between the liver N-acetylglutamate degradation and liver Nacetylglutamate concentration. N-Acetylglutamate concentration in the liver was closely correlated with the concentration of glutamate and the N-acetylglutamate synthetase activity in the liver, and excretion of urea. These results suggest that the greater synthesis and the lower degradation rate of N-acetylglutamate in the liver of rats given the lower quality of protein increase the liver concentration of N-acetylglutamate and stimulate urea synthesis. PMID:16022195

  6. Dietary protein distribution positively influences 24-h muscle protein synthesis in healthy adults.

    PubMed

    Mamerow, Madonna M; Mettler, Joni A; English, Kirk L; Casperson, Shanon L; Arentson-Lantz, Emily; Sheffield-Moore, Melinda; Layman, Donald K; Paddon-Jones, Douglas

    2014-06-01

    The RDA for protein describes the quantity that should be consumed daily to meet population needs and to prevent deficiency. Protein consumption in many countries exceeds the RDA; however, intake is often skewed toward the evening meal, whereas breakfast is typically carbohydrate rich and low in protein. We examined the effects of protein distribution on 24-h skeletal muscle protein synthesis in healthy adult men and women (n = 8; age: 36.9 ± 3.1 y; BMI: 25.7 ± 0.8 kg/m2). By using a 7-d crossover feeding design with a 30-d washout period, we measured changes in muscle protein synthesis in response to isoenergetic and isonitrogenous diets with protein at breakfast, lunch, and dinner distributed evenly (EVEN; 31.5 ± 1.3, 29.9 ± 1.6, and 32.7 ± 1.6 g protein, respectively) or skewed (SKEW; 10.7 ± 0.8, 16.0 ± 0.5, and 63.4 ± 3.7 g protein, respectively). Over 24-h periods on days 1 and 7, venous blood samples and vastus lateralis muscle biopsy samples were obtained during primed (2.0 μmol/kg) constant infusion [0.06 μmol/(kg⋅min)] of l-[ring-(13)C6]phenylalanine. The 24-h mixed muscle protein fractional synthesis rate was 25% higher in the EVEN (0.075 ± 0.006%/h) vs. the SKEW (0.056 ± 0.006%/h) protein distribution groups (P = 0.003). This pattern was maintained after 7 d of habituation to each diet (EVEN vs. SKEW: 0.077 ± 0.006 vs. 0.056 ± 0.006%/h; P = 0.001). The consumption of a moderate amount of protein at each meal stimulated 24-h muscle protein synthesis more effectively than skewing protein intake toward the evening meal. PMID:24477298

  7. Fibronectin fragments alter matrix protein synthesis in cartilage tissue cultured in vitro.

    PubMed

    Xie, D; Hui, F; Homandberg, G A

    1993-11-15

    We reported earlier that fibronectin fragments (Fn-f) added to bovine articular cartilage cultured in serum-free culture causes marked protease expression with resultant proteoglycan (PG) degradation and release into the culture media. We have further characterized the effects of Fn-f by studies of the effects on proteoglycan, collagen, general protein, and DNA synthesis and reversibility of the cartilage damage. We report here that the most active Fn-f, a 29-kDa amino-terminal Fn-f, when added to a 1 microM concentration, depressed PG and general protein synthesis in cartilage by over 50% within 24 h, as measured by sulfate and methionine/cysteine incorporation, respectively. This same Fn-f decreased PG synthesis throughout the full thickness cartilage section as shown by autoradiography. PG and general protein synthesis were significantly depressed within 24 h by 29-kDa Fn-f concentrations as low as 10 nM. Synthesis rates were effected by 100-fold lower Fn-f concentrations than was induction of proteinases. Removal of the 29-kDa Fn-f allowed a gain to supernormal levels of PG and protein synthesis. Cartilage damaged to the extent of removal of over 50% of the total PG did not replace PG after over 4 weeks in 10% serum-Dulbecco's modified Eagle minimum with or without added TGF-b1 and rIGF-a. These data show that while the effects of Fn-f on elevating protease expression and depressing PG synthesis are reversible, the resultant cartilage damage is apparently irreversible in vitro. Therefore, if Fn-f-mediated cartilage damage occurs as part of cartilage disease processes, the pathologic effects would be quite significant. PMID:8239647

  8. Marine Natural Meroterpenes: Synthesis and Antiproliferative Activity

    PubMed Central

    Simon-Levert, Annabel; Menniti, Christophe; Soulère, Laurent; Genevière, Anne-Marie; Barthomeuf, Chantal; Banaigs, Bernard; Witczak, Anne

    2010-01-01

    Meroterpenes are compounds of mixed biogenesis, isolated from plants, microorganisms and marine invertebrates. We have previously isolated and determined the structure for a series of meroterpenes extracted from the ascidian Aplidium aff. densum. Here, we demonstrate the chemical synthesis of three of them and their derivatives, and evaluate their biological activity on two bacterial strains, on sea urchin eggs, and on cancerous and healthy human cells. PMID:20390109

  9. Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis 1

    PubMed Central

    Travis, R. L.; Jordan, W. R.; Huffaker, R. C.

    1969-01-01

    The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems. PMID:16657182

  10. Synthesis of pharmacologically active indoles.

    PubMed

    Hishmat, O H; Ebeid, M Y; Nakkady, S S; Fathy, M M; Mahmoud, S S

    1999-06-01

    Formylation of 6-methoxy-1-methyl and 5-methyl,2,3-diphenyl-1H-indole (Ib and IX) gave the 5- and 6- carboxaldehyde derivatives (II and X) respectively, which were treated with ethyl cyanoacetate to form the corresponding 2-cyano-3-substituted acrylic acid ethyl ester (III and XI). The latter compounds reacted with hydrazine hydrate, urea and thiourea to form the corresponding 5-amino-4-substituted 2,4,dihydropyrazol-3- one (IV), 6-indolyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile s (V and XII) and 6-indolyl-4-oxo-2-thixo-1,2,3,4-tetrahydropyrimidine-5-ca rbonitriles (VI and XIII). Reaction of the 5- and 6-carboxaldehyde derivatives with malononitrile afforded the 2-substituted malononitrile derivatives (VII and XIV). VII and XIV reacted readilly with aromatic ketones to give the 2-amino4,6-disubstituted nicotinonitriles (VIII a,b and XVa,b). The biological activity of compounds Ia, Ib, II, III, IX and X was tested for antiinflammatory, ulcerogenic and antispasmodic activities. PMID:10464975

  11. The influence of the ratio of protein energy to total energy in the feed on the activity of protein synthesis in vitro, the level of ribosomal RNA and the RNA-DNA ratio in white trunk muscle of Atlantic cod (Gadus morhua).

    PubMed

    Lied, E; Rosenlund, G

    1984-01-01

    Cod (Gadus morhua) were fed diets containing protein energy to total energy levels (PE/TE) of 10.0, 20.6, 29.6, 38.4, 56.2 and 74.1% for 21 days. Ribosomes were isolated from the white trunk muscle tissue, the capacity for protein synthesis in vitro determined and related to muscle tissue wet weight rRNA and DNA. Protein concentrations of less than 47.4% PE/TE in the diets reduce the ribosomal capacity for protein synthesis per g wet weight and per mg DNA, and the tissue contents of rRNA and ratio of rRNA/DNA. The capacity for muscle protein synthesis in vitro is a significant and sensitive parameter of protein inadequacy in fish diets. PMID:6200270

  12. Coumarin heterocyclic derivatives: chemical synthesis and biological activity.

    PubMed

    Medina, Fernanda G; Marrero, Joaquín G; Macías-Alonso, Mariana; González, Magdalena C; Córdova-Guerrero, Iván; Teissier García, Ariana G; Osegueda-Robles, Soraya

    2015-09-23

    This review highlights the broad range of science that has arisen from the synthesis of coumarin-linked and fused heterocycle derivatives. Specific topics include their synthesis and biological activity. PMID:26151411

  13. 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.

  14. Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

    PubMed Central

    Simonetti, Leandro; Duffy, Tomas; Longhi, Silvia A.; Gómez, Karina A.; Hoebeke, Johan; Levin, Mariano J.; Smulski, Cristian R.

    2012-01-01

    The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope. PMID:22570698

  15. Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

    PubMed Central

    Drocourt, J L; Dieffenbach, C W; Ts'o, P O; Justesen, J; Thang, M N

    1982-01-01

    The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease. PMID:7079179

  16. Do maise and wheat chloroplast protein synthesis elongation factor, EF-Tu, protect Rubisco activase from thermal aggregation and inactivation?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize (Zea mays L.) chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in the development of heat tolerance. The precursor of this protein (pre-EF-Tu) has been shown to display chaperone activity, as it protected heat labile citrate synthase and malate dehydrogenase from the...

  17. 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. PMID:16014383

  18. Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

    PubMed Central

    Fogg, Mark H.; Teterina, Natalya L.; Ehrenfeld, Ellie

    2003-01-01

    Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation. PMID:14557626

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

    PubMed

    León-López, Liliana; Márquez-Mota, Claudia C; Velázquez-Villegas, Laura A; Gálvez-Mariscal, Amanda; Arrieta-Báez, Daniel; Dávila-Ortiz, Gloria; Tovar, Armando R; Torres, Nimbe

    2015-09-01

    Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition. PMID:26243665

  20. 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

  1. Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity

    PubMed Central

    Halliday, M; Radford, H; Sekine, Y; Moreno, J; Verity, N; le Quesne, J; Ortori, C A; Barrett, D A; Fromont, C; Fischer, P M; Harding, H P; Ron, D; Mallucci, G R

    2015-01-01

    Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative

  2. Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.

    PubMed

    Halliday, M; Radford, H; Sekine, Y; Moreno, J; Verity, N; le Quesne, J; Ortori, C A; Barrett, D A; Fromont, C; Fischer, P M; Harding, H P; Ron, D; Mallucci, G R

    2015-01-01

    Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative

  3. Acute effects of ethanol in the control of protein synthesis in isolated rat liver cells

    SciTech Connect

    Girbes, T.; Susin, A.; Ayuso, M.S.; Parrilla, R.

    1983-10-01

    The acute effect of ethanol on hepatic protein synthesis is a rather controversial issue. In view of the conflicting reports on this subject, the effect of ethanol on protein labeling from L-(/sup 3/H)valine in isolated liver cells was studied under a variety of experimental conditions. When tracer doses of the isotope were utilized, ethanol consistently decreased the rate of protein labeling, regardless of the metabolic conditions of the cells. This inhibition was not prevented by doses of 4-methylpyrazole large enough to abolish all the characteristic metabolic effects of ethanol, and it was not related to perturbations on the rates of L-valine transport and/or proteolysis. When ethanol was tested in the presence of saturating doses of L-(/sup 3/H)valine no effect on protein labeling was observed. These observations suggest that the ethanol effect in decreasing protein labeling from tracer doses of the radioactive precursor does not reflect variations in the rate of protein synthesis but reflects changes in the specific activity of the precursor. These changes probably are secondary to variations in the dimensions of the amino acid pool utilized for protein synthesis. Even though it showed a lack of effect when tested alone, in the presence of saturating doses of the radioactive precursor ethanol inhibited the stimulatory effects on protein synthesis mediated by glucose and several gluconeogenic substrates. This effect of ethanol was not prevented by inhibitors of alcohol dehydrogenase, indicating that a shift of the NAD system to a more reduced state is not the mediator of its action. It is suggested that ethanol probably acted by changing the steady-state levels of some common effector(s) generated from the metabolism of all these fuels or else by preventing the inactivation of a translational repressor.

  4. Reduced protein synthesis in schizophrenia patient-derived olfactory cells

    PubMed Central

    English, J A; Fan, Y; Föcking, M; Lopez, L M; Hryniewiecka, M; Wynne, K; Dicker, P; Matigian, N; Cagney, G; Mackay-Sim, A; Cotter, D R

    2015-01-01

    Human olfactory neurosphere-derived (ONS) cells have the potential to provide novel insights into the cellular pathology of schizophrenia. We used discovery-based proteomics and targeted functional analyses to reveal reductions in 17 ribosomal proteins, with an 18% decrease in the total ribosomal signal intensity in schizophrenia-patient-derived ONS cells. We quantified the rates of global protein synthesis in vitro and found a significant reduction in the rate of protein synthesis in schizophrenia patient-derived ONS cells compared with control-derived cells. Protein synthesis rates in fibroblast cell lines from the same patients did not differ, suggesting cell type-specific effects. Pathway analysis of dysregulated proteomic and transcriptomic data sets from these ONS cells converged to highlight perturbation of the eIF2α, eIF4 and mammalian target of rapamycin (mTOR) translational control pathways, and these pathways were also implicated in an independent induced pluripotent stem cell-derived neural stem model, and cohort, of schizophrenia patients. Analysis in schizophrenia genome-wide association data from the Psychiatric Genetics Consortium specifically implicated eIF2α regulatory kinase EIF2AK2, and confirmed the importance of the eIF2α, eIF4 and mTOR translational control pathways at the level of the genome. Thus, we integrated data from proteomic, transcriptomic, and functional assays from schizophrenia patient-derived ONS cells with genomics data to implicate dysregulated protein synthesis for the first time in schizophrenia. PMID:26485547

  5. Synthesis of Hydrogen-Bond Surrogate α-helices as Inhibitors of Protein-Protein Interactions

    PubMed Central

    Miller, Stephen E.; Thomson, Paul F.; Arora, Paramjit S.

    2014-01-01

    The α-helix is a prevalent secondary structure in proteins and critical in mediating protein-protein interactions (PPIs). Peptide mimetics that adopt stable helices have become powerful tools for the modulation of PPIs in vitro and in vivo. Hydrogen-bond surrogate (HBS) α-helices utilize a covalent bond in place of an N-terminal i to i+4 hydrogen bond and have been used to target and disrupt PPIs that become dysregulated in disease states. These compounds have improved conformational stability and cellular uptake as compared to their linear peptide counterparts. The protocol presented here describes current methodology for the synthesis of HBS α-helical mimetics. The solid phase synthesis of HBS helices involves solid phase peptide synthesis with three key steps involving incorporation of N-allyl functionality within the backbone of the peptide, coupling of a secondary amine, and a ring-closing metathesis step. PMID:24903885

  6. Modulation by estrogen of synthesis of specific uterine proteins.

    PubMed

    Skipper, J K; Eakle, S D; Hamilton, T H

    1980-11-01

    The contemporary procedure for high resolution two dimensional gel electrophoresis was extended to include an initial nondenaturing dimension of electrophoresis. Use of the resulting three dimensional procedure revealed that the previously described single peak of estrogen-induced protein in the uterus of the rat contains at least three distinct proteins whose rates of synthesis are regulated by estrogen. These proteins were localized within partial protein maps, thereby providing definitive operational definitions for the detection and identification of each. It was unambiguously demonstrated that each of the three proteins is continuously synthesized in control uteri. These findings cast doubt on the simplistic hypothesis that estrogen induces a single key protein that triggers a "cascade" of sequential transcriptional events in the uterus. Our finding that the major uterine protein induced by estrogen is also synthesized in liver and muscle cells is significant in that it points to a more general cellular function for the protein, rather than a unique role within uterine cells. Finally, our procedure for three dimensional gel electrophoresis opens new avenues for the detection of minor proteins in heterogeneous protein mixtures, such as those from the tissues of higher animals. PMID:7428041

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

    PubMed

    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

    2016-06-16

    Whether protein synthesis and cellular stress response pathways interact to control stem cell function is currently unknown. Here we show that mouse 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 tumorigenesis. Mechanistically, we show that inhibition of post-transcriptional cytosine-5 methylation locks tumour-initiating 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

  8. Antibiotics in development targeting protein synthesis.

    PubMed

    Sutcliffe, Joyce A

    2011-12-01

    The resolution of antibiotic-ribosomal subunit complexes and antibacterial-protein complexes at the atomic level has provided new insights into modifications of clinically relevant antimicrobials and provided new classes that target the protein cellular apparatus. New chemistry platforms that use fragment-based drug design or allow novel modifications in known structural classes are being used to design new antibiotics that overcome known resistance mechanisms and extend spectrum and potency by circumventing ubiquitous efflux pumps. This review provides details on seven antibiotics in development for treatment of moderate-to-severe community-acquired bacterial pneumonia and/or acute bacterial skin and skin structure infections: solithromycin, cethromycin, omadacycline, CEM-102, GSK1322322, radezolid, and tedizolid. Two antibiotics of the oxazolidinone class, PF-02341272 and AZD5847, are being developed as antituberculosis agents. Only three antibiotics that target the protein cellular machinery, TP-434, GSK2251052, and plazomicin, have a spectrum that encompasses multidrug-resistant Gram-negative pathogens. These compounds provide hope for treating key pathogens that cause serious disease in both the community and the hospital. PMID:22191530

  9. Chemical Synthesis of Proteins with Non-Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization.

    PubMed

    Reddy, Post Sai; Dery, Shahar; Metanis, Norman

    2016-01-18

    Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site. PMID:26636774

  10. 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.

  11. AMP-activated protein kinase and carbohydrate response element binding protein: A study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken liv...

  12. Mutation in MRPS34 Compromises Protein Synthesis and Causes Mitochondrial Dysfunction

    PubMed Central

    Richman, Tara R.; Ermer, Judith A.; Davies, Stefan M. K.; Perks, Kara L.; Viola, Helena M.; Shearwood, Anne-Marie J.; Hool, Livia C.; Rackham, Oliver; Filipovska, Aleksandra

    2015-01-01

    The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age. PMID:25816300

  13. 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.

  14. A novel role of cytosolic protein synthesis inhibition in aminoglycoside ototoxicity.

    PubMed

    Francis, Shimon P; Katz, Joshua; Fanning, Kathryn D; Harris, Kimberly A; Nicholas, Brian D; Lacy, Michael; Pagana, James; Agris, Paul F; Shin, Jung-Bum

    2013-02-13

    Ototoxicity is a main dose-limiting factor in the clinical application of aminoglycoside antibiotics. Despite longstanding research efforts, our understanding of the mechanisms underlying aminoglycoside ototoxicity remains limited. Here we report the discovery of a novel stress pathway that contributes to aminoglycoside-induced hair cell degeneration. Modifying the previously developed bioorthogonal noncanonical amino acid tagging method, we used click chemistry to study the role of protein synthesis activity in aminoglycoside-induced hair cell stress. We demonstrate that aminoglycosides inhibit protein synthesis in hair cells and activate a signaling pathway similar to ribotoxic stress response, contributing to hair cell degeneration. The ability of a particular aminoglycoside to inhibit protein synthesis and to activate the c-Jun N-terminal kinase (JNK) pathway correlated well with its ototoxic potential. Finally, we report that a Food and Drug Administration-approved drug known to inhibit ribotoxic stress response also prevents JNK activation and improves hair cell survival, opening up novel strategies to prevent and treat aminoglycoside ototoxicity. PMID:23407963

  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. 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.

  17. 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

  18. Blue copper proteins: Synthesis, spectra, and structures of CuIN3(SR) and CuIIN3(SR) active site analogues

    PubMed Central

    Thompson, Jeffery S.; Marks, Tobin J.; Ibers, James A.

    1977-01-01

    The reaction of Cu(SR) or [Cu(SR)][ClO4] derivatives (SR = p-nitrobenzenethiolate or O-ethylcysteinate) with potassium hydrotris(3,5-dimethyl-1-pyrazolyl)borate produces redox pairs of the stoichiometry CuIN3(SR) and CuIIN3(SR). These complexes are well-defined synthetic approximations to the proposed N3S binding sites of blue (type 1) copper electron transfer proteins. The compounds were investigated by a variety of chemical and spectral (optical, resonance Raman, and electron paramagnetic resonance) techniques; the complex K[Cu(hydrotris(3,5-dimethyl-1-pyrazolyl)borate)(p- NO2C6H4S]-2 acetone was also studied by single-crystal x-ray diffraction methods. The spectrochemical characteristics of the CuIIN3(SR) species are in large part similar to the native system and thus provide some perspective regarding the origin of the unique type 1 spectral parameters and electron transfer properties. PMID:16592426

  19. Global protein synthesis in human trophoblast is resistant to inhibition by hypoxia

    PubMed Central

    Williams, S.F.; Fik, E.; Zamudio, S.; Illsley, N.P.

    2012-01-01

    Placental growth and function depend on syncytial cell processes which require the continuing synthesis of cellular proteins. The substantial energy demands of protein synthesis are met primarily from oxidative metabolism. Although the responses of individual proteins produced by the syncytiotrophoblast to oxygen deprivation have been investigated previously, there is no information available on global protein synthesis in syncytiotrophoblast under conditions of hypoxia. These studies were designed to test the hypothesis that syncytial protein synthesis is decreased in a dose-dependent manner by hypoxia. Experiments were performed to measure amino acid incorporation into proteins in primary syncytiotrophoblast cells exposed to oxygen concentrations ranging from 0 to 10%. Compared to cells exposed to normoxia (10% O2), no changes were observed following exposure to 5% or 3% O2, but after exposure to 1% O2, protein synthesis after 24 and 48 h decreased by 24% and 23% and with exposure to 0% O2, by 65% and 50%. As a consequence of these results, we hypothesized that global protein synthesis in conditions of severe hypoxia was being supported by glucose metabolism. Additional experiments were performed therefore to examine the role of glucose in supporting protein synthesis. These demonstrated that at each oxygen concentration there was a significant, decreasing linear trend in protein synthesis as glucose concentration was reduced. Under conditions of near-anoxia and in the absence of glucose, protein synthesis was reduced by >85%. Even under normoxic conditions (defined as 10% O2) and in the presence of oxidative substrates, reductions in glucose were accompanied by decreases in protein synthesis. These experiments demonstrate that syncytiotrophoblast cells are resistant to reductions in protein synthesis at O2 concentrations greater than 1%. This could be explained by our finding that a significant fraction of protein synthesis in the syncytiotrophoblast is

  20. 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.

  1. 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. PMID:27246774

  2. Synthesis and phosphorylation of the glial fibrillary acidic protein during brain development: A tissue slice study

    SciTech Connect

    Noetzel, M.J. )

    1990-01-01

    Brain slices were incubated with either (3H) amino acids or (32P) orthophosphate in order to characterize the synthesis and phosphorylation of the glial fibrillary acidic protein (GFAP) in the rat nervous system. The incorporation of (3H) amino acids into GFAP was found to increase significantly during early postnatal development, reaching a peak of activity on day 5 of life and then declining over the next 2 weeks. Concomitant with this peak of synthetic activity the content of GFAP in rat brain was also observed to increase dramatically. GFAP continued to accumulate in brain through postnatal day 30 despite a decrease in the synthesis of the protein. These results indicate that the increase in GFAP during the first month of life cannot be ascribed solely to the rate of GFAP synthesis. The findings are consistent with the hypothesis that during later stages of astrocytic development the accumulation of GFAP may be primarily dependent upon a low rate of protein degradation. The pattern of GFAP phosphorylation in the developing rat brain differed from that observed for the incorporation of (3H) amino acids. The peak incorporation of 32P into GFAP occurred on postnatal day 10 at a time when synthesis of the protein had declined by 43%. These findings suggest that during development phosphorylation of GFAP is mediated by factors different from those directing its synthesis. In addition, phosphorylation of GFAP did not alter its solubility in cytoskeletal preparations indicating that GFAP phosphorylation is probably not a major regulatory mechanism in disassembly of the astroglial filaments.

  3. 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.

  4. Synthesis of Nanogel-Protein Conjugates

    PubMed Central

    Chacko, Reuben T.; Maynard, Heather D.; Thayumanavan, S.

    2014-01-01

    The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics. PMID:24761162

  5. Synthesis of Nanogel-Protein Conjugates.

    PubMed

    Matsumoto, Nicholas M; González-Toro, Daniella C; Chacko, Reuben T; Maynard, Heather D; Thayumanavan, S

    2013-04-21

    The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics. PMID:24761162

  6. Synthesis and biological activity of polyprenols.

    PubMed

    Zhang, Qiong; Huang, Lixin; Zhang, Caihong; Xie, Pujun; Zhang, Yaolei; Ding, Shasha; Xu, Feng

    2015-10-01

    The polyprenols and their derivatives are highlighted in this study. These lipid linear polymers of isoprenoid residues are widespread in nature from bacteria to human cells. This review primarily presents the synthesis and biological activities of polyprenyl derivatives. Attention is focused on the synthesis and biological activity of dolichols, polyprenyl ester derivatives and polyprenyl amines. Other polyprenyl derivatives, such as oxides of polyprenols, aromatic polyprenols, polyprenyl bromide and polyprenyl sulphates, are mentioned. It is noted that polyprenyl phosphates and polyprenyl-linked glycosylation have better antibacterial, gene therapy and immunomodulating performance, whereas polyprenyl amines have better for antibacterial and antithrombotic activity. Dolichols, polyprenyl acetic esters, polyprenyl phosphates and polyprenyl-linked glycosylation have pharmacological anti-tumour effects. Finally, the postulated prospect of polyprenols and their derivatives are discussed. Further in vivo studies on the above derivatives are needed. The compatibility of polyprenols and their derivatives with other drugs should be studied, and new preparations of polyprenyl derivatives, such as hydrogel glue and release-controlled drugs, are suggested for future research and development. PMID:26358482

  7. Voluntary Exercise Regionally Augments Rates of Cerebral Protein Synthesis

    PubMed Central

    Nadel, Jeffrey; Huang, Tianjian; Xia, Zengyan; Burlin, Thomas; Zametkin, Alan; Smith, Carolyn Beebe

    2016-01-01

    Exercise is a natural form of neurophysiologic stimulation that has known benefits for mental health, maintenance of cerebral function, and stress reduction. Exercise is known to induce an upregulation of brain-derived neurotrophic factor and this is thought to be involved in associated increases in neural plasticity. Protein synthesis is also an essential component of adaptive plasticity. We hypothesized that exercise may stimulate changes in brain protein synthesis as part of its effects on plasticity. Here, we applied the quantitative autoradiographic L-[1-14C] leucine method to the in vivo determination of regional rates of cerebral protein synthesis (rCPS) in adult rats following a seven day period of voluntary wheel-running and their sedentary counterparts. In four of 21 brain regions examined, the mean values of rCPS in the exercised rats were statistically significantly higher than in sedentary controls; regions affected were paraventricular hypothalamic nucleus, ventral hippocampus as a whole, CA1 pyramidal cell layer in ventral hippocampus, and frontal cortex. Increases in rCPS approached statistical significance in dentate gyrus of the ventral hippocampus. Our results affirm the value of exercise in encouraging hippocampal and possibly cortical neuroplasticity, and also suggest that exercise may modulate stimulation of stress-response pathways. Ultimately, our study indicates that measurement of rCPS with PET might be used as a marker of brain response to exercise in human subjects. PMID:24016692

  8. Protein synthesis in liposomes with a minimal set of enzymes.

    PubMed

    Murtas, Giovanni; Kuruma, Yutetsu; Bianchini, Paolo; Diaspro, Alberto; Luisi, Pier Luigi

    2007-11-01

    In a significant step towards the construction of the semi-synthetic minimal cell, a protein expression system with a minimal set of pure and specific enzymes is required. A novel cell-free transcription and translation system named PURESYSTEM (PS), consisting of a specified set of 36 enzymes and ribosomes, has been entrapped in POPC liposomes for protein synthesis. The PS has been used to transcribe and translate an Enhanced Green Fluorescent Protein (EGFP) gene from plasmid DNA. The synthesis is confirmed by the EGFP fluorescence emitting liposomes on fluorometric analysis and on confocal microscopy analysis. Furthermore the PS encapsulated into POPC liposomes can drive the expression of the plsB and plsC genes encoding for the sn-glycerol-3-phosphate acyltransferase (GPAT) and 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) involved in the first step of the "salvage pathway" for synthesis of POPC. The expression of GPAT and LPAAT in liposomes would in principle allow the production of the cell boundary from within. PMID:17850764

  9. Effects of pressure overload and insulin on protein turnover in the perfused rat heart. Prostaglandins are not involved although their synthesis is stimulated by insulin.

    PubMed Central

    Smith, D M; Sugden, P H

    1987-01-01

    A modified anterogradely perfused rat heart preparation is described in which all the cardiac output passes through the coronary circulation. Such a preparation develops hypertensive aortic pressures. Hypertensive aortic pressures or insulin stimulate the rate of cardiac protein synthesis and inhibit the rate of protein degradation. Aortic pressure and insulin may be important in the regulation of cardiac nitrogen balance in vivo. By abolishing cardiac prostaglandin synthesis with 4-biphenylacetate, we were able to investigate the possible involvement of prostaglandins in the modulation of protein turnover by pressure overload or insulin. There was no evidence of any involvement. However, insulin stimulated and cycloheximide inhibited cardiac prostaglandin synthesis. These findings are consonant with an enzyme involved in prostaglandin synthesis being short-lived and prostaglandin synthesis being rapidly influenced by activators and inhibitors of protein synthesis and degradation. PMID:3307762

  10. Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.

    PubMed

    Hackeng, T M; Griffin, J H; Dawson, P E

    1999-08-31

    The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis. PMID:10468563

  11. The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

    PubMed Central

    Stacey, D W; Dobrowolski, S F; Piotrkowski, A; Harter, M L

    1994-01-01

    The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity. Images PMID:7813447

  12. 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.

  13. The feed contaminant deoxynivalenol affects the intestinal barrier permeability through inhibition of protein synthesis.

    PubMed

    Awad, Wageha A; Zentek, Jürgen

    2015-06-01

    Deoxynivalenol (DON) has critical health effects if the contaminated grains consumed by humans or animals. DON can have negative effects on the active transport of glucose and amino acids in the small intestine of chickens. As the underlying mechanisms are not fully elucidated, the present study was performed to delineate more precisely the effects of cycloheximide (protein synthesis inhibitor, CHX) and DON on the intestinal absorption of nutrients. This was to confirm whether DON effects on nutrient absorption are due to an inhibition of protein synthesis. Changes in ion transport and barrier function were assessed by short-circuit current (Isc) and transepithelial ion conductance (Gt) in Ussing chambers. Addition of D-glucose or L-glutamine to the luminal side of the isolated mucosa of the jejunum increased (P < 0.001) the Isc compared with basal conditions in the control tissues. However, the Isc was not increased by the glucose or glutamine addition after pre-incubation of tissues with DON or CHX. Furthermore, both DON and CHX reduced Gt, indicating that the intestinal barrier is compromised and consequently induced a greater impairment of the barrier function. The remarkable similarity between the activity of CHX and DON on nutrient uptake is consistent with their common ability to inhibit protein synthesis. It can be concluded that the decreases in transport activity by CHX was evident in this study using the chicken as experimental model. Similarly, DON has negative effects on the active transport of some nutrients, and these can be explained by its influence on protein synthesis. PMID:24888376

  14. Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy

    PubMed Central

    Moore, Claire E.J.; Wang, Xuemin; Xie, Jianling; Pickford, Jo; Barron, John; Regufe da Mota, Sergio; Versele, Matthias; Proud, Christopher G.

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy. PMID:26795954

  15. Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis.

    PubMed

    Savoie, A; Lavastre, V; Pelletier, M; Hajto, T; Hostanska, K; Girard, D

    2000-12-01

    The plant lectin Viscum album agglutinin-I (VAA-I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA-I induces de novo protein synthesis of metabolically 35S-labeled human neutrophils when used at low concentrations (< 100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining) approaches, we found that VAA-I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA-I was also found to reverse the delaying effect of GM-CSF on neutrophil apoptosis and to inhibit GM-CSF-induced de novo protein synthesis. In contrast to GM-CSF, VAA-I does not induce tyrosine phosphorylation by itself and does not alter the GM-CSF-induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA-1077, and z-VAD-FMK, only the latter (inhibitor of caspases-1, -3, -4, and -7) was found to inhibit VAA-I-induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 +/- 1.3 to 54 +/- 3.2% (n=4). Furthermore, we confirm that caspases are involved in VAA-I-induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase-3-dependent. Such degradation was reversed by the z-VAD-FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA-I is a caspase-dependent mechanism that does not involve tyrosine phosphorylation events, G-proteins, PKCs, and PLA2. In addition, we conclude that at least caspase-3 is involved. Correlation between VAA-I-induced neutrophil apoptosis and VAA-I-induced inhibition of de novo protein synthesis is discussed. PMID:11129652

  16. 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

  17. RX-P873, a Novel Protein Synthesis Inhibitor, Accumulates in Human THP-1 Monocytes and Is Active against Intracellular Infections by Gram-Positive (Staphylococcus aureus) and Gram-Negative (Pseudomonas aeruginosa) Bacteria

    PubMed Central

    Buyck, Julien M.; Peyrusson, Frédéric; Tulkens, Paul M.

    2015-01-01

    The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies (Emax values) and bacteriostatic concentrations (Cs values) on the basis of Hill equations of the concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/liter (S. aureus) and 2 to 8 mg/liter (P. aeruginosa), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units in infected THP-1 cells, respectively, after 24 h. Cs values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, and daptomycin for S. aureus; ciprofloxacin and ceftazidime for P. aeruginosa). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus. Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species. PMID:26014952

  18. Estrogen Regulates Protein Synthesis and Actin Polymerization in Hippocampal Neurons through Different Molecular Mechanisms

    PubMed Central

    Briz, Victor; Baudry, Michel

    2014-01-01

    Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists (PPT and DPN, respectively) failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways. PMID:24611062

  19. 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

  20. 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

  1. Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption

    PubMed Central

    Fujiwara, Kei; Doi, Nobuhide

    2016-01-01

    Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20–30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL). PMID:27128597

  2. 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.

  3. Lymphocyte protein synthesis is increased with the progression of HIV-associated disease to AIDS.

    PubMed

    Caso, G; Garlick, P J; Gelato, M C; McNurlan, M A

    2001-12-01

    HIV infection has been shown to affect lymphocyte function and to reduce lymphocyte responsiveness in vitro to mitogenic stimulation, but little is known about lymphocyte metabolism in vivo and how it is affected during the course of the disease. This study investigated the metabolic activity of lymphocytes in vivo through the progression of HIV-associated disease. Lymphocyte protein synthesis was measured with L-[(2)H(5)]phenylalanine (45 mg/kg body weight) in healthy volunteers (n=7), in patients who were HIV-positive (n=7) but asymptomatic, and in patients with AIDS (n=8). The rates of lymphocyte protein synthesis [expressed as a percentage of lymphocyte protein, i.e. fractional synthesis rate (FSR)] were not altered in HIV-positive patients compared with healthy controls (7.9+/-1.28% and 9.1+/-0.53%/day respectively), but were significantly elevated in AIDS patients (14.0+/-1.16%/day; P<0.05). The serum concentration of the cytokine tumour necrosis factor-alpha (TNF-alpha) increased with the progression of the disease, and TNF-alpha levels were significantly higher in AIDS patients (6.81+/-0.88 ng/l) than in healthy controls (3.09+/-0.27 ng/l; P<0.05). Lymphocyte protein FSR was positively correlated with serum TNF-alpha concentration (r=0.55, P=0.009) and negatively correlated with CD4(+) lymphocyte count (r=-0.70, P=0.004). The elevation of lymphocyte protein synthesis in AIDS patients suggests a higher rate of turnover of lymphocytes. This may be associated with a generalized activation of the immune system, which is also reflected by the elevated serum TNF-alpha concentration in the late stages of HIV-associated disease. PMID:11724643

  4. Protein Needs of Physically Active Children.

    PubMed

    Volterman, Kimberly A; Atkinson, Stephanie A

    2016-05-01

    Current Dietary Reference Intakes (DRI) for protein for children and youth require revision as they were derived primarily on nitrogen balance data in young children or extrapolated from adult values; did not account for the possible influence of above average physical activity; and did not set an upper tolerable level of intake. Revision of the protein DRIs requires new research that investigates: 1) long-term dose-response to identify protein and essential amino acid requirements of both sexes at various pubertal stages and under differing conditions of physical activity; 2) the acute protein needs (quantity and timing) following a single bout of exercise; 3) the potential adverse effects of chronic high intakes of protein; and 4) new measurement techniques (i.e., IAAO or stable isotope methodologies) to improve accuracy of protein needs. While active individuals may require protein in excess of current DRIs, most active Canadian children and youth have habitual protein intakes that exceed current recommendations. PMID:27137165

  5. Possible involvement of phospholipase C and protein kinase C in stimulatory actions of L-leucine and its keto acid, alpha-ketoisocaproic acid, on protein synthesis in RLC-16 hepatocytes.

    PubMed

    Yagasaki, Kazumi; Morisaki-Tsuji, Naoko; Miura, Atsuhito; Funabiki, Ryuhei

    2002-11-01

    Effects of leucine and related compounds on protein synthesis were studied in RLC-16 hepatocytes. The incorporation of [(3)H] tyrosine into cellular protein was measured as an indexof protein synthesis. In leucine-depleted RLC-16 cells, L-leucineand its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipase A(2) and C canceled stimulatory actions of L-leucine and KIC on protein synthesis, suggesting a possible involvement of either arachidonic acid metabolism by phospholipase A(2), cyclooxygenase or lipoxygenase, or phosphatidylinositol degradation by phospholipase C in the stimulatory actions of L-leucine and KIC.Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of protein kinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in RLC-16 cells via activation of phospholipase C and production of diacylglycerol and inositol triphosphate from phosphatidylinositol, which in turn activate protein kinase C. PMID:19003115

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

    PubMed

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

    2016-01-01

    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. PMID:27571057

  7. Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.

    PubMed

    Schinn, Song-Min; Broadbent, Andrew; Bradley, William T; Bundy, Bradley C

    2016-06-25

    A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems. PMID:27085957

  8. Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity.

    PubMed

    Morales, Yalemi; Nitzel, Damon V; Price, Owen M; Gui, Shanying; Li, Jun; Qu, Jun; Hevel, Joan M

    2015-06-12

    Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner. PMID:25911106

  9. Castration differentially alters basal and leucine-stimulated tissue protein synthesis in skeletal muscle and adipose tissue.

    PubMed

    Jiao, Qianning; Pruznak, Anne M; Huber, Danuta; Vary, Thomas C; Lang, Charles H

    2009-11-01

    Reduced testosterone as a result of catabolic illness or aging is associated with loss of muscle and increased adiposity. We hypothesized that these changes in body composition occur because of altered rates of protein synthesis under basal and nutrient-stimulated conditions that are tissue specific. The present study investigated such mechanisms in castrated male rats (75% reduction in testosterone) with demonstrated glucose intolerance. Over 9 wk, castration impaired body weight gain, which resulted from a reduced lean body mass and preferential sparing of adipose tissue. Castration decreased gastrocnemius weight, but this atrophy was not associated with reduced basal muscle protein synthesis or differences in plasma IGF-I, insulin, or individual amino acids. However, oral leucine failed to normally stimulate muscle protein synthesis in castrated rats. In addition, castration-induced atrophy was associated with increased 3-methylhistidine excretion and in vitro-determined ubiquitin proteasome activity in skeletal muscle, changes that were associated with decreased atrogin-1 or MuRF1 mRNA expression. Castration decreased heart and kidney weight without reducing protein synthesis and did not alter either cardiac output or glomerular filtration. In contradistinction, the weight of the retroperitoneal fat depot was increased in castrated rats. This increase was associated with an elevated rate of basal protein synthesis, which was unresponsive to leucine stimulation. Castration also decreased whole body fat oxidation. Castration increased TNFα, IL-1α, IL-6, and NOS2 mRNA in fat but not muscle. In summary, the castration-induced muscle wasting results from an increased muscle protein breakdown and the inability of leucine to stimulate protein synthesis, whereas the expansion of the retroperitoneal fat depot appears mediated in part by an increased basal rate of protein synthesis-associated increased inflammatory cytokine expression. PMID:19755668

  10. 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.

  11. Respective influences of age and weaning on skeletal and visceral muscle protein synthesis in the lamb.

    PubMed Central

    Attaix, D; Aurousseau, E; Bayle, G; Rosolowska-Huszcz, D; Arnal, M

    1988-01-01

    1. The influences of age and weaning on muscle protein synthesis were studied in vivo, by injecting a large dose of [3H]valine into 1-, 5- and 8-week-old suckling or 8-week-old weaned lambs. 2. The fractional rates of protein synthesis, in red- and white-fibre-type skeletal muscles or striated and smooth visceral muscles, were in 8-week-old suckling animals 24-37% of their values at 1 week of age. This developmental decline was related to decreased capacities for protein synthesis, i.e. RNA/protein ratios. 3. At 8 weeks of age, suckling and weaned lambs had similar fractional synthesis rates, capacities for protein synthesis and efficiencies of protein synthesis (i.e. rates of protein synthesis relative to RNA) in skeletal muscles. 4. In contrast, visceral-muscle fractional synthesis rates were lower in 8-week-old suckling lambs than in weaned animals, owing to decreased efficiencies of protein synthesis. It was concluded that developmental factors and the change to a solid diet, or weaning in itself, or both, affect differently skeletal and visceral muscle protein synthesis in the immature lamb. PMID:3223952

  12. 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

  13. Marginal B-6 intake affects protein synthesis in rat tissues

    SciTech Connect

    Sampson, D.A.; Kretsch, M.J.; Young, L.A.; Jansen, G.R.

    1986-03-05

    The role of vitamin B-6 in amino acid metabolism suggests that inadequate B-6 intake may impair protein synthesis. To test this hypothesis, 30 male rats (initially 227 g) were fed AIN76A diets that contained control, marginal or devoid levels of B-6 (5.8, 1.2 or 0.1 mg B-6/kg diet, by analysis) ad libitum for 9 weeks. Protein synthesis rates (PSRs) were measured in liver, kidney and calf muscle using a flooding dose of /sup 3/H-phenylalanine. Marginal and control groups ate and gained weight at similar rates. The marginal diet did not elevate xanthurenic acid (XA) excretion following a tryptophan load. However, marginal B-6 intake did depress liver PSR by 29% (2182 vs 1549 mg/day, P<.05), liver wet weight by 15% (19.0 vs 16.1 g, P<.05) and muscle PSR by 23% (3.0 vs 2.3%/day, P<.10). Unexpectedly, marginal B-6 intake increased PSR in kidney 47% (90 vs 132 mg/day, P<.05). The devoid diet, which increased XA excretion following a tryptophan load by more than 3-fold, depressed PSRs 56% in liver and 31% in muscle. However, the devoid diet decreased food intake by 40% (25.0 vs 15.0 g/day); therefore effects of devoid B-6 intake on PSRs may have been confounded by deficits in protein-energy intake in devoid vs control groups. These data demonstrate that marginal B-6 intake alters protein synthesis in tissues of the rat.

  14. Chloroplast protein synthesis: thylakoid bound polysomes synthesize thylakoid proteins

    SciTech Connect

    Hurewitz, J.; Jagendorf, A.T.

    1986-04-01

    Previous work indicated more polysomes bound to pea thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus the major effect of light in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus translation initiation and termination probably control the cycling of bound ribosomes. While only 3 to 6% of total RNA is in bound polysomes the incorporation of /sup 3/H-Leu into thylakoids was proportional to the amount of this bound RNA. When Micrococcal nuclease-treated thylakoids were added to labeled runoff translation products of stroma ribosomes, less than 1% of the label adhered to the added membranes; but 37% of the labeled products made by thylakoid polysomes were bound. These data support the concept that stroma ribosomes are recruited into thylakoid proteins.

  15. Oxidant-specific regulation of protein synthesis in Candida albicans.

    PubMed

    Sundaram, Arunkumar; Grant, Chris M

    2014-06-01

    Eukaryotic cells typically respond to stress conditions by inhibiting global protein synthesis. The initiation phase is the main target of regulation and represents a key control point for eukaryotic gene expression. In Saccharomyces cerevisiae and mammalian cells this is achieved by phosphorylation of eukaryotic initiation factor 2 (eIF2α). We have examined how the fungal pathogen Candida albicans responds to oxidative stress conditions and show that oxidants including hydrogen peroxide, the heavy metal cadmium and the thiol oxidant diamide inhibit translation initiation. The inhibition in response to hydrogen peroxide and cadmium largely depends on phosphorylation of eIF2α since minimal inhibition is observed in a gcn2 mutant. In contrast, translation initiation is inhibited in a Gcn2-independent manner in response to diamide. Our data indicate that all three oxidants inhibit growth of C. albicans in a dose-dependent manner, however, loss of GCN2 does not improve growth in the presence of hydrogen peroxide or cadmium. Examination of translational activity indicates that these oxidants inhibit translation at a post-initiation phase which may account for the growth inhibition in a gcn2 mutant. As well as inhibiting global translation initiation, phosphorylation of eIF2α also enhances expression of the GCN4 mRNA in yeast via a well-known translational control mechanism. We show that C. albicans GCN4 is similarly induced in response to oxidative stress conditions and Gcn4 is specifically required for hydrogen peroxide tolerance. Thus, the response of C. albicans to oxidative stress is mediated by oxidant-specific regulation of translation initiation and we discuss our findings in comparison to other eukaryotes including the yeast S. cerevisiae. PMID:24699161

  16. Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

    SciTech Connect

    Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.

    1988-08-01

    Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. (3H)leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis.

  17. Continuous cultivation of fission yeast: analysis of single-cell protein synthesis kinetics

    SciTech Connect

    Agar, D.W.; Bailey, J.E.

    1981-01-01

    A fundamental problem in microbial reactor analysis is identification of the relation between environment and individual cell metabolic activity. Population balance equations provide a link between experimental measurements of composition frequency functions in microbial populations on the one hand and macromolecule synthesis kinetics and cell division control parameters for single cells on the other. Flow microfluorometry measurements of frequency functions for single-cell protein content in Schizosaccharomyces pombe in balanced exponential growth were analyzed by 2 different methods. One approach utilizes the integrated form of the population balance equation known as the Collins-Richmond equation, and the other method involves optimization of parameters in assumed kinetic and cell division functional forms to fit measured frequency functions with corresponding model solutions. Both data interpretation techniques indicate that rates of protein synthesis increase most in low-protein-content cells as the population specific growth rate increases, leading to parabolic single-cell protein synthesis kinetics at large specific growth rates. Utilization of frequency function data for an asynchronous population is in this case a far more sensitive method for determination of single-cell kinetics than is monitoring the metabolic dynamics of a single cell or, equivalently, synchronous culture analyses.

  18. Coq7p relevant residues for protein activity and stability.

    PubMed

    Busso, Cleverson; Ferreira-Júnior, José Ribamar; Paulela, Janaina A; Bleicher, Lucas; Demasi, Marilene; Barros, Mario H

    2015-12-01

    Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center. PMID:26497406

  19. Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

    PubMed Central

    Moreno, A; Mendez, R; de Haro, C

    1991-01-01

    We have developed and characterized translationally active cell-free systems from Artemia embryos at different developmental times. The optimized lysates from 16 h-developed embryos incorporated radiolabelled amino acids into polypeptides for up to 120 min. The polypeptides synthesized ranged in Mr from 150,000 to 10,000, suggesting that the endogenous mRNA was capable of directing the synthesis of complete polypeptides. Similar results were obtained by using lysates from early developmental stages; even the cell-free system prepared from 1 h-developed embryos was partially active in protein synthesis. Furthermore, all these lysates were capable of re-initiation, as demonstrated by inhibition of initiation with the inhibitors edeine and 7-methylguanosine 5'-triphosphate. Because we found no endogenous protein-synthetic activity in the corresponding lysates from undeveloped embryos, we have used cell-free translation systems from 0 h- and 16 h-developed Artemia embryos to analyse the mechanisms limiting protein synthesis at very early developmental stages. Undeveloped-embryo lysates supplemented with nuclease-treated reticulocyte lysate were capable of translating endogenous mRNAs to give products with a wide spectrum of Mr values, but lysates of 16 h-developed embryos supplemented in this way were not further stimulated. The nuclease-treated lysate appeared to be unnecessary 5 h after resumption of development. These results suggested that a component(s) limiting translation in the undeveloped-embryo lysate was provided by the nuclease-treated reticulocyte lysate, and that this component(s) no longer limited protein synthesis after development. In view of these results, partially fractionated reticulocyte lysates were tested for restoration of protein-synthetic activity in the undeveloped embryo lysate. A high-salt ribosomal wash devoid of ribosomal subunits, which is considered a crude polypeptide-initiation-factor preparation, also restored translation activity

  20. Ligands for FKBP12 Increase Ca2+ Influx and Protein Synthesis to Improve Skeletal Muscle Function*

    PubMed Central

    Lee, Chang Seok; Georgiou, Dimitra K.; Dagnino-Acosta, Adan; Xu, Jianjun; Ismailov, Iskander I.; Knoblauch, Mark; Monroe, Tanner O.; Ji, RuiRui; Hanna, Amy D.; Joshi, Aditya D.; Long, Cheng; Oakes, Joshua; Tran, Ted; Corona, Benjamin T.; Lorca, Sabina; Ingalls, Christopher P.; Narkar, Vihang A.; Lanner, Johanna T.; Bayle, J. Henri; Durham, William J.; Hamilton, Susan L.

    2014-01-01

    Rapamycin at high doses (2–10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca2+ influx to enhance refilling of sarcoplasmic reticulum Ca2+ stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function. PMID:25053409

  1. 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

  2. Requirement of de novo synthesis of the OdhI protein in penicillin-induced glutamate production by Corynebacterium glutamicum.

    PubMed

    Kim, Jongpill; Fukuda, Hirohisa; Hirasawa, Takashi; Nagahisa, Keisuke; Nagai, Kazuo; Wachi, Masaaki; Shimizu, Hiroshi

    2010-04-01

    We found that penicillin-induced glutamate production by Corynebacterium glutamicum is inhibited when a de novo protein synthesis inhibitor, chloramphenicol, is added simultaneously with penicillin. When chloramphenicol was added 4 h after penicillin addition, glutamate production was essentially unaffected. (3)H-Leucine incorporation experiments revealed that protein synthesis continued for 1 h after penicillin addition and then gradually decreased. These results suggest that de novo protein synthesis within 4 h of penicillin treatment is required for the induction of glutamate production. To identify the protein(s) necessary for penicillin-induced glutamate production, proteome analysis of penicillin-treated C. glutamicum cells was performed with two-dimensional gel electrophoresis. Of more than 500 proteins detected, the amount of 13 proteins, including OdhI (an inhibitory protein for 2-oxoglutarate dehydrogenase complex), significantly increased upon penicillin treatment. Artificial overexpression of the odhI gene resulted in the decreased specific activity of the 2-oxoglutarate dehydrogenase complex and increased glutamate production without any triggers. These results suggest that the de novo synthesis of OdhI is the necessary factor for penicillin-induced glutamate overproduction by C. glutamicum. Moreover, continuous glutamate production was achieved by overexpression of odhI without any triggers. Thus, the odhI-overexpressing strain of C. glutamicum can be useful for efficient glutamate production. PMID:19956942

  3. 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.

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

    PubMed

    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. PMID:27554410

  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. Synthesis and trafficking of prion proteins in cultured cells.

    PubMed Central

    Taraboulos, A; Raeber, A J; Borchelt, D R; Serban, D; Prusiner, S B

    1992-01-01

    Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc. Images PMID:1356522

  7. 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...

  8. : Synthesis, Characterization, and Enhanced Photocatalytic Activity

    NASA Astrophysics Data System (ADS)

    Gao, Xiaoming; Fu, Feng; Li, Wenhong

    2014-12-01

    3D hierarchical microspheres of Cu-loaded Bi2WO6 are successfully prepared by the hydrothermal synthesis method on a large scale. The as-prepared samples are characterized by UV-Vis DRS, BET, XRD, XPS, and SEM. The results reveal that the light absorption of Cu-loaded Bi2WO6 has higher intensity in the visible range and a bathochromic shift of the absorption edge compared to that of pure Bi2WO6. The photocatalytic activity is evaluated by phenol removal from aqueous solution under visible-light irradiation. The results demonstrate that loaded Cu significantly enhances the photocatalytic activity of Bi2WO6, for the loaded Cu acts as the electron receptor on the surface of Bi2WO6, and inhibits the recombination of photogenerated electron-hole. The content of loaded Cu has an impact on the catalytic activity, and the 1.0 wt.% Cu-loaded Bi2WO6 exhibits the best photocatalytic activity in the degradation of phenol. Furthermore, the reaction kinetics of phenol removal from aqueous solution over the Cu-loaded Bi2WO6 is established by the way of the Langmuir-Hinshelwood model. The results indicate that the process of photodegradation of phenol on Cu-loaded Bi2WO6 match the Langmuir-Hinshelwood kinetic model.

  9. Interferon Production and Protein Synthesis in Chick Cells

    PubMed Central

    Friedman, Robert M.

    1966-01-01

    Friedman, Robert M. (National Cancer Institute, Bethesda, Md.). Interferon production and protein synthesis in chick cells. J. Bacteriol. 91:1224–1229. 1966.—Overnight incubation of chick embryo fibroblasts (CEF) at 4 C before infection with live Semliki Forest virus (SFV) increased virus yields but decreased interferon production. The same findings were noted when CEF were incubated for 4 hr with p-fluorophenylalanine (FPA) before infection with live SFV or inactivated Chikungunya virus. In both systems incorporation of C14-leucine into protein appeared to be increased after pretreatment at 4 C or with FPA. Protein synthesis could be raised in CEF incubated in 0.5% serum after trypsinization by increasing the concentration of serum. CEF in 10% serum had higher rates of C14-leucine incorporation than did cells in 1.5% serum, but again the cells with the apparently high rate of incorporation produced less interferon. These findings may be related to the mechanism of cellular control over interferon production. PMID:5929753

  10. 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. PMID:25671290

  11. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  12. 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. PMID:27260286

  13. Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

    PubMed Central

    Du, X; Thiem, S M

    1997-01-01

    Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection. PMID:9311875

  14. 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

  15. Electrochemical template synthesis of multisegment nanowires: fabrication and protein functionalization.

    PubMed

    Wildt, Bridget; Mali, Prashant; Searson, Peter C

    2006-12-01

    Multisegment nanowires represent a unique platform for engineering multifunctional nanoparticles for a wide range of applications. For example, the optical and magnetic properties of nanowires can be tailored by modifying the size, shape, and composition of each segment. Similarly, surface modification can be used to tailor chemical and biological properties. In this article, we report on recent work on electrochemical template synthesis of nanogap electrodes, the fabrication of multisegment nanowires with embedded catalysts, and the selective functionalization of multisegment nanowires with proteins. PMID:17129026

  16. Dihydrolipoic acid activates oligomycin-sensitive thiol groups and increases ATP synthesis in mitochondria.

    PubMed

    Zimmer, G; Mainka, L; Krüger, E

    1991-08-01

    Investigations with dihydrolipoic acid in rat heart mitochondria and mitoplasts reveal an activation of ATP-synthase up to 45%, whereas ATPase activities decrease by 36%. In parallel with an increase in ATP synthesis oligomycin-sensitive mitochondrial -SH groups are activated at 2-4 nmol dihydrolipoic acid/mg protein. ATPase activation by the uncouplers carbonylcyanide-p-trifluoromethoxyphenylhydrazone and oleate is diminished by dihydrolipoic acid, and ATP synthesis depressed by oleate is partially restored. No such efficiency of dihydrolipoic acid is seen with palmitate-induced ATPase activation or decrease of ATP synthesis. This indicates different interference of oleate and palmitate with mitochondria. In addition to its known coenzymatic properties dihydrolipoic acid may act as a substitute for coenzyme A, thereby diminishing the uncoupling efficiency of oleate. Furthermore, dihydrolipoic acid is a very potent antioxidant, shifting the -SH-S-S- equilibrium in mitochondria to the reduced state and improving the energetic state of cells. PMID:1832845

  17. 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. PMID:26568031

  18. 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. PMID:26118653

  19. 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.

  20. Gene Activation in Eukaryotes: Are Nuclear Acidic Proteins the Cause or the Effect?

    PubMed Central

    Pederson, Thoru

    1974-01-01

    Nuclear acidic proteins have been implicated in the positive control of gene transcription in eukaryotes. This hypothesis was examined in greater detail by analysis of these proteins during experimental gene activation by a technique for fractionating nuclei into chromatin and the ribonucleoprotein particles that contain heterogeneous nuclear RNA. When synthesis of rat-liver heterogeneous nuclear RNA was stimulated by administration of hydrocortisone, there was a parallel increase in the labeling of acidic proteins in ribonucleoprotein particles. However, there was no detectable effect on the labeling of either acidic chromatin proteins or histones. Thus, the nuclear acidic proteins that respond to the hormone are concerned with a post-transcriptional event, namely the assembly and processing of ribonucleoprotein particles that contain heterogeneous RNA, rather than with direct gene activation. Increases in synthesis of “chromatin” acidic proteins during gene activation observed by others may reflect the presence of these ribonucleoprotein particles in crude chromatin preparations. Images PMID:4522777

  1. Protein synthesis in isolated rabbit forelimb muscles. The possible role of metabolites of arachidonic acid in the response to intermittent stretching.

    PubMed Central

    Smith, R H; Palmer, R M; Reeds, P J

    1983-01-01

    Protein synthesis was measured in isolated intact rabbit muscles by the incorporation of [3H]phenylalanine added at a high concentration (2.5 mM) to the incubation medium. Intermittent mechanical stretching substantially increased the rate of protein synthesis relative to that in control muscles incubated under a constant tension. Indomethacin and meclofenamic acid, inhibitors of the enzyme cyclo-oxygenase, which converts free arachidonic acid into the prostaglandins, prostacyclins and thromboxanes, decreased the rate of protein synthesis in intermittently stretched muscles, but had no effect on synthesis rates in the unstimulated controls. Arachidonic acid at concentrations of 0.2 and 1.0 microM gave a highly significant increase in the rate of protein synthesis in muscles incubated under a constant tension. The ability of arachidonic acid to increase protein-synthesis rates was abolished by the addition of indomethacin. Activation of protein synthesis by intermittent stretching persisted for 10-20 min after the stretch stimulation had ceased. Indomethacin, added either during the initial incubation with intermittent stretching or during the subsequent period when protein synthesis was measured after stimulation had ceased, decreased protein-synthesis rates. This decrease was similar whether indomethacin was present during the initial, final or entire incubation period. In experiments analogous with those in (4) above, when Ca2+ was withheld and EGTA added for the entire incubation, rates of protein synthesis were again decreased. The rates of protein synthesis observed when Ca2+ was present during either an initial stimulation phase or a final, unstimulated, measurement phase were similar, and were intermediate between control rates and those in muscles incubated without Ca2+ for the whole experiment. Two prostaglandins, F2 alpha (2.8 microM) and A1 (28 microM), increased rates of protein synthesis in unstimulated muscles, but prostaglandins E2 and D2 and the

  2. Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

    PubMed Central

    Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J.; Alani, Sara; Bocchetta, Maurizio

    2015-01-01

    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. PMID:25283437

  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. PMID:25283437

  4. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  5. Evolution, structure, and synthesis of vertebrate egg-coat proteins

    PubMed Central

    Litscher, Eveline S.; Wassarman, Paul M.

    2015-01-01

    All vertebrate eggs are surrounded by an extracellular coat that supports growth of oocytes, protects oocytes, eggs, and early embryos, and participates in the process of fertilization. In mammals (platypus to human beings) the coat is called a zona pellucida (ZP) and in non-mammals (molluscs to birds), a vitelline envelope (VE). The ZP and VE are composed of just a few proteins that are related to one another and possess a common motif, called the zona pellucida domain (ZPD). The ZPD arose more than ~600 million years ago, consists of ~260 amino acids, and has 8 conserved Cys residues that participate in 4 intramolecular disulfides. It is likely that egg-coat proteins are derived from a common ancestral gene. This gene duplicated several times during evolution and gave rise to 3–4 genes in fish, 5 genes in amphibians, 6 genes in birds, and 3–4 genes in mammals. Some highly divergent sequences, N- and C-terminal to the ZPD, have been identified in egg-coat proteins and some of these sequences may be under positive Darwinian selection that drives evolution of the proteins. These and other aspects of egg-coat proteins, including their structure and synthesis, are addressed in this review. PMID:26504367

  6. A Network Synthesis Model for Generating Protein Interaction Network Families

    PubMed Central

    Sahraeian, Sayed Mohammad Ebrahim; Yoon, Byung-Jun

    2012-01-01

    In this work, we introduce a novel network synthesis model that can generate families of evolutionarily related synthetic protein–protein interaction (PPI) networks. Given an ancestral network, the proposed model generates the network family according to a hypothetical phylogenetic tree, where the descendant networks are obtained through duplication and divergence of their ancestors, followed by network growth using network evolution models. We demonstrate that this network synthesis model can effectively create synthetic networks whose internal and cross-network properties closely resemble those of real PPI networks. The proposed model can serve as an effective framework for generating comprehensive benchmark datasets that can be used for reliable performance assessment of comparative network analysis algorithms. Using this model, we constructed a large-scale network alignment benchmark, called NAPAbench, and evaluated the performance of several representative network alignment algorithms. Our analysis clearly shows the relative performance of the leading network algorithms, with their respective advantages and disadvantages. The algorithm and source code of the network synthesis model and the network alignment benchmark NAPAbench are publicly available at http://www.ece.tamu.edu/bjyoon/NAPAbench/. PMID:22912671

  7. Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

    PubMed Central

    Roffé, Martín; Beraldo, Flávio Henrique; Bester, Romina; Nunziante, Max; Bach, Christian; Mancini, Gabriel; Gilch, Sabine; Vorberg, Ina; Castilho, Beatriz A.; Martins, Vilma Regina; Hajj, Glaucia Noeli Maroso

    2010-01-01

    Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrPC) into an infectious isoform (PrPSc). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrPC interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrPC–STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrPC–STI1 binding is corrupted in neuronal cell lines persistently infected with PrPSc, as well as in primary cultured hippocampal neurons acutely exposed to PrPSc. Consistent with this, high levels of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation were found in PrPSc-infected cells and in neurons acutely exposed to PrPSc. These data indicate that modulation of protein synthesis is critical for PrPC–STI1 neurotrophic functions, and point to the impairment of this process during PrPSc infection as a possible contributor to neurodegeneration. PMID:20615969

  8. First total synthesis of prasinic acid and its anticancer activity.

    PubMed

    Chakor, Narayan; Patil, Ganesh; Writer, Diana; Periyasamy, Giridharan; Sharma, Rajiv; Roychowdhury, Abhijit; Mishra, Prabhu Dutt

    2012-11-01

    The first total synthesis of prasinic acid is being reported along with its biological evaluation. The ten step synthesis involved readily available and cheap starting materials and can easily be transposed to large scale manufacturing. The crucial steps of the synthesis included the formation of two different aromatic units (7 and 9) and their coupling reaction. The synthetic prasinic acid exhibited moderate antitumor activity (IC(50) 4.3-9.1 μM) in different lines of cancer cells. PMID:23031589

  9. Aging accentuates alcohol-induced decrease in protein synthesis in gastrocnemius.

    PubMed

    Korzick, Donna H; Sharda, Daniel R; Pruznak, Anne M; Lang, Charles H

    2013-05-15

    The present study sought to determine whether the protein catabolic response in skeletal muscle produced by chronic alcohol feeding was exaggerated in aged rats. Adult (3 mo) and aged (18 mo) female F344 rats were fed a nutritionally complete liquid diet containing alcohol (36% of total calories) or an isocaloric isonitrogenous control diet for 20 wk. Muscle (gastrocnemius) protein synthesis, as well as mTOR and proteasome activity did not differ between control-fed adult and aged rats, despite the increased TNF-α and IL-6 mRNA and decreased IGF-I mRNA in muscle of aged rats. Compared with alcohol-fed adult rats, aged rats demonstrated an exaggerated alcohol-induced reduction in lean body mass and protein synthesis (both sarcoplasmic and myofibrillar) in gastrocnemius. Alcohol-fed aged rats had enhanced dephosphorylation of 4E-BP1, as well as enhanced binding of raptor with both mTOR and Deptor, and a decreased binding of raptor with 4E-BP1. Alcohol feeding of both adult and aged rats reduced RagA binding to raptor. The LKB1-AMPK-REDD1 pathway was upregulated in gastrocnemius from alcohol-fed aged rats. These exaggerated alcohol-induced effects in aged rats were associated with a greater decrease in muscle but not circulating IGF-I, but no further increase in inflammatory mediators. In contrast, alcohol did not exaggerate the age-induced increase in atrogin-1 and MuRF1 mRNA or the increased proteasome activity. Our results demonstrate that, compared with adult rats, the gastrocnemius from aged rats is more sensitive to the catabolic effects of alcohol on protein synthesis, but not protein degradation, and this exaggerated response may be AMPK-dependent. PMID:23535459

  10. 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

  11. Pushing the threshold: How NMDAR antagonists induce homeostasis through protein synthesis to remedy depression.

    PubMed

    Raab-Graham, Kimberly F; Workman, Emily R; Namjoshi, Sanjeev; Niere, Farr

    2016-09-15

    Healthy neurons have an optimal operating range, coded globally by the frequency of action potentials or locally by calcium. The maintenance of this range is governed by homeostatic plasticity. Here, we discuss how new approaches to treat depression alter synaptic activity. These approaches induce the neuron to recruit homeostatic mechanisms to relieve depression. Homeostasis generally implies that the direction of activity necessary to restore the neuron's critical operating range is opposite in direction to its current activity pattern. Unconventional antidepressant therapies-deep brain stimulation and NMDAR antagonists-alter the neuron's "depressed" state by pushing the neuron's current activity in the same direction but to the extreme edge. These therapies rally the intrinsic drive of neurons in the opposite direction, thereby allowing the cell to return to baseline activity, form new synapses, and restore proper communication. In this review, we discuss seminal studies on protein synthesis dependent homeostatic plasticity and their contribution to our understanding of molecular mechanisms underlying the effectiveness of NMDAR antagonists as rapid antidepressants. Rapid antidepressant efficacy is likely to require a cascade of mRNA translational regulation. Emerging evidence suggests that changes in synaptic strength or intrinsic excitability converge on the same protein synthesis pathways, relieving depressive symptoms. Thus, we address the question: Are there multiple homeostatic mechanisms that induce the neuron and neuronal circuits to self-correct to regulate mood in vivo? Targeting alternative ways to induce homeostatic protein synthesis may provide, faster, safer, and longer lasting antidepressants. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease. PMID:27125595

  12. Serum stimulation of plasma protein synthesis in culture is selective and rapidly reversible.

    PubMed

    Plant, P W; Liang, T J; Pindyck, J; Grieninger, G

    1981-10-27

    Primary hepatocyte monolayers, derived from chick embryos, can be cultured from the onset in a completely chemically defined medium, free of added hormones. The liver cells synthesize and secrete a wide spectrum of plasma proteins for several days in this serum-free environment. Addition of fetal bovine serum elicits a 3-5-fold increase in the production of certain plasma proteins: fibrinogen, albumin, and the alpha1-globulin M. This effect of serum is selective; transferrin and plasminogen syntheses are enhanced less than 1.5-fold. Significant stimulation is observed with 0.1% fetal bovine serum, and half-maximal values for individual plasma proteins are obtained with concentrations ranging between 0.4 and 1%. The stimulatory activity of serum shows no developmental or species specificity. Plasma is active as serum derived from the same blood sample. The hepatocytes respond rapidly to serum, significant changes in albumin synthesis occurring less than 1 h after serum addition or removal. The effect of short exposure is fully reversible. These results establish the capacity of low concentrations of serum to stimulate plasma protein synthesis and underscore the importance of studying the effects of hormones and other factors under serum-free conditions. The findings suggest that, in addition to the classical hormones, ubiquitous but as yet uncharacterized serum components play a role in controlling this major hepatic function. PMID:7284395

  13. Isolation and partial characterization of a protein synthesis inhibitor from brine shrimp embryos.

    PubMed

    Warner, A H; Shridhar, V; Finamore, F J

    1977-09-01

    Encysted embryos of the brine shrimp, Artemia salina, contain an inhibitor of protein synthesis that appears to be important in translational control. In cyst homogenates, the inhibitor appears to be partitioned almost equally between the cytosol and ribosome fractions and it has been purified from both fractions to near homogeneity. In a cell-free protein-synthesizing system derived from Artemia cysts, with poly(U) as messenger, the protein inhibits polyphenylalanine synthesis proportional to inhibitor concentration up to about 75% inhibition, and the primary site of action appears to be at the elongation step. The inhibitor activity is not altered by 50-150 mM KCl in the reaction mixture, but it is slightly more effective at 5 mM MgCl2 than at 10 mM MgCl2. The inhibitor is a heat-labile protein of 130000 molecular weight and is devoid of hydrolase activity. Our data indicate that the inhibitor is not elongation factor EF-1 or EF-2, but we are studying the possibility that it may be a modified form of elongation factor EF-2. PMID:907902

  14. Synthesis of several membrane proteins during developmental aggregation in Myxococcus xanthus.

    PubMed

    Orndorff, P E; Dworkin, M

    1982-01-01

    We have examined the pattern of synthesis of several membrane proteins during the aggregation phase of development in Myxococcus xanthus. Development was initiated by plating vegetative cells on polycarbonate filters placed on top of an agar medium that supported fruiting body formation. At various times during aggregation a filter was removed, the cells were pulse-labeled with [35S]methionine, and the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rate of synthesis of numerous individual proteins changed during aggregation; we concentrated on six whose pattern of synthesis was greatly altered during aggregation. The rate of synthesis of five of the six proteins increased considerably during aggregation; that of the remaining protein was curtailed and appeared to be regulated by nutrient conditions. Three of the five major membrane proteins that increased during aggregation had a unique pattern of synthesis that was displayed only under conditions that are are required for development - high cell density, nutrient depletion, and a solid (agar) surface. The remaining two proteins were not unique to development; the appearance of one protein could be induced under conditions of high cell density, whereas the other could be induced by placing the cells on a solid agar surface. All of the five major proteins that appeared during development did so during the preaggregation stage, and the synthesis of four of the five proteins appeared to be curtailed late in aggregation. The synthesis of the remaining protein continued throughout aggregation. PMID:6798022

  15. 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.

  16. Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal and cancer tissues

    SciTech Connect

    Abou-Khalil, W.H.; Arimura, G.K.; Yunis, A.A.; Abou-Khalil, S.

    1986-06-13

    The effect of the mitochondrial dye rhodamine 123 (Rho 123) on protein synthesis (PS) activity was investigated in mitochondria isolated from liver and from both chloroma and erythroleukemia tumors. Incorporation of labelled leucine into mitochondrial protein was used to measure the rate of PS. While PS specific activity was much higher in hematopoietic tumors mitochondria as compared to that of liver, the addition of increased concentration of Rho 123 in all tested organelles resulted in increased inhibition of PS to reach 75-82% with 10..mu..g/ml of the dye. Similar results were obtained with 10 ..mu..g/ml of chloramphenicol, the specific inhibitor of mitochondrial PS. Moreover, under the conditions of the study, the addition of Rho 123 to mitochondria did not trigger any ATPase activity, thus eliminating any competition for the energy source ATP between PS and ATPase.

  17. Application of electroimmunoassay to the study of plasma protein synthesis in cultured hepatocytes.

    PubMed

    Grieninger, G; Pindyck, J; Hertzberg, K M; Mosesson, M W

    1979-01-01

    Electroimmunoassay has been applied to the study of plasma protein synthesis and secretion in liver cell cultures. The assay is performed on unconcentrated samples of culture medium containing the secreted plasma proteins and yields results within 2 hours. The characteristics of plasma protein production by the cultured hepatocytes coupled with the sensitivity of this assay permit the study of plasma protein in synthesis and its regulation by hormones and other agents without the routine use of radioisotopes. PMID:518014

  18. Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis.

    PubMed

    Watford, Malcolm; Wu, Guoyao

    2005-04-01

    High intracellular glutamine levels have been implicated in promoting net protein synthesis and accretion in mammalian skeletal muscle. Little is known regarding glutamine metabolism in uricotelic species but chicken breast muscle exhibits high rates of protein accretion and would be predicted to maintain high glutamine levels. However, chicken breast muscle expresses high glutaminase activity and here we report that chicken breast muscle also expresses low glutamine synthetase activity (0.07+/-0.01 U/g) when compared to leg muscle (0.50+/-0.04 U/g). Free glutamine levels were 1.38+/-0.09 and 9.69+/-0.12 nmol/mg wet weight in breast and leg muscles of fed chickens, respectively. Glutamine levels were also lower in dove breast muscle (4.82+/-0.35 nmol/mg wet weight) when compared to leg muscle (16.2+/-1.0 nmol/mg wet weight) and much lower (1.80+/-0.46 nmol/mg wet weight) in lizard leg muscle. In fed chickens, rates of fractional protein synthesis were higher in leg than in breast muscle, and starvation (48 h) resulted in a decrease in both glutamine content and rate of protein synthesis in leg muscle. Thus, although tissue-specific glutamine metabolism in uricotelic species differs markedly from that in ureotelic animals, differences in rates of skeletal muscle protein synthesis are associated with corresponding differences in intramuscular glutamine content. PMID:15763516

  19. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  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. 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.

  2. Protein-directed synthesis of highly monodispersed, spherical gold nanoparticles and their applications in multidimensional sensing.

    PubMed

    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

  3. 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

  4. 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.

  5. 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. PMID:25355192

  6. STIMULATION OF MUSCLE PROTEIN SYNTHESIS BY GLUCOSE IN NEONATES IS AMP KINASE INDEPENDENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Muscle protein synthesis is elevated in the neonate, in part due to an elevated response to the rise in amino acids and insulin after a meal. Recent evidence suggests that glucose may also play a role in the regulation of protein synthesis. AMP kinase has been recognized as an energy sensor and a ...

  7. 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...

  8. 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…

  9. 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…

  10. MODULATION OF MUSCLE PROTEIN SYNTHESIS BY INSULIN IS MAINTAINED DURING NEONATAL ENDOTOXEMIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sepsis promotes insulin resistance and reduces protein synthesis in skeletal muscle of adults. The effect of sepsis on insulin-stimulated muscle protein synthesis has not been determined in neonates, a highly anabolic population that is uniquely sensitive to insulin. Overnight fasted neonatal pigs w...

  11. Somatotropin enhanced muscle protein synthesis in growing pigs is not modulated by insulin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic, 7-day treatment of growing pigs with porcine somatotropin (ST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that enhances translation initiation. This study aimed to determine whether the ST-induced increase in skeletal muscle protein synthesis was media...

  12. LOCAL IGF-I ENHANCES THE SENSITIVITY OF MUSCLE PROTEIN SYNTHESIS TO INSULIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle protein synthesis in the immature muscle is highly sensitive to insulin and nutrients. We hypothesized that this sensitivity of protein synthesis to insulin is attributable to local IGFs that are expressed at a significant level by immature skeletal muscle. To test the hypothesis, t...

  13. 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. PMID:21562168

  14. Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

    PubMed Central

    Jones, T. L.; Ort, D. R.

    1997-01-01

    Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state. PMID:12223667

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

    PubMed Central

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

    2015-01-01

    Canis lupus familiaris, the domesticated dog, is capable of extreme endurance performance. The ability to perform sustained aerobic exercise is dependent on a well-developed mitochondrial reticulum. In this study we examined the cumulative muscle protein and DNA synthesis in groups of athletic dogs at the onset of an exercise training program and following a strenuous exercise training program. We hypothesized that both at the onset and during an exercise training program there would be greater mitochondrial protein synthesis rates compared with sedentary control with no difference in mixed or cytoplasmic protein synthesis rates. Protein synthetic rates of three protein fractions and DNA synthesis were determined over 1 wk using 2H2O in competitive Alaskan Huskies and Labrador Retrievers trained for explosive device detection. Both groups of dogs had very high rates of skeletal muscle protein synthesis in the sedentary state [Alaskan Huskies: Mixed = 2.28 ± 0.12, cytoplasmic (Cyto) = 2.91 ± 0.10, and mitochondrial (Mito) = 2.62 ± 0.07; Labrador Retrievers: Mixed = 3.88 ± 0.37, Cyto = 3.85 ± 0.06, and Mito = 2.92 ± 0.20%/day]. Mitochondrial (Mito) protein synthesis rates did not increase at the onset of an exercise training program. Exercise-trained dogs maintained Mito protein synthesis during exercise training when mixed (Mixed) and cytosolic (Cyto) fractions decreased, and this coincided with a decrease in p-RpS6 but also a decrease in p-ACC signaling. Contrary to our hypothesis, canines did not have large increases in mitochondrial protein synthesis at the onset or during an exercise training program. However, dogs have a high rate of protein synthesis compared with humans that perhaps does not necessitate an extra increase in protein synthesis at the onset of aerobic exercise training. PMID:25614602

  16. 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

  17. DEVELOPMENTAL REGULATION OF PROTEIN KINASE B ACTIVATION IS ISOFORM SPECIFIC IN SKELETAL MUSCLE OF NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial activation of the insulin signaling pathway that leads to translation initiation is enhanced in skeletal muscle of the neonate and decreases with development in parallel with the developmental decline in muscle protein synthesis. Our previous study showed that the activity of protei...

  18. 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

  19. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice.

    PubMed

    You, Jae-Sung; Anderson, Garrett B; Dooley, Matthew S; Hornberger, Troy A

    2015-09-01

    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 that the

  20. Arginine Depletion by Arginine Deiminase Does Not Affect Whole Protein Metabolism or Muscle Fractional Protein Synthesis Rate in Mice

    PubMed Central

    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. PMID:25775142

  1. 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. PMID:24993823

  2. Protein turnover, amino acid requirements and recommendations for athletes and active populations.

    PubMed

    Poortmans, J R; Carpentier, A; Pereira-Lancha, L O; Lancha Jr, A

    2012-10-01

    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 ((13)C-lysine, (15)N-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

  3. 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

  4. Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

    PubMed Central

    Gong, Z Y; Brandhorst, B P

    1988-01-01

    An increased level of unpolymerized tubulin caused by depolymerization of microtubules in sea urchin larvae resulted in a rapid loss of tubulin mRNA, which was prevented by nearly complete inhibition of protein synthesis. Results of an RNA run-on assay indicated that inhibition of protein synthesis does not alter tubulin gene transcription. Analysis of the decay of tubulin mRNA in embryos in which RNA synthesis was inhibited by actinomycin D indicated that inhibition of protein synthesis prevents the destabilization of tubulin mRNA. The effect was similar whether mRNA was maintained on polysomes in the presence of emetine or anisomycin or displaced from the polysomes in the presence of puromycin or pactamycin; thus, the stabilization of tubulin mRNA is not dependent on the state of the polysomes after inhibition of protein synthesis. Even after tubulin mRNA declined to a low level after depolymerization of microtubules, it could be rescued by treatment of embryos with inhibitors of protein synthesis. Tubulin mRNA could be induced to accumulate prematurely in gastrulae but not in plutei if protein synthesis was inhibited, an observation that is indicative of the importance of the autogenous regulation of tubulin mRNA stability during embryogenesis. Possible explanations for the role of protein synthesis in the control of mRNA stability are discussed. Images PMID:3211150

  5. 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. PMID:26698196

  6. 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. PMID:27194793

  7. Synthesis and Structural Characterization of Carboxyethylpyrrole-Modified Proteins: Mediators of Age-related Macular Degeneration

    PubMed Central

    Lu, Liang; Gu, Xiaorong; Hong, Li; Laird, James; Jaffe, Keeve; Choi, Jaewoo; Crabb, John; Salomon, Robert G.

    2009-01-01

    Protein modifications in which the ε-amino group of lysyl residues is incorporated into a 2-(ω-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina (“wet AMD”) and geographic retinal atrophy (“dry AMD”). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, e.g., through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy. PMID:19786352

  8. A quantitative autoradiographic method for the measurement of local rates of brain protein synthesis

    SciTech Connect

    Dwyer, B.E.; Donatoni, P.; Wasterlain, C.G.

    1982-05-01

    We have developed a new method for measuring local rates of brain protein synthesis in vivo. It combines the intraperitoneal injection of a large dose of low specific activity amino acid with quantitative autoradiography. This method has several advantages: 1) It is ideally suited for young or small animals or where immobilizing an animal is undesirable. 2 The amino acid injection ''floods'' amino acid pools so that errors in estimating precursor specific activity, which is especially important in pathological conditions, are minimized. 3) The method provides for the use of a radioautographic internal standard in which valine incorporation is measured directly. Internal standards from experimental animals correct for tissue protein content and self-absorption of radiation in tissue sections which could vary under experimental conditions.

  9. Simvastatin represses protein synthesis in the muscle-derived C₂C₁₂ cell line with a concomitant reduction in eukaryotic initiation factor 2B expression.

    PubMed

    Tuckow, Alexander P; Jefferson, Sarah J; Kimball, Scot R; Jefferson, Leonard S

    2011-03-01

    Statins are a widely prescribed class of cholesterol lowering drugs whose use is frequently associated with muscle-related ailments. A number of mechanisms have been implicated in statin-induced myotoxicity including alterations in both protein synthesis and protein degradation. The objective of the present study was to explore the mechanism(s) contributing to the statin-induced reduction in protein synthesis in the muscle-derived C₂C₁₂ cell line. Cells were treated with 10 μM simvastatin or vehicle alone for 24 h in 1% serum. Cells exposed to simvastatin exhibited reduced rates of protein synthesis, as evidenced by [(35)S]methionine and [(35)S]cysteine incorporation into protein. The reduction in protein synthesis occurred with a concomitant decrease in expression and activity of eukaryotic initiation factor 2B (eIF2B), a regulated and rate-controlling guanine nucleotide exchange factor known to affect global rates of protein synthesis. The reductions in protein synthesis and eIF2B expression were prevented by coincubation with mevalonate. Simvastatin treatment also resulted in a proteasome-sensitive reduction in the protein expression of all the subunits of the eIF2B heteropentameric complex. Finally, increased phosphorylation of the catalytic ε-subunit at Ser(535) was observed, an event consistent with an observed reduction in eIF2B activity. These results suggest that repression of eIF2B expression and activity may contribute, at least in part, to the statin-induced reduction in protein synthesis. PMID:21224482

  10. Calcium absorption and calcium-binding protein synthesis: solanum malacoxylon reverses strontium inhibition.

    PubMed

    Wasserman, R H

    1974-03-15

    The ingestion of diets containing high concentrations of stable strontium inhibits calcium absorption and intestinal calcium-binding protein synthesis and, as shown by others, does so by inhibiting the conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, the active form of vitamin D. The addition of the South American plant Solanum malacoxylon to strontium-containing diets counteracts the inhibitory action of dietary strontium, thereby indicating that the plant contains a factor which can mimic the action of 1,25-dihydroxycholecalciferol and representing the first such factor identified in a botanical source. PMID:4812040

  11. 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

  12. 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.

  13. Isolation and characterization of an endogenous inhibitor of protein synthesis in Escherichia coli K-12.

    PubMed Central

    Clark, V L

    1979-01-01

    A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of beta-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of beta-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of beta-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation. PMID:104965

  14. Rates of synthesis of prealbumin and retinol-binding protein during acute inflammation in the rat.

    PubMed

    Felding, P; Fex, G

    1985-04-01

    The rates of synthesis of prealbumin (PA), retinol-binding protein (RBP), and other plasma proteins were measured in primary monolayer cultures of rat hepatocytes isolated from normal rats and from rats 18 h after induction of an inflammatory reaction by subcutaneous injection of croton oil. The inflammatory pattern of protein synthesis seemed to persist in the isolated hepatocytes for 1-2 days. This pattern included significantly decreased rates of synthesis of PA. The rate of synthesis of RBP was probably also decreased, but significantly less than the rate of PA synthesis. The results support the idea that it is mainly the decreased rate of PA synthesis which is responsible for the decreased plasma concentration of PA, and its ligand RBP and retinol during inflammation. PMID:4039519

  15. LEUCINE STIMULATION OF SKELETAL MUSCLE PROTEIN SYNTHESIS DURING PROLONGED LEUCINE INFUSION IS DEPENDENT ON AMINO ACID AVAILABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine stimulates protein synthesis in cultured cells, mature rats and neonatal pigs. We have reported that leucine infusion increases protein synthesis in skeletal muscle of neonatal pigs during a 60-min infusion. When leucine infusion was prolonged for 120 min, however, protein synthesis was no...

  16. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were...

  17. 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.

  18. Silver nanoparticles: green synthesis and their antimicrobial activities.

    PubMed

    Sharma, Virender K; Yngard, Ria A; Lin, Yekaterina

    2009-01-30

    This review presents an overview of silver nanoparticles (Ag NPs) preparation by green synthesis approaches that have advantages over conventional methods involving chemical agents associated with environmental toxicity. Green synthetic methods include mixed-valence polyoxometallates, polysaccharide, Tollens, irradiation, and biological. The mixed-valence polyoxometallates method was carried out in water, an environmentally-friendly solvent. Solutions of AgNO(3) containing glucose and starch in water gave starch-protected Ag NPs, which could be integrated into medical applications. Tollens process involves the reduction of Ag(NH(3))(2)(+) by saccharides forming Ag NP films with particle sizes from 50-200 nm, Ag hydrosols with particles in the order of 20-50 nm, and Ag colloid particles of different shapes. The reduction of Ag(NH(3))(2)(+) by HTAB (n-hexadecyltrimethylammonium bromide) gave Ag NPs of different morphologies: cubes, triangles, wires, and aligned wires. Ag NPs synthesis by irradiation of Ag(+) ions does not involve a reducing agent and is an appealing procedure. Eco-friendly bio-organisms in plant extracts contain proteins, which act as both reducing and capping agents forming stable and shape-controlled Ag NPs. The synthetic procedures of polymer-Ag and TiO(2)-Ag NPs are also given. Both Ag NPs and Ag NPs modified by surfactants or polymers showed high antimicrobial activity against gram-positive and gram-negative bacteria. The mechanism of the Ag NP bactericidal activity is discussed in terms of Ag NP interaction with the cell membranes of bacteria. Silver-containing filters are shown to have antibacterial properties in water and air purification. Finally, human and environmental implications of Ag NPs to the ecology of aquatic environment are briefly discussed. PMID:18945421

  19. Inhibition of protein synthesis or mTOR in the basolateral amygdala blocks retrieval-induced memory strengthening.

    PubMed

    Pedroso, Thiago R; Jobim, Paulo F C; Carvalho, Leonardo M; Christoff, Raissa R; Maurmann, Natasha; Reolon, Gustavo K; Werenicz, Aline; Roesler, Rafael

    2013-11-01

    Fear memory retrieval can lead to either reconsolidation (accompanied or not by strengthening of the memory trace) or extinction. Here, we show that non-reinforced retrieval of inhibitory avoidance (IA) conditioning can induce memory strengthening assessed in a subsequent retention test trial. Infusion of the protein synthesis inhibitor cycloheximide or the mTOR inhibitor rapamycin into the rat basolateral complex of the amygdala (BLA) after a reactivation (retrieval) session impaired retrieval-induced strengthening. Intra-BLA infusion of the mRNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) after retrieval had no effect. These findings provide the first evidence suggesting that non-reinforced IA retrieval can lead to memory strengthening through a mechanism dependent on protein synthesis and mTOR activity in the BLA. PMID:23649124

  20. R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome

    PubMed Central

    Qin, Mei; Huang, Tianjian; Kader, Michael; Krych, Leland; Xia, Zengyan; Burlin, Thomas; Zeidler, Zachary; Zhao, Tingrui

    2015-01-01

    Background: Fragile X syndrome (FXS) is the most common known inherited form of intellectual disability and the single genomic cause of autism spectrum disorders. It is caused by the absence of a fragile X mental retardation gene (Fmr1) product, FMRP, an RNA-binding translation suppressor. Elevated rates of protein synthesis in the brain and an imbalance between synaptic signaling via glutamate and γ-aminobutyric acid (GABA) are both considered important in the pathogenesis of FXS. In a mouse model of FXS (Fmr1 knockout [KO]), treatment with R-baclofen reversed some behavioral and biochemical phenotypes. A remaining crucial question is whether R-baclofen is also able to reverse increased brain protein synthesis rates. Methods: To answer this question, we measured regional rates of cerebral protein synthesis in vivo with the L-[1-14C]leucine method in vehicle- and R-baclofen–treated wildtype and Fmr1 KO mice. We further probed signaling pathways involved in the regulation of protein synthesis. Results: Acute R-baclofen administration corrected elevated protein synthesis and reduced deficits on a test of social behavior in adult Fmr1 KO mice. It also suppressed activity of the mammalian target of rapamycin pathway, particularly in synaptosome-enriched fractions, but it had no effect on extracellular-regulated kinase 1/2 activity. Ninety min after R-baclofen treatment, we observed an increase in metabotropic glutamate receptor 5 expression in the frontal cortex, a finding that may shed light on the tolerance observed in human studies with this drug. Conclusions: Our results suggest that treatment via activation of the GABA (GABA receptor subtype B) system warrants further study in patients with FXS. PMID:25820841

  1. Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis

    PubMed Central

    Munday, Diane C.; Wu, Weining; Smith, Nikki; Fix, Jenna; Noton, Sarah Louise; Galloux, Marie; Touzelet, Olivier; Armstrong, Stuart D.; Dawson, Jenna M.; Aljabr, Waleed; Easton, Andrew J.; Rameix-Welti, Marie-Anne; de Oliveira, Andressa Peres; Simabuco, Fernando M.; Ventura, Armando M.; Hughes, David J.; Barr, John N.; Fearns, Rachel; Digard, Paul

    2014-01-01

    ABSTRACT The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is

  2. Functions of AMP-activated protein kinase in adipose tissue

    PubMed Central

    Daval, Marie; Foufelle, Fabienne; Ferré, Pascal

    2006-01-01

    AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Its functions have been extensively studied in muscles and liver. AMPK stimulates pathways which increase energy production (glucose transport, fatty acid oxidation) and switches off pathways which consume energy (lipogenesis, protein synthesis, gluconeogenesis). This has led to the concept that AMPK has an interesting pharmaceutical potential in situations of insulin resistance and it is indeed the target of existing drugs and hormones which improve insulin sensitivity. Adipose tissue is a key player in energy metabolism through the release of substrates and hormones involved in metabolism and insulin sensitivity. Activation of AMPK in adipose tissue can be achieved through situations such as fasting and exercise. Leptin and adiponectin as well as hypoglycaemic drugs are activators of adipose tissue AMPK. This activation probably involves changes in the AMP/ATP ratio and the upstream kinase LKB1. When activated, AMPK limits fatty acid efflux from adipocytes and favours local fatty acid oxidation. Since fatty acids have a key role in insulin resistance, especially in muscles, activating AMPK in adipose tissue might be found to be beneficial in insulin-resistant states, particularly as AMPK activation also reduces cytokine secretion in adipocytes. PMID:16709632

  3. 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.

  4. Taxol induces paraptosis independent of both protein synthesis and MAPK pathway.

    PubMed

    Sun, Qingrui; Chen, Tongsheng; Wang, Xiaoping; Wei, Xunbin

    2010-02-01

    Our recent studies have shown that high concentration of taxol induced a caspase-independent paraptosis-like cell death and cytoplasmic vacuolization derived predominantly from endoplasmic reticulum (ER) swelling in human lung carcinoma cell lines (ASTC-a-1). In this report, we further explored the relationship between taxol-induced cell death and vacuolization, and the roles of protein synthesis, mitogen-activated protein kinase kinases (MEK), c-jun N-terminal kinase (JNK) and P38 in taxol-induced paraptosis. Enhanced green fluorescent protein (EGFP) was used to probe the cell morphological change, while ER-targeted red fluorescent protein (er-RFP) was used to probe ER spatial distribution. Real-time monitoring of the ER swelling dynamics during the formation of vacuolization inside single living cells co-expressing EGFP and er-RFP further demonstrated that taxol-induced cytoplasmic vacuolization was from the ER restructuring due to fusion and swelling. PI staining showed that taxol-induced vacuolization was not necrosis. These results further demonstrated that the taxol-induced cell death was neither apoptosis nor necrosis, and fitted the criteria of paraptosis characterized by cytoplasmic vacuolization, caspase-independence, lack of apoptotic morphology and insensitivity to broad caspase inhibitor. Our data further indicated that taxol-induced paraptosis required neither protein synthesis nor the participation of MEK, JNK, and P38, which was different from the insulin-like growth factor I receptor (IGFIR)-induced paraptosis. These results suggest that high concentration of taxol activates an alternative paraptotic cell death pathway. PMID:19918793

  5. Leucine alleviates dexamethasone-induced suppression of muscle protein synthesis via synergy involvement of mTOR and AMPK pathways.

    PubMed

    Wang, Xiao J; Yang, Xin; Wang, Ru X; Jiao, Hong C; Zhao, Jing P; Song, Zhi G; Lin, Hai

    2016-07-01

    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

  6. Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway.

    PubMed

    Suryawan, Agus; Nguyen, Hanh V; Almonaci, Rosemarie D; Davis, Teresa A

    2012-02-28

    Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine's action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg(-1)·h(-1)) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2α and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation. PMID:22675606

  7. Intestinal mucosa in diabetes: synthesis of total proteins and sucrase-isomaltase

    SciTech Connect

    Olsen, W.A.; Perchellet, E.; Malinowski, R.L.

    1986-06-01

    The effects of insulin deficiency on nitrogen metabolism in muscle and liver have been extensively studied with recent in vivo demonstration of impaired protein synthesis in rats with streptozotocin-induced diabetes. Despite the significant contribution of small intestinal mucosa to overall protein metabolism, the effect of insulin deficiency on intestinal protein synthesis have not been completely defined. The authors studied the effects of streptozotocin-induced diabetes on total protein synthesis by small intestinal mucosa and on synthesis of a single enzyme protein of the enterocyte brush-border membrane sucrase-isomaltase. They used the flood-dose technique to minimize the difficulties of measuring specific radioactivity of precursor phenylalanine and determined incorporation into mucosal proteins and sucrase-isomaltase 20 min after injection of the labeled amino acid. Diabetes did not alter mucosal mass as determined by weight and content of protein and DNA during the 5 days after injection of streptozotocin. Increased rates of sucrase-isomaltase synthesis developed beginning on day 3, and those of total protein developed on day 5. Thus intestinal mucosal protein synthesis is not an insulin-sensitive process.

  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. 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

  10. A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes.

    PubMed

    Madin, K; Sawasaki, T; Ogasawara, T; Endo, Y

    2000-01-18

    Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself. PMID:10639118

  11. 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

  12. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    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

  13. 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...

  14. Phenylketonuria: brain phenylalanine concentrations relate inversely to cerebral protein synthesis

    PubMed Central

    de Groot, Martijn J; Sijens, Paul E; Reijngoud, Dirk-Jan; Paans, Anne M; van Spronsen, Francjan J

    2015-01-01

    In phenylketonuria, elevated plasma phenylalanine concentrations may disturb blood-to-brain large neutral amino acid (LNAA) transport and cerebral protein synthesis (CPS). We investigated the associations between these processes, using data obtained by positron emission tomography with l-[1-11C]-tyrosine (11C-Tyr) as a tracer. Blood-to-brain transport of non-Phe LNAAs was modeled by the rate constant for 11C-Tyr transport from arterial plasma to brain tissue (K1), while CPS was modeled by the rate constant for 11C-Tyr incorporation into cerebral protein (k3). Brain phenylalanine concentrations were measured by magnetic resonance spectroscopy in three volumes of interest (VOIs): supraventricular brain tissue (VOI 1), ventricular brain tissue (VOI 2), and fluid-containing ventricular voxels (VOI 3). The associations between k3 and each predictor variable were analyzed by multiple linear regression. The rate constant k3 was inversely associated with brain phenylalanine concentrations in VOIs 2 and 3 (adjusted R2=0.826, F=19.936, P=0.021). Since brain phenylalanine concentrations in these VOIs highly correlated with each other, the specific associations of each predictor with k3 could not be determined. The associations between k3 and plasma phenylalanine concentration, K1, and brain phenylalanine concentrations in VOI 1 were nonsignificant. In conclusion, our study shows an inverse association between k3 and increased brain phenylalanine concentrations. PMID:25352046

  15. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility

    PubMed Central

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

    2016-01-01

    Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues. PMID:27468805

  16. 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. PMID:24395411

  17. Chemical Synthesis of a Glycopeptide Derived from Skp1 for Probing Protein Specific Glycosylation.

    PubMed

    Chinoy, Zoeisha S; Schafer, Christopher M; West, Christopher M; Boons, Geert-Jan

    2015-08-10

    Skp1 is a cytoplasmic and nuclear protein, best known as an adaptor of the SCF family of E3-ubiquitin ligases that label proteins for their degradation. Skp1 in Dictyostelium is posttranslationally modified on a specific hydroxyproline (Hyp) residue by a pentasaccharide, which consists of a Fucα1,2-Galβ-1,3-GlcNAcα core, decorated with two α-linked Gal residues. A glycopeptide derived form Skp1 was prepared to characterize the α-galactosyltransferase (AgtA) that mediates the addition of the α-Gal moieties, and to develop antibodies suitable for tracking the trisaccharide isoform of Skp1 in cells. A strategy was developed for the synthesis of the core trisaccharide-Hyp based on the use of 2-naphthylmethyl (Nap) ethers as permanent protecting groups to allow late stage installation of the Hyp moiety. Tuning of glycosyl donor and acceptor reactivities was critical for achieving high yields and anomeric selectivities of glycosylations. The trisaccharide-Hyp moiety was employed for the preparation of the glycopeptide using microwave-assisted solid phase peptide synthesis. Enzyme kinetic studies revealed that trisaccharide-Hyp and trisaccharide-peptide are poorly recognized by AgtA, indicating the importance of context provided by the native Skp1 protein for engagement with the active site. The trisaccharide-peptide was a potent immunogen capable of generating a rabbit antiserum that was highly selective toward the trisaccharide isoform of full-length Skp1. PMID:26179871

  18. 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

  19. 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. PMID:25096519

  20. Synthesis, characterization and biological activities of curcumin nanospheres.

    PubMed

    Arunraj, T R; Sanoj Rejinold, N; Mangalathillam, Sabitha; Saroj, Soumya; Biswas, Raja; Jayakumar, R

    2014-02-01

    Curcumin is one of the most versatile compounds obtained from Curcuma longa. The major obstacle in the therapeutic use of curcumin is its aqueous solubility. To enhance its aqueous solubility and biological activities, we prepared curcumin nanospheres (CNSs) by wet milling-solvent evaporation technique without any surfactants. In this study, we have focused on the synthesis, characterization and biological effects of CNSs. DLS and SEM analyses showed 50-80 nm spherical shaped CNSs with a zeta potential of -31.65 mV. FTIR revealed that there were no structural changes to CNSs. Antibacterial and antifungal studies proved that CNSs were much more effective than curcumin against Escherichia coil, Staphylococcus aureus and Candida albicans. Antioxidant activity of CNSs showed promising result for therapeutic applications. The in vitro anti-inflammatory studies proved that CNSs possessed enhanced anti-inflammatory effect against protein denaturation. Cytotoxicity and uptake of CNSs showed more toxicity on cancer cells (T47D, MG63, A375) sparing normal HDF and IEC cell lines. Skin permeation studies showed CNSs retained at different layers of pig skin. These results give clear evidence for their use against microbial and fungal skin infections as well as cancer treatment. PMID:24738332

  1. Cardiac protein synthesis and degradation during thyroxine-induced left ventricular hypertrophy.

    PubMed

    Parmacek, M S; Magid, N M; Lesch, M; Decker, R S; Samarel, A M

    1986-11-01

    Assessment of cardiac protein metabolism in thyroxine-induced left ventricular hypertrophy requires measurements of both protein synthesis and degradation. In vivo protein degradative rates can best be measured as the difference between rates of protein synthesis and growth. Accordingly, rates of left ventricular protein accumulation were determined in growing rabbits, and in animals administered intravenous L-thyroxine (200 micrograms X kg-1 X day-1) for up to 15 days. Left ventricular protein fractional synthetic rates in euthyroid and thyroxine-treated rabbits were measured by continuous infusion of [3H]leucine (200 mu Ci/h X 6 h), and results converted to milligrams protein synthesized and degraded per day. Thyroxine administration produced left ventricular hypertrophy by increasing the rate of total protein synthesis (35.7 +/- 2.0, 71.0 +/- 7.0, and 62.6 +/- 4.0 mg of left ventricular protein synthesized per day for 0-, 3-, and 9-day, thyroxine-treated rabbits, respectively). However, the increased rate of total protein synthesis was greater than the measured rate of total protein accumulation (8.1 vs. 15.9 mg protein/day for euthyroid and thyroxine-treated animals), indicating that left ventricular protein degradative rates were increased as well. These studies indicate that accelerated proteolysis may be important in the molecular and architectural remodeling of the rapidly hypertrophying heart during thyrotoxicosis. PMID:2946236

  2. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins.

    PubMed

    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

  3. 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. PMID:7864110

  4. The protein quality control system manages plant defence compound synthesis.

    PubMed

    Pollier, Jacob; Moses, Tessa; González-Guzmán, Miguel; De Geyter, Nathan; Lippens, Saskia; Vanden Bossche, Robin; Marhavý, Peter; Kremer, Anna; Morreel, Kris; Guérin, Christopher J; Tava, Aldo; Oleszek, Wieslaw; Thevelein, Johan M; Campos, Narciso; Goormachtig, Sofie; Goossens, Alain

    2013-12-01

    Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues. PMID:24213631

  5. Altered cerebral protein synthesis in fragile X syndrome: studies in human subjects and knockout mice

    PubMed Central

    Qin, Mei; Schmidt, Kathleen C; Zametkin, Alan J; Bishu, Shrinivas; Horowitz, Lisa M; Burlin, Thomas V; Xia, Zengyan; Huang, Tianjiang; Quezado, Zenaide M; Smith, Carolyn Beebe

    2013-01-01

    Dysregulated protein synthesis is thought to be a core phenotype of fragile X syndrome (FXS). In a mouse model (Fmr1 knockout (KO)) of FXS, rates of cerebral protein synthesis (rCPS) are increased in selective brain regions. We hypothesized that rCPS are also increased in FXS subjects. We measured rCPS with the ℒ-[1-11C]leucine positron emission tomography (PET) method in whole brain and 10 regions in 15 FXS subjects who, because of their impairments, were studied under deep sedation with propofol. We compared results with those of 12 age-matched controls studied both awake and sedated. In controls, we found no differences in rCPS between awake and propofol sedation. Contrary to our hypothesis, FXS subjects under propofol sedation had reduced rCPS in whole brain, cerebellum, and cortex compared with sedated controls. To investigate whether propofol could have a disparate effect in FXS subjects masking usually elevated rCPS, we measured rCPS in C57Bl/6 wild-type (WT) and KO mice awake or under propofol sedation. Propofol decreased rCPS substantially in most regions examined in KO mice, but in WT mice caused few discrete changes. Propofol acts by decreasing neuronal activity either directly or by increasing inhibitory synaptic activity. Our results suggest that changes in synaptic signaling can correct increased rCPS in FXS. PMID:23299245

  6. Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression.

    PubMed

    Kurotani, Atsushi; Takagi, Tetsuo; Toyama, Mitsutoshi; Shirouzu, Mikako; Yokoyama, Shigeyuki; Fukami, Yasuo; Tokmakov, Alexander A

    2010-04-01

    High-throughput cell-free protein synthesis is being used increasingly in structural/functional genomics projects. However, the factors determining expression success are poorly understood. Here, we evaluated the expression of 3066 human proteins and their domains in a bacterial cell-free system and analyzed the correlation of protein expression with 39 physicochemical and structural properties of proteins. As a result of the bioinformatics analysis performed, we determined the 18 most influential features that affect protein amenability to cell-free expression. They include protein length; hydrophobicity; pI; content of charged, nonpolar, and aromatic residues;, cysteine content; solvent accessibility; presence of coiled coil; content of intrinsically disordered and structured (alpha-helix and beta-sheet) sequence; number of disulfide bonds and functional domains; presence of transmembrane regions; PEST motifs; and signaling sequences. This study represents the first comprehensive bioinformatics analysis of heterologous protein synthesis in a cell-free system. The rules and correlations revealed here provide a plethora of important insights into rationalization of cell-free protein production and can be of practical use for protein engineering with the aim of increasing expression success.-Kurotani, A., Takagi, T., Toyama, M., Shirouzu, M., Yokoyama, S., Fukami, Y., Tokmakov, A. A. Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression. PMID:19940260

  7. Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

    PubMed Central

    Imbra, R J; Karin, M

    1987-01-01

    The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C. Images PMID:3600629

  8. 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.

  9. 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

  10. Pushing the Boundaries of Chemical Protein Synthesis: The Case of Ubiquitin Chains and Polyubiquitinated Peptides and Proteins.

    PubMed

    Meledin, Roman; Mali, Sachitanand M; Brik, Ashraf

    2016-02-01

    Chemical synthesis offers unique opportunities to prepare proteins with precise control of the atomic composition. Thanks to recent breakthroughs in synthetic methods, the preparation of large and complex proteins composed of 200-300 residues has now become possible. With these advances, a unique toolbox has been created to enable chemical biologists to investigate proteins that are difficult or even impossible to achieve otherwise, such as posttranslationally modified proteins and proteins composed of d-amino acids. In this review we describe the latest achievements in constructing protein conjugates of record sizes, such as those that are involved in the ubiquitin system. PMID:26477936

  11. Biodegradable nanoparticles for protein delivery: analysis of preparation conditions on particle morphology and protein loading, activity and sustained release properties.

    PubMed

    Coleman, Jason; Lowman, Anthony

    2012-01-01

    PLGA particles have been extensively used as a sustained drug-delivery system, but there are multiple drawbacks when delivering proteins. The focus of this work is to address the most significant disadvantages to the W/O/W double emulsion procedure and demonstrate that simple changes to this procedure can have significant changes to particle size and dispersity and considerable improvements to protein loading, activity and sustained active protein release. A systematic approach was taken to analyze the effects of the following variables: solvent miscibility (dichloromethane (DCM), ethyl acetate, acetone), homogenization speed (10 000-25 000 rpm), PLGA concentration (10-30 mg/ml) and additives in both the organic (sucrose acetate isobutyrate (SAIB)) and aqueous (bovine serum albumin (BSA)) phases. Increasing solvent miscibility decreased particle size, dispersity and protein denaturation, while maintaining adequate protein loading. Increasing solvent miscibility also lowered the impact of homogenization on particle size and dispersity and protein activity. Changes to PLGA concentration demonstrated a minimum impact on particle size and dispersity, but showed an inverse relationship between protein encapsulation efficiency and particle protein weight percent. Most particles tested provided sustained release of active protein over 60 days. Increasing solvent miscibility resulted in increases in the percent of active protein released. When subjected to synthesis conditions with DCM as the solvent, BSA as a stabilizer resulted in the maximum stabilization of protein at a concentration of 100 mg/ml. At this concentration, BSA allowed for increases in the total amount of active protein delivered for all three solvents. The benefit of SAIB was primarily increased protein loading. PMID:21639993

  12. Synthesis and protein degradation capacity of photoactivated enediynes.

    PubMed

    Fouad, Farid S; Wright, Justin M; Plourde, Gary; Purohit, Ajay D; Wyatt, Justin K; El-Shafey, Ahmed; Hynd, George; Crasto, Curtis F; Lin, Yiqing; Jones, Graham B

    2005-11-25

    [structure: see text] The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer from diyl radicals produced from enediynes to form captodatively stabilized carbon centered aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding, a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were prepared and used to target specific proteins, showing good correlation between affinity and photoinduced protein degrading activity. The findings have potential applications in the design of artificial chemical proteases and add to our understanding of the mechanism of action of the clinically important enediyne antitumor antibiotics. PMID:16292807

  13. 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. PMID:7852311

  14. Mechanisms underlying the protein-kinase mediated regulation of the HERG potassium channel synthesis

    PubMed Central

    Krishnan, Yamini; Li, Yan; Zheng, Renjian; Kanda, Vikram; McDonald, Thomas V.

    2012-01-01

    The HERG (human ether-a-go-go related gene) potassium channel aids in repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cAMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo. PMID:22613764

  15. Peroxidase synthesis and activity in the interaction of soybean with Phytophthora megasperma f. sp. glycinea (Pmg)

    SciTech Connect

    Chibbar, R.N.; Esnault, R.; Lee, D.; van Huystee, R.B.; Ward, E.W.B.

    1986-04-01

    Changes, in peroxidase (EC1.11.1.7) have been reported following infection. However, determinations of biosynthesis of quantities of the peroxidase protein molecule have not been madeexclamation In this study hypocotyl of soybean seedlings (Glycine max; cv Harosoy, susceptible; cv Harosoy 63, resistant) were inoculated with zoospores of Pmg. Incorporation of /sup 35/S-methionine (supplied with inoculum) in TCA precipitates was measured. Peroxidase synthesis was measured by immuno precipitation using antibodies against a cationic and an anionic peroxidase derived from peanut cells. Specific peroxidase activity increased rapidly from 5 to 9 h following infection in the resistant reaction but not in the susceptible reaction or the water controls. There was increased synthesis of the anionic peroxidase but not of the cationic peroxidase in the resistant reaction. The anionic peroxidase did not increase in the susceptible until 15 h. The ratio of peroxidase synthesis to total protein synthesis decreased in inoculated tissues compared to control. Peroxidase synthesis is, therefore, a relative minor host response to infection.

  16. Long-lived crowded-litter mice have an age-dependent increase in protein synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation

    PubMed Central

    Bruns, Danielle R.; Peelor, Frederick F.; Biela, Laurie M.; Miller, Richard A.; Hamilton, Karyn L.; Miller, Benjamin F.

    2014-01-01

    Increasing mouse litter size [crowded litter (CL)] presumably imposes a transient nutrient stress during suckling and extends lifespan through unknown mechanisms. Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling but maintained protein synthesis, suggesting maintenance of existing cellular structures. We hypothesized that CL would exhibit similar synthetic and signaling responses to other long-lived models and, by comparing synthesis of new protein to new DNA, that insight may be gained into the potential preservation of existing cellular structures in the CL model. Protein and DNA synthesis was assessed in gastroc complex, heart, and liver of 4- and 7-mo CL mice. We also examined mTORC1 signaling in 3- and 7-mo aged animals. Compared with controls, 4-mo CL had greater DNA synthesis in gastroc complex with no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than controls in heart and greater protein synthesis and mTORC1 substrate phosphorylation across tissues. The increased new protein-to-new DNA synthesis ratio suggests that new proteins are synthesized more so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls. We propose that, in CL, protein synthesis shifts from being directed toward new cells (4 mo) to maintenance of existing cellular structures (7 mo), independently of decreased mTORC1. PMID:25205819

  17. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  18. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  19. 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.

  20. Oxygen tension limits nitric oxide synthesis by activated macrophages.

    PubMed Central

    McCormick, C C; Li, W P; Calero, M

    2000-01-01

    Previous studies have established that constitutive calcium-dependent ('low-output') nitric oxide synthase (NOS) is regulated by oxygen tension. We have investigated the role of oxygen tension in the synthesis of NO by the 'high-output' calcium-independent NOS in activated macrophages. Hypoxia increased macrophage NOS gene expression in the presence of one additional activator, such as lipopolysaccharide or interferon-gamma, but not in the presence of both. Hypoxia markedly reduced the synthesis of NO by activated macrophages (as measured by accumulation of nitrite and citrulline), such that, at 1% oxygen tension, NO accumulation was reduced by 80-90%. The apparent K(m) for oxygen calculated from cells exposed to a range of oxygen tensions was found to be 10.8%, or 137 microM, O(2) This value is considerably higher than the oxygen tension in tissues, and is virtually identical to that reported recently for purified recombinant macrophage NOS. The decrease in NO synthesis did not appear to be due to diminished arginine or cofactor availability, since arginine transport and NO synthesis during recovery in normoxia were normal. Analysis of NO synthesis during hypoxia as a function of extracellular arginine indicated that an altered V(max), but not K(m)(Arg), accounted for the observed decrease in NO synthesis. We conclude that oxygen tension regulates the synthesis of NO in macrophages by a mechanism similar to that described previously for the calcium-dependent low-output NOS. Our data suggest that oxygen tension may be an important physiological regulator of macrophage NO synthesis in vivo. PMID:10970783

  1. Synthesis of dental matrix proteins and viability of odontoblast-like cells irradiated with blue LED.

    PubMed

    Alonso, Juliana Rosa Luiz; Turrioni, Ana Paula Silveira; Basso, Fernanda Gonçalves; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-04-01

    To evaluate the effect of irradiation with light-emitting diode (LED; 455 nm) on the viability and synthesis of dentin matrix proteins by odontoblast-like cells, MDPC-23 cells were cultivated (10(4) cells/cm(2)) in 24-well culture plates. After 12 h incubation in Dulbecco's modified Eagle's medium (DMEM), the cells were submitted to nutritional restriction by means of reducing the concentration of fetal bovine serum (FBS) for an additional 12 h. Cells were irradiated one single time with one of the following energy densities (EDs): 0.5, 2, 4, 10, or 15 J/cm(2) and irradiance fixed at 20 mW/cm(2). Non-irradiated cells served as control. After 72 h, cells were evaluated with regard to viability (methylthiazol tetrazolium technique (MTT)), mineralization nodule (MN) formation, total protein (TP) production, alkaline phosphatase activity (ALP), and collagen synthesis (Sircol), n = 8. The data were submitted to Kruskal-Wallis and Mann-Whitney tests (p > 0.05). There was no statistical difference between the viability of cells irradiated or not (control), for all the EDs. However, an increase in TP was observed for all the EDs when compared with the control group. A reduced ALP activity was seen in all irradiated groups, except for the ED of 0.5 J/cm(2), which did not differ from the control. There was no difference between the irradiated groups and control regarding collagen synthesis, with the exception of the ED of 10 J/cm(2), which inhibited this cell function. Significant reduction in MN occurred only for the EDs of 0.5 and 2 J/cm(2). The single irradiation with blue LED (455 nm), irradiance of 20 mW/cm(2), and energy densities ranging from 0.5 to 15 J/cm(2) exerted no effective biostimulatory capacity on odontoblast-like cells. PMID:26873499

  2. Glucagon-like peptide-1 receptor agonists increase pancreatic mass by induction of protein synthesis.

    PubMed

    Koehler, Jacqueline A; Baggio, Laurie L; Cao, Xiemin; Abdulla, Tahmid; Campbell, Jonathan E; Secher, Thomas; Jelsing, Jacob; Larsen, Brett; Drucker, Daniel J

    2015-03-01

    Glucagon-like peptide-1 (GLP-1) controls glucose homeostasis by regulating secretion of insulin and glucagon through a single GLP-1 receptor (GLP-1R). GLP-1R agonists also increase pancreatic weight in some preclinical studies through poorly understood mechanisms. Here we demonstrate that the increase in pancreatic weight following activation of GLP-1R signaling in mice reflects an increase in acinar cell mass, without changes in ductal compartments or β-cell mass. GLP-1R agonists did not increase pancreatic DNA content or the number of Ki67(+) cells in the exocrine compartment; however, pancreatic protein content was increased in mice treated with exendin-4 or liraglutide. The increased pancreatic mass and protein content was independent of cholecystokinin receptors, associated with a rapid increase in S6 phosphorylation, and mediated through the GLP-1R. Rapamycin abrogated the GLP-1R-dependent increase in pancreatic mass but had no effect on the robust induction of Reg3α and Reg3β gene expression. Mass spectrometry analysis identified GLP-1R-dependent upregulation of Reg family members, as well as proteins important for translation and export, including Fam129a, eIF4a1, Wars, and Dmbt1. Hence, pharmacological GLP-1R activation induces protein synthesis, leading to increased pancreatic mass, independent of changes in DNA content or cell proliferation in mice. PMID:25277394

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

    PubMed

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-03-01

    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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells.

    PubMed

    Jiang, Qian; He, Liuqin; Hou, Yongqing; Chen, Jiashun; Duan, Yehui; Deng, Dun; Wu, Guoyao; Yin, Yulong; Yao, Kang

    2016-09-01

    Alpha-ketoglutarate (AKG), a key intermediate in the Krebs cycle, has been reported to promote protein synthesis through activating mechanistic targeting of rapamycin (mTOR) in enterocytes. The study tested the hypothesis that AKG may enhance growth and milk protein synthesis in porcine mammary epithelial cells (PMECs). PMECs were cultured for 96 h in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing prolactin (2 µg/ml) and AKG (0 or 1.5 mM). At the end of 96-h culture, the abundance of apoptosis-related proteins (caspase-3, caspase-9), milk-specific proteins (α-lactalbumin and β-casein), mTOR signaling proteins (mTOR, p-mTOR, PERK, p-PERK, eIF2a, P70S6K and p-P70S6K), and endoplasmic reticulum stress (ERS)-associated proteins (BiP and CHOP) in PMEC were determined. Addition of AKG dose-dependently enhanced cell viability in the absence or presence of prolactin, with optimal concentrations of AKG being at 1.0 and 1.5 mM, respectively. In the presence of prolactin, addition of 1.5 mM AKG: (1) decreased (P < 0.05) the abundance of caspase-3 and caspase-9 by 21 and 39 %; (2) enhanced (P < 0.05) the phosphorylation of p-mTOR and p-P70S6K by 39 and 89 %, respectively; (3) increased (P < 0.05) the production of β-casein and α-lactalbumin by 16 and 20 %, respectively; (4) attenuated (P < 0.05) the expression of CHOP by 34 % but promoted (P < 0.05) the expression of BiP by 46 %; (5) increased (P < 0.05) the secretion of lactose by 15 %, when compared to the 0 mM AKG group. Rapamycin (50 nM; an inhibitor of mTOR) attenuated (P < 0.05) the stimulatory effect of AKG on mTOR signaling and syntheses of milk protein and lactose, while relieving (P < 0.05) an inhibitory effect of AKG on expression of proteins related to ERS. Collectively, our results indicate that AKG enhances milk protein production by modulating mTOR and ERS signaling pathways in PMECs. PMID:27188418

  10. Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

    PubMed Central

    Richter, Uwe; Lahtinen, Taina; Marttinen, Paula; Suomi, Fumi

    2015-01-01

    Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes. PMID:26504172

  11. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  12. Petunia nectar proteins have ribonuclease activity.

    PubMed

    Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

    2010-06-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  13. Petunia nectar proteins have ribonuclease activity

    PubMed Central

    Hillwig, Melissa S.; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W.; MacIntosh, Gustavo C.

    2010-01-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  14. Synthesis of protein-containing polymers in organic solvents.

    PubMed

    Yang, Z; Williams, D; Russell, A J

    1995-01-01

    Subtilisin has been modified with polyethylene glycol (PEG) monomethacrylate (MW 8000) by reductive alkylation, and incorporated into polymethyl methacrylate durring free-radical initiated polymerization. The activity and stability of the PEG-modified enzymes have been determined in aqueous buffer and organic solvents. The K(m) and V(max) values for unmodified, singly and doubly modified subtilisin were compared in these environments, and the half-lives of both modified enzymes were remarkably high (up to 2 months). The protein-containing polymer was analyzed for activity and polymer properties, and our results indicate that active subtilisin can be incorporated into polymethyl methacrylate during polymerization in organic solvents while retaining its activity and stability. (c) 1995 John Wiley & Sons, Inc. PMID:18623046

  15. Synthesis and antiplatelet activity of antithrombotic thiourea compounds: biological and structure-activity relationship studies.

    PubMed

    Lourenço, André Luiz; Saito, Max Seidy; Dorneles, Luís Eduardo Gomes; Viana, Gil Mendes; Sathler, Plínio Cunha; Aguiar, Lúcia Cruz de Sequeira; de Pádula, Marcelo; Domingos, Thaisa Francielle Souza; Fraga, Aline Guerra Manssour; Rodrigues, Carlos Rangel; de Sousa, Valeria Pereira; Castro, Helena Carla; Cabral, Lucio Mendes

    2015-01-01

    The incidence of hematological disorders has increased steadily in Western countries despite the advances in drug development. The high expression of the multi-resistance protein 4 in patients with transitory aspirin resistance, points to the importance of finding new molecules, including those that are not affected by these proteins. In this work, we describe the synthesis and biological evaluation of a series of N,N'-disubstituted thioureas derivatives using in vitro and in silico approaches. New designed compounds inhibit the arachidonic acid pathway in human platelets. The most active thioureas (compounds 3d, 3i, 3m and 3p) displayed IC50 values ranging from 29 to 84 µM with direct influence over in vitro PGE2 and TXA2 formation. In silico evaluation of these compounds suggests that direct blockage of the tyrosyl-radical at the COX-1 active site is achieved by strong hydrophobic contacts as well as electrostatic interactions. A low toxicity profile of this series was observed through hemolytic, genotoxic and mutagenic assays. The most active thioureas were able to reduce both PGE2 and TXB2 production in human platelets, suggesting a direct inhibition of COX-1. These results reinforce their promising profile as lead antiplatelet agents for further in vivo experimental investigations. PMID:25903367

  16. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

    PubMed Central

    Areta, José L; Burke, Louise M; Ross, Megan L; Camera, Donny M; West, Daniel W D; Broad, Elizabeth M; Jeacocke, Nikki A; Moore, Daniel R; Stellingwerff, Trent; Phillips, Stuart M; Hawley, John A; Coffey, Vernon G

    2013-01-01

    Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n= 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-13C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1–12 h recovery (88–148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31–48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass. PMID:23459753

  17. 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. PMID:26792192

  18. Auxin Reduces the Synthesis of Major Vacuolar Proteins in Tobacco Mesophyl Protoplast

    PubMed Central

    Meyer, Yves; Chartier, Yvette; Alibert, Gilbert

    1987-01-01

    We have established that polypeptides whose synthesis is reduced by 2,4-dichlorophenoxyacetic acid during in vitro culture of tobacco mesophyll protoplasts are secreted into the vacuole where they constitute the bulk of labeled proteins. In addition, these proteins continue to be synthesized in protoplast-derived cultured cells and their synthesis is strictly correlated with the size of the cell, i.e. with vacuolar size. Images Fig. 1 Fig. 2 Fig. 3 PMID:16665313

  19. 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

  20. 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. PMID:21960964

  1. 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

  2. Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications.

    PubMed

    Chong, Shaorong

    2014-01-01

    During the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology. PMID:25271714

  3. Overview of Cell-Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

    PubMed Central

    Chong, Shaorong

    2014-01-01

    During early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owning to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate and wheat germ extracts to recent insect and human cell extracts to defined systems reconstituted from purified recombinant components. Though each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This article provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology. PMID:25271714

  4. The cytotoxicity of the parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins.

    PubMed Central

    Anouja, F; Wattiez, R; Mousset, S; Caillet-Fauquet, P

    1997-01-01

    Autonomous parvoviruses exert lytic and cytostatic effects believed to contribute to their antineoplastic activity. Studies with inducible clones have demonstrated a direct involvement of parvovirus nonstructural proteins (NS) in oncolysis. Human and rat fibroblasts have been stably transfected with MVM(p) (minute virus of mice prototype strain) NS genes cloned under the control of a hormone-inducible promoter. Dexamethasone-induced synthesis of the NS proteins in sensitive transformed cells results in cell killing within a few days. From these sensitive cell lines have been isolated some NS-resistant clones that also prove resistant to MVM(p) infection, suggesting that cell factors modulate NS cytotoxicity. We have previously reported that factors involved in cell cycle regulation may contribute to this modulation, since NS toxicity requires cell proliferation and correlates with a cell cycle perturbation leading to an arrest in phase S/G2. In addition to its role in cytotoxicity, NS1 can regulate transcription driven by parvovirus and nonparvovirus promoters. Since phosphorylation is a critical event in controlling the activity of many proteins, notably transcription factors and cell cycle-regulated proteins, we have examined the effect of NS1 on the synthesis and phosphorylation of cell proteins. Our results indicate that NS1 interferes, within 7 h of induction, with phosphorylation of a protein of about 14 kDa (p14). Cell synchronization has enabled us to show that phosphorylation of this protein occurs in early S phase and is prevented when NS1 is induced. This early effect of NS1 on p14 phosphorylation may be directly linked to cytotoxicity and is probably related to the previously reported inhibition of cell DNA synthesis. Late in the induction period (24 h), NS1 also alters the synthesis of a 50-kDa protein and a 35-kDa protein (p50 and p35, respectively). Microsequencing of p35 reveals sequence homology with beta-tubulin. These effects of NS1, observed

  5. Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-lactones).

    PubMed

    Ann, Jihyae; Yoon, Suyoung; Baek, Jisoo; Kim, Da Hye; Lewin, Nancy E; Hill, Colin S; Blumberg, Peter M; Lee, Jeewoo

    2015-01-27

    DAG-lactones afford a synthetically accessible, high affinity platform for probing structure activity relationships at the C1 regulatory domain of protein kinase C (PKC). Given the central role of PKC isoforms in cellular signaling, along with their differential biological activities, a critical objective is the design of isoform selective ligands. Here, we report the synthesis of a series of DAG-lactones varying in their side chains, with a particular focus on linoleic acid derivatives. We evaluated their selectivity for PKC epsilon versus PKC alpha both under standard lipid conditions (100% phosphatidylserine, PS) as well as in the presence of a nuclear membrane mimetic lipid mixture (NML). We find that selectivity for PKC epsilon versus PKC alpha tended to be enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead compound, report a selectivity of 32-fold. PMID:25437619

  6. Protein synthesis in distal axons is not required for growth cone responses to guidance cues

    PubMed Central

    Roche, Florence K.; Marsick, Bonnie M.; Letourneau, Paul C.

    2009-01-01

    Recent evidence suggests growth cone responses to guidance cues require local protein synthesis. Using chick neurons, we investigated whether protein synthesis is required for growth cones of several types to respond to guidance cues. First, we found that global inhibition of protein synthesis stops axonal elongation after two hr. When protein synthesis inhibitors were added 15 min before adding guidance cues, we found no changes in the typical responses of retinal, sensory and sympathetic growth cones. In the presence of cycloheximide or anisomycin, ephrin-A2, slit-3, and semaphorin3A still induced growth cone collapse and loss of actin filaments, NGF and NT-3 still induced growth cone protrusion and increased F-actin, and sensory growth cones turned toward an NGF source. In compartmented chambers that separated perikarya from axons, axons grew for 24-48 hr in the presence of cycloheximide and responded to negative and positive cues. Our results indicate that protein synthesis is not strictly required in the mechanisms for growth cone responses to many guidance cues. Differences between our results and other studies may exist because of different cellular metabolic levels in in vitro conditions, and a difference in when axonal functions become dependent on local protein synthesis. PMID:19158291

  7. 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...

  8. Synthesis of europium-activated calcium tungstate phosphor

    NASA Astrophysics Data System (ADS)

    Popovici, Elisabeth-Jeanne; Forgaciu, Flavia; Nemes, Miloslava; Ursu, Veronica

    1998-07-01

    The purpose of this study is to establish the way in which different synthesis conditions influence on the structural and luminescent characteristics of europium activated calcium tungstate powder phosphor. CaWO4:Eu3+ samples were prepared by thermal synthesis from mixtures consisting of precipitated-CaWO4, equivalent amounts of Eu2O3 and WO3 (activating system) and CaCl2 or Na2WO4 as flux. Calcination was performed at 800 - 1000 degree(s)C for 2 h, in air. The crystalline structure (XRD-patterns) and luminescent characteristics (emission and excitation spectra of phosphor samples were determined and interpreted.

  9. 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.

  10. Mechanism of IL-1 induced inhibition of protein synthesis in skeletal muscle.

    PubMed

    Cooney, R N; Maish, G O; Gilpin, T; Shumate, M L; Lang, C H; Vary, T C

    1999-04-01

    Chronic interleukin (IL)-1 administration is associated with negative nitrogen balance and the loss of lean body mass. To elucidate the molecular mechanism(s) by which IL-1 modulates protein metabolism in muscle, we investigated the effects of chronic (6 day) IL-1alpha infusion on protein synthesis in Individual muscles (gastrocnemius, soleus, heart) compared with saline-infused control rats. IL-1 significantly decreased muscle weight, protein content, and the rate of protein synthesis in gastrocnemius (fast-twitch muscle). IL-1 had no effect on these parameters in the heart, whereas only the rate of protein synthesis was reduced in soleus (slow-twitch muscle). The reduction in gastrocnemius protein synthesis was not the result of a decrease in total RNA content, but was associated with a diminished translational efficiency. The diminished translational efficiency correlated with a 40% reduction in the epsilon-subunit of eukaryotic initiation factor 2B (elF2Bepsilon) in gastrocnemius from IL-1 -treated animals. However, the content of the alpha-subunit of elF2 (elF2alpha) was unaffected. In contrast, the elF2alpha content in heart was increased by IL-1, although elF2Bepsilon levels were unchanged. Reductions in skeletal muscle protein synthesis were not associated with a concomitant reduction in circulating or tissue content of insulin-like growth factor I. In summary, the IL-1-induced decrease in gastrocnemius protein synthesis appears to be regulated at the level of RNA translation via a reduction in elF2Bepsilon. These findings support a regulatory role for IL-1 as a mediator of muscle protein synthesis and the alterations in body composition observed in catabolic states where this cytokine is overexpressed. PMID:10220298

  11. [Effect of fibronectin on the synthesis of extracellular matrix proteins in periodontal ligament cells].

    PubMed

    Wan, L; Wu, Z; Zhou, Y

    1996-11-01

    Immunofluorescence staining method and fluorescence spectrophotometry were used to study the synthesis of extracellular matrix proteins in periodontal ligament cells (PDL cells) when exogenous fibronectin (FN) existed. The results showed that the right amount of exogenous FN (0.044 mumol/l) could increase the amount of type I collagen and type III collagen in PDL cells (P < 0.01), inhibit the synthesis of FN itself (P < 0.01). It suggested that exogenous FN can effect the synthesis of extracellular matrix proteins so as to promote a new connective tissue attachment formation. PMID:9592289

  12. AMP-activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

    PubMed Central

    Li, Yen-Hsing; Luo, Jia; Mosley, Yung-Yi C.; Hedrick, Victoria E.; Paul, Lake N.; Chang, Julia; Zhang, GuangJun; Wang, Yu-Kuo; Banko, Max R.; Brunet, Anne; Kuang, Shihuan; Wu, Jen-Leih; Chang, Chun-Ju; Scott, Matthew P.; Yang, Jer-Yen

    2015-01-01

    Summary The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated and how energy sensor regulates Hh pathway is not clear. AMP-activated Protein Kinase (AMPK) is an important sensor of energy stores that controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This in turn leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency. PMID:26190112

  13. The activated glucocorticoid receptor modulates presumptive autoregulation of ribosomal protein S6 protein kinase, p70 S6K.

    PubMed

    Shah, O Jameel; Iniguez-Lluhi, Jorge A; Romanelli, Angela; Kimball, Scot R; Jefferson, Leonard S

    2002-01-25

    Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated. PMID:11705993

  14. The substrate for long-lasting memory: if not protein synthesis, then what?

    PubMed Central

    Routtenberg, Aryeh

    2011-01-01

    The prevailing textbook view that de novo protein synthesis is required for memory (e.g., Bear, 2006) is seriously flawed and the 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 (Routtenberg and Rekart, 2005). 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 (Routtenberg and Rekart, 2005), 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

  15. 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

  16. Intense pulsed light induces synthesis of dermal extracellular proteins in vitro.

    PubMed

    Cuerda-Galindo, E; Díaz-Gil, G; Palomar-Gallego, M A; Linares-GarcíaValdecasas, R

    2015-09-01

    Intense pulsed light (IPL) devices have been shown to be highly effective for the skin rejuvenation. In our study, we try to elucidate effects of IPL in fibroblast proliferation, in gene expression, and in extracellular matrix protein production. 1BR3G human skin fibroblasts were used to test the effects of an IPL device (MiniSilk FT, Deka®). Fibroblasts were divided into three groups: group 1 was irradiated with filter 800-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm) twice; group 2 was irradiated with filter 550-1200 nm (double pulse 5 ms + 5 ms, delay 10 ms, fluence 13 J/cm2) twice; and group 3 was irradiated with filter 550-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm2) twice. To determine changes in gene expression, messenger RNA (mRNA) levels for collagen types I and III and metalloproteinase 1 (MMP-1) were performed 48 h after irradiation. To determine changes in hyaluronic acid, versican, and decorin, mRNA and ELISA tests were performed after 48 h of treatment. In addition to this, a Picro-Sirius red staining for collagen was made. The study showed an increase of mRNA and hyaluronic acid, decorin, and versican production. With RT-PCR assays, an increase mRNA for collagen type I, type III, and MMP-1 was observed. Collagen and hyaluronic synthesis was increased in all groups with no differences among them, while decorin and versican synthesis was higher in those groups irradiated with 550-1200-nm filters with no dependence of type pulse or total energy dose. IPL applied in vitro cultured cells increases fibroblasts activity. Synthesis of extracellular proteins seems to be produced more specifically in determined wavelengths, which could demonstrate a biochemical mechanism light depending. PMID:26188855

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

    PubMed Central

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês CR; 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

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.02860.001 PMID:24948515

  18. 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

    2014-01-01

    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. PMID:24948515

  19. Synthesis and biological evaluation of optically active Ki16425.

    PubMed

    Sato, Takanao; Sugimoto, Kenji; Inoue, Asuka; Okudaira, Shinichi; Aoki, Junken; Tokuyama, Hidetoshi

    2012-07-01

    An enantionselective synthesis of both enantiomers of Ki16425, which possesses selective LPA antagonistic activity, was achieved. The isoxazole core was constructed by a 1,3-dipolar cycloaddition of nitrile oxide with alkyne and condensation with the optically active α-phenethyl alcohol segment, which was prepared by an enantioselective reduction of arylmethylketone. Biological evaluation of both enantiomers of Ki16425 revealed that the (R)-isomer showed much higher antagonistic activity for LPA(1) and LPA(3) receptors. PMID:22658556

  20. Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

    PubMed Central

    Nishiuchi, Yuji; Inui, Tatsuya; Nishio, Hideki; Bódi, József; Kimura, Terutoshi; Tsuji, Frederick I.; Sakakibara, Shumpei

    1998-01-01

    The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor. PMID:9811837

  1. Insulin regulates milk protein synthesis at multiple levels in the bovine mammary gland.

    PubMed

    Menzies, Karensa K; Lefèvre, Christophe; Macmillan, Keith L; Nicholas, Kevin R

    2009-05-01

    The role of insulin in milk protein synthesis is unresolved in the bovine mammary gland. This study examined the potential role of insulin in the presence of two lactogenic hormones, hydrocortisone and prolactin, in milk protein synthesis. Insulin was shown to stimulate milk protein gene expression, casein synthesis and (14)C-lysine uptake in mammary explants from late pregnant cows. A global assessment of changes in gene expression in mammary explants in response to insulin was undertaken using Affymetrix microarray. The resulting data provided insight into the molecular mechanisms stimulated by insulin and showed that the hormone stimulated the expression of 28 genes directly involved in protein synthesis. These genes included the milk protein transcription factor, ELF5, translation factors, the folate metabolism genes, FOLR1 and MTHFR, as well as several genes encoding enzymes involved in catabolism of essential amino acids and biosynthesis of non-essential amino acids. These data show that insulin is not only essential for milk protein gene expression, but stimulates milk protein synthesis at multiple levels within bovine mammary epithelial cells. PMID:19107532

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

    PubMed

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

    2016-01-01

    Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. PMID:27117251

  3. 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.

  4. 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.

  5. 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

  6. Genome activation by raspberry bushy dwarf virus coat protein.

    PubMed

    Macfarlane, Stuart A; McGavin, Wendy J

    2009-03-01

    Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation. PMID:19218221

  7. Protein synthesis in cancer patients with inflammatory response: investigations with [15N]glycine.

    PubMed

    McMillan, D C; Preston, T; Fearon, K C; Burns, H J; Slater, C; Shenkin, A

    1994-01-01

    It has been proposed that the increase in amino acid flux and derived protein synthesis rates observed in weight-losing cancer patients may contribute to an ongoing negative energy balance. The mediators and tissues responsible for such apparent increased protein synthesis have not been clearly identified. The aim of this study was to examine the relationship between protein synthetic rates in whole-body, skeletal muscle, and circulating cortisol concentrations in healthy subjects (n = 6) and cancer patients with evidence of an inflammatory response (n = 6). Protein synthetic rates were measured with a primed continuous 20-h infusion of [15N]glycine. Skeletal muscle was biopsied at laparotomy. Serum cortisol, resting energy expenditure, plasma proteins, nitrogen metabolites in urine, and skeletal muscle free amino acids were also measured. Derived whole-body and skeletal muscle protein synthetic rates in the cancer group were increased significantly (by 70 and 93%, respectively, p < 0.05). Circulating concentrations of cortisol, fibrinogen, and C-reactive protein were also significantly increased in the cancer group and indicated the presence of an inflammatory response. However, there was no significant increase in resting energy expenditure. Mechanisms by which apparent increases in whole-body and skeletal protein synthesis do not result in an increase in resting energy expenditure are discussed. We conclude that glycine utilization is increased in cancer patients but that rates of protein synthesis derived from [15N]glycine kinetics may not be valid in such patients. PMID:7919675

  8. 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. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure–function relationships, kinase signalling networks and kinase inhibitor drugs. PMID:26350765

  9. 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. PMID:15333735

  10. Coat protein activation of alfalfa mosaic virus replication is concentration dependent.

    PubMed

    Guogas, Laura M; Laforest, Siana M; Gehrke, Lee

    2005-05-01

    Alfalfa mosaic virus (AMV) and ilarvirus RNAs are infectious only in the presence of the viral coat protein; therefore, an understanding of coat protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV coat protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that coat protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining coat protein's function in the earliest stages of AMV replication. To detect coat-protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by coat protein is concentration dependent; that is, replication was strongly stimulated at low coat protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing coat protein mRNA translation or disrupting coat protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of coat protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that coat protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local coat protein concentrations and low coat protein occupancy on the multiple binding sites present in the 3' untranslated regions of the viral RNAs. PMID:15827190

  11. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase