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Sample records for microsomal protein synthesis

  1. 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.

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

    PubMed Central

    Johnson, Terry C.; Belytschko, Gail

    1969-01-01

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

  3. Cell-free synthesis of membrane proteins: tailored cell models out of microsomes.

    PubMed

    Fenz, Susanne F; Sachse, Rita; Schmidt, Thomas; Kubick, Stefan

    2014-05-01

    Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  4. VARIANCE OF MICROSOMAL PROTEIN AND ...

    EPA Pesticide Factsheets

    Differences in the pharmacokinetics of xenobiotics among humans makes them differentially susceptible to risk. Differences in enzyme content can mediate pharmacokinetic differences. Microsomal protein is often isolated fromliver to characterize enzyme content and activity, but no measures exist to extrapolate these data to the intact liver. Measures were developed from up to 60 samples of adult human liver to characterize the content of microsomal protein and cytochrome P450 (CYP) enzymes. Statistical evaluations are necessary to estimate values far from the mean value. Adult human liver contains 52.9 - 1.476 mg microsomal protein per g; 2587 - 1.84 pmoles CYP2E1 per g; and 5237 - 2.214 pmols CYP3A per g (geometric mean - geometric standard deviation). These values are useful for identifying and testing susceptibility as a function of enzyme content when used to extrapolate in vitro rates of chemical metabolism for input to physiologically based pharmacokinetic models which can then be exercised to quantify the effect of variance in enzyme expression on risk-relevant pharmacokinetic outcomes.

  5. Multiple functions of microsomal triglyceride transfer protein

    PubMed Central

    2012-01-01

    Microsomal triglyceride transfer protein (MTP) was first identified as a major cellular protein capable of transferring neutral lipids between membrane vesicles. Its role as an essential chaperone for the biosynthesis of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins was established after the realization that abetalipoproteinemia patients carry mutations in the MTTP gene resulting in the loss of its lipid transfer activity. Now it is known that it also plays a role in the biosynthesis of CD1, glycolipid presenting molecules, as well as in the regulation of cholesterol ester biosynthesis. In this review, we will provide a historical perspective about the identification, purification and characterization of MTP, describe methods used to measure its lipid transfer activity, and discuss tissue expression and function. Finally, we will review the role MTP plays in the assembly of apoB-lipoprotein, the regulation of cholesterol ester synthesis, biosynthesis of CD1 proteins and propagation of hepatitis C virus. We will also provide a brief overview about the clinical potentials of MTP inhibition. PMID:22353470

  6. [Microsomal triglyceride transfer protein and abetalipoproteinemia].

    PubMed

    Berriot-Varoqueaux, N; Aggerbeck, L P; Samson-Bouma, M

    2000-05-01

    Microsomal triglyceride transfer protein (MTP) is a dimeric protein complex consisting of protein disulfide isomerase and a unique 97 kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between vesicles. It was recently demonstrated that abetalipoproteinemia, a disease characterized as an inability to produce chylomicrons and very low density lipoproteins in the intestine and liver, respectively, is the result of a genetic absence of MTP. Downstream effects resulting from this defect, include very low plasma cholesterol and triglyceride levels, absence of plasma apolipoprotein B and a lipid malabsorption syndrome, leading to lipo-soluble vitamin deficiencies. A low fat diet is instituted to eliminate the diarrhea. In addition, a therapy with vitamins A and E is essential to prevent patients from developing secondary effects such as neuropathy, muscle weakness, and retinopathy.

  7. Membrane Structure: Lipid-Protein Interactions in Microsomal Membranes*

    PubMed Central

    Trump, Benjamin F.; Duttera, Sue M.; Byrne, William L.; Arstila, Antti U.

    1970-01-01

    The relationships of phospholipid to membrane structure and function were examined in hepatic microsomes. Findings indicate that normal microsomal membrane structure is dependent on lipid-protein interactions and that it correlates closely with glucose-6-phosphatase activity. Modification of most phospholipid with phospholipase-C is associated with widening of the membrane which can be reversed following readdition of phospholipid. Images PMID:4317915

  8. Selenium-containing proteins of rat kidney and liver microsomes

    SciTech Connect

    Viljoen, A.J.; Motchnik, P.A.; Tappel, A.L. )

    1989-12-01

    Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with ({sup 75}Se)selenite. More than 85% of the {sup 75}Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several {sup 75}Se-containing proteins in addition to glutathione peroxidase. {sup 75}Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The {sup 75}Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the {sup 75}Se was associated with the 55 kDa subunit 3 hr after injection. The amount of {sup 75}Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.

  9. Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins.

    PubMed

    Mathias, Rommel A; Chen, Yuan-Shou; Kapp, Eugene A; Greening, David W; Mathivanan, Suresh; Simpson, Richard J

    2011-08-01

    elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.

  10. [Effect of electron irradiation on the structure of microsomal membrane proteins].

    PubMed

    Gorbenko, G P; Diubko, T S; Krupin, V D

    1998-01-01

    Using tryptophan fluorescence quenching by iodide and acrylamide the effect of electrons with the energy 5 Mev on the structure of microsomal membrane proteins has been studied. Conformation of microsomal proteins were found to change upon irradiation.

  11. The role of the microsomal triglygeride transfer protein in abetalipoproteinemia.

    PubMed

    Berriot-Varoqueaux, N; Aggerbeck, L P; Samson-Bouma, M; Wetterau, J R

    2000-01-01

    The microsomal triglyceride transfer protein (MTP) is a dimeric lipid transfer protein consisting of protein disulfide isomerase and a unique 97-kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between membranes. It was recently demonstrated that abetalipoproteinemia, a hereditary disease characterized as an inability to produce chylomicrons and very low-density lipoproteins in the intestine and liver, respectively, results from mutations in the gene encoding the 97-kDa subunit of the microsomal triglyceride transfer protein. Downstream effects resulting from this defect include malnutrition, very low plasma cholesterol and triglyceride levels, altered lipid and protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated, abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological abnormalities.

  12. Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

    PubMed

    Radominska-Pyrek, A; Strosznajder, J; Dabrowiecki, Z; Goracci, G; Chojnacki, T; Horrocks, L A

    1977-01-01

    The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.

  13. Effects of a short-term fast on albumin synthesis studied in vivo, in the perfused liver, and on amino acid incorporation by hepatic microsomes

    PubMed Central

    Rothschild, Marcus A.; Oratz, Murray; Mongelli, Joseph; Schreiber, Sidney S.

    1968-01-01

    Carbonate-14C was used to label the hepatic intracellular arginine pool and direct measurement of albumin synthesis was made in six rabbits before and after an 18-36 hr fast. 18 perfusion studies were performed with livers derived from fed and fasted rabbits (18-24 hr). Microsomal amino acid-incorporating ability with leucine-3H and phenylalanine-14C was compared in 17 studies, using microsomes isolated from livers taken from fed and fasted rabbits and from isolated perfused livers whose donors were fed and fasted. Albumin synthesis is rapidly inhibited by fasting. Albumin synthesis decreased 33% in vivo and 53% in the perfused liver. The microsomes from perfused livers taken from fed animals did not demonstrate a significantly reduced capacity to incorporate leucine-3H or phenylalanine-14C into protein. Microsomes derived from perfused and nonperfused livers whose donors were fasted incorporated 32-54% less tracer than microsomes obtained from fed donor rabbits. Microsomes separated from perfused livers removed from fed and fasted rabbits responded to polyuridylic acid stimulation and phenylalanine-14C incorporation rose from 58 to 171%. An 18-36 hr fast inhibits albumin production in vivo and in the perfused liver. The microsomal system is less active in the fasted state and perfusion per se does not inhibit the microsomal response. PMID:5725276

  14. Increase of translatable mRNA for major microsomal proteins in n-alkane-grown Candida maltosa

    SciTech Connect

    Sunairi, M.; Watabe, K.; Takagi, M.; Yano, K.

    1984-12-01

    In an n-alkane-assimilating Candida sp., transfer from glucose- to n-alkane-containing medium induced changes in the microsomal proteins, and several distinctive polypeptides were demonstrated in the solubilized microsomal fraction derived from n-alkane-grown cells. Long-term-labeling and pulse-labeling experiments in vivo demonstrated the synthesis of the specific microsomal polypeptides. The polypeptides were synthesized as in vitro translation products directed by polyadenylated RNA extracted from n-alkane-grown cells. Two major polypeptides were partially purified from the microsomal fraction from n-alkane-grown cells, and antiserum was prepared in a rabbit. Immunoprecipitation of these two polypeptides was accompanied by an increase in the amount of translatable mRNA. The molecular weights of the polypeptides derived from long-term-labeling, pulse-labeling and in vitro translation experiments appeared to be identical.

  15. IN VITRO STIMULATION OF ENZYME SECRETION AND THE SYNTHESIS OF MICROSOMAL MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

    PubMed Central

    Meldolesi, Jacopo; Cova, Dario

    1971-01-01

    Several mechanisms have been suggested to explain how secretory cells remove from the plasmalemma the excess membrane resulting from the insertion of granule membrane during exocytosis: intact patches of membrane may be internalized and then reutilized within the cell; alternatively these membranes may be either disassembled to subunits or degraded. In the latter case new membranes should be synthetized at other sites of the cell, probably in the rough-surfaced endoplasmic reticulum (RER) and the Golgi complex. In the present research, membrane subfractions were obtained from rough microsomes (derived from fragmented and resealed RER cisternae) and from smooth microsomes (primarily contributed by Golgi stacks and vesicles) of the guinea pig pancreas by incubation at 4°C for 4 hr in 0.0005 M puromycin at high ionic strength followed by mild (pH 7.8) alkaline extraction with 0.2 M NaHCO3. Such treatments release the majority of nonmembrane components of both microsomal fractions (i.e., contained secretory enzymes, ribosomes, and absorbed proteins of the cell sap) and allow the membranes to be recovered by centrifugation. The effect of in vitro stimulation of enzyme secretion (brought about in pancreas slices by 0.0001 M carbamoyl choline) on the rate of synthesis of the phospholipid (PLP) and protein of these membranes was then investigated. In agreement with previous data, we observed that in stimulated slices the synthesis of microsomal PLP was greatly increased. In contrast, the synthesis of microsomal membrane proteins was unchanged. These results suggest that exocytosis is not coupled with an increased rate of synthesis of complete ER and Golgi membranes and are, therefore, consistent with the view that excess plasma membrane is preserved and reutilized, either as discrete membrane patches or as membrane macromolecules, throughout the secretory cycle. PMID:4329615

  16. Haplotype-based identification of a microsomal transfer protein marker associated with the human lifespan

    PubMed Central

    Geesaman, Bard J.; Benson, Erica; Brewster, Stephanie J.; Kunkel, Louis M.; Blanché, Hélène; Thomas, Gilles; Perls, Thomas T.; Daly, Mark J.; Puca, Annibale A.

    2003-01-01

    We previously reported a genomewide linkage study for human longevity using 308 long-lived individuals (LLI) (centenarians or near-centenarians) in 137 sibships and identified statistically significant linkage within chromosome 4 near microsatellite D4S1564. This interval spans 12 million bp and contains ≈50 putative genes. To identify the specific gene and gene variants impacting lifespan, we performed a haplotype-based fine-mapping study of the interval. The resulting genetic association study identified a haplotype marker within microsomal transfer protein as a modifier of human lifespan. This same variant was tested in a second cohort of LLI from France, and although the association was not replicated, there was evidence for statistical distortion in the form of Hardy–Weinberg disequilibrium. Microsomal transfer protein has been identified as the rate-limiting step in lipoprotein synthesis and may affect longevity by subtly modulating this pathway. This study provides proof of concept for the feasibility of using the genomes of LLI to identify genes impacting longevity. PMID:14615589

  17. Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide*

    PubMed Central

    Iqbal, Jahangir; Walsh, Meghan T.; Hammad, Samar M.; Cuchel, Marina; Tarugi, Patrizia; Hegele, Robert A.; Davidson, Nicholas O.; Rader, Daniel J.; Klein, Richard L.; Hussain, M. Mahmood

    2015-01-01

    Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion. PMID:26350457

  18. Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.

    PubMed

    Quast, Robert B; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R; Wüstenhagen, Doreen A; Kele, Péter; Schiller, Stefan M; Kubick, Stefan

    2016-09-27

    Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

  19. Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

    PubMed Central

    Quast, Robert B.; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R.; Wüstenhagen, Doreen A.; Kele, Péter; Schiller, Stefan M.; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system. PMID:27670253

  20. Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo.

    PubMed

    Eresheim, Christine; Plieschnig, Julia; Ivessa, N Erwin; Schneider, Wolfgang J; Hermann, Marcela

    2014-06-01

    In contrast to mammals, in the chicken major sites of lipoprotein synthesis and secretion are not only the liver and intestine, but also the kidney and the embryonic yolk sac. Two key components in the assembly of triglyceride-rich lipoproteins are the microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). We have analyzed the expression of MTP in the embryonic liver, small intestine, and kidney, and have studied the expression of MTP in, and the secretion of apoB from, the developing yolk sac (YS). Transcript and protein levels of MTP increase during embryogenesis in YS, liver, kidney, and small intestine, and decrease in YS, embryonic liver, and kidney after hatching. In small intestine, the MTP mRNA level rises sharply during the last trimester of embryo development (after day 15), while MTP protein is detectable only after hatching (day 21). In the YS of 15- and 20-day old embryos, apoB secretion was detected by pulse-chase metabolic radiolabeling experiments and subsequent immunoprecipitation. Taken together, our data reveal the importance of coordinated production of MTP and apoB in chicken tissues capable of secreting triglyceride-rich lipoproteins even before hatching.

  1. Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

    PubMed

    Sellers, Jeremy A; Hou, Li; Schoenberg, Daniel R; Batistuzzo de Medeiros, Silvia R; Wahli, Walter; Shelness, Gregory S

    2005-04-08

    Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

  2. Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation.

    PubMed Central

    Kaderbhai, M A; Harding, V J; Karim, A; Austen, B M; Kaderbhai, N N

    1995-01-01

    A procedure is described for the preparation of rough membrane vesicles of endoplasmic-reticular origin from the pancreas of sheep. These isolated membranes translocate, process and glycosylate in vitro-translated heterologous proteins in a manner comparable with that exhibited by dog pancreatic microsomes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7864829

  3. Microsomal membrane proteins and vanadate-sensitive ATPase from Vicia faba root tips after clinostat treatment.

    PubMed

    Bramer, M; Hunte, C; Schulz, M; Schnabl, H

    1996-07-01

    Microsomal and soluble protein fractions from Vicia faba root tips were used for SDS-PAGE and Western-immunoblot analysis with anti-ubiquitin antibodies after 9 h clinostat treatment of the plants. In contrast to soluble proteins omnilateral gravistimulation (9 h) resulted in an enhanced proteolytic capacity for microsomal proteins. The increase of vanadate-sensitive ATPase activity was 83% after 9 h clinostat treatment, when the enzyme activity was measured directly after membrane preparation. Enhanced ATPase activity was correlated with the appearance of a polypeptide of about 100 kDa and its fragments (93 and 80 kDa). ATPases are not the only membrane bound proteins, which are changed during clinostat treatment, as several ubiquitinated polypeptides were also affected. A 1 h storage of microsomal fractions led to a shift of band intensities on ubiquitin-specific Western-blots. The demonstrated effect could not be observed, when fractions were isolated in the presence of protease inhibitors. In accordance with the polypeptide analysis omnilateral gravistimulation resulted in an enhanced capacity to degrade specific microsomal ubiquitin-conjugates, whereas the soluble ubiquitin-pool was not visibly affected.

  4. New lipid modulating drugs: the role of microsomal transport protein inhibitors.

    PubMed

    Rizzo, Manfredi; Wierzbicki, Anthony S

    2011-01-01

    Microsomal triglyceride transfer protein (MTP) is involved in the synthesis of very low density lipoprotein in the liver. Its deficiency results in abetalipoproteinemia. MTP inhibitors target the assembly and secretion of apolipoprotein B-containing lipoproteins. These agents may potentially play a role, alone or in combination, in the treatment of hypercholesterolemia or hypertriglyceridaemia. Clinical applications of MTP inhibitors initially focused primarily on high-dose monotherapy in order to produce substantial reductions in LDL-cholesterol levels but these proved to induce significant hepatic steatosis and transaminase elevations. However, likely orphan indications for MTP inhibitors, where a different risk-benefit profile applies, include patients with homozygous familial hypercholesterolemia where statins often show a low response. Development of MTP inhibitors has continued to enter clinical trials at lower doses or in formulations aimed at utilizing their efficacy while avoiding their side effects. These have shown promising results in reducing cholesterol, triglycerides and apolipoprotein B with a far lower incidence of, often, transient side-effects. The clinical efficacy and safety of MTP inhibition in patients with hyperlipidaemia remains to be fully determined and to be proven in both surrogate and clinical endpoint trials but there may be a role for these agents in orphan indications for rarer severe hyperlipidaemias.

  5. Structure-function analyses of microsomal triglyceride transfer protein missense mutations in abetalipoproteinemia and hypobetalipoproteinemia subjects.

    PubMed

    Walsh, Meghan T; Di Leo, Enza; Okur, Ilyas; Tarugi, Patrizia; Hussain, M Mahmood

    2016-11-01

    We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their functional effects. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. H297Q, D384A, and G661A mutants also supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP's three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, novel mutations that cause severe hypolipidemia probably exist in other genes in these patients, and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

  6. Further studies on the stimulation of protein synthesis in androgen-dependent tissues by testosterone

    PubMed Central

    Mainwaring, W. I. P.; Wilce, P. A.

    1972-01-01

    1. By using centrifugation through a discontinuous sucrose gradient, four microsomal fractions are obtained from the prostate gland. 2. Administration of androgens to castrated rats stimulates protein synthesis in all fractions, particularly in the heavy rough fraction. 3. Androgens also increase the content of protein, RNA and phospholipid in the heavy rough fraction. 4. Time-course experiments in vivo show that androgens induce a rapid increase in the synthesis of ribosomal precursor RNA preceding the synthesis of new microsomal fraction and the increase in protein synthesis. PMID:4655422

  7. Stch encodes the 'ATPase core' of a microsomal stress 70 protein.

    PubMed Central

    Otterson, G A; Flynn, G C; Kratzke, R A; Coxon, A; Johnston, P G; Kaye, F J

    1994-01-01

    The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements. Images PMID:8131751

  8. Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes

    PubMed Central

    Leslie, Ruth A.; Mansbridge, J. N.

    1970-01-01

    1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85–95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [14C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction. PMID:5451910

  9. Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia.

    PubMed

    Sharp, D; Blinderman, L; Combs, K A; Kienzle, B; Ricci, B; Wager-Smith, K; Gil, C M; Turck, C W; Bouma, M E; Rader, D J

    1993-09-02

    The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.

  10. Functional Investigation of Iron-Responsive Microsomal Proteins, including MirC, in Aspergillus fumigatus

    PubMed Central

    Mulvihill, Eoin D.; Moloney, Nicola M.; Owens, Rebecca A.; Dolan, Stephen K.; Russell, Lauren; Doyle, Sean

    2017-01-01

    The functionality of many microsome-associated proteins which exhibit altered abundance in response to iron limitation in Aspergillus fumigatus is unknown. Here, we generate and characterize eight gene deletion strains, and of most significance reveal that MirC (AFUA_2G05730) contributes to the maintenance of intracellular siderophore [ferricrocin (FC)] levels, augments conidiation, confers protection against oxidative stress, exhibits an intracellular localization and contributes to fungal virulence in the Galleria mellonella animal model system. FC levels were unaffected following deletion of all other genes encoding microsome-associated proteins. MirC does not appear to play a role in either siderophore export from, or uptake into, A. fumigatus. Label-free quantitative proteomic analysis unexpectedly revealed increased abundance of siderophore biosynthetic enzymes. In addition, increased expression of hapX (7.2 and 13.8-fold at 48 and 72 h, respectively; p < 0.001) was observed in ΔmirC compared to wild-type under iron-replete conditions by qRT-PCR. This was complemented by significantly elevated extracellular triacetylfusarinine C (TAFC; p < 0.01) and fusarinine C (FSC; p < 0.05) siderophore secretion. We conclude that MirC plays an important role in FC biosynthesis and contributes to the maintenance of iron homeostasis in A. fumigatus. PMID:28367141

  11. Functional Investigation of Iron-Responsive Microsomal Proteins, including MirC, in Aspergillus fumigatus.

    PubMed

    Mulvihill, Eoin D; Moloney, Nicola M; Owens, Rebecca A; Dolan, Stephen K; Russell, Lauren; Doyle, Sean

    2017-01-01

    The functionality of many microsome-associated proteins which exhibit altered abundance in response to iron limitation in Aspergillus fumigatus is unknown. Here, we generate and characterize eight gene deletion strains, and of most significance reveal that MirC (AFUA_2G05730) contributes to the maintenance of intracellular siderophore [ferricrocin (FC)] levels, augments conidiation, confers protection against oxidative stress, exhibits an intracellular localization and contributes to fungal virulence in the Galleria mellonella animal model system. FC levels were unaffected following deletion of all other genes encoding microsome-associated proteins. MirC does not appear to play a role in either siderophore export from, or uptake into, A. fumigatus. Label-free quantitative proteomic analysis unexpectedly revealed increased abundance of siderophore biosynthetic enzymes. In addition, increased expression of hapX (7.2 and 13.8-fold at 48 and 72 h, respectively; p < 0.001) was observed in ΔmirC compared to wild-type under iron-replete conditions by qRT-PCR. This was complemented by significantly elevated extracellular triacetylfusarinine C (TAFC; p < 0.01) and fusarinine C (FSC; p < 0.05) siderophore secretion. We conclude that MirC plays an important role in FC biosynthesis and contributes to the maintenance of iron homeostasis in A. fumigatus.

  12. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes

    PubMed Central

    Caldwell, Charles R.

    1987-01-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32°C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30°C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32°C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14°C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32°C which could account for the complex temperature dependence of the barley root plasma membrane ATPase. PMID:16665545

  13. The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

    PubMed Central

    Graham, A. B.; Woodcock, B. G.; Wood, G. C.

    1974-01-01

    After force-feeding a protein-free diet to male rats for 5–7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed. PMID:4214255

  14. Identification of cytosolic and microsomal bile acid-binding proteins in rat ileal enterocytes

    SciTech Connect

    Lin, M.C.; Kramer, W.; Wilson, F.A. )

    1990-09-05

    Studies were performed to determine the subcellular fractions and proteins involved in the intracellular transport of bile acids in rat ileal cells. The photolabile derivative 7,7-azo-taurocholate inhibited the Na(+)-dependent uptake of taurocholate into rat ileal enterocytes reversibly in the dark and irreversibly following photolysis. When photolabeled cells were submitted to subcellular fractionation, greatest radioactivity was found in the soluble protein (SP) fraction with decreasing radioactivity in the brush-border-(BBM), basolateral-(BLM), mitochondria-(MT), microsome-(MC), and Golgi-(GO) enriched fractions. Following trichloroacetic acid precipitation, delipidation, and correction for loss of marker enzyme activity, protein bound radioactivity was in SP greater than BBM greater than MC greater than BLM greater than GO greater than MT. When photolabeled cells were first fractionated and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 99-kDa polypeptide was associated with BBM, 54- and 59-kDa polypeptides with BLM, 14-, 35-, 43-, 59-, and 68-kDa polypeptides with SP and a 20-kDa polypeptide with MC fractions. Immunoprecipitation with known antisera identified the 68-kDa polypeptide as albumin and the 43-kDa polypeptide as actin. No precipitation on the 14-kDa polypeptide was noted with anti-hepatic and anti-intestinal fatty acid-binding proteins. No precipitation of the 35-kDa polypeptide occurred with antibody to the hepatic cytosolic bile acid-binding protein. These studies reveal a previously unrecognized 20-kDa microsomal, and 14- and 35-kDa cytosolic bile acid-binding polypeptides which may be involved in the transcellular movement of bile acids.

  15. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L. ) microsomal and soluble protein tryptophanyl residues in vitro

    SciTech Connect

    Caldwell, C.R. )

    1993-03-01

    The in vitro effects of ultraviolet B (280--320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen species. The optimum wavelengths for loss of tryptophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[degrees]C, which influenced protein structure and tryptophan photosensitivity. 29 refs., 6 figs., 1 tab.

  16. Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein

    PubMed Central

    Lee, Richard G.; Fu, Wuxia; Graham, Mark J.; Mullick, Adam E.; Sipe, Donna; Gattis, Danielle; Bell, Thomas A.; Booten, Sheri; Crooke, Rosanne M.

    2013-01-01

    Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr−/− mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile. PMID:23220583

  17. Chemical Synthesis of Proteins

    PubMed Central

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

    2010-01-01

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

  18. [Plasmatic membrane protein synthesis in cells of the regenerating liver].

    PubMed

    Pospelov, A V; Gorelova, N V

    1978-05-01

    Protein synthesis in the cells of the regenerating rat liver was studied. The rate of 3H-glycine incorporation into the total proteins of the liver, those of microsomal fraction, proteins of hyaloplasm, and plasmatic membrane proteins, soluble and non-soluble in 0.05 M K2CO3, was determined. The rate of 3H-glycine incorporation into soluble proteines of plasma membranes became maximal one hour after partial hepatectomy. The peak of the rate of synthesis of proteins of other fractions fell on the end of the G1-period. A sharp increase of the synthesis rate of plasma membrane proteins seems to be one of the earliest biochemical events in the regenerating liver hepatocytes ready for division.

  19. A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP).

    PubMed

    Pons, Véronique; Rolland, Corinne; Nauze, Michel; Danjoux, Marie; Gaibelet, Gérald; Durandy, Anne; Sassolas, Agnès; Lévy, Emile; Tercé, François; Collet, Xavier; Mas, Emmanuel

    2011-07-01

    Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTTP) deficiency. We report two patients with new MTTP mutations. We studied their functional consequences on the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in HepG2 and HeLa cells to investigate their association with protein disulfide isomerase (PDI) and their localization at the endoplasmic reticulum. These children have a severe abetalipoproteinemia. Both of them had also a mild hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237-28A>G mutations within the MTTP gene. mRNA analysis revealed abnormal splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 (Δ6-MTTP) introduced a frame shift in the reading frame and a premature stop codon at position 234. Despite the fact that Δ6-MTTP and Δ10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP triglyceride transfer activity. These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI and responsible for the loss of function of MTTP, thereby explaining the severe abetalipoproteinemia phenotype of these children.

  20. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    PubMed Central

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  1. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues.

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-06-03

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity.

  2. Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes.

    PubMed

    Goldman, Jennifer L; Koen, Yakov M; Rogers, Steven A; Li, Kelin; Leeder, James S; Hanzlik, Robert P

    2016-10-01

    The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [(14)C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs.

  3. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

    PubMed

    Caldwell, C R

    1987-07-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

  4. Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia.

    PubMed

    Khatun, Irani; Walsh, Meghan T; Hussain, M Mahmood

    2013-06-01

    Mutations in microsomal triglyceride transfer protein (MTP) cause abetalipoproteinemia (ABL), characterized by the absence of plasma apoB-containing lipoproteins. In this study, we characterized the effects of various MTP missense mutations found in ABL patients with respect to their expression, subcellular location, and interaction with protein disulfide isomerase (PDI). In addition, we characterized functional properties by analyzing phospholipid and triglyceride transfer activities and studied their ability to support apoB secretion. All the mutants colocalized with calnexin and interacted with PDI. We found that R540H and N780Y, known to be deficient in triglyceride transfer activity, also lacked phospholipid transfer activity. Novel mutants S590I and G746E did not transfer triglycerides and phospholipids and did not assist in apoB secretion. In contrast, D384A displayed both triglyceride and phospholipid transfer activities and supported apoB secretion. These studies point out that ABL is associated with the absence of both triglyceride and phospholipid transfer activities in MTP.

  5. Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia.

    PubMed

    Ohashi, K; Ishibashi, S; Osuga, J; Tozawa, R; Harada, K; Yahagi, N; Shionoiri, F; Iizuka, Y; Tamura, Y; Nagai, R; Illingworth, D R; Gotoda, T; Yamada, N

    2000-08-01

    Abetalipoproteinemia (ABL) is an inherited disease characterized by the virtual absence of apolipoprotein B (apoB)-containing lipoproteins from plasma. Only limited numbers of families have been screened for mutations in the microsomal triglyceride transfer protein (MTP) gene. To clarify the genetic basis of clinical diversity of ABL, mutations of the MTP gene have been screened in 4 unrelated patients with ABL. Three novel mutations have been identified: a frameshift mutation caused by a single adenine deletion at position 1389 of the cDNA, and a missense mutation, Asn780Tyr, each in homozygous forms; and a splice site mutation, 2218-2A-->G, in a compound heterozygous form. The frameshift and splice site mutations are predicted to encode truncated forms of MTP. When transiently expressed in Cos-1 cells, the Asn780Tyr mutant MTP bound protein disulfide isomerase (PDI) but displayed negligible MTP activity. It is of interest that the patient having the Asn780Tyr mutation, a 27-year-old male, has none of the manifestations characteristic of classic ABL even though his plasma apoB and vitamin E were virtually undetectable. These results indicated that defects of the MTP gene are the proximal cause of ABL.

  6. Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia

    SciTech Connect

    Narcisi, T.M.E.; Shoulders, C.C.; Chester, S.A.

    1995-12-01

    Elevated plasma levels of apolipoprotein B (apoB)-containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. 49 refs., 4 figs., 5 tabs.

  7. In vitro synthesis of nitroxide free radicals by hog liver microsomes

    SciTech Connect

    Valvis, I.I.; Lischick, D.; Shen, D.; Sofer, S.S. )

    1990-01-01

    The in vitro biooxidation of 4-hydroxy-2,2,6,6-tetra methylpiperidine (TEMP), 4-hydroxy-2,2,4,4-tetra methyl-1,3-oxazolidine (TEMO) and diphenylamine (DPA) by hog liver microsomes to their respective nitroxide free radicals, 4-hydroxy-2,2,6,6-tetra methylpiperidine-1-oxyl (TEMPO), 2,2,4,4-tetra methyl-1,3-oxazolidine-1-oxyl (TEMOO), and diphenylnitroxide (DPNO) has been investigated. For extending the life span of the liver microsomes, a calcium alginate immobilization procedure was used. The biooxidation rates of the above amines to their respective nitroxide metabolites were measured by means of oxygen uptake at 37 degrees C and pH 7.4. N-octylamine was found to be an activator in the biooxidation of the amines. The formation of the nitroxide radicals was identified by E.S.R. spectroscopy.

  8. Microsomal triglyceride transfer protein (MTP) gene mutations in Canadian subjects with abetalipoproteinemia.

    PubMed

    Wang, J; Hegele, R A

    2000-03-01

    Abetalipoproteinemia (ABL) is an extremely rare autosomal recessive disorder, which is characterized by defective assembly and secretion of plasma apolipoprotein (apo) B-containing lipoproteins. ABL results from mutations in the gene encoding the microsomal triglyceride transfer protein (MTP). We sequenced the MTP gene in six Canadian subjects with ABL, of whom four were found to be simple homozygotes and two were found to be compound heterozygotes for MTP gene mutations. Of the 8 MTP gene mutations identified, 6 had not been previously reported, including two new nonsense mutations (K448X and K842X), two new missense mutations (S590I and G746E), one new frameshift mutation (1820del1) and one new splice donor site mutation (G1770A). Despite appropriate treatment with high doses of fat-soluble vitamins in all subjects, there was a wide variation in the progression and severity of the clinical phenotypes. For example, the presence of severe retinopathy and neuropathy did not correlate with the type and position of the mutation, but rather with the age at diagnosis and onset of treatment with fat-soluble vitamins. These findings suggest that genetic and non-genetic factors can modulate the clinical impact of mutant MTP in ABL patients.

  9. Contemporary aspects of the biology and therapeutic regulation of the microsomal triglyceride transfer protein.

    PubMed

    Hooper, Amanda J; Burnett, John R; Watts, Gerald F

    2015-01-02

    The microsomal triglyceride transfer protein (MTP), the product of the MTTP gene, is essential for the assembly and secretion of apolipoprotein B-containing lipoproteins, but when defective causes abetalipoproteinemia. Abetalipoproteinemia is a rare autosomal recessive disorder characterized by the inability to produce chylomicrons or very low-density lipoproteins, with the absence of apolipoprotein B-containing lipoproteins in the circulation. Knowledge of the molecular basis for abetalipoproteinemia has led to the development of therapies for dyslipidemia that inhibit MTP. Partial MTP inhibition using small molecule inhibitors, such as lomitapide, can effectively lower plasma low-density lipoprotein-cholesterol and apolipoprotein B levels, but is associated with gastrointestinal side effects and hepatic steatosis, whose long-term sequelae remain unclear; lomitapide has accordingly only been approved as a treatment for homozygous familial hypercholesterolemia. Intestine-specific inhibitors of MTP decrease chylomicron biogenesis and improve insulin sensitivity in experimental animals and, while overcoming hepatic steatosis, may have significant gastrointestinal side effects that could limit their use in humans. We review contemporary aspects of the biology and therapeutic regulation of MTP and their significance for lipid metabolism and cardiovascular disease.

  10. Microsomal transfer protein (MTP) inhibition-a novel approach to the treatment of homozygous hypercholesterolemia.

    PubMed

    Sirtori, Cesare R; Pavanello, Chiara; Bertolini, Stefano

    2014-11-01

    Homozygous familial hypercholesterolemia (HoFH) represents the most severe lipoprotein disorder, generally attributable to mutation(s) of the low-density lipoprotein receptor (LDL-R), i.e. autosomal dominant hypercholesterolemia type 1 (ADH1). Much lower percentages are due to alterations of apolipoprotein B (ADH2), or gain-of-function mutations of proprotein convertase subtilisin/kexin type 9 (PCSK9) (ADH3). In certain geographical areas a significant number of patients may be affected by an autosomal recessive hypercholesterolemia (ARH). Mutations may be also combined (two mutations of the same gene, compound heterozygosity), or two in different genes (double heterozygosity). Among the most innovative therapeutic approaches made available recently, inhibitors of the microsomal transfer protein (MTP) system have shown a high clinical potential. MTP plays a critical role in the assembly/secretion of very-low-density lipoproteins (VLDL), and its absence leads to apo B deficiency. MTP antagonists dramatically lower LDL-cholesterol (LDL-C) in animals, although a reported increase of liver fat delayed their clinical development. Lomitapide, the best-studied MTP inhibitor, reduces LDL-C by 50% or more in HoFH patients, with modest, reversible, liver steatosis. Recent US approval has confirmed an acceptable tolerability, provided patients adhere to a strictly low-fat regimen. There are no clinical data on atherosclerosis reduction/regression, but animal models provide encouraging results.

  11. An intrinsic gut leptin-melanocortin pathway modulates intestinal microsomal triglyceride transfer protein and lipid absorption.

    PubMed

    Iqbal, Jahangir; Li, Xiaosong; Chang, Benny Hung-Junn; Chan, Lawrence; Schwartz, Gary J; Chua, Streamson C; Hussain, M Mahmood

    2010-07-01

    Fat is delivered to tissues by apoB-containing lipoproteins synthesized in the liver and intestine with the help of an intracellular chaperone, microsomal triglyceride transfer protein (MTP). Leptin, a hormone secreted by adipose tissue, acts in the brain and on peripheral tissues to regulate fat storage and metabolism. Our aim was to identify the role of leptin signaling in MTP regulation and lipid absorption using several mouse models deficient in leptin receptor (LEPR) signaling and downstream effectors. Mice with spontaneous LEPR B mutations or targeted ablation of LEPR B in proopiomelanocortin (POMC) or agouti gene related peptide (AGRP) expressing cells had increased triglyceride in plasma, liver, and intestine. Furthermore, melanocortin 4 receptor (MC4R) knockout mice expressed a similar triglyceride phenotype, suggesting that leptin might regulate intestinal MTP expression through the melanocortin pathway. Mechanistic studies revealed that the accumulation of triglyceride in the intestine might be secondary to decreased expression of MTP and lipid absorption in these mice. Surgical and chemical blockade of vagal efferent outflow to the intestine in wild-type mice failed to alter the triglyceride phenotype, demonstrating that central neural control mechanisms were likely not involved in the observed regulation of intestinal MTP. Instead, we found that enterocytes express LEPR, POMC, AGRP, and MC4R. We propose that a peripheral, local gut signaling mechanism involving LEPR B and MC4R regulates intestinal MTP and controls intestinal lipid absorption.

  12. Cardiac Expression of Microsomal Triglyceride Transfer Protein Is Increased in Obesity and Serves to Attenuate Cardiac Triglyceride Accumulation

    PubMed Central

    Bartels, Emil D.; Nielsen, Jan M.; Hellgren, Lars I.; Ploug, Thorkil; Nielsen, Lars B.

    2009-01-01

    Obesity causes lipid accumulation in the heart and may lead to lipotoxic heart disease. Traditionally, the size of the cardiac triglyceride pool is thought to reflect the balance between uptake and β-oxidation of fatty acids. However, triglycerides can also be exported from cardiomyocytes via secretion of apolipoproteinB-containing (apoB) lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP); the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism remain unknown, we investigated how cardiac lipoprotein synthesis affects cardiac expression of triglyceride metabolism-controlling genes, insulin sensitivity, and function in obese mice. Heart-specific ablation of MTP-A in mice using Cre-loxP technology impaired upregulation of MTP expression in response to increased fatty acid availability during fasting and fat feeding. This resulted in cardiac triglyceride accumulation but unaffected cardiac insulin-stimulated glucose uptake. Long-term fat-feeding of male C57Bl/6 mice increased cardiac triglycerides, induced cardiac expression of triglyceride metabolism-controlling genes and attenuated heart function. Abolishing cardiac triglyceride accumulation in fat-fed mice by overexpression of an apoB transgene in the heart prevented the induction of triglyceride metabolism-controlling genes and improved heart function. The results suggest that in obesity, the physiological increase of cardiac MTP expression serves to attenuate cardiac triglyceride accumulation albeit without major effects on cardiac insulin sensitivity. Nevertheless, the data suggest that genetically increased lipoprotein secretion prevents development of obesity-induced lipotoxic heart disease. PMID:19390571

  13. 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.

  14. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  15. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

    PubMed Central

    Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  16. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  17. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein.

    PubMed

    Suzuki, Takashi; Brown, Judy J; Swift, Larry L

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5'-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5'-UTR for MTP-A. We generated reporter constructs in which the 5'-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5'-UTR, but not by the MTP-A 5'-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity.

  18. Efficacy and Safety of a Microsomal Triglyceride Transfer Protein Inhibitor in Homozygous Familial Hypercholesterolemia

    PubMed Central

    Cuchel, M; Meagher, EA; du Toit, Theron H.; Blom, DJ; Marais, AD; Hegele, RA; Averna, M; Sirtori, C; Shah, PK; Gaudet, D; Stefanutti, C; Vigna, GB; Du Plessis, AME; Propert, Kathleen J.; Sasiela, WJ; Bloedon, LT; Rader, DJ

    2015-01-01

    Background Patients with homozygous familial hypercholesterolemia (HoFH) respond inadequately to existing drugs. We conducted a phase 3 study to assess the efficacy and safety of the microsomal triglyceride transfer protein inhibitor lomitapide in adults with HoFH. Methods Twenty-nine subjects enrolled into a single-arm, open-label study and maintained current lipid lowering therapy from six weeks before baseline through at least week 26. Lomitapide dose was escalated based on safety and tolerability from 5 mg to a maximum of 60 mg/day. The primary endpoint was mean percent change from baseline in LDL-C at week 26, after which patients remained on lomitapide through week 78 for safety assessment. Findings Twenty-three subjects completed weeks 26 and 78. The median dose of lomitapide was 40 mg/day. LDL-C was reduced by 50% from baseline at week 26 (4·3 ± 2·5 mmol/L vs. 8·7 ± 2·9 mmol/L, p<0.0001). Eight subjects achieved LDL-C <2·6 mmol/L at this time point. LDL-C was reduced by 44% at week 56 and 38% at week 78 (p<0.0001 for both). Gastrointestinal symptoms were the most common adverse event. Four patients had aminotransaminase > 5× ULN that resolved after dose reduction or temporary interruption of lomitapide. No subject permanently discontinued treatment due to liver abnormalities. Liver fat content assessed by nuclear magnetic resonance spectroscopy (NMRS; n=20) was 1·0 ± 1·3 % at baseline, 8·6 ± 8·1% at week 26 and remained stable up to week 78 (8·3± 5·3%). Interpretation These data demonstrate that lomitapide had a robust and durable efficacy in lowering LDL-C in patients with HoFH with an acceptable safety and tolerability profile. PMID:23122768

  19. Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

    PubMed Central

    Liang, Zhe; Xie, Yan; Dominguez, Jessica A.; Breed, Elise R.; Yoseph, Benyam P.; Burd, Eileen M.; Farris, Alton B.

    2014-01-01

    Background Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. Methods Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. Results In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. Conclusions Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice. PMID:25010671

  20. No association between microsomal triglyceride transfer protein (MTP) haplotype and longevity in humans.

    PubMed

    Nebel, Almut; Croucher, Peter J P; Stiegeler, Rieke; Nikolaus, Susanna; Krawczak, Michael; Schreiber, Stefan

    2005-05-31

    Human longevity is a multifactorial condition with a significant genetic contribution. A recent association study in two independent samples of long-lived U.S. Caucasians [long-lived individuals (LLI)] identified a SNP haplotype of the microsomal triglyceride transfer protein (MTP, 4q25) that was underrepresented among LLI when compared with younger controls. This suggested that variation in the MTP gene might modify human longevity. Because of its function in lipid metabolism, the MTP gene product could plausibly play a pivotal role in the physiology of aging. However, the association observed in the U.S. samples could not be replicated by the same authors in a larger French LLI sample. We have therefore investigated the MTP "risk" haplotype in our own collection of 1,589 German nonagenarians, centenarians, and appropriately matched controls. No statistically significant differences were observed between LLI and controls at the allele, genotype, or haplotype level. This indicates that a noteworthy influence of the respective MTP haplotype on human longevity in the German population is unlikely. Furthermore, in comparison with all other U.S. and European samples analyzed, the MTP "risk" haplotype was found to be overrepresented only in the U.S. controls. This implies that the putative association is more likely to reflect recent changes in the genetic structure of the U.S. Caucasian population as a whole, rather than genetic effects upon survival to old age. In our view, the original study therefore highlights potential problems that arise when the case-control design is used as a means to map longevity genes in humans.

  1. Microgravity Induces Changes in Microsome-Associated Proteins of Arabidopsis Seedlings Grown on Board the International Space Station

    PubMed Central

    Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

    2014-01-01

    The “GENARA A” experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected. PMID:24618597

  2. Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station.

    PubMed

    Mazars, Christian; Brière, Christian; Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

    2014-01-01

    The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.

  3. The complex between retinol and retinol-binding protein is formed in the rough microsomes of liver following repletion of vitamin A-depleted rats.

    PubMed

    Smith, J E; DeMoor, L M; Handler, C E; Green, E L; Ritter, S J

    1998-03-12

    Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is primarily synthesized in the rough endoplasmic reticulum of the liver. RBP then passes through the smooth endoplasmic reticulum and into the Golgi apparatus where vesicles form and transport the protein to the cell membrane. When rats were depleted of their vitamin A stores, RBP accumulated in the liver microsomes, particularly in the rough microsomes. To identify the organelle(s) where retinol initially binds to RBP, vitamin A-depleted rats were given an i.v. injection of [3H]retinol suspended in Tween 40. After intervals of 2, 3, 4, 5, 6, 8, 10, 15 and 20 min, liver fractions enriched in rough and smooth microsomes and Golgi apparatus were prepared. The retinol/RBP complex (holoRBP) was detected in the rough microsomes within 3 min post injection. HoloRBP later appeared in the smooth microsomes and Golgi fraction, and then the serum at time intervals consistent with the known secretion rate for RBP. HoloRBP was detected in the rough microsomes at all times after 3 min, whether or not the complex was present in the other subcellular fractions. Thus, the holoRBP complex can form in the rough endoplasmic reticulum of the liver.

  4. Prevention of hepatic fibrosis with liver microsomal triglyceride transfer protein deletion in liver fatty acid binding protein null mice.

    PubMed

    Newberry, Elizabeth P; Xie, Yan; Kennedy, Susan M; Graham, Mark J; Crooke, Rosanne M; Jiang, Hui; Chen, Anping; Ory, Daniel S; Davidson, Nicholas O

    2017-03-01

    Blocking hepatic very low-density lipoprotein secretion through genetic or pharmacologic inhibition of microsomal triglyceride transfer protein (Mttp) causes hepatic steatosis, yet the risks for developing hepatic fibrosis are poorly understood. We report that liver-specific Mttp knockout mice (Mttp-LKO) exhibit both steatosis and fibrosis, which is exacerbated by a high-transfat/fructose diet. When crossed into germline liver fatty acid (FA) binding protein null mice (Mttp-LKO, i.e., double knockout mice) hepatic steatosis was greatly diminished and fibrosis prevented, on both low-fat and high-fat diets. The mechanisms underlying protection include reduced long chain FA uptake, shifts in FA distribution (lipidomic profiling), and metabolic turnover, specifically decreased hepatic 18:2 FA and triglyceride species and a shift in 18:2 FA use for oxidation versus incorporation into newly synthesized triglyceride. Double knockout mice were protected against fasting-induced hepatic steatosis (a model of enhanced exogenous FA delivery) yet developed steatosis upon induction of hepatic de novo lipogenesis with fructose feeding. Mttp-LKO mice, on either the liver FA binding protein null or Apobec-1 null background (i.e., apolipoprotein B100 only) exhibited only subtle increases in endoplasmic reticulum stress, suggesting that an altered unfolded protein response is unlikely to account for the attenuated phenotype in double knockout mice. Acute, antisense-mediated liver FA binding protein knockdown in Mttp-LKO mice also reduced FA uptake, increased oxidation versus incorporation of 18:2 species with complete reversal of hepatic steatosis, increased hepatic injury, and worsened fibrosis. Perturbing exogenous hepatic FA use modulates both hepatic steatosis and fibrosis in the setting of hepatic Mttp deletion, adding new insight into the pathophysiological mechanisms and consequences of defective very low-density lipoprotein secretion. (Hepatology 2017;65:836-852). © 2016 by

  5. Characteristics of the active oxygen in covalent binding of the pesticide methoxychlor to hepatic microsomal proteins.

    PubMed

    Kupfer, D; Bulger, W H; Nanni, F J

    1986-08-15

    This study examined the characteristics of the active oxygen species involved in generation of the reactive intermediate of methoxychlor which covalently binds to liver microsomal proteins. The possibility that the active oxygen participating in the above reaction is the superoxide anion (O2-) or a species generated from O2- was examined with the help of superoxide dismutase (SOD) and with an SOD-mimetic agent, CuDIPS [Cu2+(3,5-diisopropylsalicylic acid)2]. It was observed that, whereas CuDIPS inhibited covalent binding of methoxychlor metabolite(s), SOD did not. However, ZnDIPS [Zn2+(3,5-diisopropylsalicylic acid)2], which exhibits no SOD-mimetic activity, did not inhibit covalent binding. Furthermore, both CuDIPS and ZnDIPS had little or no effect on the formation of demethylated (polar) metabolites of methoxychlor, demonstrating that the inhibition of covalent binding by CuDIPS was not merely due to a general inhibition of the hepatic monooxygenase system. These findings suggested that O2- was involved in covalent binding, but was not accessible to SOD. Additional support for O2- involvement stems from the observation that alpha-tocopheryl acid succinate markedly inhibited covalent binding of methoxychlor. The possibility that hydrogen peroxide (H2O2) was involved in covalent binding of methoxychlor appears unlikely. Catalase had no effect on covalent binding when NADPH was the cofactor, and the use of H2O2 in place of NADPH did not yield covalent binding. Certain scavengers of hydroxyl radical (ethanol, t-butanol and benzoate) inhibited, and other known scavengers (DMSO and mannitol) did not inhibit, covalent binding. EDTA stimulated binding, desferal (desferrioxamine) exhibited no effect on binding, and diethylenetriaminepentaacetic acid (DETAPAC) inhibited binding. A possible explanation for this observation is that the Fe2+ needed for generation of X OH is much more easily obtained from Fe3+-EDTA than from Fe3+-desferal, which resists reduction. The

  6. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated depression of rat testicular heme synthesis and microsomal cytochrome P-450.

    PubMed

    Tofilon, P J; Piper, W N

    1982-11-15

    Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chloracne, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 microgram/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular delta-aminolevulinic acid (ALA) synthase were detected. The incorporation of [14C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.

  7. Profiling the progression of cancer: separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis.

    PubMed

    O'Neil, Kimberly A; Miller, Fred R; Barder, Timothy J; Lubman, David M

    2003-07-01

    The heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following work describes the development of a technique for the characterization of membrane subproteomes from five different breast epithelial cell lines. Intact membrane proteins are separated by hydrophobicity in the first dimension using nonporous reversed-phase high-performance liquid chromatography (RP-HPLC) to generate unique chromatographic profiles. Fractions of eluent are further separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to create distinct banding patterns. This hybrid liquid phase/gel phase method circumvents issues of membrane protein precipitation and provides a simple strategy aimed at isolating and characterizing a traditionally underrepresented protein class. Membrane protein profiles are created that discriminate between microsomal fractions of breast epithelial cells in different stages of neoplastic progression. Proteins are subsequently identified using matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) mass fingerprinting and MALDI-quadrupole time of flight - tandem mass spectrometry (QTOF-MS/MS) peptide sequencing. Furthermore, as this strategy preserves intact protein structure, further characterization can be performed on proteins producing mass fingerprint spectra and fragmentation spectra that did not result in database protein identifications. The coupling of nonporous RP-HPLC with SDS-PAGE provides a useful alternative to two-dimensional PAGE (2-D-PAGE) for membrane protein analysis.

  8. Microsomal triacylglycerol transfer protein (MTP) is required to expand tracheal lumen in Drosophila in a cell-autonomous manner.

    PubMed

    Baer, Magdalena M; Palm, Wilhelm; Eaton, Suzanne; Leptin, Maria; Affolter, Markus

    2012-12-15

    The Drosophila tracheal system is a useful model for dissecting the molecular mechanisms controlling the assembly and expansion of tubular organs. We have identified microsomal triacylglycerol transfer protein (MTP) as a new player involved in the lumen expansion in unicellular tubes. MTP is an endoplasmic reticulum resident protein that can transfer triglycerides and phospholipids between membranes in vitro. MTP lipid transfer activity is crucial for the assembly and secretion of apoB family lipoproteins, which are carriers of lipids between different tissues. Here we describe an unexpected role of MTP in tracheal development, which we postulate to be independent of its known function in lipoprotein secretion. We propose that, in tracheal cells, MTP is involved in regulation of de novo apical membrane delivery to the existing lumen and thus promotes proper expansion of the larval tracheal system.

  9. Effect of Mangifera indica L. extract (QF808) on protein and hepatic microsome peroxidation.

    PubMed

    Martínez, G; Giuliani, A; León, O S; Pérez, G; Núñez Selles, A J

    2001-11-01

    The antioxidant activities of QF808, a steam bark extract of Mangifera indica L., were studied on hydroxyl-mediated oxidation of bovine serum albumin (BSA) and in a hepatic microsome system. The extract was effective in reducing the oxidation of BSA, since its half- maximal inhibition concentration (IC(50)) was 0.0049% w/v in the inhibition of carbonyl group formation and lower than 0.0025% w/v in the inhibition of sulfhydryl group loss. QF808 inhibited lipid peroxidation which was initiated enzymatically by reduced nicotinamide adenine dinucleotide phosphate (NADPH), IC(50)= 0.00075% w/v, or non-enzymatically by ascorbic acid, IC(50) = 0.0126% w/v. The extract tested did not inhibit NADPH-dependent cytochrome P-450 reductase activity, since it had no effect on the oxidation rate of NADPH. These results suggest that QF808 has an antioxidant activity, probably due to its ability to scavenge free radicals involved in microsome lipid peroxidation. In addition, QF808 antioxidant profile in vitro is probably similar to its principal polyphenolic component, mangiferin, a glycosylated xanthone.

  10. Discovery of microsomal triglyceride transfer protein (MTP) inhibitors with potential for decreased active metabolite load compared to dirlotapide.

    PubMed

    Robinson, Ralph P; Bartlett, Jeremy A; Bertinato, Peter; Bessire, Andrew J; Cosgrove, Judith; Foley, Patrick M; Manion, Tara B; Minich, Martha L; Ramos, Brenda; Reese, Matthew R; Schmahai, Theodore J; Swick, Andrew G; Tess, David A; Vaz, Alfin; Wolford, Angela

    2011-07-15

    Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4'-alkyl and 4'-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.

  11. Proteomics and gene expression analyses of squalene-supplemented mice identify microsomal thioredoxin domain-containing protein 5 changes associated with hepatic steatosis.

    PubMed

    Ramírez-Torres, Adela; Barceló-Batllori, Sílvia; Martínez-Beamonte, Roberto; Navarro, María A; Surra, Joaquín C; Arnal, Carmen; Guillén, Natalia; Acín, Sergio; Osada, Jesús

    2012-12-21

    Squalene is an abundant hydrocarbon present in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE-knock-out mice. To study its effects on microsomal proteins, 1g/kg/day of squalene was administered to those mice. After 10 weeks, hepatic fat content was assessed and protein extracts of microsomal enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by peptide fingerprinting and MSMS analysis. Squalene administration modified the expression of thirty-one proteins involved in different metabolic functions and increased the levels of those involved in vesicle transport, protein folding and redox status. Only mRNA levels of 9 genes (Arg1, Atp5b, Cat, Hyou1, Nipsnap1, Pcca, Pcx, Pyroxd2, and Txndc5) paralleled these findings. No such mRNA changes were observed in wild-type mice receiving squalene. Thioredoxin domain-containing protein 5 (TXNDC5) protein and mRNA levels were significantly associated with hepatic fat content in apoE-ko mice. These results suggest that squalene action may be executed through a complex regulation of microsomal proteins, both at the mRNA and post-transcriptional levels and the presence of apoE may change the outcome. Txndc5 reflects the anti-steatotic properties of squalene and the sensitivity to lipid accumulation. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes.

    PubMed

    Love, Joseph D; Suzuki, Takashi; Robinson, Delia B; Harris, Carla M; Johnson, Joyce E; Mohler, Peter J; Jerome, W Gray; Swift, Larry L

    2015-01-01

    Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology.

  13. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes

    PubMed Central

    Robinson, Delia B.; Harris, Carla M.; Johnson, Joyce E.; Mohler, Peter J.; Jerome, W. Gray; Swift, Larry L.

    2015-01-01

    Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology. PMID:26267806

  14. [A substrate-type induction of liver microsomal monooxygenases by phenobarbital].

    PubMed

    Tsyrlov, I B; Gromova, O A; Rivkind, N B; Vakulin, G M; Liakhovich, V V

    1977-07-01

    A possibility of step-wise induction of microsomal monooxygenases after injection of phenobarbital in the presence of 3-methylcholanthrene-caused induction was studied. It was found that the ratio of the high- and low-spin types of cytochrome, rather than the position of the CO-peak of its reduced form is a criterion for functional specificity of hemoprotein. Induction by phenobarbital appears possible under conditions when the inductor binding to microsomal hemoprotein is lacking, since cytochrome P-488 has no binding sites for phenobarbital. It is assumed that under microsomal monooxygenases induction by phenobarbital activation of genome and subsequent protein synthesis are operated by the substrate rather than by products of its primary metabolism in microsomes.

  15. Mouse microsomal triglyceride transfer protein large subunit: cDNA cloning, tissue-specific expression, and chromosomal localization

    SciTech Connect

    Nakamuta, Makoto; Chang, Benny Hung-Junn; Hoogeveen, R.

    1996-04-15

    Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phospholipid between membranes. It is essential for the secretion of apolipoprotein B from the cell. Mutations in MTP are a major cause of abetalipoproteinemia. The mouse is a popular animal model for lipoprotein metabolism. We have cloned and sequenced mouse MTP cDNA. The DNA-deduced amino acid sequence indicates that mouse protein shows 93, 86, and 83% sequence indicates that mouse MTP contains 894 amino acids; the mouse protein shows 93, 86, and 83% sequence identity to the hamster, human, and bovine sequences, respectively. Northern blot analysis indicates that mouse MTP mRNA is expressed at high levels in the small intestine and at substantially lower levels in the liver and that it is not detectable in six other tissues examined. The mouse MTP gene has been localized to the distal region of chromosome 3 by Southern blots of interspecific backcross panels using progeny derived from matings of (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. Comparison of MTP sequences from human, bovine, hamster, and mouse indicates that the C-terminal region of MTP is better conserved than its N-terminal region. 21 refs., 2 figs.

  16. Inhibition of microsomal lipid peroxidation by cytosolic protein in presence of ADP and high concentration of Fe2+.

    PubMed

    Ramasarma, T; Muakkassah-Kelly, S; Hochstein, P

    1984-12-06

    Microsomal lipid peroxidation induced by NADPH, but not by ascorbate, was found to be inhibited by liver cytosol. This inhibition was not dependent on glutathione and was enhanced by ADP in presence of Fe2+ at a concentration of 50 microM or higher. ATP was also effective, but not AMP or cyclic AMP. The cytosolic factor appeared to be a protein as it was heat-labile (greater than 70 degrees C), was non-dialyzable and was precipitated by ammonium sulfate and acetone. It was stable for several months in frozen state and also when heated at 50 degrees C for 10 min. The inhibition by the cytosolic protein was obtained by producing a lag in the activity of lipid peroxidation and was reversed by ceruloplasmin but not by catalase, cytochrome c, hemoglobin or superoxide dismutase. This inhibitory effect by cytosol was limited to formation of lipid peroxides whereas oxygen uptake and NADPH oxidation remained unaffected. Regulation of lipid peroxidation by nucleotide-Fe complexes and cytosolic proteins is indicated by these studies.

  17. Modulation of hepatic microsomal triglyceride transfer protein (MTP) induced by S-nitroso-N-acetylcysteine in ob/ob mice.

    PubMed

    Oliveira, Claudia P M S; Alves, Venâncio A F; Lima, Vicência M R; Stefano, José Tadeu; Debbas, Victor; Sá, Sandra Valéria; Wakamatsu, Alda; Corrêa-Giannella, Maria Lúcia; de Mello, Evandro Sobroza; Havaki, Sofia; Tiniakos, Dina G; Marinos, Evangelos; de Oliveira, Marcelo G; Giannella-Neto, Daniel; Laurindo, Francisco R; Caldwell, Stephen; Carrilho, Flair J

    2007-07-15

    We evaluated the effects of a potent NO donor, S-nitroso-N-acetylcysteine (SNAC), on microsomal triglyceride transfer protein (MTP) expression in ob/ob mice. NAFLD was induced in male ob/ob mice using a methionine-choline deficient diet (MCD) concomitantly with oral SNAC fed solution (n=5) or vehicle (control; n=5) by gavage daily for 4 weeks. Livers were collected for histology and for assessing MTP by RT-qPCR, Western blot, immunohistochemistry and immunogold electron microscopy analyses. Histological analysis showed diffuse macro and microvesicular steatosis, moderate hepatocellular ballooning and moderate inflammatory infiltrate in ob/ob mice fed the MCD diet. With SNAC, mice showed a marked reduction in liver steatosis (p<0.01), in parenchymal inflammation (p=0.02) and in MTP protein immunoexpression in zone III (p=0.05). Moreover, SNAC caused reduction of MTP protein in Western blot analysis (p<0.05). In contrast, MTP mRNA content was significantly higher (p<0.05) in mice receiving SNAC. Immuno-electron microscopy showed MTP localized in the rough endoplasmic reticulum of hepatocytes in both treated and untreated groups. However with SNAC treatment, MTP was also observed surrounding fat globules. Histological improvement mediated by a nitric oxide donor is associated with significantly altered expression and distribution of MTP in this animal model of fatty liver disease. Further studies are in progress to examine possible mechanisms and to develop SNAC as a possible therapy for human fatty liver disease.

  18. 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

  19. Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function.

    PubMed

    Zeissig, Sebastian; Dougan, Stephanie K; Barral, Duarte C; Junker, Yvonne; Chen, Zhangguo; Kaser, Arthur; Ho, Madelyn; Mandel, Hannah; McIntyre, Adam; Kennedy, Susan M; Painter, Gavin F; Veerapen, Natacha; Besra, Gurdyal S; Cerundolo, Vincenzo; Yue, Simon; Beladi, Sarah; Behar, Samuel M; Chen, Xiuxu; Gumperz, Jenny E; Breckpot, Karine; Raper, Anna; Baer, Amanda; Exley, Mark A; Hegele, Robert A; Cuchel, Marina; Rader, Daniel J; Davidson, Nicholas O; Blumberg, Richard S

    2010-08-01

    Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

  20. Pharmacokinetic changes in drugs during protein-calorie malnutrition: correlation between drug metabolism and hepatic microsomal cytochrome P450 isozymes.

    PubMed

    Lee, Joo Hyun; Suh, Ok Kyung; Lee, Myung Gull

    2004-07-01

    The rats with protein-calorie malnutrition (PCM, 5% casein diet for a period of 4-week) were reported to exhibit 60 and 80% suppression in the hepatic microsomal cytochrome P450 (CYP) 1A2 and CYP2C11 levels, respectively, and 40-50% decreases in CYP2E1 and CYP3A1/2 levels compared to control (23% casein diet for a period of 4-week) based on Western blot analysis. In addition, Northern blot analysis showed that CYP1A2, CYP2E1, CYP2C11, and CYP3A1/2 mRNAs decreased in the state of PCM as well. Hence, pharmacokinetic changes of the drugs in rats with PCM [especially the area under the plasma concentration-time curve from time zero to time infinity (AUC) changes of metabolite(s)] reported from literatures were tried to explain in terms of CYP isozyme changes in the rats. Otherwise, the time-averaged nonrenal clearance (CL NR) of parent drug was compared. Pharmacokinetic changes of the drugs in other types of malnutritional state, such as kwashiorkor and marasmus, in both human and animal models were also compared. The drugs reviewed are as follows: diuretics, antibiotics, anticancer agents, antiepileptics, antiarrythmics, analgesics, xanthines, antimalarials, and miscellaneous.

  1. JTT-130, a novel intestine-specific inhibitor of microsomal triglyceride transfer protein, reduces food preference for fat.

    PubMed

    Mera, Yasuko; Hata, Takahiro; Ishii, Yukihito; Tomimoto, Daisuke; Kawai, Takashi; Ohta, Takeshi; Kakutani, Makoto

    2014-01-01

    Microsomal triglyceride transfer protein (MTP) is involved in the assembly and secretion of triglyceride-rich lipoproteins from enterocytes and hepatocytes. JTT-130 is a novel intestine-specific MTP inhibitor, which has been shown to be useful in the prevention and treatment of dyslipidemia, obesity, and diabetes. JTT-130 has also been shown to suppress food intake in a dietary fat-dependent manner in rats. However, whether JTT-130 enables changes in food preference and nutrient consumption remains to be determined. Therefore, the aim of the present study was to investigate the effects of JTT-130 on food preference in rat under free access to two different diets containing 3.3% fat (low-fat diet, LF diet) and 35% fat (high-fat diet, HF diet). JTT-130 decreased HF diet intake and increased LF diet intake, resulting in a change in ratio of caloric intake from LF and HF diets to total caloric intake. In addition, macronutrient analysis revealed that JTT-130 did not affect carbohydrate consumption but significantly decreased fat consumption (P < 0.01). These findings suggest that JTT-130 not only inhibits fat absorption, but also suppresses food intake and specifically reduces food preference for fat. Therefore, JTT-130 is expected to provide a new option for the prevention and treatment of obesity and obesity-related metabolic disorders.

  2. Synthesis, microsome-mediated metabolism, and identification of major metabolites of environmental pollutant naphtho(8,1,2-ghi)chrysene

    SciTech Connect

    Sharma, A.K.; Gowdahalli, K.; Gimbor, M.; Amin, S.

    2008-05-15

    Naphtho(8,1,2-ghi)chrysene, commonly known as naphtho(1,2-e)pyrene (N(1,2-e)P) is a widespread environmental pollutant, identified in coal tar extract, air borne particulate matter, marine sediment, cigarette smoke condensate, and vehicle exhaust. Herein, we determined the ability of rat liver microsomes to metabolize N(1,2-e)P and an unequivocal assignment of the metabolites by comparing them with independently,synthesized standards. We developed the synthesis of both the fjord region and the K-region dihydrodiols and various phenolic derivatives for metabolite identification. In summary, N(1,2-e)P trans-11, 12-dihydrodiol was the major metabolite formed along with N(1,2-e)P 4,5-trtins-dihydrodiol and 12-OH-N(1,2-e)P on exposure of rat liver microsomes to N(1,2-e)P. The presence of N(1,2-e)P in the environment and formation of fjord region dihydrodiol 14 as a major metabolite in in vitro metabolism studies strongly suggest the role of N(1,2-e)P as a potential health hazard.

  3. CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein.

    PubMed

    Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus; Broekema, Marjoleine F; de Haar, Colin; Schipper, Henk S; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

    2014-08-08

    Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.

  4. CD1d-mediated Presentation of Endogenous Lipid Antigens by Adipocytes Requires Microsomal Triglyceride Transfer Protein*

    PubMed Central

    Rakhshandehroo, Maryam; Gijzel, Sanne M. W.; Siersbæk, Rasmus; Broekema, Marjoleine F.; de Haar, Colin; Schipper, Henk S.; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

    2014-01-01

    Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and –δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis. PMID:24966328

  5. Microsomal triglyceride transfer protein is required for yolk lipid utilization and absorption of dietary lipids in zebrafish larvae.

    PubMed

    Schlegel, Amnon; Stainier, Didier Y R

    2006-12-26

    Although the absorption, transport, and catabolism of dietary lipids have been studied extensively in great detail in mammals and other vertebrates, a tractable genetic system for identifying novel genes involved in these physiologic processes is not available. To establish such a model, we monitored neutral lipid by staining fixed zebrafish larvae with oil red o (ORO). The head structures, heart, vasculature, and swim bladder stained with ORO until the yolk was consumed 6 days after fertilization (6 dpf). Thereafter, the heart and vasculature no longer had stainable neutral lipids. Following a high-fat meal, ORO stained the intestine and vasculature of 6 dpf larvae, and whole-larval triacylglycerol (TAG) and apolipoprotein B levels increased. Levels of microsomal triglyceride transfer protein (Mtp), the protein responsible for packaging TAG and betalipoproteins into lipoprotein particles, were unchanged by feeding. Since the developing zebrafish embryo expresses mtp in the yolk cell layer, liver, and intestine, we determined the effect of targeted knockdown of Mtp expression using an antisense morpholino oligonucleotide approach (Mtp MO) on the transport of yolk and dietary lipids. Mtp MO injection led to loss of Mtp expression and of lipid staining in the vasculature, heart, and head structures. Mtp MO-injected larvae were smaller than age-matched, uninjected larvae, consumed very little yolk, and did not absorb dietary neutral lipids; however, they absorbed a short chain fatty acid that does not require Mtp for transport. Importantly, the vasculature appeared unaffected in Mtp MO-injected larvae. These studies indicate that zebrafish larvae are suitable for genetic studies of lipid transport and metabolism.

  6. Microsomal metabolism of picene.

    PubMed

    Platt, K L; Petrovic, P; Seidel, A; Beermann, D; Oesch, F

    1988-01-01

    Picene, a polycyclic aromatic hydrocarbon (PAH) of environmental relevance has recently been predicted to be carcinogenic, based on quantum mechanical calculation, although in several animal studies no carcinogenicity could be detected. In order to find out if the metabolism of this PAH can provide an explanation for its lack of carcinogenicity, picene was incubated with the hepatic microsomal fraction of Sprague-Dawley rats, which had been pretreated with Aroclor 1254. Sixteen ethyl acetate-extractable metabolites could be separated by reversed-phase high-performance liquid chromatography. Comparison of the chromatographic behavior and the UV and mass spectral properties of the metabolites with those of synthetic derivatives of picene allowed the identification of trans-1,2-, -3,4-, -5,6-dihydrodiol as well as 2- and 4-phenol as microsomal metabolites of picene. At a substrate concentration of 2.7 microM and an amount of 68 micrograms microsomal protein per ml incubation volume, 4-picenol was the main microsomal metabolite with 32.2% of total metabolic conversion, followed by the 1,2-(bay-region)dihydrodiol with 16.7%, the 3,4-(M-region)dihydrodiol with 15.9%, 2-picenol with 9.1% and the 5,6-(K-region)dihydrodiol with 1.6%. In this respect the metabolism of picene is not significantly different from that of the carcinogenic PAH benzo[a]pyrene and dibenz[a,h]anthracene. The M-region dihydrodiols, potential precursors of electrophilically reactive dihydrodiol bay-region epoxides, are formed from all three PAHs at 11-16% of total metabolic conversion. From the 2.8- to 4.4-fold lower amounts of polar and water-soluble metabolites of picene as compared to dibenz[a,h]anthracene and benzo[a]pyrene it is deduced that dihydrodiol epoxides are generated from picene to a much smaller extent than from the two carcinogenic PAHs. The lacking carcinogenicity of picene could therefore result from the inability of microsomal enzymes to transform its M-region dihydrodiol to

  7. Inhibition of hepatic microsomal triglyceride transfer protein – a novel therapeutic option for treatment of homozygous familial hypercholesterolemia

    PubMed Central

    Vuorio, Alpo; Tikkanen, Matti J; Kovanen, Petri T

    2014-01-01

    Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein (LDL)-receptor gene (LDLR). Patients with homozygous FH (hoFH) have inherited a mutated LDLR gene from both parents, and therefore all their LDL-receptors are incapable of functioning normally. In hoFH, serum LDL levels often exceed 13 mmol/L and tendon and cutaneous xanthomata appear early (under 10 years of age). If untreated, this extremely severe form of hypercholesterolemia may cause death in childhood or in early adulthood. Based on recent data, it can be estimated that the prevalence of hoFH is about 1:500,000 or even 1:400,000. Until now, the treatment of hoFH has been based on high-dose statin treatment combined with LDL apheresis. Since the LDL cholesterol-lowering effect of statins is weak in this disease, and apheresis is a cumbersome treatment and not available at all centers, alternative novel pharmaceutical therapies are needed. Lomitapide is a newly introduced drug, capable of effectively decreasing serum LDL cholesterol concentration in hoFH. It inhibits the microsomal triglyceride transfer protein (MTTP). By inhibiting in hepatocytes the transfer of triglycerides into very low density lipoprotein particles, the drug blocks their assembly and secretion into the circulating blood. Since the very low density lipoprotein particles are precursors of LDL particles in the circulation, the reduced secretion of the former results in lower plasma concentration of the latter. The greatest concern in lomitapide treatment has been the increase in liver fat, which can be, however, counteracted by strictly adhering to a low-fat diet. Lomitapide is a welcome addition to the meager selection of drugs currently available for the treatment of refractory hypercholesterolemia in hoFH patients. PMID:24851052

  8. Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons.

    PubMed

    Tanchak, M A; Chrispeels, M J

    1989-11-01

    Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.

  9. Oleate-mediated stimulation of microsomal triglyceride transfer protein (MTP) gene promoter: implications for hepatic MTP overexpression in insulin resistance.

    PubMed

    Qiu, Wei; Taghibiglou, Changiz; Avramoglu, Rita Kohen; Van Iderstine, Stephen C; Naples, Mark; Ashrafpour, Homa; Mhapsekar, Shailen; Sato, Ryuichiro; Adeli, Khosrow

    2005-03-01

    Hepatic lipoprotein overproduction in a fructose-fed hamster model of insulin resistance was previously shown to be associated with a significant elevation of intracellular mass of microsomal triglyceride transfer protein (MTP) and elevated plasma levels of free fatty acids (FFA). Here, we further establish that fructose feeding and development of an insulin resistant state result in higher levels of MTP mRNA, protein mass, and lipid transfer activity. MTP protein mass was increased in fructose-fed hamster hepatocytes to 161 +/- 35.8% of control (p < 0.05), while MTP mRNA levels and MTP lipid transfer activity were increased to 147.5 +/- 30.8% (p < 0.05) and 177.5 +/- 14.5% (p < 0.05) of control levels, respectively. To identify underlying mechanisms, we also investigated the potential link between enhanced FFA flux and hepatic MTP gene expression. Direct modulation of MTP gene transcription by fatty acids was investigated by transfecting HepG2 cells with a reporter (luciferase) construct containing various base pair regions of the human MTP promoter including pMTP124 (with the sterol response element (SRE)), pMTP116, pMTP109 and pMTP100 (no SRE), and pMTP124SREKO (SRE sequences mutated). Treatment of HepG2 cells with oleic acid (360 muM) significantly increased luciferase activities in cells transfected with pMTP124 (136.6 +/- 11.0%, p < 0.05) and pMTP124SREKO (153.9 +/- 11.1%, p < 0.01) compared with control. Luciferase activity was also increased in a time and dose-dependent manner in the presence of oleic acid when transfected with pMTP124SREKO but not pMTP109 and pMTP100. Furthermore, long-term oleic acid treatment of HepG2 cells (10 days) induced higher levels of MTP mRNA (p < 0.05) confirming transcriptional stimulation of the MTP gene by oleic acid. In contrast, palmitate, arachidonic acid or linoleic acid did not significantly stimulate pMTP124 or pMTP124SREKO luciferase activity (p > 0.05). These data demonstrate that (1) MTP gene transcription may be

  10. Microsomal triglyceride transfer protein contributes to lipid droplet maturation in adipocytes.

    PubMed

    Swift, Larry L; Love, Joseph D; Harris, Carla M; Chang, Benny H; Jerome, W Gray

    2017-01-01

    Previous studies in our laboratory have established the presence of MTP in both white and brown adipose tissue in mice as well as in 3T3-L1 cells. Additional studies demonstrated an increase in MTP levels as 3T3-L1 cells differentiate into adipocytes concurrent with the movement of MTP from the juxtanuclear region of the cell to the surface of lipid droplets. This suggested a role for MTP in lipid droplet biogenesis and/or maturation. To probe the role of MTP in adipocytes, we used a Cre-Lox approach with aP2-Cre and Adipoq-Cre recombinase transgenic mice to knock down MTP expression in brown and white fat of mice. MTP expression was reduced approximately 55% in white fat and 65-80% in brown fat. Reducing MTP expression in adipose tissue had no effect on weight gain or body composition, whether the mice were fed a regular rodent or high fat diet. In addition, serum lipids and unesterified fatty acid levels were not altered in the knockdown mice. Importantly, decreased MTP expression in adipose tissue was associated with smaller lipid droplets in brown fat and smaller adipocytes in white fat. These results combined with our previous studies showing MTP lipid transfer activity is not necessary for lipid droplet initiation or growth in the early stages of differentiation, suggest that a structural feature of the MTP protein is important in lipid droplet maturation. We conclude that MTP protein plays a critical role in lipid droplet maturation, but does not regulate total body fat accumulation.

  11. Proteomic Study of Microsomal Proteins Reveals a Key Role for Arabidopsis Annexin 1 in Mediating Heat Stress-Induced Increase in Intracellular Calcium Levels*

    PubMed Central

    Wang, Xu; Ma, Xiaolong; Wang, Hui; Li, Bingjie; Clark, Greg; Guo, Yi; Roux, Stan; Sun, Daye; Tang, Wenqiang

    2015-01-01

    To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca2+]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca2+]cyt and in the response of Arabidopsis seedlings to heat stress. PMID:25587034

  12. Proteomic study of microsomal proteins reveals a key role for Arabidopsis annexin 1 in mediating heat stress-induced increase in intracellular calcium levels.

    PubMed

    Wang, Xu; Ma, Xiaolong; Wang, Hui; Li, Bingjie; Clark, Greg; Guo, Yi; Roux, Stan; Sun, Daye; Tang, Wenqiang

    2015-03-01

    To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca(2+)]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca(2+)]cyt and in the response of Arabidopsis seedlings to heat stress.

  13. The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein

    NASA Astrophysics Data System (ADS)

    Shibuya, Makiko; Hiraoki, Toshifumi; Kimura, Kunie; Fukushima, Kazuaki; Suzuki, Kuniaki

    2012-12-01

    We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy. The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins. The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and τ values, which are indices of the fluidity of the membrane. These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

  14. Etiology of fatty liver in dairy cattle: effects of nutritional and hormonal status on hepatic microsomal triglyceride transfer protein.

    PubMed

    Bremmer, D R; Trower, S L; Bertics, S J; Besong, S A; Bernabucci, U; Grummer, R R

    2000-10-01

    We conducted three experiments to determine the effects of nutritional and hormonal status on microsomal triglyceride transfer protein (MTP) activity and mass. In experiment 1, 18 nonlactating Holstein cows, 75 d before expected calving date, in their second gestation or greater were monitored from d 75 to 55 prepartum. Cows were fed a control diet from d 75 to 62 prepartum for covariable measurements. From d 61 to 55 prepartum, six cows continued to receive the control diet, six cows were restricted to 2.3 kg of grass hay/d, and six cows were fed the control diet plus 1.8 kg of concentrate/d and 500 ml of propylene glycol given 2 times/d as an oral drench. Plasma glucose and serum insulin concentrations were highest in cows that received propylene glycol and lowest in feed restricted cows. Plasma nonesterified fatty acids (NEFA) and liver triglyceride (TG) concentrations were highest in feed restricted cows and not different between cows that received the control diet and cows that received propylene glycol. Hepatic MTP activity and mass were not affected by treatment in experiment 1. In experiment 2, bovine hepatocytes isolated from the caudate process of five preruminating Holstein bull calves were incubated with either 0, 0.5, 1.0, or 2.0 mM NEFA for 48 h. Intracellular TG increased linearly as NEFA concentration in the media increased. Concentration of NEFA in the incubation media had no effect on MTP activity or mass. There was a quadratic effect of concentration of NEFA in the incubation media on MTP mRNA. In experiment 3, bovine hepatocytes isolated from the caudate process of five preruminating Holstein bull calves were incubated with 2 mM [1-14C]oleate for 24 h to accumulate TG, followed by a 36-h period of TG depletion, during which hepatocytes were incubated with no hormone, 10 nM insulin, or 10 nM glucagon. There was no effect of insulin or glucagon on intracellular TG, MTP activity or mass. Cells incubated with no hormone had higher levels of MTP m

  15. Protein chemical synthesis in drug discovery.

    PubMed

    Liu, Fa; Mayer, John P

    2015-01-01

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

  16. Chronological protein synthesis in regenerating rat liver.

    PubMed

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

    2015-07-01

    Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide.

    PubMed

    Silva, Elias C C; Masui, Douglas C; Furriel, Rosa P; McNamara, John C; Barrabin, Hector; Scofano, Helena M; Perales, Jonas; Teixeira-Ferreira, André; Leone, Francisco A; Fontes, Carlos Frederico L

    2012-11-01

    This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.

  18. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo

    PubMed Central

    Kats, Anna; Båge, Tove; Georgsson, Pierre; Jönsson, Jörgen; Quezada, Hernán Concha; Gustafsson, Anders; Jansson, Leif; Lindberg, Claes; Näsström, Karin; Yucel-Lindberg, Tülay

    2013-01-01

    The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL-1β and a cell-free mPGES-1 activity assay, as well as on inflammation-induced bone resorption in vivo, using ligature-induced experimental periodontitis in Sprague-Dawley rats. Aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) and 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) reduced IL-1β-induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES-1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase-2. In ligature-induced experimental periodontitis, alveolar bone loss, assessed by X-ray imaging, was reduced by 46% by local treatment with TH-848, compared to vehicle, without any systemic effects on PGE2, 6-keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES-1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti-inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel-Lindberg, T. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo. PMID:23447581

  19. 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)

  20. 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)

  1. 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. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  2. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  3. Proteomic analysis of microsomes from lactating bovine mammary gland.

    PubMed

    Peng, Lifeng; Rawson, Pisana; McLauchlan, Danyl; Lehnert, Klaus; Snell, Russell; Jordan, T William

    2008-04-01

    Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESI-MS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue.

  4. Analysis of mRNA With Microsomal Fractionation Using a SAGE-Based DNA Microarray System Facilitates Identification of the Genes Encoding Secretory Proteins

    PubMed Central

    Toyoda , Nobuaki; Nagai, Shigenori; Terashima, Yuya; Motomura, Kazushi; Haino, Makoto; Hashimoto, Shin-ichi; Takizawa, Hajime; Matsushima, Kouji

    2003-01-01

    In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)–based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins. PMID:12805275

  5. Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

    PubMed

    Nakamura, Kenji; Hirayama-Kurogi, Mio; Ito, Shingo; Kuno, Takuya; Yoneyama, Toshihiro; Obuchi, Wataru; Terasaki, Tetsuya; Ohtsuki, Sumio

    2016-08-01

    The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

  6. Modulation of catechol estrogen synthesis by rat liver microsomes: effects of treatment with growth hormone or testosterone

    SciTech Connect

    Quail, J.A.; Jellinck, P.H.

    1987-09-01

    The ability of GH from various mammalian species, administered to normal mature male rats by constant infusion, to decrease the hepatic 2-hydroxylation of estradiol (E2) to female levels, as measured by the release of /sup 3/H/sub 2/O from (2-3H)E2, was determined. Rat and human GH (hGH) showed the highest activity while ovine GH was inactive. PRL (0.6 IU/h X kg) administered together with hGH (0.02 IU/h X kg) did not antagonize the feminizing action of GH. Infusion of hGH into male rats decreased the affinity of estradiol 2-hydroxylase for its steroid substrate and altered the linear Lineweaver-Burk plot towards a nonlinear hyperbolic plot characteristic of the female. The apparent Michaelis-Menten constant (Km) for the reaction was 1.69 microM for males and 2.75 microM for testosterone-treated ovariectomized females. An equal mixture of liver microsomes from male and female rats gave kinetic values similar to those observed with males alone. Neonatal imprinting with androgen did not alter the magnitude of the response of female rats to treatment with testosterone and/or GH at maturity and the androgen effect could only be shown in ovariectomized animals. The results with rats of different endocrine status were corroborated by the kinetic data and by the pattern of metabolites obtained with (4-/sup 14/C)E2 when examined by TLC and autoradiography. The hormonal control of estradiol 2-hydroxylase, the key enzyme in catechol estrogen formation, and the contribution of sex-specific multiple forms of the enzyme to this reaction are discussed.

  7. 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.

  8. Leucine stimulation of skeletal muscle protein synthesis

    SciTech Connect

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

    1986-03-01

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

  9. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    PubMed Central

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  10. Modulation of protein synthesis by polyamines.

    PubMed

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

    Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis. © 2015 International Union of Biochemistry and Molecular Biology.

  11. Novel findings for the development of drug therapy for various liver diseases: Liver microsomal triglyceride transfer protein activator may be a possible therapeutic agent in non-alcoholic steatohepatitis.

    PubMed

    Fujita, Koji; Imajo, Kento; Shinohara, Yoshiyasu; Nozaki, Yuichi; Wada, Koichiro; Yoneda, Masato; Endo, Hiroki; Takahashi, Hirokazu; Abe, Yasunobu; Inamori, Masahiko; Shimamura, Takeshi; Kobayashi, Noritoshi; Kirikoshi, Hiroyuki; Kubota, Kensuke; Saito, Satoru; Nakajima, Atsushi

    2011-01-01

    The factors involved in the progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) are not fully understood and thus it is urgently needed to elucidate these factors. Steatosis is not causal in the development of NASH, but rather it sensitizes the liver to the damaging effects of second hits such that stressors innocuous to a healthy liver lead to the development of NASH in the steatotic liver. In the previous study, most of the hepatic lipid metabolite profiles were similar in the NAFL and NASH groups. However, very-low-density lipoprotein (VLDL) synthesis, especially hepatic microsomal triglyceride transfer protein (MTP) mRNA expression, was impaired in the NASH group. Moreover, NASH showed significantly higher incidence of minor alley appearance compared with NAFL, indicating the possibility of association between NASH pathogenesis and decreased congenital MTP activity. MTP is one of the enzymes that transfer triglycerides to nascent apolipoprotein B, producing VLDL and removing lipid from the hepatocyte. A growing body of literature suggests that the measurement of hepatic MTP expression may be helpful for diagnosis; and moreover, hepatic MTP activator may be a possible therapeutic agent for the treatment of NASH.

  12. Synthesis of post-translationally modified proteins.

    PubMed

    van Kasteren, Sander

    2012-10-01

    Post-translational modifications of proteins can have dramatic effect on the function of proteins. Significant research effort has gone into understanding the effect of particular modifications on protein parameters. In the present paper, I review some of the recently developed tools for the synthesis of proteins modified with single post-translational modifications at specific sites in the protein, such as amber codon suppression technologies, tag and modify, and native chemical ligation.

  13. Chemical protein synthesis (CPS) meeting 2013.

    PubMed

    Metanis, Norman

    2013-07-22

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

  14. Hepatic steatosis in hepatitis C is a storage disease due to HCV interaction with microsomal triglyceride transfer protein (MTP).

    PubMed

    Mirandola, Silvia; Bowman, David; Hussain, Mahmood M; Alberti, Alfredo

    2010-02-23

    Liver steatosis is a frequent histological feature in patients chronically infected with hepatitis C virus (HCV). The relationship between HCV and hepatic steatosis seems to be the result of both epigenetic and genetic factors. In vivo and in vitro studies have shown that HCV can alter intrahepatic lipid metabolism by affecting lipid synthesis, oxidative stress, lipid peroxidation, insulin resistance and the assembly and secretion of VLDL. Many studies suggest that HCV-related steatosis might be the result of a direct interaction between the virus and MTP. It has been demonstrated that MTP is critical for the secretion of HCV particles and that inhibition of its lipid transfer activity reduces HCV production. However, higher degrees of hepatic steatosis were found in chronic hepatitis C patients carrying the T allele of MTP -493G/T polymorphism that seems to be associated with increased MTP transcription. We propose here that liver steatosis in hepatitis C could be a storage disease induced by the effects of the virus and of its proteins on the intracellular lipid machinery and on MTP. Available data support the hypothesis that HCV may modulate MTP expression and activity through a number of mechanisms such as inhibition of its activity and transcriptional control. Initial up regulation could favour propagation of HCV while down regulation in chronic phase could cause impairment of triglyceride secretion and excessive lipid accumulation, with abnormal lipid droplets facilitating the "storage" of virus particles for persistent infection.

  15. Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

    PubMed Central

    1977-01-01

    The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal

  16. Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization.

    PubMed

    Bozcaarmutlu, Azra; Arinç, Emel

    2008-01-01

    In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

  17. Cloning of cDNAs encoding a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal dipeptidase.

    PubMed Central

    Igarashi, P; Karniski, L P

    1991-01-01

    Distinct anion transport processes have been identified in the mammalian renal proximal tubule, but none of the responsible proteins or genes have been isolated. A 43 kDa rabbit microvillus membrane protein that is immunologically related to the erythroid anion exchanger (band 3) was a candidate for a renal anion transporter. To examine the structural relationship with band 3, we cloned cDNAs encoding the 43 kDa protein. The 43 kDa band-3-like protein was purified, and a novel sequence of 24 amino acids was obtained from the N-terminus. Degenerate oligonucleotides were synthesized based on this sequence, and the polymerase chain reaction with single-sided specificity was used to amplify and clone a 1330 bp cDNA from rabbit renal cortex. Additional overlapping 272 bp and 1123 bp cDNAs were obtained by synthesizing and screening a rabbit renal cortical cDNA library. The composite sequence was 1483 bp, terminated with (A)16, and was similar in size to the principal transcript expressed in rabbit renal cortex. The single long open reading frame was predicted to encode a protein composed of 410 amino acids with a molecular mass of 45,193 Da; 15 amino acids predicted to reside at the N-terminus were absent in the mature protein and may constitute a signal peptide. There was only limited sequence similarity with human erythroid band 3. Rather, the sequence was highly similar to microsomal dipeptidase, including the presence of a signal peptide and a consensus sequence for covalent linkage to glycosylphosphatidylinositol. In summary, the 43 kDa protein from rabbit renal cortex that is recognized by a monospecific antibody to erythroid band 3 is most likely a microvillus membrane dipeptidase. Images Fig. 1. Fig. 5. Fig. 6. PMID:1741759

  18. Short-term changes in diet composition do not affect in vivo hepatic protein synthesis in rats.

    PubMed

    Estrada, Andrea Lee; Hudson, William Max; Kim, Paul Y; Stewart, Claire Marie; Peelor, Frederick F; Wei, Yuren; Wang, Dong; Hamilton, Karyn L; Miller, Benjamin F; Pagliassotti, Michael J

    2017-08-29

    Protein synthesis is critical to protein homeostasis (proteostasis) and modifications in protein synthesis influence lifespan and the development of co-morbidities associated with obesity. In the present study, we examined the acute response of liver protein synthesis to either high fat or high sucrose diets in order to elucidate nutrient-mediated regulation of hepatic protein synthesis in the absence of body fat accumulation. Total and endoplasmic reticulum-associated protein synthesis were assessed by use of the stable isotope, deuterium oxide ((2)H2O), in rats provided a control diet or diets enriched in polyunsaturated fat, saturated fat, or sucrose for 2, 4, or 7 days. The three experimental diets increased hepatic triglycerides 46-91% on day 7 and fasting insulin levels 83-117% on day 7, but did not result in differences in body weight when compared to control (n=6/diet/time). The fraction of newly synthesized proteins in total liver lysates and microsomes was not significantly different among dietary groups (n=3/diet/time). To determine whether the experimental diets provoked a transcriptional response to enhance the capacity for protein synthesis we also measured a panel of genes linked to amino acid transport, synthesis and processing. There were no significant differences in any of the genes measured among groups. Therefore, dietary treatments that have been linked to impaired proteostasis, and that promote hepatic steatosis and insulin resistance, did not result in significant changes in total or ER-associated protein synthesis in the liver over a 7-day period. Copyright © 2017, American Journal of Physiology-Endocrinology and Metabolism.

  19. 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.

  20. 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.

  1. Abetalipoproteinemia caused by maternal isodisomy of chromosome 4q containing an intron 9 splice acceptor mutation in the microsomal triglyceride transfer protein gene.

    PubMed

    Yang, X P; Inazu, A; Yagi, K; Kajinami, K; Koizumi, J; Mabuchi, H

    1999-08-01

    Uniparental disomy (UPD), a rare inheritance of 2 copies of a single chromosome homolog or a region of a chromosome from one parent, can result in various autosomal recessive diseases. Abetalipoproteinemia (ABL) is a rare autosomal recessive deficiency of apoB-containing lipoproteins caused by a microsomal triglyceride transfer protein (MTP) deficiency. In this study, we describe a patient with ABL inherited as a homozygous intron 9 splice acceptor G(-1)-to-A mutation of the transfer protein gene. This mutation alters the splicing of the mRNA, resulting in a 36 amino acids, in-frame deletion of sequence encoded by exon 10. We analyzed chromosome 4, including MTP gene (4q22-24), using short tandem repeat markers. The proband has only his mother's genes in chromosome 4q spanning a 150-centimorgan region; ie, segmental maternal isodisomy 4q21-35, probably due to mitotic recombination. Nonpaternity between the proband and his father was excluded using 6 polymorphic markers from different chromosomes (paternity probability, 0.999). Maternal isodisomy (maternal UPD 4q) was the basis for homozygosity of the MTP gene mutation in this patient.

  2. RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

    PubMed Central

    Tsai, Tsung-Yuan; Wang, Wei-Ting; Li, Hao-Kang; Chen, Wei-Ju; Tsai, Yu-Hong; Chao, Chi-Hong; Wu Lee, Yan-Hwa

    2017-01-01

    Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis. PMID:28128295

  3. Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of the N-Terminal β-Barrel in Microsomal Triglyceride Transfer Protein Function.

    PubMed

    Walsh, Meghan T; Iqbal, Jahangir; Josekutty, Joby; Soh, James; Di Leo, Enza; Özaydin, Eda; Gündüz, Mehmet; Tarugi, Patrizia; Hussain, M Mahmood

    2015-10-01

    The use of microsomal triglyceride transfer protein (MTP) inhibitors is limited to severe hyperlipidemias because of associated hepatosteatosis and gastrointestinal adverse effects. Comprehensive knowledge about the structure-function of MTP might help design new molecules that avoid steatosis. Characterization of mutations in MTP causing abetalipoproteinemia has revealed that the central α-helical and C-terminal β-sheet domains are important for protein disulfide isomerase binding and lipid transfer activity. Our aim was to identify and characterize mutations in the N-terminal domain to understand its function. We identified a novel missense mutation (D169V) in a 4-month-old Turkish male child with severe signs of abetalipoproteinemia. To study the effect of this mutation on MTP function, we created mutants via site-directed mutagenesis. Although D169V was expressed in the endoplasmic reticulum and interacted with apolipoprotein B (apoB) 17, it was unable to bind protein disulfide isomerase, transfer lipids, and support apoB secretion. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished protein disulfide isomerase heterodimerization, lipid transfer, and apoB secretion, without affecting apoB17 binding. Furthermore, mutants with preserved charges (D169E, K187R, and K189R) rescued these activities. D169V is detrimental because it disrupts an internal salt bridge leading to loss of protein disulfide isomerase binding and lipid transfer activities; however, it does not affect apoB binding. Thus, the N-terminal domain of MTP is also important for its lipid transfer activity. © 2015 American Heart Association, Inc.

  4. 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.

  5. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    PubMed

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

    2012-07-01

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

  6. 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

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

    PubMed Central

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

    2016-01-01

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

  8. Protein synthesis during encystment of Azotobacter vinelandii

    SciTech Connect

    Su, C.J.; da Cunha, A.; Wernette, C.M.; Reusch, R.N.; Sadoff, H.L.

    1987-10-01

    Proteins synthesized during the encystment of Azotobacter vinelandii were radiolabeled with (/sup 35/S) methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pulse labeling was used to demonstrate that early encystment-specific proteins were beginning to be synthesized by 2 h and reach peak levels about 12 h after initiation of encystment. One such protein was identified as a ..beta..-ketoacyl acyl-carrier protein synthase. The concentration of early proteins began to decrease at 16 h, when intermediate proteins specific to the differentiation process began to be synthesized. The cessation of synthesis of intermediate proteins began at 20 h postinitiation, and the labeling pattern of proteins then remained constant throughout the remaining 4 days of encystment.

  9. Insights into the novel hydrolytic mechanism of a diethyl 2-phenyl-2-(2-arylacetoxy)methyl malonate ester-based microsomal triglyceride transfer protein (MTP) inhibitor.

    PubMed

    Ryder, Tim; Walker, Gregory S; Goosen, Theunis C; Ruggeri, Roger B; Conn, Edward L; Rocke, Benjamin N; Lapham, Kimberly; Steppan, Claire M; Hepworth, David; Kalgutkar, Amit S

    2012-10-15

    Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)methyl)-2-phenylmalonate (JTT-130) is an intestine-specific inhibitor of MTP and does not cause increases in transaminases in short-term clinical trials in patients with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite 1. In the course of discovery efforts around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of ¹⁸O incorporation in 1 following the incubation of JTT-130 in human liver microsomes in the presence of H₂¹⁸O suggested that hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups followed by decarboxylative fragmentation to 1 and the concomitant liberation of the potentially electrophilic acrylate species. Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4'-trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)ethyl)-2-phenylmalonate (3), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene group

  10. Lipid Absorption Defects in Intestine-specific Microsomal Triglyceride Transfer Protein and ATP-binding Cassette Transporter A1-deficient Mice*

    PubMed Central

    Iqbal, Jahangir; Parks, John S.; Hussain, M. Mahmood

    2013-01-01

    We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92–95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations. PMID:24019513

  11. Mechanism of action of hypoglycemic effects of an intestine-specific inhibitor of microsomal triglyceride transfer protein (MTP) in obese rats.

    PubMed

    Sakata, Shohei; Katsumi, Sohei; Mera, Yasuko; Kuroki, Yukiharu; Nashida, Reiko; Kakutani, Makoto; Ohta, Takeshi

    2015-01-01

    Diminished insulin sensitivity in the peripheral tissues and failure of pancreatic beta cells to secrete insulin are known major determinants of type 2 diabetes mellitus. JTT-130, an intestine-specific microsomal transfer protein inhibitor, has been shown to suppress high fat-induced obesity and ameliorate impaired glucose tolerance while enhancing glucagon-like peptide-1 (GLP-1) secretion. We investigated the effects of JTT-130 on glucose metabolism and elucidated the mechanism of action, direct effects on insulin sensitivity and glucose-stimulated insulin secretion in a high fat diet-induced obesity rat model. Male Sprague Dawley rats fed a high-fat diet were treated with a single administration of JTT-130. Glucose tolerance, hyperglycemic clamp and hyperinsulinemic-euglycemic testing were performed to assess effects on insulin sensitivity and glucose-stimulated insulin secretion, respectively. Plasma GLP-1 and tissue triglyceride content were also determined under the same conditions. A single administration of JTT-130 suppressed plasma glucose elevations after oral glucose loading and increased the disposition index while elevating GLP-1. JTT-130 also enhanced glucose-stimulated insulin secretion in hyperglycemic clamp tests, whereas increased insulin sensitivity was observed in hyperinsulinemic-euglycemic clamp tests. Single-dose administration of JTT-130 decreased lipid content in the liver and skeletal muscle. JTT-130 demonstrated acute and direct hypoglycemic effects by enhancing insulin secretion and/or insulin sensitivity.

  12. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.

    PubMed

    Iqbal, Jahangir; Parks, John S; Hussain, M Mahmood

    2013-10-18

    We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92-95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations.

  13. Subcellular fractionation of rough microsomes.

    PubMed

    Sabatini, David D

    2014-09-02

    When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction. © 2014 Cold Spring Harbor Laboratory Press.

  14. Cytochromes P450 (CYP) in tropical fishes: catalytic activities, expression of multiple CYP proteins and high levels of microsomal P450 in liver of fishes from Bermuda.

    PubMed

    Stegeman, J J; Woodin, B R; Singh, H; Oleksiak, M F; Celander, M

    1997-01-01

    Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with beta-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 beta- and 16 beta-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of

  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. 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.

  17. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    NASA Astrophysics Data System (ADS)

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.

    2000-12-01

    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  18. Cloning and characterization of microsomal triglyceride transfer protein gene and its potential connection with peroxisome proliferator-activated receptor (PPAR) in blunt snout bream (Megalobrama amblycephala).

    PubMed

    Li, Jun-Yi; Zhang, Ding-Dong; Jiang, Guang-Zhen; Li, Xiang-Fei; Zhang, Chun-Nuan; Zhou, Man; Liu, Wen-Bin; Xu, Wei-Na

    2015-11-01

    Microsomal triglyceride transfer protein (MTTP), a major intracellular protein capable of transferring neutral lipids, plays a pivotal role in the assembly and secretion of apolipoprotein B-containing lipoproteins. In this study, MTTP cDNA was firstly cloned from the liver of blunt snout bream (Megalobrama amblycephala), the full-length cDNA covered 3457-bp with an open reading frame of 2661-bp, which encodes 886 amino acids, including a putative signal peptide of 24 amino acids long. After the feeding trial, a graded tissue-specific expression pattern of MTTP was observed and high expression abundance in the liver and intestine indicated its major function in lipid transport in this fish species. In addition, expression of genes encoding MTTP as well as peroxisome proliferator-activated receptor (PPAR), which are transcription factors and serve as key regulators in lipid homoeostasis, was all affected by dietary lipid and choline supplementations. Elevated dietary lipid levels significantly increased the liver, intestinal and muscle MTTP mRNA abundance. Additionally, the down-regulation of MTTP expression in the liver and muscle was observed when fish were fed with inadequate choline supplementation in high-fat diet, yet up-regulated as supplementing extra choline in diet. Expressions of PPARα and PPARβ in the liver and muscle showed similar trend of MTTP expression. The results suggested the potential connection of MTTP and PPAR in response to different dietary nutritional factors. Furthermore, extra choline supplementations could promote lipid transfer and enhance fatty acid oxidation, which indicated a molecular mechanism of choline on diminishing fat accumulation in blunt snout bream. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of N-Terminal β-Barrel in Microsomal Triglyceride Transfer Protein Function

    PubMed Central

    Walsh, Meghan T.; Iqbal, Jahangir; Josekutty, Joby; Soh, James; Di Leo, Enza; Özaydin, Eda; Gündüz, Mehmet; Tarugi, Patrizia; Hussain, M. Mahmood

    2015-01-01

    Background The use of microsomal triglyceride transfer protein (MTP) inhibitors is limited to severe hyperlipidemias due to associated hepatosteatosis and gastrointestinal adverse effects. Comprehensive knowledge about the structure-function of MTP might help design new molecules that avoid steatosis. Characterization of mutations in MTP causing abetalipoproteinemia have revealed that the central α-helical and C-terminal β-sheet domains are important for protein disulfide isomerase (PDI) binding and lipid transfer activity. Our aim was to identify and characterize mutations in the N-terminal domain to understand its function. Methods and Results We identified a novel missense mutation (D169V) in a 4-month old Turkish male with severe signs of ABL. To study the effect of this mutation on MTP function, we created mutants via site-directed mutagenesis. Although D169V was expressed in the endoplasmic reticulum and interacted with apoB17, it was unable to bind PDI, transfer lipids, and support apoB secretion. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished PDI heterodimerization, lipid transfer, and apoB secretion, without affecting apoB17 binding. Further, mutants with preserved charges (D169E, K187R, K189R) rescued these activities. Conclusions D169V is detrimental because it disrupts an internal salt bridge leading to loss of PDI binding and lipid transfer activities; however, it does not affect apoB-binding. Thus, the N-terminal domain of MTP is also important for its lipid transfer activity. PMID:26224785

  20. Kinetics and mechanism of electron transfer from dithionite to microsomal cytochrome b5 and to forms of the protein associated with charged and neutral vesicles.

    PubMed Central

    Davies, D M; Lawther, J M

    1989-01-01

    The kinetics of the dithionite reduction of calf liver microsomal cytochrome b5, both free in solution and bound to dimyristoyl phosphatidylcholine vesicles, are consistent with electron transfer between SO2- and the exposed haem edge of the protein. The vesicle membrane does not hinder the approach of SO2- to the site of electron transfer on the protein. In 0.01 M-Tris/HCl buffer, pH 8.1, ket (25 degrees C), delta H et and delta S et are estimated to be 1.44 x 10(6) M-1.s-1, 7.8 kJ.mol-1 and -92.3 J.K-1.mol-1 respectively. The cytochrome exhibits an acid dissociation, pKa 9.3 +/- 0.3, and the rate of electron transfer from dithionite to the high-pH form is about one-third of that to the neutral-pH form. The effect of ionic strength on the kinetics is consistent with a reaction between like-charged species and is discussed in terms of a number of theoretical models. In systems comprising cytochrome b5 and negatively charged vesicles, the effect of increasing the charge density of mixed dimyristoyl phosphatidylcholine/dicetyl phosphate vesicles and of increasing the concentration of dicetyl phosphate vesicles is to lower the rate of electron transfer from dithionite to the haem moiety of the cytochrome. With vesicles of high charge density, however, the kinetics are complicated by vesicle-induced conformation changes of the cytochrome. PMID:2705988

  1. SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION

    PubMed Central

    Paul, M.; Goldsmith, M. R.; Hunsley, J. R.; Kafatos, F. C.

    1972-01-01

    Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems. PMID:4656706

  2. Affinity labeling of protein synthesis factors

    SciTech Connect

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

    1986-05-01

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

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

    PubMed

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

    2014-05-01

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

  4. HT-2 toxin 4-glucuronide as new T-2 toxin metabolite: enzymatic synthesis, analysis, and species specific formation of T-2 and HT-2 toxin glucuronides by rat, mouse, pig, and human liver microsomes.

    PubMed

    Welsch, Tanja; Humpf, Hans-Ulrich

    2012-10-10

    Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared.

  5. Effect of ethionine on hepatic mitochondrial and microsomal calcium uptake

    SciTech Connect

    Agarwal, A.K.; Zinermon, W.D.; Latoni, L.

    1988-02-01

    Ethionine, an ethyl analog of methionine, produces a variety of physiological and pathological effects in animals. These range from acute effects in the liver, kidney, pancreas, and other organs to liver carcinogenesis. Female rats when injected with ethionine exhibit a rapid decrease in hepatic adenosine triphosphate levels followed by a marked inhibition of RNA and protein synthesis and accumulation of triglycerides. Since calcium transport in mitochondria and microsomes is ATP dependent, it becomes interesting to find out if ethionine administration has any effect on subcellular calcium transport. Calcium has recently gained an increased controversy regarding its role in chemical induced lethal cell damage. Certain groups believe that influx of extracellular calcium across the damaged plasma membrane might actually mediate the irreversible damage to the cell, whereas according to other, entry of calcium into the cell is secondary to the damage. The present study was carried out to investigate the calcium (/sup 45/Ca) transport in mitochondria and microsomes following ethionine administration. The effect of carbon tetrachloride on calcium uptake in ethionine treated rats was also studied.

  6. Protein ingestion increases myofibrillar protein synthesis after concurrent exercise.

    PubMed

    Camera, Donny M; West, Daniel W D; Phillips, Stuart M; Rerecich, Tracy; Stellingwerff, Trent; Hawley, John A; Coffey, Vernon G

    2015-01-01

    We determined the effect of protein supplementation on anabolic signaling and rates of myofibrillar and mitochondrial protein synthesis after a single bout of concurrent training. Using a randomized crossover design, eight healthy males were assigned to experimental trials consisting of resistance exercise (8 × 5 leg extension, 80% 1RM) followed by cycling (30 min at approximately 70% V˙O2peak) with either postexercise protein (PRO, 25-g whey protein) or placebo (PLA) ingestion. Muscle biopsies were obtained at rest and at 1 and 4 h after exercise. Akt and mTOR phosphorylation increased 1 h after exercise with PRO (175%-400%, P < 0.01) and was different from PLA (150%-300%, P < 0.001). Muscle RING finger 1 and atrogin-1 messenger RNA (mRNA) were elevated after exercise but were higher with PLA compared with those in PRO at 1 h (50%-315%, P < 0.05), whereas peroxisome proliferator-activated receptor gamma coactivator 1-alpha mRNA increased 4 h after exercise (620%-730%, P < 0.001), with no difference between treatments. Postexercise rates of myofibrillar protein synthesis increased above rest in both trials (75%-145%, P < 0.05) but were higher with PRO (67%, P < 0.05), whereas mitochondrial protein synthesis did not change from baseline. Our results show that a concurrent training session promotes anabolic adaptive responses and increases metabolic/oxidative mRNA expression in the skeletal muscle. PRO ingestion after combined resistance and endurance exercise enhances myofibrillar protein synthesis and attenuates markers of muscle catabolism and thus is likely an important nutritional strategy to enhance adaptation responses with concurrent training.

  7. Antibiotics that target protein synthesis.

    PubMed

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

    2011-01-01

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

  8. Semi-synthesis of thioamide containing proteins.

    PubMed

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

    2015-05-14

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

  9. Circadian regulation of intestinal lipid absorption by apolipoprotein AIV involves forkhead transcription factors A2 and O1 and microsomal triglyceride transfer protein.

    PubMed

    Pan, Xiaoyue; Munshi, Mohamed Khalid; Iqbal, Jahangir; Queiroz, Joyce; Sirwi, Alaa Ahmed; Shah, Shrenik; Younus, Abdullah; Hussain, M Mahmood

    2013-07-12

    We have shown previously that Clock, microsomal triglyceride transfer protein (MTP), and nocturnin are involved in the circadian regulation of intestinal lipid absorption. Here, we clarified the role of apolipoprotein AIV (apoAIV) in the diurnal regulation of plasma lipids and intestinal lipid absorption in mice. Plasma triglyceride in apoAIV(-/-) mice showed diurnal variations similar to apoAIV(+/+) mice; however, the increases in plasma triglyceride at night were significantly lower in these mice. ApoAIV(-/-) mice absorbed fewer lipids at night and showed blunted response to daytime feeding. To explain reasons for these lower responses, we measured MTP expression; intestinal MTP was low at night, and its induction after food entrainment was less in apoAIV(-/-) mice. Conversely, apoAIV overexpression increased MTP mRNA in hepatoma cells, indicating transcriptional regulation. Mechanistic studies revealed that sequences between -204/-775 bp in the MTP promoter respond to apoAIV and that apoAIV enhances expression of FoxA2 and FoxO1 transcription factors and their binding to the identified cis elements in the MTP promoter at night. Knockdown of FoxA2 and FoxO1 abolished apoAIV-mediated MTP induction. Similarly, knockdown of apoAIV in differentiated Caco-2 cells reduced MTP, FoxA2, and FoxO1 mRNA levels, cellular MTP activity, and media apoB. Moreover, FoxA2 and FoxO1 expression showed diurnal variations, and their expression was significantly lower in apoAIV(-/-) mice. These data indicate that apoAIV modulates diurnal changes in lipid absorption by regulating forkhead transcription factors and MTP and that inhibition of apoAIV expression might reduce plasma lipids.

  10. Mechanism and regulation of eukaryotic protein synthesis.

    PubMed Central

    Merrick, W C

    1992-01-01

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

  11. Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: monitoring the early steps in protein unfolding.

    PubMed

    Arnesano, F; Banci, L; Bertini, I; Koulougliotis, D

    1998-12-01

    One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the oxidized state of the A-form of rat microsomal cytochrome b5 (cyt b5). The protein rapidly starts losing the heme at denaturant concentrations larger than approximately 2.0 M and a largely unfolded protein is eventually obtained. An estimate of the unfolding kinetics is obtained and, by use of a two-state model (folded left and right arrow unfolded), a value for DeltaG degrees. Below this concentration, small (protein core maintains its overall structure. The analysis of the two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY) maps has allowed the determination of the solution structure of the protein in the presence of 2 M GdmCl. By use of 1199 meaningful NOESY constraints (obtained from the assignment of 75% of the total protons) and 166 pseudocontact shifts, a family of 40 structures has been obtained through the program PSEUDYANA. The family was further refined through restrained energy minimization and the final root mean square deviation (RMSD) values with respect to the average structure are 0.67 +/- 0.10 A and 1.14 +/- 0.11 A for the backbone and heavy atoms, respectively. The quality of the present structure is equivalent to that of the one obtained recently for the native form [Arnesano et al. (1998) Biochemistry 37, 173-184], thus allowing a meaningful comparison between the two structures. Upon addition of 2 M GdmCl, significant local structural differences are induced to the protein backbone segments comprising residues 33-38 (helix alpha2) and 62-64 (end of helix alpha4-beginning of helix alpha5) while the overall folding scheme of the protein is still maintained. These protein regions form part of the "pocket

  12. Elongation factors in protein synthesis.

    PubMed

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

    1993-01-01

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

  13. Effects of salt on the pattern of protein synthesis in barley roots

    SciTech Connect

    Hurkman, W.J.; Tanaka, C.K.

    1987-03-01

    The effect of salt stress on the incorporation of (/sup 3//sub 5/S)methionine into protein was examined in roots of barley (Hordeum vulgare L.cv California Mariout 72). Plants were grown in nutrient solution with or without 200 millimolar NaCl. Roots of intact plants were labeled in vivo and proteins were extracted and analyzed by fluorography of two-dimensional gels. Although the protein patterns for control and salt-stressed plants were qualitatively similar, the net synthesis of a number of proteins was quantitatively changed. The most striking change was a significant increase of label in two protein pairs that had pls of approximately 6.3 and 6.5. Each pair consisted of proteins of approximately 26 and 27 kilodaltons (kD). In roots of control plants, the 27-kD proteins were more heavily labeled in the microsomal fraction relative to the 26-kD proteins, whereas the 26-kD proteins were enriched in the post 178,000g supernatant fraction; in roots of salt treated plants, the 26- and 27-kD proteins were more intensely labeled in both fractions. Labeling of the 26- and 27-kD proteins returned to control levels when salt-stressed plants were transferred to nutrient solution without NaCl. No cross-reaction was detected between the antibody to the 26-kD protein from salt-adapted tobacco cells and the 26- and 27-kD proteins of barley.

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

    PubMed

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

    2014-11-01

    In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

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

  16. 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.

  17. Failure of leucine to stimulate protein synthesis in vivo.

    PubMed Central

    McNurlan, M A; Fern, E B; Garlick, P J

    1982-01-01

    The effect of 100 mumol of leucine on protein synthesis in several tissues was assessed in the intact rat. Leucine had no immediate effect on protein synthesis in gastrocnemius muscle, heart, jejunal serosa, jejunal mucosa or liver in rats which were fed, starved for 2 days or deprived of dietary protein for 9 days. Leucine treatment for 1 h also failed to stimulate protein synthesis in tissues of 2-day-starved animals. PMID:7126170

  18. Protein synthesis by ribosomes with tethered subunits.

    PubMed

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

    2015-08-06

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

  19. Glucocorticoid effects on hippocampal protein synthesis

    SciTech Connect

    Schlatter, L.K.

    1988-01-01

    Following subcutaneous injection of rats with 5 mg corticosterone, hippocampal slices in vitro show increased ({sup 35}S)-methionine labeling of a cytosolic protein with an apparent molecular weight (M{sub r}) of 35,000 and an isoelectric point (IEP) of 6.6. This labeling is temporally consistent with a transcriptional event, and is steroid- and tissue-specific. The pear serum concentration of steroid occurs one hour or less following the injection. Maximal labeling of this protein is reached whenever serum corticosterone values are approximately 100 ng/ml. When endogenous corticosterone levels are elevated to 100 ng/ml through stressors or exogenous ACTH injections the same maximal increase in synthesis of the 35,000 M{sub r} protein is observed. Adrenalectomy prevents the observed response from occurring following stressor application or ACTH injections. Comparison of the increases observed after administration of the type 2 receptor agonist RU 28362 and aldosterone, which has a higher affinity for the type 1 receptor, shows a 50-fold greater sensitivity of the response to the type 2 receptor agonist. Synthesis of this protein following serum increases of steroid possibly correlates to the theorized function of the type 2 receptor feedback regulation. The similar protein in the liver has an IEP of 6.8 and a slightly higher M{sub r}. A second hippocampal protein with an M{sub r} of 46,000 and an IEP of 6.2 is also increased in labeling. Two additional liver proteins, one of Mr 53,000 (IEP of 6.2) and the other with an M{sub r} of 45,000 (IEP of 8.7-7.8) are increased in the liver following glucocorticoid administration.

  20. Inhibitory effect of some imidazole antifungal compounds on the synthesis of 16-ene-C19-steroid catalyzed by pig testicular microsomes.

    PubMed

    Nakajin, S; Takahashi, K; Shinoda, M

    1991-01-01

    The activity of the enzyme (16-ene-C19-steroid synthesizing enzyme) responsible for the conversion of C21-steroids to 16-ene-C19-steroids, which was localized on pig testicular microsomes, was inhibited by some typical imidazole antifungal compounds such as clotrimazole, econazole, miconazole and ketoconazole which are known to be universal inhibitors of cytochrome P-450-dependent enzymes. The 50% inhibitory concentrations of clotrimazole, econazole and miconazole were 0.29, 0.36 and 1.25 microM, respectively for 16-ene-C19-steroid synthesizing enzyme activity. Clotrimazole was the most powerful inhibitor of all the compounds examined, which shows the competitive inhibition for 16-ene-C19-steroid synthesizing enzyme activity. The Ki-value was 0.26 microM for its activity. The degree of the inhibition by these imidazole compounds was very similar to the inhibition of 17 alpha-hydroxylase and C17,20-lyase activities on pig testicular microsomes.

  1. 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.

  2. 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.

  3. The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology.

    PubMed Central

    Friedberg, T; Holler, R; Löllmann, B; Arand, M; Oesch, F

    1996-01-01

    Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather

  4. Engineering the prion protein using chemical synthesis.

    PubMed

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

    2001-11-01

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

  5. Microsomal triglyceride transfer protein -164 T > C gene polymorphism and risk of cardiovascular disease: results from the EPIC-Potsdam case-cohort study.

    PubMed

    di Giuseppe, Romina; Pechlivanis, Sonali; Fisher, Eva; Arregui, Maria; Weikert, Beate; Knüppel, Sven; Buijsse, Brian; Fritsche, Andreas; Willich, Stefan N; Joost, Hans-Georg; Boeing, Heiner; Moebus, Susanne; Weikert, Cornelia

    2013-01-29

    The microsomal triglyceride transfer protein (MTTP) is encoded by the MTTP gene that is regulated by cholesterol in humans. Previous studies investigating the effect of MTTP on ischemic heart disease have produced inconsistent results. Therefore, we have tested the hypothesis that the rare allele of the -164T > C polymorphism in MTTP alters the risk of cardiovascular disease (CVD), depending on the cholesterol levels. The -164T > C polymorphism was genotyped in a case-cohort study (193 incident myocardial infarction (MI) and 131 incident ischemic stroke (IS) cases and 1 978 non-cases) nested within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study, comprising 27 548 middle-aged subjects. The Heinz Nixdorf Recall study (30 CVD cases and 1 188 controls) was used to replicate our findings. Genotype frequencies were not different between CVD and CVD free subjects (P = 0.79). We observed an interaction between the -164T > C polymorphism and total cholesterol levels in relation to future CVD. Corresponding stratified analyses showed a significant increased risk of CVD (HR(additve) = 1.38, 95% CI: 1.07 to 1.78) for individuals with cholesterol levels <200 mg/dL in the EPIC-Potsdam study. HR(additive) was 1.06, 95% CI: 0.33 to 3.40 for individuals in the Heinz Nixdorf Recall study. A borderline significant decrease in CVD risk was observed in subjects with cholesterol levels ≥ 200 mg/dL (HR(additve) = 0.77, 95% CI: 0.58 to 1.03) in the EPIC-Potsdam study. A similar trend was observed in the independent cohort (HR(additve) = 0.60, 95% CI: 0.29 to 1.25). Our study suggests an interaction between MTTP -164T > C functional polymorphism with total cholesterol levels. Thereby risk allele carriers with low cholesterol levels may be predisposed to an increased risk of developing CVD, which seems to be abolished among risk allele carriers with high cholesterol levels.

  6. High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

    PubMed Central

    Quast, Robert B.; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041

  7. 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

  8. Protein synthesis in synaptosomes: a proteomics analysis.

    PubMed

    Jiménez, C R; Eyman, M; Lavina, Z Scotto; Gioio, A; Li, K W; van der Schors, R C; Geraerts, W P M; Giuditta, A; Kaplan, B B; van Minnen, J

    2002-05-01

    A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

  9. Radiation inactivation analysis of rat liver microsomal glucose 6-phosphatase

    SciTech Connect

    Ness, G.C.; Sample, C.E.; McCreery, M.J.; Sukalski, K.A.; Nordlie, R.C.

    1986-05-01

    Attempts to obtain the molecular weight of microsomal glucose-6-phosphatase based on solubilization and purification have yielded widely divergent results. Since radiation inactivation analysis can be used to obtain molecular weights of proteins within the native membrane environments, this technique was applied. Identical target sizes of about 70 kd for both glucose 6-phosphate phosphohydrolase and carbamyl phosphate:glucose phosphotransferase were observed. This value was unaffected by adding deoxycholate, which disrupts the microsomal membranes, to the microsomal suspensions prior to irradiation. The data suggest that the glucose 6-phosphate transport function and the glucose 6-phosphate phosphohydrolase activity of microsomal glucose 6-phosphatase either residue on a single polypeptide or on two covalently linked polypeptides.

  10. Metabolic activation of 2-methylfuran by rat microsomal systems

    SciTech Connect

    Ravindranath, V.; Boyd, M.R.

    1985-05-01

    2-Methylfuran (2-MF), a constituent of cigarette smoke and coffee, causes necrosis of liver, lungs, and kidneys in rodents. 2-MF is metabolically activated by mixed-function oxidases to acetylacrolein, a reactive metabolite that binds covalently to microsomal protein. The hepatic microsomal metabolism of 2-MF to reactive metabolite required the presence of NADPH and oxygen and was dependent on incubation time and substrate concentration. The microsomal metabolism of 2-MF was inducible by pretreatment of rats with phenobarbital and was inhibited by piperonyl butoxide and N-octyl imidazole, which indicates that the metabolism of 2-MF may be mediated by cytochrome P-450. Acetylacrolein was a potent inhibitor of mixed-function oxidase and completely inhibited the microsomal metabolism of 2-MF, indicating that 2-MF is a suicide substrate for the enzyme. The sulfhydryl nucleophile cysteine was a better trapping agent of the reactive metabolite of 2-MF than N-acetylcysteine or glutathione. Lysine decreased the covalent binding of 2-MF metabolites, presumably by reacting with the aldehyde group of acetylacrolein. In addition, in the presence of NADPH, 2-MF was bioactivated by both pulmonary and renal cortical microsomes to reactive metabolites that were covalently bound to microsomal proteins.

  11. Cyclosporin metabolism by human gastrointestinal mucosal microsomes.

    PubMed Central

    Webber, I R; Peters, W H; Back, D J

    1992-01-01

    The in vitro metabolism of the immunosuppressant cyclosporin (CsA) by human gastrointestinal mucosal microsomes has been studied. Macroscopically normal intestinal (n = 4) and liver (n = 2) tissue was obtained from kidney transplant donors, and microsomes prepared. Intestinal metabolism was most extensive with duodenal protein (15% conversion to metabolites M1/M17 after 2 h incubation at 37 degrees C; metabolite measurement by h.p.l.c). Western blotting confirmed the presence of P-4503A (enzyme subfamily responsible for CsA metabolism) in duodenum and ileum tissue, but not in colon tissue. The results of this study indicate that the gut wall may play a role in the first-pass metabolism of CsA, and could therefore be a contributory factor to the highly variable oral bioavailability of CsA. PMID:1389941

  12. 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…

  13. Protein synthesis during sleep consolidates cortical plasticity in vivo

    PubMed Central

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

    2012-01-01

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

  14. 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.

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

    PubMed Central

    Kourtis, Nikos; Tavernarakis, Nektarios

    2017-01-01

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

  16. 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…

  17. In vitro metabolism of BIIB021, an inhibitor of heat shock protein 90, in liver microsomes and hepatocytes of rats, dogs, and humans and recombinant human cytochrome P450 isoforms.

    PubMed

    Xu, Lin; Woodward, Caroline; Khan, Samina; Prakash, Chandra

    2012-04-01

    Inhibition of heat shock protein 90 (HSP90) results in the degradation of oncoproteins that drive malignant progression and induce cell death, thus making HSP90 a potential target of cancer therapy. 6-Chloro-9-(4-methoxy-3, 5-dimethyl-pyridin-2-ylmethyl)-9H-purin-2-ylamine (BIIB021), a synthetic HSP90 inhibitor, exhibited promising antitumor activity in preclinical models. It is currently in phase II clinical trials for the oral treatment of breast cancer. The objective of this study was to obtain both quantitative and qualitative metabolic profiles of [(14)C]BIIB021 in rat, dog, and human liver microsomes and hepatocytes to provide support for in vivo safety and clinical studies. The metabolites of [(14)C]BIIB021 were identified using liquid chromatography-tandem mass spectrometry coupled with radiometric detection. BIIB021 was extensively metabolized in both liver microsomes and hepatocytes. The major oxidative metabolic pathways identified for all species were due to hydroxylation (M7) and O-demethylation (M2) of the methoxy-dimethylpyridine moiety. The majority of M7 in dog hepatocytes was further conjugated to form the glucuronide (M4). Oxidative dechlorination (M6), monooxygenation (M10), and oxidative N-dealkylation of the methoxy-dimethylpyridine moiety (M11 and M12) were observed as the minor metabolic pathways in hepatocytes of all three species. A glutathione conjugate (M18) was also identified in all species. Its formation was catalyzed, in part, by soluble glutathione transferase via direct displacement of the chlorine on the amino-chloropurine moiety. Subsequent minor secondary metabolites M13, M14, M15, and M17 were observed in human, dog, and rat hepatocytes. Results from incubations of BIIB021 with human recombinant cytochrome P450 (P450) isoforms and a P450 antibody inhibition study in human liver microsomes suggested that the formation of M7 is mainly catalyzed by CYP2C19 and CYP3A4, whereas the formation of minor metabolite M2 in human liver

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

    PubMed

    Garrett, R J; Jackson, M A

    1979-05-01

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

  19. Synthesis of Endosperm Proteins in Wheat Seed during Maturation 1

    PubMed Central

    Flint, Dennis; Ayers, George S.; Ries, Stanley K.

    1975-01-01

    The time of synthesis, the molecular weight, and the relative glutamine-glutamate and proline to leucine ratios of the endosperm proteins of wheat (Triticum aestivum L. cv. Logan) were determined using a sodium dodecyl sulfate-polyacrylamide gel technique. In general, synthesis of most proteins occurred through much of the maturation of the seed, but past 20 days the rate of synthesis of the high molecular weight proteins declined more rapidly than those of lower molecular weight. The synthesis of at least one protein occurred only late in seed maturation. Several of the high molecular weight proteins had glutamate-glutamine to leucine ratios higher than the remainder of the proteins. No evidence for proteins of a polyglutamine-glutamate and/or proline nature was found. PMID:16659308

  20. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    PubMed

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  1. [Influence of the fermentative cross-seams on the structure of the microsomal membrane].

    PubMed

    Zhikhareva, V O; Lapshin, E N; Devichenskiĭ, V M; Dobretsov, G E; Spirin, M M

    1983-01-01

    In rat liver microsomes freezing with subsequent thawing led to irreversible redistribution of protein-lipid packing. This redistribution was detected by a change in the efficiency of energy transfer between protein aromatic groups of membrane protein and lipid-soluble fluorescent probe pyrene. Transglutaminase pretreatment of microsomes prevented the irreversible redistribution. The enzyme is shown to bind no more than 15 per cent of the whole membrane protein. This smaller part of the microsomal protein is supposed to play the decisive role in the movements of its remaining part.

  2. Subcellular targets of cadmium nephrotoxicity: cadmium binding to renal membrane proteins in animals with or without protective metallothionein synthesis.

    PubMed Central

    Nordberg, G F; Jin, T; Nordberg, M

    1994-01-01

    Nephrotoxic effects of cadmium exposure are well established in humans and experimental animals. An early manifestation of such toxicity is calciuria a few hours after injection of CdMT in rats. Protection against calciuria and other adverse effects such as proteinuria (occurring later) is offered by pretreatment with Cd, which effectively induces metallothionein synthesis. In the present experiment, one group of animals was given pretreatment with CdCl2 to induce metallothionein synthesis. The comparison group was left without pretreatment. The distribution of Cd from a normally nephrotoxic dose of 109CdMT was studied by gel chromatography in subcellular fractions of kidney cortex in both groups. In the pretreated animals, 109Cd in the plasma membrane and microsome fractions of renal cortical cells was mainly bound to metallothionein and other low molecular weight proteins at 4 hr. In nonpretreated animals the major part of 109Cd was bound to high molecular weight proteins. These findings indicate that membrane proteins may be important targets for Cd when inducing nephrotoxicity and that sequestering of Cd by metallothionein (and other low molecular weight proteins) may be a mechanism of protection. PMID:7843096

  3. ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS

    EPA Science Inventory

    Introduction

    Membrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit ...

  4. ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS

    EPA Science Inventory

    Introduction

    Membrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit ...

  5. Radiation inactivation analysis of rat liver microsomal glucose-6-phosphatase

    SciTech Connect

    Ness, G.C.; Sukalski, K.A.; Sample, C.E.; Pendleton, L.C.; McCreery, M.J.; Nordlie, R.C.

    1989-05-05

    Radiation inactivation analysis was utilized to estimate the sizes of the units catalyzing the various activities of hepatic microsomal glucose-6-phosphatase. This technique revealed that the target molecular weights for mannose-6-P phosphohydrolase, glucose-6-P phosphohydrolase, and carbamyl-P:glucose phosphotransferase activities were all about Mr 75,000. These results are consistent with the widely held view that all of these activities are catalyzed by the same protein or proteins. Certain observations indicate that the molecular organization of microsomal glucose-6-phosphatase is better described by the conformational hypothesis which envisions the enzyme as a single covalent structure rather than by the substrate transport model which requires the participation of several physically separate polypeptides. These include the findings: (1) that the target sizes for glucose-6-P phosphohydrolase and carbamyl-P:glucose phosphotransferase activities were not larger than that for mannose-6-P phosphohydrolase in intact microsomes and (2) that the target size for glucose-6-P phosphohydrolase in disrupted microsomes was not less than that observed in intact microsomes. These findings are most consistent with a model for glucose-6-phosphatase of a single polypeptide or a disulfide-linked dimer which spans the endoplasmic reticulum with the various activities of this multifunctional enzyme residing in distinct protein domains.

  6. 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

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

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

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

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

    PubMed

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

    2014-11-01

    Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

  9. Endotoxemia reduces skeletal muscle protein synthesis in neonates.

    PubMed

    Orellana, Renan A; O'Connor, Pamela M J; Nguyen, Hanh V; Bush, Jill A; Suryawan, Agus; Thivierge, M Carole; Fiorotto, Marta L; Davis, Teresa A

    2002-11-01

    Protein synthesis in skeletal muscle is reduced by as much as 50% as early as 4 h after a septic challenge in adults. However, the effect of sepsis on muscle protein synthesis has not been determined in neonates, a highly anabolic population whose muscle protein synthesis rates are elevated and uniquely sensitive to insulin and amino acid stimulation. Neonatal piglets (n = 10/group) were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 microg. kg(-1). h(-1)]. Plasma amino acid and glucose concentrations were kept at the fed level by infusion of dextrose and a balanced amino acid mixture. Fractional protein synthesis rates were determined by use of a flooding dose of [(3)H]phenylalanine. LPS infusion produced a septic-like state, as indicated by an early and sustained elevation in body temperature, heart rate, and plasma tumor necrosis factor-alpha, interleukin-1, cortisol, and lactate concentrations. Plasma levels of insulin increased, whereas glucose and amino acids decreased, suggesting the absence of insulin resistance. LPS significantly reduced protein synthesis in longissimus dorsi muscle by only 11% and in gastrocnemius by only 15%, but it had no significant effect in masseter and cardiac muscles. LPS increased protein synthesis in the liver (22%), spleen (28%), kidney (53%), jejunum (19%), diaphragm (21%), lung (50%), and skin (13%), but not in the stomach, pancreas, or brain. These findings suggest that, when substrate supply is maintained, skeletal muscle protein synthesis in neonates compared with adults is relatively resistant to the catabolic effects of sepsis.

  10. Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes.

    PubMed

    Allister, Emma M; Borradaile, Nica M; Edwards, Jane Y; Huff, Murray W

    2005-06-01

    Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK(erk)) pathway. We hypothesized that naringenin acts via a similar mechanism. Inhibition of MAPK kinase (MEK) 1/2 in HepG2 cells significantly attenuated the naringenin- and insulin-induced reduction in MTP expression. Both naringenin and insulin increased ERK1/2 phosphorylation, which was completely inhibited by MEK1/2 inhibition and enhanced by inhibition of MAPK(p38), a negative regulator of MAPK(erk) activity. Inhibition of MEK1/2 significantly attenuated both the naringenin- and insulin-induced decrease in apoB100 secretion demonstrating a direct link between MAPK(erk) activation and apoB100 secretion. Furthermore, both compounds increased MAPK(p38) activation, and therefore inhibition of MAPK(p38) amplified thenaringenin- and insulin-induced decrease in apoB100 secretion. We conclude that MAPK(erk) signaling in hepatocytes is critical for inhibition of apoB100 secretion by naringenin and insulin. Therefore, naringenin may prove useful for activating insulin-signaling pathways important for regulation of hepatocyte lipid homeostasis.

  11. Sex-specific association of fatty acid binding protein 2 and microsomal triacylglycerol transfer protein variants with response to dietary lipid changes in the 3-mo Medi-RIVAGE primary intervention study.

    PubMed

    Gastaldi, Marguerite; Dizière, Sophie; Defoort, Catherine; Portugal, Henri; Lairon, Denis; Darmon, Michel; Planells, Richard

    2007-12-01

    The dietary guidelines targeted at reducing cardiovascular risk lead to largely heterogeneous responses in which genetic determinants are largely involved. We evaluated the effect of fatty acid binding protein 2 (FABP2) Ala54Thr and microsomal triacylglycerol transfer protein (MTTP) -493G/T allelic variations on plasma lipid markers, at baseline and on the response to the 3-mo Medi-RIVAGE primary prevention study. Subjects with moderate cardiovascular disease risk (n = 169) were advised to reduce total and saturated dietary fats and to increase intake of monounsaturated and polyunsaturated fats. They were genotyped for FABP2 Ala54Thr and MTTP -493G/T allelic variations, and plasma was processed for cardiovascular risk marker analyses. At baseline, men and women homozygous for Thr54 presented a significant opposite profile for plasma oleic acid (18:1), triacylglycerol-rich lipoprotein (TRL) cholesterol, and TRL phospholipids. In addition, all Thr/Thr men presented higher 18:1 values than did women. For the MTTP -493G/T polymorphism, although all TT subjects presented high apolipoprotein B-48, a genotype x sex interaction was present for palmitic acid, linolenic acid, eicosatrienoic acid, and insulin. The prudent diet clearly improved plasma lipid markers. FABP2 genotype did not interact much with the amplitude of the response. However, for MTTP polymorphism, men homozygous for the T allele displayed a significantly more pronounced response than did men carrying the G allele, which is particularly evident by their larger decrease in the Framingham score. These 2 polymorphic loci are thus differently associated with the baseline lipid markers as well as with the response to nutritional recommendations, but both presented a marked sex-specific profile, with the response to diet being particularly efficient in men homozygous for the MTTP -493T allele.

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

    PubMed

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

    2017-03-09

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

  13. Dendritic protein synthesis in the normal and diseased brain

    PubMed Central

    Swanger, Sharon A.; Bassell, Gary J.

    2015-01-01

    Synaptic activity is a spatially-limited process that requires a precise, yet dynamic, complement of proteins within the synaptic micro-domain. The maintenance and regulation of these synaptic proteins is regulated, in part, by local mRNA translation in dendrites. Protein synthesis within the postsynaptic compartment allows neurons tight spatial and temporal control of synaptic protein expression, which is critical for proper functioning of synapses and neural circuits. In this review, we discuss the identity of proteins synthesized within dendrites, the receptor-mediated mechanisms regulating their synthesis, and the possible roles for these locally synthesized proteins. We also explore how our current understanding of dendritic protein synthesis in the hippocampus can be applied to new brain regions and to understanding the pathological mechanisms underlying varied neurological diseases. PMID:23262237

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

    PubMed Central

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

    2010-01-01

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

  15. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

    PubMed

    Guenthner, T M; Hammock, B D; Vogel, U; Oesch, F

    1981-04-10

    Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

  16. Regulation of muscle protein synthesis in humans.

    PubMed

    Phillips, Bethan E; Hill, Derek S; Atherton, Philip J

    2012-01-01

    Investigations into the regulation of muscle protein synthesis (MPS) are a cornerstone of understanding the control of muscle mass. Rates of MPS are finely tuned according to levels of activity, nutrient availability and health status. For instance, rates of MPS are positively regulated by exercise and nutrition, and negatively regulated by inactivity (e.g. disuse), ageing (i.e. sarcopenia) and in muscle-wasting related diseases (e.g. cancer). Skeletal muscles display a high degree of intrinsic regulation. Increases in MPS after exercise occur independently of the systemic milieu for example growth hormone/testosterone concentrations. In the absence of exercise, increases in MPS after feeding are of finite duration despite enduring precursor availability; that is muscles can sense they are 'full'. Intriguingly, exercise delays this 'muscle-full' response to allow for building and repair. In contrast, muscle-wasting conditions exhibit a premature 'muscle-full' response to nutrition and exercise (i.e. anabolic resistance), which may cause atrophy. Observations of 'dissociations' between MPS and anabolic signalling pathways have cast doubt on how much we understand of the molecular regulation of human MPS. Anabolic and anticatabolic interventions in health and disease should be aimed at manipulating the 'muscle-full' set point to maximize muscle maintenance/hypertrophy.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2016-02-01

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

  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. Hyperoxia: Influence on Lung Mechanics and Protein Synthesis

    PubMed Central

    Gacad, Gerardo; Massaro, Donald

    1973-01-01

    We studied the time-course of the influence of in vivo hyperoxia on lung mechanics and on protein synthesis. After 24 h of exposure to greater than 98% O2 at 1 atm there were no alterations in descending pressure-volume curves (air or saline) of lungs excised from O2-exposed rats compared to control rats. After 48 h of hyperoxia there was a decrease in lung compliance. To study protein synthesis, as indicated by L-[U-24C] leucine incorporation into protein, lung slices were incubated with L-[U-14C]leucine and surface-active material then obtained by ultracentrifugation of lung homogenates. We measured radioactivity in total protein and in protein in the surface-active fraction. There were no alterations in incorporation after 12 h of hypertoxia. After 24 h of hyperoxia there were significant decreases (P<0.05) in L-[U-14C]leucine incorporation into total protein and into protein of the surface-active fraction. After 48 h of hyperoxia incorporation into protein of the surface-active fraction was decreased to a greater extent than incorporation into total protein, 63±4% and 75±5%, respectively, (P<0.025). These studies show that hyperoxia produces a major decrease in protein synthesis, including synthesis of protein in a surface-active fraction, before the onset of any detectable changes in the static compliance of excised lungs. PMID:4739291

  1. 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.

  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. 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.

  4. 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.

  5. [Detection of synthesized microsomal hemoproteins (cytochrome P-448) using autofluorography].

    PubMed

    Chasovnikova, O B; Tsyrlov, I B

    1986-01-01

    Simple and informative method for the elucidation of de novo synthesized forms of microsomal cytochrome P-448 induced by 3-methylcholanthrene and 2,3,7,8-tetrachlordibenzo-p-dioxine has been developed. The method is based on gel fluorography upon electrophoretic separation of microsomal proteins obtained from the liver of rats pre-treated with the inducers of monooxygenase system components and then with 14C-leucine. At least two forms of cytochrome P-448 (with molecular weight of 56000 and 53000) were shown to be de novo synthesized under the influence of 3-methylcholanthrene and 2,3,7,8-tetrachlodbibenzo-p-dioxine.

  6. Discovery, Synthesis and Biological Evaluation of Novel SMN Protein Modulators

    PubMed Central

    Xiao, Jingbo; Marugan, Juan J.; Zheng, Wei; Titus, Steve; Southall, Noel; Cherry, Jonathan J.; Evans, Matthew; Androphy, Elliot J.; Austin, Christopher P.

    2011-01-01

    Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure property relationships including microsomal stability, cell permeability and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models, and a starting point for clinical development. PMID:21819082

  7. Improved Cell-Free RNA and Protein Synthesis System

    PubMed Central

    Li, Jun; Gu, Liangcai; Aach, John; Church, George M.

    2014-01-01

    Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G, and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency. PMID:25180701

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

    PubMed

    He, Mingyue

    2008-01-01

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

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

    PubMed

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

    1982-11-01

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

  10. Regulation of ribosomal protein synthesis in Vibrio cholerae.

    PubMed

    Allen, Todd D; Watkins, Tonya; Lindahl, Lasse; Zengel, Janice M

    2004-09-01

    We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.

  11. Regulation of Ribosomal Protein Synthesis in Vibrio cholerae

    PubMed Central

    Allen, Todd D.; Watkins, Tonya; Lindahl, Lasse; Zengel, Janice M.

    2004-01-01

    We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria. PMID:15317799

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

    PubMed

    Wong, J T; Bronskill, P M

    1979-07-18

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

  13. 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.

  14. Predictors of muscle protein synthesis after severe pediatric burns

    USDA-ARS?s Scientific Manuscript database

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

  15. 9-Fluorenylmethyloxycarbonyl/ tbutyl-based convergent protein synthesis.

    PubMed

    Barlos, K; Gatos, D

    1999-01-01

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

  16. Chemical synthesis and biophysical applications of membrane proteins.

    PubMed

    Zuo, Chao; Tang, Shan; Zheng, Ji-Shen

    2015-07-01

    Chemical synthesis or semi-synthesis of membrane proteins can provide unique molecular tools, such as site-specific isotope labeling or post-translationally modified membrane proteins to gain insight into their biophysical and functional characteristics. However, during preparation, purification, and ligation of transmembrane peptides, tremendous challenges are encountered owing to their hydrophobic nature. This review focuses on the recent advances in chemical synthesis strategies of membrane proteins. These strategies help to solubilize the hydrophobic transmembrane peptide sequences under standard purification and chemical ligation conditions to improve their handling properties. Biophysical and functional studies of synthetic membrane proteins are reviewed as well. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  17. Serine/threonine ligation for the chemical synthesis of proteins.

    PubMed

    Lee, Chi Lung; Li, Xuechen

    2014-10-01

    Advances in the development of efficient peptide ligation methods have enabled the total synthesis of complex proteins to be successfully undertaken. Recently, a Ser/Thr ligation has emerged as a new tool in synthetic protein chemistry. The chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment gives rise to an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. Ser/Thr ligation has been used for the synthesis of the human erythrocyte acylphosphatase protein and MUC1 glycopeptide segments, semisynthesis of peptoid/PEG-RNase S protein hybrids, and cyclic peptide synthesis including cyclic tetrapeptides, cyclomontanin B, yunnanin C, mahafacyclin B, and daptomycin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

    PubMed Central

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

    2016-01-01

    In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2015-07-08

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

  1. 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.

  2. Retinal protein synthesis in relationship to environmental lighting

    SciTech Connect

    Hollyfield, J.G.; Anderson, R.E.

    1982-11-01

    A series of in vivo and in vitro experiments using Xenopus laevis juvenile toads was conducted to probe the relationship between environmental lighting and protein synthesis in the retina. Autoradiographic and biochemical analyses indicated that measurable changes in protein synthesis did not occur during a normal diurnal cycle when animals were conditioned to 12 hr light followed by 12 hr darkness each day (LD). However, when retinas from animals maintained in continuous darkness (DD) for 3 days were incubated with /sup 3/H-leucine, there was a 40% reduction in the specific radioactivity of total retinal proteins compared with retinas from animals maintained in continuous light (LL) for 3 days or on the LD cycle. Retinas from DD animals injected with /sup 3/H-leucine showed a 48% reduction in protein synthesis compared with retinas of LL animals. In autoradiographs of retinas from in vivo or in vitro experiments, grain counts were 40% lower in the total retinas of the DD animals compared with retinas of LL animals. This reduction occurred throughout the entire retina and was not restricted to any specific cell type. There was also a 35% reduction in the rate of radioactive band displacement in the rod outer segments of DD animals, although the percent of /sup 3/H-leucine incorporated into opsin relative to total retinal protein was the same for both groups. We conclude from these studies that fluctuations in the rate of protein synthesis during the normal light-dark cycle are not detectable. However, major differences in protein synthesis are evident when animals are stressed with continuous darkness for several days. This effect is not restricted to any particular retinal layer but occurs throughout the entire retina. Moreover, prolonged darkness affects protein synthesis in extraocular tissues as well.

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

    PubMed

    Feduchi, E; Cosín, M; Carrasco, L

    1985-03-01

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

  4. 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. 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.

  6. 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.

  7. Regulation of platelet activating factor synthesis: modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes

    SciTech Connect

    Lenihan, D.J.; Lee, T.C.

    1984-05-16

    1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase plays an important regulatory role in the biosynthesis of platelet activating factor, a potent bioactive mediator. The authors tested the hypothesis that the activity of acetyltransferase may be modulated by enzymatic phosphorylation and dephosphorylation. The results showed that acetyltransferase activity in rat spleens was 2- to 3-fold higher in microsomes isolated in the presence of F/sup -/ than in those isolated in the presence of Cl/sup -/. The microsomal acetyltransferase could be activated by preincubation of microsomes, isolated in the presence of Cl/sup -/, with ATP, Mg/sup 2 +/, and the soluble fraction from rat spleen. Addition of phosphatidylserine, diacylglycerols, plus Ca/sup 2 +/ further enhanced the activity. The increase in the activity of acetyltranferase was abolished by treatment of the activated microsomes with alkaline phosphatase. Conversely, the activity of acetyltransferase can be reactivated in the alkaline phosphatase-treated microsomes with incubation conditions that favor phosphorylation. Therefore, the findings suggest that acetyltransferase activity is regulated by reversible activation/inactivation through phosphorylation/dephosphorylation.

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

    PubMed

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

    1996-12-13

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

  9. His6 tag-assisted chemical protein synthesis.

    PubMed

    Bang, Duhee; Kent, Stephen B H

    2005-04-05

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

  10. His6 tag-assisted chemical protein synthesis

    PubMed Central

    Bang, Duhee; Kent, Stephen B. H.

    2005-01-01

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

  11. His6 tag-assisted chemical protein synthesis

    NASA Astrophysics Data System (ADS)

    Bang, Duhee; Kent, Stephen B. H.

    2005-04-01

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

  12. 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

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

    PubMed

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

    2016-06-28

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

  14. Astrocytic glucose-6-phosphatase and the permeability of brain microsomes to glucose 6-phosphate.

    PubMed Central

    Forsyth, R J; Bartlett, K; Burchell, A; Scott, H M; Eyre, J A

    1993-01-01

    Cells from primary rat astrocyte cultures express a 36.5 kDa protein that cross-reacts with polyclonal antibodies to the catalytic subunit of rat hepatic glucose-6-phosphatase on Western blotting. Glucose-6-phosphate-hydrolysing activity of the order of 10 nmol/min per mg of total cellular protein can be demonstrated in cell homogenates. This activity shows latency, and is localized to the microsomal fraction. Kinetic analysis shows a Km of 15 mM and a Vmax. of 30 nmol/min per mg of microsomal protein in disrupted microsomes. Approx. 40% of the total phosphohydrolase activity is specific glucose-6-phosphatase, as judged by sensitivity to exposure to pH 5 at 37 degrees C. Previous reports that the brain microsomal glucose-6-phosphatase system does not distinguish glucose 6-phosphate and mannose 6-phosphate are confirmed in astrocyte microsomes. However, we demonstrate significant phosphomannose isomerase activity in brain microsomes, allowing for ready interconversion between mannose 6-phosphate and glucose 6-phosphate (Vmax. 15 nmol/min per mg of microsomal protein; apparent Km < 1 mM; pH optimum 5-6 for the two-step conversion). This finding invalidates the past inference from the failure of brain microsomes to distinguish mannose 6-phosphate and glucose 6-phosphate that the cerebral glucose-6-phosphatase system lacks a 'glucose 6-phosphate translocase' [Fishman and Karnovsky (1986) J. Neurochem. 46, 371-378]. Furthermore, light-scattering experiments confirm that a proportion of whole brain microsomes is readily permeable to glucose 6-phosphate. Images Figure 1 PMID:8395816

  15. Immunolocalization of a microsomal prostaglandin E synthase in rabbit kidney.

    PubMed

    Fuson, Amanda L; Komlosi, Peter; Unlap, Tino M; Bell, P Darwin; Peti-Peterdi, János

    2003-09-01

    PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2 synthase (mPGES), the terminal enzyme of PGE2 synthesis, which converts COX-derived PGH2 to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2 in descending thin limb and collecting tubule and a paracrine effect of PGE2 on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.

  16. Microsome composition-based model as a mechanistic tool to predict nonspecific binding of drugs in liver microsomes.

    PubMed

    Poulin, Patrick; Haddad, Sami

    2011-10-01

    The purpose of this study was to investigate the ability of the microsome composition-based model to predict the unbound fraction determined in vitro in microsomal incubation system (fuinc ). Another objective was to make a comparative assessment between the proposed mechanistic method and three empirical methods published in the literature, namely the models of Austin et al. (2002, Drug Metab Dispos 30:1497-1503), Turner et al. [2007, Drug Metab Rev 38(S1):162], and Halifax and Houston (2006, Drug Metab Rev 34:724-726), which are based solely on physicochemical properties. The assessment was confined by the availability of measured fuinc data in rat and human at diverse microsomal protein concentrations for 132 compounds. The proposed microsome composition-based model can be viewed as a combination of two distinct processes, namely the nonspecific binding to neutral lipids and the ionic binding to acidic phospholipids. Across methods, the maximum success rate in predicting fuinc of all compounds was 98%, 91%, and 84% with predictions falling within threefold, twofold, and 1.5-fold error of the observed fuinc , respectively. The statistical analyses suggest that the prediction models are more effective at computing fuinc (i) for rat as compared with human, and (ii) for acids and neutral drugs as compared with strong basic drugs. In addition, on the basis of the comparisons made using all datasets, the method that made use of microsome composition data compares well with those methods that relied solely on physicochemistry. The sensitivity analysis demonstrated the importance of the compound properties and physiological parameters reflective of specific mechanistic determinants relevant to prediction of fuinc values of drugs. Overall, the results obtained with our proposed model demonstrate a significant step toward the development of a generic and mechanistic model of fuinc for liver microsomes, which should provide rationale extrapolation procedures of hepatic

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

    PubMed

    Takai, Kazuyuki; Endo, Yaeta

    2010-01-01

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

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

    PubMed

    Scanlon, Denis B; Karas, John A

    2011-01-01

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

  19. Compounds affecting membranes that inhibit protein synthesis in yeast.

    PubMed

    Alonso, M A; Vázquez, D; Carrasco, L

    1979-12-01

    The regulation of translation has been investigated in yeast cells by means of ionophores and other compounds affecting the ionic concentration inside the cell. Treatment of a variety of cells with these compounds produces a drastic inhibition in the protein-synthesizing activity of the cell. Protein synthesis in yeast is strongly inhibited by amphotericin B and nystatin. Mammalian cells are blocked in their translation capacity by gramicidin D, nigericin, monensin, nystatin, A23187, and bromolasalocid. The effects of these compounds on protein synthesis in Escherichia coli and Staphylococcus aureus were also investigated. Amphotericin B is a powerful inhibitor of both protein and ribonucleic acid syntheses in yeast cells at concentrations that do not affect the transport of the labeled amino acid or nucleoside precursor. The analysis of the polysomal profiles in yeast spheroplasts could indicate that initiation is the target of amphotericin B action on translation. Studies on the reversion of the protein synthesis blockade by amphotericin B by increasing the potassium concentration in the medium suggest that changes in the potassium concentration in cellular cytoplasm might be responsible, at least in part, for the inhibition of protein synthesis.

  20. Compounds affecting membranes that inhibit protein synthesis in yeast.

    PubMed Central

    Alonso, M A; Vázquez, D; Carrasco, L

    1979-01-01

    The regulation of translation has been investigated in yeast cells by means of ionophores and other compounds affecting the ionic concentration inside the cell. Treatment of a variety of cells with these compounds produces a drastic inhibition in the protein-synthesizing activity of the cell. Protein synthesis in yeast is strongly inhibited by amphotericin B and nystatin. Mammalian cells are blocked in their translation capacity by gramicidin D, nigericin, monensin, nystatin, A23187, and bromolasalocid. The effects of these compounds on protein synthesis in Escherichia coli and Staphylococcus aureus were also investigated. Amphotericin B is a powerful inhibitor of both protein and ribonucleic acid syntheses in yeast cells at concentrations that do not affect the transport of the labeled amino acid or nucleoside precursor. The analysis of the polysomal profiles in yeast spheroplasts could indicate that initiation is the target of amphotericin B action on translation. Studies on the reversion of the protein synthesis blockade by amphotericin B by increasing the potassium concentration in the medium suggest that changes in the potassium concentration in cellular cytoplasm might be responsible, at least in part, for the inhibition of protein synthesis. PMID:394675

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

    PubMed

    Kawarasaki, Y; Nakano, H; Yamane, T

    1994-10-01

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

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

    PubMed

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

    2005-02-01

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

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

    PubMed

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

    2010-01-01

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

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

    PubMed

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

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. 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.

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

    PubMed

    2017-07-01

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

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

    PubMed

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

    2016-06-03

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

  8. 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

  9. Rabies virus protein synthesis in infected BHK-21 cells.

    PubMed Central

    Madore, H P; England, J M

    1977-01-01

    Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions. PMID:558341

  10. Covalent binding of chlorotrianisene (TACE) metabolite(s) to rat hepatic microsomal components

    SciTech Connect

    Bulger, W.H.; Juedes, M.J.; Kupfer, D.

    1986-03-01

    TACE, an estrogen, is a member of the triarylethylene series of compounds which includes the antiestrogens clomiphene and tamoxifen. TACE has been used as a therapeutic estrogen and has been identified as a contaminant in the pesticide methoxychlor (M) and is presumably one of the factors responsible for the estrogenic properties of technical M. The possibility that like M, TACE is activated to covalently bind to microsomal proteins, was examined. (/sup 3/H)TACE was incubated with liver microsomes from phenobarbital (Pb)-treated male rats and NADPH. Microsomes were precipitated with ethanol and trapped on glass-fiber filter. The filter was washed with ethanol, hexane, and methanol:ether mixtures. The residue was solubilized from the filter by incubating (1hr, 37/sup 0/) With 2% SDS and the radioactivity and protein contents were determined. The solubilized samples were also subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Binding of TACE metabolites to microsomal components in the presence of NADPH was 350 pmol/30 min/mg protein. PAGE analysis revealed radioactivity in a region of 50-55K daltons, suggesting covalent binding to protein(s). When compared to incubations with control microsomes, binding was markedly enhanced by microsomes Pb treated rats.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. 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)

  13. 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)

  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. Translational Control of Specific Uterine Protein Synthesis After Estrogen Induction

    PubMed Central

    DeAngelo, Anthony B.; Fujimoto, George I.

    1973-01-01

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

  16. Effect of dietary zinc on endogenous free radical production in rat lung microsomes.

    PubMed

    Bray, T M; Kubow, S; Bettger, W J

    1986-06-01

    The objective of this study was to investigate the effect of dietary zinc on endogenous production of free radicals in lung and liver microsomes. Male weanling rats were fed a zinc-deficient basal diet containing less than 1.1 ppm zinc, or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 100 ppm zinc. The isolated microsomes (100,000 X g precipitate) of lung and liver were incubated with 0.1 M PBN (spin trap) and 0.3 mM NADPH (cofactor) at 37 degrees C for 1.0 h. A carbon-centered free radical (aN = 16.0 G, aH beta = 3.4 G) was trapped in both lung and liver microsomes. There was a significant increase in the concentration of carbon-centered free radicals generated in lung microsomes in animals fed a zinc-deficient diet. Dietary zinc status did not significantly affect the concentration of free radicals in liver microsomes. The amount of free radicals generated is proportional to microsomal protein concentration and is linear with protein concentration between 5 and 20 mg per milliliter of incubate. The free radicals formed in the microsomal system were dependent on the presence of NADPH. Carbon monoxide inhibited 40-50% of the free radical production in both lung and liver microsomes. The results suggest that dietary zinc deficiency stimulates the production of endogenous free radicals in rat lung microsomes by an NADPH- and cytochrome P-450-dependent system.

  17. Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans

    PubMed Central

    Carlile, David J; Hakooz, Nancy; Bayliss, Martin K; Houston, J Brian

    1999-01-01

    Aims To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. Methods V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Results Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) μl min−1 mg−1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1–5 μm is substantially lower than the value determined in microsomes based on total concentrations (56 μm), correction for the in vitro binding reduces this value to 20 μm and 6 μm in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. Conclusions An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent. PMID:10383540

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

    PubMed Central

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

    1997-01-01

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

  19. Frog Foam Nest Protein Diversity and Synthesis.

    PubMed

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

    2016-08-01

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

  20. 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…

  1. 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…

  2. 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)

  3. Initiation of protein-primed picornavirus RNA synthesis

    PubMed Central

    Paul, Aniko V.; Wimmer, Eckard

    2015-01-01

    Plus strand RNA viruses use different mechanisms to initiate the synthesis of their RNA chains. The Picornaviridae family constitutes a large group of plus strand RNA viruses that possess a small terminal protein (VPg) covalently linked to the 5’-end of their genomes. The RNA polymerases of these viruses use VPg as primer for both minus and plus strand RNA synthesis. In the first step of the initiation reaction the RNA polymerase links a UMP to the hydroxyl group of a tyrosine in VPg using as template a cis-replicating element (cre) positioned in different regions of the viral genome. In this review we will summarize what is known about the intiation reaction of protein-primed RNA synthesis by the RNA polymerases of the Picornaviridae. As an example we will use the RNA polymerase of poliovirus, the prototype of Picornaviridae. We will also discuss models of how these nucleotidylylated protein primers might be used, together with viral and cellular replication proteins and other cis-replicating RNA elements, during minus and plus strand RNA synthesis. PMID:25592245

  4. 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…

  5. 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…

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

    USDA-ARS?s Scientific Manuscript database

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

  7. Pin1 and PKMζ Sequentially Control Dendritic Protein Synthesis

    PubMed Central

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

    2010-01-01

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

  8. Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers.

    PubMed

    Pytharopoulou, Sofia; Kouvela, Ekaterini C; Sazakli, Eleni; Leotsinidis, Michel; Kalpaxis, Dimitrios L

    2006-10-25

    Protein synthesis down-regulation is a life-saving mechanism for many organisms exposed to xenobiotics that threaten normal life. The present study was designed to assess the spatial and seasonal variability of global protein synthesis, determined in the microsomal fraction of digestive glands from caged Mytilus galloprovincialis mussels exposed for 30 days in a relatively clean region and two unevenly polluted areas (Stations 1 and 2) along the Gulf of Patras (Greece). The in vivo activity of translating ribosomes was evaluated by analyzing the translating ribosomes, polysome content, which may serve as an indicator of the efficiency of the protein-synthesizing machinery. To correlate with classical biomonitoring strategies, various biomarkers were measured in digestive glands, including metallothionein content, heavy-metal content, and lysosomal membrane stability. In parallel, gill cells were examined for micronucleus frequency. Metal ion concentrations were also estimated in the surrounding waters as a measure of metal exposure. Substantially lower polysome content was recorded in caged mussels collected from Station 1, in particular during the winter and spring sampling. As verified by chemical analysis of the seawater and measurement of other biomarkers, Station 1 was more contaminated than Station 2. Polysome content was found negatively correlated with metallothionein levels, micronucleus frequency and cytosolic Cu and Hg in all seasons. In addition, negative correlations were obtained between polysome content and lysosomal membrane stability in winter and spring. A progressive increase in polysomes was observed from winter to autumn, in particular in samples from Station 1. A non-uniform trend was detected in 80S ribosomal monosomes, whereas the seasonal changes in ribosomal subunits were opposite to those found in polysome content. Comparisons between seasonal and local site-specific influences on polysome content provides evidence that winter and spring

  9. Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

    PubMed

    Guenthner, T M; Oesch, F

    1983-12-25

    A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

  10. ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER

    PubMed Central

    Amar-Costesec, Alain; Beaufay, Henri; Wibo, Maurice; Thinès-Sempoux, Denise; Feytmans, Ernest; Robbi, Mariette; Berthet, Jacques

    1974-01-01

    Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-β-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction. PMID:4150489

  11. Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs.

    PubMed

    O'Connor, Pamela M J; Bush, Jill A; Suryawan, Agus; Nguyen, Hanh V; Davis, Teresa A

    2003-01-01

    Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.

  12. 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.

  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. From Asymmetric Exclusion Processes to Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Dong, Jiajia; Schmittmann, Beate; Zia, Royce K. P.

    2006-03-01

    Protein production rates are clearly vital for all biological systems. Thus, there is considerable interest in understanding the origins of these rates, as well as in manipulating them, especially for physiological and pharmaceutical applications. Since some codons are ``fast'' and others ``slow,'' we propose to exploit these differences and modify the production rate for any specific protein by replacing codons in the associated mRNA by their synonymous counterparts. As an illustration, we study a simple model of protein production: the one-dimensional driven lattice gas, also known as the totally asymmetric simple exclusion process (TASEP). We investigate systematically the effects on the overall current (the protein production rate) of having one or two slow/fast sites (i.e., codons) in an otherwise homogeneous lattice. The currents show a non-trivial dependence on the location of a single ``defect'' as well as on the separation between two defects. We discuss the implications for more realistic models of protein production.

  15. Protein Synthesis Driven by Dynamical Stochastic Transcription.

    PubMed

    Innocentini, Guilherme C P; Forger, Michael; Radulescu, Ovidiu; Antoneli, Fernando

    2016-01-01

    In this manuscript, we propose a mathematical framework to couple transcription and translation in which mRNA production is described by a set of master equations, while the dynamics of protein density is governed by a random differential equation. The coupling between the two processes is given by a stochastic perturbation whose statistics satisfies the master equations. In this approach, from the knowledge of the analytical time-dependent distribution of mRNA number, we are able to calculate the dynamics of the probability density of the protein population.

  16. Protein Synthesis Using A Reconstituted Cell-Free System

    PubMed Central

    Tuckey, Corinna; Asahara, Haruichi; Zhou, Ying; Chong, Shaorong

    2014-01-01

    Most cell free protein synthesis systems are based on cell extracts, which often contain undesirable activities. The reconstituted systems, by contrast, are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. This unit describes the use of a particular commercial reconstituted system, "PURExpress®" This system allows in vitro synthesis of proteins from mRNA and circular and linear DNA templates, as well as co-translational labeling of proteins. Unique to this system, all recombinant protein components of the system are His-tagged, allowing purification of the synthesized untagged protein by removing the rest of the system’s components. Newly synthesized proteins can often be visible on a SDS-PAGE gel and directly assayed for their functions without labeling and purification. Certain components of the system, such as ribosomes or release factors, can be omitted for specific applications. Such "delta" versions of the system are well suited for studies of bacterial translation, assays of ribosome functions, incorporation of unnatural amino acids and ribosome display of protein libraries. PMID:25271715

  17. 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

  18. Ribosomal history reveals origins of modern protein synthesis.

    PubMed

    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.

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

    PubMed Central

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

    1987-01-01

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

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

    PubMed

    Pace, Douglas A; Manahan, Donal T

    2006-01-01

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

  1. Glucocorticoid action on protein synthesis and protein breakdown in isolated skeletal muscles

    PubMed Central

    McGrath, Josephine A.; Goldspink, David F.

    1982-01-01

    The direct actions of glucocorticoid hormones on protein turnover were studied in isolated soleus muscles. These steroids were found to decrease the rates of both protein synthesis and protein breakdown within 3 h and 4 h respectively. Synthetic steroids (e.g. dexamethasone) were found to be more potent than naturally secreted hormones (e.g. cortisol) in inducing these changes, but only at concentrations in vitro less than 10nm. PMID:7150268

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

    PubMed

    Verdier, Jérôme; Thompson, Richard D

    2008-09-01

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

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

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  4. Protein synthesis rate is the predominant regulator of protein expression during differentiation

    PubMed Central

    Kristensen, Anders R; Gsponer, Joerg; Foster, Leonard J

    2013-01-01

    External perturbations, by forcing cells to adapt to a new environment, often elicit large-scale changes in gene expression resulting in an altered proteome that improves the cell's fitness in the new conditions. Steady-state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system-level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub-structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo-complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account. PMID:24045637

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

    PubMed

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

    2011-04-01

    N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Hepatic microsomal. omega. -oxidation of leukotriene B/sub 4/

    SciTech Connect

    Mukhtar, H.; Bik, D.P.; Das, M.; Bickers, D.R.

    1986-03-01

    In this study the authors investigated the metabolism of LTB/sub 4/ by rat hepatic microsomes. /sup 3/H-LTB/sub 4/ (9 ..mu..M) was incubated for 20 min in the presence3 of oxygen, NADPH and liver microsomes (1.5 mg). Incubation was followed by extraction and analysis by HPLC. Metabolite identification was based on cochromatography with reference standards. At least three radioactive peaks were observed; two of which were ..omega..-oxidation products 20-OH-LTB/sub 4/ and 20-COOH-LTB/sub 4/. The rate of formation of 20-OH-LTB/sub 4/ (14.6 nmoles/min/mg protein) was higher than that of 20-COOH-LTB/sub 4/ (2.5 nmoles/min/mg protein). The third radioactive peak remains unidentified. Product formation was negligible with boiled microsomes. LTB/sub 4/ ..omega..-hydroxylase activity required NADPH and oxygen, was linear with respect to incubation time and protein, and was maximal at pH 7.4. Enzyme activity was inhibited (>90%) by carbon monoxide, SKF 525A (1 mM), but was not affected by ..cap alpha..-naphthoflavone. Phenobarbital (PB, 80 mg/kg for 3 days), AROCLOR 1254 (100 mg/kg for 3 days) or 3-methylcholanthrene (40 mg/kg for 3 days) administration to rats resulted in only slight (<20%) or no increase in LTB/sub 4/ ..omega..-hydroxylase activity. However, the formation of the unidentified peak was increased (>100%) by PB treatment. These results suggest that ..omega..-oxidation is the major pathway for biotransformation of LTB/sub 4/ in liver microsomes and that this reaction is mediated by cytochrome P-450.

  7. 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.

  8. 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.

  9. When Too Much ATP Is Bad for Protein Synthesis.

    PubMed

    Pontes, Mauricio H; Sevostyanova, Anastasia; Groisman, Eduardo A

    2015-08-14

    Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration. Magnesium (Mg(2+)), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counterion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg(2+) limitation because ATP is the most abundant nucleotide triphosphate in the cell, and Mg(2+) is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg(2+) limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg(2+) to indispensable cellular processes. Copyright © 2015. Published by Elsevier Ltd.

  10. 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.

  11. Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts 1

    PubMed Central

    Vasconcelos, Aurea C.

    1976-01-01

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

  12. Oral contraceptive use alters the balance of platelet prostaglandin and thromboxane synthesis.

    PubMed

    Schorer, A E; Gerrard, J M; White, J G; Krivit, W

    1978-07-01

    The ability of platelet microsomes to generate platelet aggregating activity on addition of arachidonic acid was evaluated in women taking oral contraceptives and in controls taking no medication but matched for age, sex, and family history. Oral contraceptive users generated significantly more platelet aggregating activity per 100 ug of platelet microsomal protein than controls. Variation in generation of platelet aggregating activity during the menstrual cycle was also observed with highest activity during the third week. These studies show an altered balance of platelet prostaglandin and thromboxane synthesis in oral contraceptive users which may contribute to their increased incidence of thromboembolic phenomena.

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

    PubMed

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

    2009-05-01

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

  14. The Roles of RNA in the Synthesis of Protein

    PubMed Central

    Moore, Peter B.; Steitz, Thomas A.

    2011-01-01

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

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

    PubMed Central

    Jacobson, A; Favreau, M

    1983-01-01

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

  16. Voluntary exercise regionally augments rates of cerebral protein synthesis.

    PubMed

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

    2013-11-06

    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-(14)C]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. Published by Elsevier B.V.

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

    PubMed

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

    2014-02-01

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

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

    SciTech Connect

    Camarero, J A; Mitchell, A R

    2005-01-20

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

  19. Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase.

    PubMed

    Ged, C; Umbenhauer, D R; Bellew, T M; Bork, R W; Srivastava, P K; Shinriki, N; Lloyd, R S; Guengerich, F P

    1988-09-06

    A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the

  20. Covalent Modification of Microsomal Lipids by Thiobenzamide Metabolites in Vivo

    PubMed Central

    Ji, Tao; Ikehata, Keisuke; Koen, Yakov M.; Esch, Steven W.; Williams, Todd D.; Hanzlik, Robert P.

    2008-01-01

    Thiobenzamide (TB) is hepatotoxic in rats causing centrolobular necrosis, steatosis, cholestasis and hyperbilirubinemia. It serves as a model compound for a number of thiocarbonyl compounds that undergo oxidative bioactivation to chemically reactive metabolites. The hepatotoxicity of TB is strongly dependent on the electronic character of substituents in the meta- and para- positions, with Hammett rho values ranging from −4 to −2. On the other hand ortho substituents which hinder nucleophilic addition to the benzylic carbon of S-oxidized TB metabolites abrogate the toxicity and protein covalent binding of TB. This strong linkage between the chemistry of TB and its metabolites and their toxicity suggests that this model is a good one for probing the overall mechanism of chemically-induced biological responses. While investigating the protein covalent binding of TB metabolites we noticed an unusually large amount of radioactivity associated with the lipid fraction of rat liver microsomes. Thin layer chromatography showed that most of the radioactivity was contained in a single spot more polar than the neutral lipids but less polar than the phospholipid fractions. Mass spectral analyses aided by the use of synthetic standards identified the material as N-benzimidoyl derivatives of typical microsomal phosphatidylethanolamine (PE) lipids. Quantitative analysis indicated that up to 25% of total microsomal PE became modified within 5 h after a hepatotoxic dose of TB. Further studies will be required to determine the contribution of lipid modification to the hepatotoxicity of thiobenzamide. PMID:17381136

  1. Characterization of a novel ACTH inducible cytochrome P-450 from rat adrenal microsomes

    SciTech Connect

    Otto, S.A.; Marcus, C.M.; Jefcoate, C.R. )

    1990-02-26

    In rat adrenal cortex 7,12 dimethylbenz(a)anthracene (DMBA) causes massive necrosis that is dependent of ACTH. This is related to an ACTH inducible adrenal microsomal cytochrome P-450 that catalyzes hydrocarbon metabolism. Rat adrenal microsomes, catalyze the formation of DMBA 3,4 diol a precursor of the bay region reactive electrophile DMBA 3,4 diol 1,2 oxide. Both DMBA metabolism and a 57Kd protein have disappeared from microsomes 30 days after hypophysectomy, but are restored by 14 days treatment with ACTH. Dexamethasone which fully suppresses ACTH only partially suppresses this activity. The 57 Kd protein was partially purified to a single major band in one step from solubilized microsomes by h.p.l.c. chromatography using detergent elution from a novel column that mimics phospholipid membranes. This preparation exhibits a specific content of 2 nm P-450/mg protein and a turnover number of 1,500pm DMBA/nm P-450/minutes. A polyclonal antisera raised against this preparation provides a single western blot corresponding to the 57Kd ACTH sensitive protein. This antibody did not blot microsomal P-450 c21, nor did selected antibodies from known families react with this adrenal P-450 protein, suggesting substantial sequence differences from known P-450's.

  2. 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.

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

    PubMed

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

    2015-02-01

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

  4. 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

  5. Microsomal metabolism of NDMA and analogs

    SciTech Connect

    Wade, D.; Yang, C.S.

    1987-05-01

    The metabolism of N-nitrosodimethylamine (NDMA), dimethylamine (DMA), N-nitro-DMA (N x NO/sub 2/ x DMA), N-nitrosodiethylamine (NDEA), and diethylamine (DEA) was studied using control, acetone (Ac)-, butylated hydroxytoluene (BHT)-, pregnenolone 16- ..cap alpha..-carbonitrile (PCN)-, and phenobarbital (PB)-induced rat liver microsomes. At low substrate concentrations, the NDMA demethylase activity of Ac-induced microsomes was 5-fold greater than that of control, BHT-, and PCN-induced microsomes. The rate of NDMA denitrosation was ca. 10% that of demethylation. N x NO/sub 2/ x DMA was metabolized to HCHO, but not to NO/sub 2//sup -/, and the rate of metabolism was greatest with Ac-induced microsomes; the K/sub m/ and V/sub max/ of Ac-induced microsomes were similar to those of NDMA. For the dealkylation of NDEA, Ac- and BHT-induced microsomes were twice as active as the control. Ratios of dealkylation/denitrosation for NDEA remained constant over a broad range of low substrate concentrations. BHT- or Ac-treatment appeared to cause a selective increase in the ability of microsomes to denitrosate NDEA. The activity of all microsome preparations with the amines, DMA and DEA was less than that with the nitrosamine or nitramine substrates. The results suggest that both the N-nitroso and N-nitro compounds are good substrates for microsomal P-450; the amines, which bear positive charges, are not. Denitrosation appeared to be a more important pathway with NDEA than with NDMA.

  6. 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

  7. Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. I. Nuclear-directed protein synthesis.

    PubMed

    Heude, M; Chanet, R; Moustacchi, E

    1975-04-01

    The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phage haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the "petite" recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of "petites" in stationary phase cells (increase of the frequency of "petites" during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.

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

    PubMed Central

    2012-01-01

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

  9. Protein synthesis in vitro by Micrococcus luteus.

    PubMed Central

    Farwell, M A; Rabinowitz, J C

    1991-01-01

    Bacillus subtilis and related gram-positive bacteria which have low to moderate genomic G + C contents are unable to efficiently translate mRNA derived from gram-negative bacteria, whereas Escherichia coli and other gram-negative bacteria are able to translate mRNA from both types of organisms. This phenomenon has been termed translational species specificity. Ribosomes from the low-G + C-content group (low-G + C group) of gram-positive organisms (B. subtilis and relatives) lack an equivalent to Escherichia ribosomal protein S1. The requirement for S1 for translation in E. coli (G. van Dieijen, P. H. van Knippenberg, J. van Duin, B. Koekman, and P. H. Pouwels, Mol. Gen. Genet. 153:75-80, 1977) and its specific role (A.R. Subramanian, Trends Biochem. Sci. 9:491-494, 1984) have been proposed. The group of gram-positive bacteria characterized by high genomic G + C content (formerly Actinomyces species and relatives) contain S1, in contrast to the low-G + C group (K. Mikulik, J. Smardova, A. Jiranova, and P. Branny, Eur. J. Biochem. 155:557-563, 1986). It is not known whether members of the high-G + C group are translationally specific, although there is evidence that one genus, Streptomyces, can express Escherichia genes in vivo (M. J. Bibb and S. N. Cohen, Mol. Gen. Genet. 187:265-277, 1985; J. L. Schottel, M. J. Bibb, and S. N. Cohen, J. Bacteriol. 146:360-368, 1981). In order to determine whether the organisms of this group are translationally specific, we examined the in vitro translational characteristics of a member of the high-G + C group, Micrococcus luteus, whose genomic G + C content is 73%. A semipurified coupled transcription-translation system of M. luteus translates Escherichia mRNA as well as Bacillus and Micrococcus mRNA. Therefore, M. luteus is translationally nonspecific and resembles E. coli rather than B. subtilis in its translational characteristics. Images PMID:2045372

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

    PubMed

    Liu, Han-Hsuan; Cline, Hollis T

    2016-07-06

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

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

    PubMed Central

    Liu, Han-Hsuan

    2016-01-01

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

  12. 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

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

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

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

  14. Synthesis and secretion of proteins by released malarial parasites.

    PubMed

    Elmendorf, H G; Bangs, J D; Haldar, K

    1992-06-01

    Controlled mechanical homogenization of Plasmodium falciparum-infected erythrocytes releases parasites of a quality sufficient for studying the export of newly synthesized plasmodial proteins. Protein synthesis occurs within intact released parasites as defined by resistance of acid-insoluble incorporation of radiolabel to high levels of exogenously added EDTA, hexokinase, and RNaseA. While exogenously added ATP and erythrocyte cytosol were not essential for biosynthetic activity at levels comparable to that seen in infected erythrocytes, the addition of an extracellular ATP regenerating system (ARS) stimulated the synthesis of parasite proteins. Conversely, parasite viability and biosynthetic activity are decreased by the addition of a non-hydrolyzable ATP analogue (ATP gamma S), ADP, or ATP in the absence of a regenerating system. These data suggest a metabolic interdependence between extracellular energy metabolism and biosynthetic functions within the parasite. The export of a predominant subset of proteins was retarded in the presence of Brefeldin A, indicating the existence of a classical secretory pathway characteristic of that seen in higher eukaryotic cells. Interestingly, a Brefeldin A-insensitive component of export was also consistently observed; this may suggest the existence of an additional alternative secretory mechanism in malaria.

  15. The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass.

    PubMed Central

    Millward, D J; Garlick, P J; Nnanyelugo, D O; Waterlow, J C

    1976-01-01

    The effects of growth-suppressing and muscle-wasting treatments on muscle protein turnover and amino acid concentrations were determined in vivo. All treatments depressed protein synthesis and some treatments depressed protein breakdown. Only prolonged starvation increased protein breakdown. Muscle protein mass is regulated primarily through alterations in protein synthesis in all except emergency conditions. The increased concentrations of the branched-chain amino acids indicate that they are unlikely to be involved in this regulation. PMID:133677

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

    PubMed

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

    2015-01-01

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

  17. 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

  18. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    PubMed

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    1991-01-01

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

  1. Monocyte cytokine synthesis in response to extracellular cell stress proteins suggests these proteins exhibit network behaviour.

    PubMed

    Kaiser, Frank; Steptoe, Andrew; Thompson, Stephen; Henderson, Brian

    2014-01-01

    Human peripheral blood monocytes were exposed to single or pairs of cell stress proteins (CSPs), specifically Hsp10, Hsp27, Hsp60 and Hsp70-the former two having anti-inflammatory actions while the latter pair being assumed to be pro-inflammatory in activity. This study was to test if these proteins exhibited any network behaviour. To control for possible lipopolysaccharide contamination, polymyxin B was used. Surprisingly, at concentrations higher than 1 μg/ml, polymyxin B itself could induce cytokine synthesis. A number of commercial sources of the molecular chaperones were tested, and marked variations in monocyte cytokine synthesis were found. All four CSPs stimulated the same profile of IL-1/IL-6 synthesis and IL-10/TNF-α synthesis although the kinetics of production of these two pairs of cytokines were very different. A key question was whether extracellular molecular chaperones exhibited network behaviour. To test this, monocytes were cultured with suboptimal concentrations of single CSP and pairs of CSP to look for additive, synergistic or antagonistic cell responses. The major finding was that pairs of molecular chaperones, including chaperones thought to stimulate monocyte cytokine synthesis, could produce significant antagonistic cellular responses. This demonstrates that extracellular CSPs constitute an additional potent layer within the complex cytokine network and furthermore suggests that monocytes have evolved to dampen their immune responses upon exposure to extracellular networks of CSPs-perhaps as a mechanism for protecting cells against detrimental cellular stress responses.

  2. 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

  3. Protein synthesis in tomato-fruit locule tissue

    PubMed Central

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

    1967-01-01

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

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

    PubMed Central

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

    1967-01-01

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

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

    SciTech Connect

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

    1987-05-01

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

  6. Activation of misonidazole by rat liver microsomes and purified NADPH-cytochrome c reductase.

    PubMed

    McManus, M E; Lang, M A; Stuart, K; Strong, J

    1982-02-15

    Rat liver microsomes and purified NADPH-cytochrome c reductase metabolized [14C]misonidazole anaerobically to a reactive intermediate that covalently binds to tissue macromolecules. Air strongly inhibited the binding whereas carbon monoxide had no effect, indicating that misonidazole is activated via reduction and not by cytochrome P-450-dependent oxidation. Both systems showed an absolute requirement for NADPH and were stimulated by flavine (FAD) and paraquat. The apparent Km for misonidazole binding to microsomal protein was 0.74 mM the apparent Vmax was 0.64 nmole 14C bound . mg-1 . min-1. At a single substrate concentration, nitrofurantoin, nitrofurazone and desmethylmisonidazole inhibited the covalent binding of misonidazole to microsomal protein by 47, 26, and 38% respectively. The effect of nitrofurantoin on the kinetics of misonidazole binding gave a complex interaction indicative of uncompetitive inhibition. Glutathione reduced the binding of misonidazole to microsomal protein below the level observed for boiled microsomes while ascorbic acid had no effect. Compared to nitrofurantoin and paraquat, misonidazole was a poor stimulator of superoxide production as measured by adrenochrome formation.

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

    PubMed

    Mavelli, Fabio; Marangoni, Roberto; Stano, Pasquale

    2015-06-01

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

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

    PubMed

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

    2007-01-01

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

  9. Quantitative aspects of protein fractional synthesis rates in ruminants.

    PubMed

    Lescoat, P; Sauvant, D; Danfaer, A

    1997-01-01

    Protein fractional synthesis rate (FSR) is a key-factor in the characterisation of ruminant metabolism. Published data from the literature were collected and statistically analysed to isolate the factors influencing FSR. FSR varied largely depending on the tissue considered, over a range from 1 to 20. FSR, with the plasma as the precursor pool for protein synthesis, was halved compared to that of the intracellular pool. The method for supplying the amino acid also significantly affects FSR since the flooding dose technique gave higher FSR estimates than the constant infusion technique. The choice of the labelled amino acid infused influenced FSR. There is a ranking order depending on the tissue or organ. The protein and energy levels of the diets significantly increased FSR, which raises the question of the body nitrogen requirements. Moreover, FSR values were dependent on the physiological status of the animals. To conclude, FSR values should be determined simultaneously with other biological parameters in order to obtain a realistic quantitative estimate of the nitrogen turnover rates during intermediary metabolism.

  10. Creating a completely "cell-free" system for protein synthesis.

    PubMed

    Smith, Mark Thomas; Bennett, Anthony M; Hunt, Jeremy M; Bundy, Bradley C

    2015-01-01

    Cell-free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell-free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell-free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell-free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell-free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems' protein synthesis capabilities. Lyophilization provides the additional benefit of long-term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely "cell-free" systems. © 2015 American Institute of Chemical Engineers.

  11. 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.

  12. Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S

    PubMed Central

    1986-01-01

    The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K- dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events. PMID:2939094

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

    PubMed

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

    2009-07-20

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

  14. 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. Copyright © 2016 the American Physiological Society.

  15. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway

    SciTech Connect

    Bernhard, W.R.; Thoma, S.; Botella, J.; Somerville, C.R. )

    1991-01-01

    A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.

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

    PubMed

    Pace, Douglas A; Manahan, Donal T

    2007-04-01

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

  17. Co-purification of microsomal epoxide hydrolase with the warfarin-sensitive vitamin K1 oxide reductase of the vitamin K cycle.

    PubMed

    Guenthner, T M; Cai, D; Wallin, R

    1998-01-15

    Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level or epoxide hydrolase activity against two xenobiotic epoxide substrates. The K(m) values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25-35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

  18. ATP-regulated chloride conductance in endoplasmic reticulum (ER)-enriched pig pancreas microsomes.

    PubMed

    Bégault, B; Anagnostopoulos, T; Edelman, A

    1993-11-07

    The Cl- conductance of endoplasmic reticulum-enriched pancreatic microsomes was identified. Its regulation by nucleotides was investigated by measuring the rate of cation ionophore-induced microsome swelling in the presence of an inward Cl- gradient. The conductance was solubilized and reconstituted into liposomes. The Cl- conductance in intact microsomes was inhibited by stilbene (10(-4) M) and indanyloxyacetic acid (10(-5) M) derivatives. ATP increased Cl- conductance with half-maximal stimulation at 8 x 10(-6) M. Other trinucleotides (GTP, CTP and UTP) were without effect at 10(-4) M. The non-hydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate (AppCH2p) increased Cl- conductance with a potency similar to that of ATP. The same concentration of adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) which is a substrate for kinases, had no effect. ATP stimulation of Cl- conductance was inhibited by stilbene derivatives. The data suggest the presence of at least one ATP-binding site, and show that the ATP does not need to be hydrolyzed and that its spatial conformation is important for activating the Cl- conductance. Solubilized microsomal proteins reconstituted into liposomes retained their stilbene-inhibited, ATP-stimulated Cl- conductance. A 167 kDa protein was detected by anti-CFTR antibodies in the intact microsomes, but not in the solubilized proteins. The 64 kDa protein (a component of a ubiquitous Cl- channel) was detected in the both intact and solubilized microsomes. These results suggest that this Cl- conductance is not a CFTR protein.

  19. 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. © 2014 Wiley Periodicals, Inc.

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

    PubMed Central

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

    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. J. Cell. Physiol. 230: 1064–1074, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:25283437

  1. Colostrum enhances the nutritional stimulation of vital organ protein synthesis in neonatal pigs.

    PubMed

    Burrin, D G; Davis, T A; Ebner, S; Schoknecht, P A; Fiorotto, M L; Reeds, P J

    1997-07-01

    Our objective was to determine the relative importance of the macronutrient components of colostrum in the stimulation of vital organ protein synthesis in neonatal pigs. We studied colostrum-deprived newborn pigs within 4-6 h after birth (unfed) and three groups fed for 24 h mature milk, colostrum, or a formula containing a macronutrient composition comparable to that of colostrum. We measured protein synthesis in vivo using a flooding dose of 3H-phenylalanine. The fractional rates of protein synthesis (Ks) in the brain, heart, lung, kidney and spleen were significantly higher in all fed groups than in the unfed newborns. Among the three fed groups, brain and heart protein synthesis rates were greater in colostrum-fed than in either milk- or formula-fed pigs. Kidney and spleen protein synthesis rates in colostrum- and formula-fed pigs were not significantly different, but both were higher than in milk-fed pigs. The stimulation of kidney protein synthesis in response to feeding was primarily a consequence of greater protein synthetic efficiency; however, protein synthetic capacity in the heart, lung and spleen was generally greater in colostrum- and formula-fed pigs than in unfed newborns. Our results suggest that the predominant stimulus for vital organ protein synthesis in colostrum-fed neonatal pigs is nutrient intake. However, there was a specific stimulation of both brain and heart protein synthesis in colostrum-fed pigs that cannot be attributed to macronutrients.

  2. 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.

  3. 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

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

    PubMed

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

    2012-10-25

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

  5. Porcine colostrum and milk stimulate visceral organ and skeletal muscle protein synthesis in neonatal piglets.

    PubMed

    Burrin, D G; Shulman, R J; Reeds, P J; Davis, T A; Gravitt, K R

    1992-06-01

    Our objective was to determine the relative contributions of protein synthesis and protein absorption in the rapid accretion of gastrointestinal protein in suckling piglets during the early neonatal period. We measured the rates of tissue protein synthesis using a flooding dose of L-[4-3H]phenylalanine in various visceral and peripheral tissues of neonatal piglets fed water, mature milk or colostrum for 6 h. The jejunal and ileal protein synthesis rates in piglets fed either colostrum or milk were three- to fourfold higher than in piglets fed water. The increased jejunal and ileal protein synthesis could not, however, account for the differences in protein mass between the colostrum-fed and water-fed groups. The relative abundance of IgG, a major porcine colostral protein, in jejunal tissue was markedly higher in piglets fed colostrum than in piglets fed either milk or water. The fractional protein synthesis rates in liver, kidney, spleen and skeletal muscle and the absolute protein synthesis rates in liver and spleen were also greater in piglets fed colostrum than in those fed milk or water. Increased endogenous protein synthesis made only a minor contribution to the increased intestinal protein accretion in neonatal piglets fed colostrum. A much larger proportion of this increase seemed to be a result of absorption and retention of ingested immunoglobulins.

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

    PubMed

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

    2006-06-01

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

  7. Semisynthetic tRNA complement mediates in vitro protein synthesis.

    PubMed

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

    2015-04-08

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

  8. Molecular insights into protein synthesis with proline residues.

    PubMed

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

    2016-12-01

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

  9. 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.

  10. Spectroscopic quantitation of cytochrome P-450 in human lung microsomes.

    PubMed

    Wheeler, C W; Guenthner, T M

    1990-01-01

    The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.

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

    PubMed

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

    2009-05-01

    Propranolol administration has been demonstrated to improve cardiac work, decrease energy expenditure, and attenuate lipolysis in burned patients; however, its effect on wound healing has not been reported. In rabbits, a partial-thickness skin donor site wound was created on the back, and catheters were placed in the carotid artery and jugular vein. A nasogastric feeding tube was placed for enteral feeding. On day 5 after injury, stable isotope tracers were infused to determine protein and DNA kinetics in the wound. Propranolol hydrochloride was injected in 1 group during the tracer infusion to decrease heart rate, and the other group without propranolol injection served as a control. The propranolol infusion decreased heart rate by 21%. The protein fractional synthetic rate in the wound was greater in the propranolol group (8.6 +/- 0.9 vs 6.1 +/- 0.5%/day, P < .05). Wound protein fractional breakdown rates were not significantly different. The rate of protein deposition (synthesis - breakdown) was increased in the propranolol group (5.0 +/- 1.2 vs 2.8 +/- 0.7%/day, P = .07). Wound DNA fractional synthetic rates were comparable. The protein fractional synthetic rate was correlated with percent decrease in heart rate, but expression of the beta-adrenergic receptors and downstream signaling cascades in local wounds were not affected after propranolol treatment. Propranolol infusion increased wound protein synthetic rate and tended to increase wound protein deposition rate, which might be beneficial to wound healing. These changes might reflect a systemic response to the beta-adrenergic blockade.

  12. 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

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

    PubMed

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

    2015-10-21

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

  14. Continuous cell-free protein synthesis using glycolytic intermediates as energy sources.

    PubMed

    Kim, Ho-Cheol; Kim, Tae-Wan; Park, Chang-Gil; Oh, In-Seok; Park, Kyungmoon; Kim, Dong-Myung

    2008-05-01

    In this work, we demonstrate that glycolytic intermediates can serve as efficient energy sources to regenerate ATP during continuous-exchange cell-free (CECF) protein synthesis reactions. Through the use of an optimal energy source, approximately 10 mg/ml of protein was generated from CECF protein synthesis reaction at greatly reduced reagent costs. Compared with the conventional reactions utilizing phosphoenol pyruvate as an energy source, the described method yields 10-fold higher productivity per unit reagent cost, making the techniques of CECF protein synthesis more realistic alternative for rapid protein production.

  15. ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER

    PubMed Central

    Beaufay, Henri; Amar-Costesec, Alain; Thinès-Sempoux, Denise; Wibo, Maurice; Robbi, Mariette; Berthet, Jacques

    1974-01-01

    Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, β-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity. PMID:4150490

  16. Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff.

    PubMed

    Cao, Shuai; Dhungel, Pragyesh; Yang, Zhilong

    2017-09-01

    Many viral infections cause host shutoff, a state in which host protein synthesis is globally inhibited. Emerging evidence from vaccinia and influenza A virus infections indicates that subsets of cellular proteins are resistant to host shutoff and continue to be synthesized. Remarkably, the proteins of oxidative phosphorylation, the cellular-energy-generating machinery, are selectively synthesized in both cases. Identifying mechanisms that drive selective protein synthesis should facilitate understanding both viral replication and fundamental cell biology. Copyright © 2017 American Society for Microbiology.

  17. Comparison of protein synthesis and degradation in incubated and perfused muscle.

    PubMed Central

    Clark, A S; Mitch, W E

    1983-01-01

    Rates of muscle protein synthesis and degradation measured in the perfused hindquarter were compared with those in incubated epitrochlearis muscles. With fed or starved mature rats, results without insulin treatment were identical. With insulin treatment, protein synthesis in perfused hindquarters was greater, though protein degradation was the same. Thus rates of muscle protein degradation estimated by these two methods in vitro correspond closely. PMID:6349623

  18. 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

  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. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  20. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.

    PubMed

    Blackburn, Matthew C; Petrova, Ekaterina; Correia, Bruno E; Maerkl, Sebastian J

    2016-04-20

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

  1. Effect of Acyclovir on Viral Protein Synthesis in Cells Infected with Herpes Simplex Virus Type 1

    PubMed Central

    Furman, Phillip A.; McGuirt, Paul V.

    1983-01-01

    The effect of the antiviral agent 9-(2-hydroxyethoxymethyl)guanine (acyclovir) on herpes simplex virus type 1 protein synthesis during virus replication was examined. Treatment of infected cells with acyclovir markedly affected the amounts of the four major glycosylated and certain non-glycosylated viral polypeptides synthesized; other viral polypeptides were made in normal amounts. The reduced amount of late protein synthesis was most likely due to the inhibition of progeny viral DNA synthesis by acyclovir. Images PMID:6301368

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

    PubMed Central

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael C.

    2014-01-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications. PMID:24959531

  3. 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…

  4. 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…

  5. 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…

  6. Translate to divide: сontrol of the cell cycle by protein synthesis

    PubMed Central

    Polymenis, Michael; Aramayo, Rodolfo

    2015-01-01

    Protein synthesis underpins much of cell growth and, consequently, cell multiplication. Understanding how proliferating cells commit and progress into the cell cycle requires knowing not only which proteins need to be synthesized, but also what determines their rate of synthesis during cell division. PMID:28357283

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  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. 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…

  11. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Long-term leucine induced stimulation of muscle protein synthesis is amino acid dependent

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  15. Effects of aging and life-prolonging diet on thyroid regulation of protein synthesis.

    PubMed

    Gromakova, I A; Konovalenko, O A

    2004-03-01

    The effect of thyroxin on the intensity of protein synthesis in rats of different age was studied during natural aging and in rats maintained on a low-caloric diet inhibiting aging. The intensity of protein synthesis decreased and the reaction to hormonal stimulus was absent in animals fed life-prolonging diet.

  16. 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.

  17. 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

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

    PubMed

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

    2015-04-01

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

  19. Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards.

    PubMed

    Clapper, M L; Tew, K D

    1989-06-15

    Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WRr and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.

  20. 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.

  1. Effects of Amino Acids on Protein Synthesis by Cellular and Subcellular Preparations from Ischaemic Livers

    PubMed Central

    Cajone, F.; Schiaffonati, L.

    1974-01-01

    The effects of liver ischaemia on protein synthesis have been studied in tissue slices from ischaemic livers, incubated in vitro. There is a rapid decrease in protein synthesis after the onset of ischaemia, which is more severe in slices than in any cell-free preparation. Liver slices from ischaemic livers do not respond to the presence of a full complement of amino acids with an increase in protein synthesis. The presence of amino acids in the incubation medium does not protect the state of aggregation of the polysomes in the slices from ischaemic livers, as occurs in normal liver slices. Isolated ribosomes, obtained from ischaemic livers and incubated with normal purified factors, show a reduced—but still evident—increase in their protein synthesis in the presence of a full complement of amino acids. This suggests that the decay of cell sap factors also plays a role in the impairment of protein synthesis caused by ischaemia. PMID:4447791

  2. Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis.

    PubMed

    Nagano, Hikaru; Sugihara, Shouhei; Takagi, Hisanori; Ogasawara, Tomio; Endo, Yaeta; Takai, Kazuyuki

    2008-01-01

    The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution of protein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryotic elongation factors (eEFs) 1A, 1B, and 2. The eEF1A and eEF2 fractions supported polyphenylalanine synthesis.

  3. In vivo downregulation of protein synthesis in the snail Helix apersa during estivation.

    PubMed

    Pakay, Julian L; Withers, Philip C; Hobbs, Andrew A; Guppy, Michael

    2002-07-01

    Protein synthesis is downregulated during metabolic depression in a number of systems where the metabolic depression is effected by obvious extrinsic cues. The metabolic depression of the estivating land snail Helix apersa occurs in the absence of any obvious physiological stress and has an intrinsic component independent of temperature, pH, O(2) status, or osmolality. We show that this metabolic depression is accompanied by a downregulation of protein synthesis in vivo. The rate of protein synthesis decreases in two major tissues during estivation: to 23% and 53% of the awake rate in hepatopancreas and foot muscle, respectively. We show from calculations of the theoretical contribution of protein synthesis to total O(2) consumption that the depression of protein synthesis must be a significant, obligate, in vivo component of metabolic depression in H. aspersa.

  4. N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

    PubMed

    Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

    2010-07-01

    Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

  5. Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor.

    PubMed

    Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

    2011-10-01

    Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

  6. Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor*

    PubMed Central

    Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G.; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

    2011-01-01

    Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism. PMID:21749988

  7. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes

    PubMed Central

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md.; Gregory, Brian; Samsel, Leigh; Zengel, Janice M.; Lindahl, Lasse

    2013-01-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle. PMID:24109599

  8. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

    PubMed

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md; Gregory, Brian; Samsel, Leigh; Zengel, Janice M; Lindahl, Lasse

    2013-12-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

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

    PubMed

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

    2015-01-01

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

  10. Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase.

    PubMed

    Valenzuela, M A; López, J; Depix, M; Mancilla, M; Kettlun, A M; Catalán, L; Chiong, M; Garrido, J; Traverso-Cori, A

    1989-01-01

    1. Apyrase (ATP: diphosphohydrolase) has been found in the microsomal fraction of rat salivary gland, mammary gland and uterus. 2. This enzyme, already described in plant tissue, is mainly present as a soluble polypeptide in tubers of Solanum tuberosum. 3. A fraction of this enzyme is associated with the microsomal fraction with a higher specific activity than the soluble one, for either ATP or ADP as substrate. 4. Apyrase bound to microsomes from rat and potato tissues was characterized in its substrate specificity and effect of inhibitors. 5. The Km values for ATP and ADP, optimum pH and metal ion requirement were determined. 6. A characteristic common to the microsomal and soluble apyrases is the stimulatory effect of a potato activator protein of soluble plant apyrase. 7. The microsomal-bound apyrase from rat and potato tissues were solubilized and subjected to size-exclusion chromatography. 8. The mammary gland and salivary gland apyrases eluted as molecular aggregates, in contrast to the uterus and potato enzyme.

  11. Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation.

    PubMed

    Abas, Lindy; Luschnig, Christian

    2010-06-15

    Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  12. Evidence for microsomal variation of opioid sites not coupled to N/sub i/

    SciTech Connect

    Spain, J.W.; Coscia, C.J.

    1986-03-05

    Previously, the authors demonstrated a multi-step association of variation of opioid agonists to bovine hippocampal synaptic membranes (SPM). A slowly formed high affinity state is most sensitive to the GTP analog, Gpp(NH)p. Multi-step association can be inferred from the assoc.-time dependent rate of dissociation seen for agonists, i.e., following a brief incubation period the rate of dissociation is more rapid than following a longer association. When 1 nM /sup 3/H-DADL (in the presence of 20 nM unlabeled DAGO) was incubated with microsomal membranes for 7, 12, 20, or 60 min, the off-rate remained constant, suggesting a bimolecular interaction. The monophasic rate of association to microsomes contrasted with the observed biphasic association to SPM. Computer simulation of binding models indicated that, while binding kinetics for SPM can best be simulated by a three-step sequential equilibrium, the pattern for microsomes represents a bimolecular interaction. In contrast to the profound increase in rate of dissociation from SPM's in the presence of Gpp(NH)p, microsome kinetics are not affected. The authors proposed that for SPM's the slowly formed state with a high sensitivity to Gpp(NH)p is a form of the ternary complex consisting of receptor, ligand and GTP binding protein. These results suggest that microsomal membranes, which they believe are primarily internal sites, do not form the ternary complex and are not coupled to N/sub i/.

  13. Alphavirus RNA synthesis and non-structural protein functions

    PubMed Central

    Rupp, Jonathan C.; Sokoloski, Kevin J.; Gebhart, Natasha N.

    2015-01-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field. PMID:26219641

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

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

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

  15. Altered Protein Synthesis is a Trigger for Long-term Memory Formation

    PubMed Central

    Klann, Eric; Sweatt, J. David

    2008-01-01

    Summary There is ongoing debate concerning whether new protein synthesis is necessary for, or even contributes to, memory formation and storage. This review summarizes a contemporary model proposing a role for altered protein synthesis in memory formation and its subsequent stabilization. One defining aspect of the model is that altered protein synthesis serves as a trigger for memory consolidation. Thus, we propose that specific alterations in the pattern of neuronal protein translation serve as an initial event in long-term memory formation. These specific alterations in protein read-out result in the formation of a protein complex that then serves as a nidus for subsequent perpetuating reinforcement by a positive feedback mechanism. The model proposes this scenario as a minimal but requisite component for long-term memory formation. Our description specifies three aspects of prevailing scenarios for the role of altered protein synthesis in memory that we feel will help clarify what, precisely, is typically proposed as the role for protein translation in memory formation. First, that a relatively short initial time window exists wherein specific alterations in the pattern of proteins translated (not overall protein synthesis) is involved in initializing the engram. Second, that a self-perpetuating positive feedback mechanism maintains the altered pattern of protein expression (synthesis or recruitment) locally. Third, that other than the formation and subsequent perpetuation of the unique initializing proteins, ongoing constitutive protein synthesis is all that is minimally necessary for formation and maintenance of the engram. We feel that a clear delineation of these three principles will assist in interpreting the available experimental data, and propose that the available data are consistent with a role for protein synthesis in memory. PMID:17919940

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

    PubMed Central

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

    1994-01-01

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

  17. Design, synthesis, and evaluation of an alpha-helix mimetic library targeting protein-protein interactions.

    PubMed

    Shaginian, Alex; Whitby, Landon R; Hong, Sukwon; Hwang, Inkyu; Farooqi, Bilal; Searcey, Mark; Chen, Jiandong; Vogt, Peter K; Boger, Dale L

    2009-04-22

    The design and solution-phase synthesis of an alpha-helix mimetic library as an integral component of a small-molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate alpha-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 x 20 x 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i + 4, i + 7 amino acid side chains of an alpha-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long-term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead alpha-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an alpha-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

  18. A unified view of the initiation of protein synthesis.

    PubMed

    Nakamoto, Tokumasa

    2006-03-17

    The mechanism of the initiation of protein synthesis is discussed in terms of two different hypotheses in which each emphasized a different possible element of the process: the Shine-Dalgarno (SD) hypothesis ascribed an essential role to recognition of the SD segment by the ribosomal RNA; it is supported by a variety of experiments but conflicting evidence negates its obligatory nature. In contrast, our hypothesis highlighted the role of the structure of the mRNA and proposes that the initiation codon is selected by virtue of its unique accessibility. The rationale for the importance of accessibility in the selection of the initiation site is discussed. An analysis and a recapitulation of the initiation process and ribosomal specificity are presented. The apparent conflicts with the SD hypothesis are resolved in a unified mechanism where accessibility is the dominant factor.

  19. Testicular proteins, nucleic acids and their synthesis following gamma irradiation

    SciTech Connect

    Bansal, M.R.; Kaul, A.; Nehru, B. )

    1989-01-01

    The effects of two doses (250 and 1000 rads) of local gamma irradiation on testes of adult rats are reported after 1, 2, 4 and 16 weeks. There was a significant increase in DNA content per gm testes at 1 weeks; a gradual decrease at 2 and 4 week intervals was followed by a trend towards recovery at 16 weeks post-irradiation. The rate of synthesis of both DNA and RNA as studied by the incorporation of (3H)- thymidine and (3H)-uridine, showed similar results. Total protein content per gm testis declined with both doses and at all post-irradiation intervals. Histological observation showed loss of spermatogenic cells suggestive of DNA loss.

  20. Design and synthesis of a protein. beta. -turn mimetic

    SciTech Connect

    Olson, G.L.; Voss, M.E.; Hill, D.E.; Kahn, M.; Madison, V.S.; Cook, C.M. )

    1990-01-03

    A nine-membered-ring lactam system (1) has been chosen as a framework for the development of non-peptide molecules to mimic structural features of peptide and protein {beta}-turns. The synthesis of model di- and tetrapeptide mimetics starting from 1,5-cyclooctadiene derivatives is reported. In the model dipeptide mimetic (9), the amide linkages is trans (NMR, X-ray) and functional groups at positions adjacent to the lactam amide bond correspond closely to the side-chain positions of residues i + 1 and i + 2 of classical type II{prime} {beta}-turns. In the model tetrapeptide mimetic (30), all four side chains of low-energy trans amide conformers of the mimetic are well matched to their peptide counterparts.

  1. 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.

  2. Development of QSAR models for microsomal stability: identification of good and bad structural features for rat, human and mouse microsomal stability

    NASA Astrophysics Data System (ADS)

    Hu, Yongbo; Unwalla, Ray; Denny, R. Aldrin; Bikker, Jack; Di, Li; Humblet, Christine

    2010-01-01

    High throughput microsomal stability assays have been widely implemented in drug discovery and many companies have accumulated experimental measurements for thousands of compounds. Such datasets have been used to develop in silico models to predict metabolic stability and guide the selection of promising candidates for synthesis. This approach has proven most effective when selecting compounds from proposed virtual libraries prior to synthesis. However, these models are not easily interpretable at the structural level, and thus provide little insight to guide traditional synthetic efforts. We have developed global classification models of rat, mouse and human liver microsomal stability using in-house data. These models were built with FCFP_6 fingerprints using a Naïve Bayesian classifier within Pipeline Pilot. The test sets were correctly classified as stable or unstable with satisfying accuracies of 78, 77 and 75% for rat, human and mouse models, respectively. The prediction confidence was assigned using the Bayesian score to assess the applicability of the models. Using the resulting models, we developed a novel data mining strategy to identify structural features associated with good and bad microsomal stability. We also used this approach to identify structural features which are good for one species but bad for another. With these findings, the structure-metabolism relationships are likely to be understood faster and earlier in drug discovery.

  3. Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

    USDA-ARS?s Scientific Manuscript database

    UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

  4. Fate of a terminal olefin with Drosophila microsomes and its inhibitory effects on some P-450 dependent activities.

    PubMed

    Cuany, A; Helvig, C; Amichot, M; Pflieger, P; Mioskowski, C; Salaun, J P; Pauron, D; Larroque, C; Berge, J B

    1995-01-01

    In vitro bioassays were used to analyze the metabolism of the 11-dodecenoic acid (11-DDNA) by microsomes prepared from Drosophila melanogaster RalDDTR strain. 11-DDNA is metabolized to 11,12-epoxylauric acid (epoxyLA) in a NADPH-dependent way. The microsomal production of epoxyLA reaches a plateau very quickly, suggesting the occurrence of an enzyme inactivation process. After incubation of microsomes with (1-14C)11-DDNA, three proteins of Mr approximately 50 kDa were labeled. 11-DDNA inhibits the microsomal metabolism of lauric acid and 7-ethoxycoumarin in a time and NADPH-dependent process. An inhibition of metabolites generated from DDT and testosterone was also obtained but at higher concentrations. These results are discussed according to the fact that RalDDTR is an insecticide resistant strain characterized as a high metabolizer of the insecticide DDT and also of lauric acid, testosterone, and ethoxycoumarin.

  5. The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes.

    PubMed

    Marcolongo, Paola; Fulceri, Rosella; Giunti, Roberta; Margittai, Eva; Banhegyi, Gabor; Benedetti, Angelo

    2012-09-21

    A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.

  6. 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.

  7. Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs.

    PubMed

    Davis, T A; Fiorotto, M L; Beckett, P R; Burrin, D G; Reeds, P J; Wray-Cahen, D; Nguyen, H V

    2001-05-01

    We recently demonstrated in neonatal pigs that, with amino acids and glucose maintained at fasting levels, the stimulation of protein synthesis in longissimus dorsi muscle with feeding can be reproduced by a physiological rise in insulin alone. In the current report, we determine whether the response of protein synthesis to insulin in the neonatal pig is 1) present in muscles of different fiber types, 2) proportional in myofibrillar and sarcoplasmic proteins, 3) associated with increased translational efficiency and ribosome number, and 4) present in other peripheral tissues and in viscera. Hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs infused with 0, 30, 100, or 1,000 ng. kg(-0.66). min(-1) of insulin to reproduce insulin levels present in fasted, fed, refed, and supraphysiological conditions, respectively. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. Insulin increased protein synthesis in gastrocnemius muscle and, to a lesser degree, masseter muscle. The degree of stimulation of protein synthesis by insulin was similar in myofibrillar and sarcoplasmic proteins. Insulin increased translational efficiency but had no effect on ribosome number in muscle. All of these insulin-induced changes in muscle protein synthesis decreased with age. Insulin also stimulated protein synthesis in cardiac muscle and skin but not in liver, intestine, spleen, pancreas, or kidney. The results support the hypothesis that insulin mediates the feeding-induced stimulation of myofibrillar and sarcoplasmic protein synthesis in muscles of different fiber types in the neonate by increasing the efficiency of translation. However, insulin does not appear to be involved in the feeding-induced stimulation of protein synthesis in visceral tissues. Thus different mechanisms regulate the growth of peripheral and visceral tissues in the neonate.

  8. Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

    PubMed

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

    2016-09-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P < 0.01). Myofibrillar protein synthesis rates increased after ingesting MCas-35 (P < 0.01) and were higher after ingesting MCas-35 (0.050% ± 0.005%/h) than after WPH-35 (0.032% ± 0.004%/h) (P = 0.03). The postprandial increase in plasma leucine concentrations was greater after ingesting Whey-35 than after WPH-60 (peak value: 580 ± 18 compared with 378 ± 10 μM, respectively; P < 0.01), despite similar leucine contents (4.4 g leucine). Nevertheless, the ingestion of WPH-60 increased myofibrillar protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an

  9. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    PubMed

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  10. Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes.

    PubMed

    Yang, Fang; Yang, Hong; Ramesh, Aramandla; Goodwin, J Shawn; Okoro, Emmanuel U; Guo, ZhongMao

    2016-01-01

    We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and

  11. Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes

    PubMed Central

    Yang, Fang; Yang, Hong; Ramesh, Aramandla; Goodwin, J. Shawn; Okoro, Emmanuel U.; Guo, ZhongMao

    2016-01-01

    We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of NAD(P)H: quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites

  12. Enantioselective liver microsomal sulphoxidation of albendazole in cattle: effect of nutritional status.

    PubMed

    Virkel, G; Lifschitz, A; Soraci, A; Sansinanea, A; Lanusse, C

    2000-04-01

    1. The enantioselective liver microsomal sulphoxidation of the benzimidazole anthelmintic, albendazole (ABZ), by cattle liver microsomes has been investigated. The influence of nutritional condition on this biotransformation process was also characterized. 2. ABZ was oxidized to its sulphoxide metabolite (ABZSO) in a NADPH concentration-dependent reaction and the (+) and (-) ABZSO enantiomers formed were identified. 3. Vmax (0.27 nmol ABZSO formed per min x mg(-1) microsomal protein) and Km (15.10 microM) for ABZ sulphoxidation by cattle liver microsomes were obtained. Different Vmax (0.11 and 0.16 nmol x min(-1) x mg(-1)) and Km (9.40 and 26.70 microM) characterized the enantioselective formation of (+) and (-) ABZSO antipodes, respectively. 4. Free fatty acid (FFA) concentrations and beta-hydroxybutyrate concentrations (beta-OHB) in serum and liver homogenates were significantly higher in feed-restricted (poor nutritional condition) compared with control animals in an optimal nutritional status. Serum protein concentrations and liver cytosolic glucose 6-phosphate dehydrogenase (G6PD) activity were significantly lower in the feed-restricted compared with control calf. 5. Animal nutritional condition affected the pattern of ABZ sulphoxidation. A higher Km for (total) ABZSO and (+) ABZSO production was observed in the calf subjected to a period of undernutrition. 6. A nutritionally induced impairment in the affinity of microsomal mixed-function oxidases responsible of ABZ oxidation may be responsible for the observed changes in the liver microsomal sulphoxidation of ABZ in the feed-restricted calf. Furthermore, undernutrition may affect primarily the FMO-mediated formation of (+) ABZSO. These in vitro observations agree with the changes observed in vivo following the administration of ABZ to the calf subjected to a dietary restriction.

  13. Inhibitory capacity of human serum on induced microsomal lipoperoxidation.

    PubMed

    Hicks, J J; Medina-Navarro, R

    1995-01-01

    The capacity of human serum for inhibiting in vitro the membrane lipoperoxidation induced by a controlled system (ADP/NADPH + H+/Fe3+) was demonstrated. A concentration of 8 nmol of malondialdehyde was produced in 20 min in rat liver microsomes (1.5 mg of protein) after exposure to an induced lipoperoxidation mixture. Addition of 100 microliters (13.89 mg of protein) of human serum decreased malondialdehyde production nearly 50%. An increase of 25.97% of the inhibitory capacity of serum was obtained by the in vitro addition of 10 microliters/ml of vitamin E. Ten volunteers were supplemented with 400 mg of vitamin E and 1 g of vitamin C/daily for 2 weeks. Their serum inhibitory capacity increased in 12% (p < 0.05). The serum inhibitory capacity for microsomal lipoperoxidation is described herein, and we propose its utilization as an index to determine the individual nonspecific antioxidative defenses against free radical injury and lipoperoxidation in relation to exposure to air pollutants, tobacco smoke, and several acute and chronic diseases, including the hypoxia-reperfusion phenomena.

  14. [Protein and RNA synthesis during the transition of potato tuber meristematic tissue from rest to growth].

    PubMed

    Korableva, N P; Ladyzhenskaia, E P; Metlitskiĭ, L V

    1976-01-01

    The rate of RNA and protein synthesis in resting and growing meristem tissues from isolated tuber growth points under their incubation with labelled precursors is studied. It is found that the label incorporation into RNA is low during deep resting, protein synthesis being rather intensive. Both RNA and protein synthesis is intensified with the beginning of growth period. More homogenous in their metabolic rate set of RNAs is found in resting tissues in contrast with growing ones. The data on fractionation of total RNA preparation on MAK column and in sucrose density gradient showed that synthesis of high molecular weight RNA with metabolic period of 4 hours is stimulated in growing meristems. It is possible that synthesis of certain fractions of RNA and protein is one of regulation mechanisms for resting in meristem tissues.

  15. Cell-free synthesis system suitable for disulfide-containing proteins.

    PubMed

    Matsuda, Takayoshi; Watanabe, Satoru; Kigawa, Takanori

    2013-02-08

    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 (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. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    SciTech Connect

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

    1986-03-01

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

  17. Leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs

    PubMed Central

    Suryawan, A.; Orellana, R. A.; Fiorotto, M. L.; Davis, T. A.

    2012-01-01

    The postprandial rise in amino acids and insulin independently stimulate 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 protein synthesis in piglets. Leucine increases muscle protein synthesis by modulating the activation of mammalian target of rapamycin complex 1 (mTORC1) and signaling components of translation initiation. Leucine increases the phosphorylation of mTOR, 70-kDa ribosomal protein S6 kinase-1 (S6K1), eukaryotic initiation factor (eIF) 4E-binding protein-1 (4EBP1), and eIF4G, decreases eIF2α phosphorylation, and increases the association of eIF4E with eIF4G. However, leucine does not affect the upstream activators of mTOR, that is, protein kinase B (PKB), adenosine monophosphate (AMP)-activated protein kinase (AMPK), and tuberous sclerosis complex 1/2 (TSC1/2), or the activation translation of elongation regulator, eukaryotic elongation factor 2 (eEF2). Leucine’s action can be replicated by α-ketoisocaproate (KIC) but not by norleucine. Interference by rapamycin with the raptor-mTOR interaction blocks leucine-induced muscle protein synthesis. The acute leucine-induced stimulation of muscle protein synthesis is not maintained for prolonged periods, despite continued activation of mTOR signaling, because circulating amino acids fall as they are utilized for protein synthesis. However, when circulating amino acid levels are maintained, the leucine-induced stimulation of muscle protein synthesis is maintained for prolonged periods. Thus, leucine acts as a nutrient signal to stimulate translation initiation but whether this translates into a prolonged increase in protein synthesis depends on the sustained availability of all amino acids. PMID:20935141

  18. Design, synthesis, and characterization of protein-based nanostructures

    NASA Astrophysics Data System (ADS)

    Modica, Justin Alan

    This thesis outlines a modular method for the bottom-up fabrication of molecular architectures and functional molecular assemblies too large for common organic synthesis and too small for current lithographic techniques. The construction of these assemblies relies on the reactions of bifunctional recombinant proteins with small molecule linkers that exploit the rapid kinetics and superior selectivity of enzymes. The selectivity of the bond forming reactions yield precisely-defined covalent assemblies with dimensions on the order of 10 - 100 nm. Preequilibration of reactants ensure rapid kinetics at nano- to low micromolar concentrations even as the molecules approach MDa molecular weights. Consequently, this 'dock-and-lock' strategy offers a superior alternative to standard bioconjugation chemistries available to fabricate biomolecular assemblies. Using this strategy, we are able to prepare and isolate discrete, monodisperse molecules that have linear, cyclic and star geometries. Our ability to isolate these discreet molecular objects offers a distinct advantage over non-covalent biomolecular self-assembly systems as the sizes, and therefore structures, of our assemblies are not concentration dependant. Furthermore, the modularity of our approach allows the incorporation of functional protein domains into architectures that retain their native functionality even in the context of a very complex molecular environment. We show that oligomers of the functional domains and their contraction upon treatment with a calcium stimulus is linearly proportional to the degree of oligomerization thus providing a means toward rational functional nanomaterials design.

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

    PubMed

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

    2014-08-01

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

  20. Microsomal metabolism of trenbolone acetate metabolites ...

    EPA Pesticide Factsheets

    Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

  1. 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.

  2. On the role of protein synthesis in the circadian clock of Neurospora crassa.

    PubMed Central

    Dunlap, J C; Feldman, J F

    1988-01-01

    Inhibitors of protein synthesis reset the biological clocks of many organisms. This has been interpreted to mean either that the synthesis per se of proteins is a step in the oscillatory feedback loop or merely that certain unstable protein(s) are required at certain times of the cycle to complete the feedback loop. We report here that Neurospora strains bearing the clock mutation frq-7 are relatively insensitive to the resetting action of the protein-synthesis-inhibitor cycloheximide. Protein synthesis itself in this mutant is inhibited by the drug to the same extent as in wild type. Since the clock of frq-7 continues to run relatively unimpeded even in the virtual absence of protein synthesis, it is unlikely that synthesis per se can be a part of the feedback cycle. Rather, we suggest that for normal operation of the Neurospora clock, certain protein(s) with a high turnover rate are required daily and, thus, must be resynthesized each day (at least) during discrete times in the cycle. The frq-7 mutation simultaneously alters several distinct clock characteristics--period length, temperature compensation, and resetting by cycloheximide. A model is presented to unify these observations. PMID:2963337

  3. 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

  4. Activite succino-deshydrogenasique des microsomes et mode d'incorporation du succinate 2,3-(14)C dans les acides gras des microsomes de foie de rat in vitro.

    PubMed

    Rous, S; Aubry, L; Bonini, F

    1970-03-16

    The incorporations of 2,3-(14)C-succinate 2-(14)C-acetate into fatty acids of different cellular fractions of rat liver were studied. Acetate was incorporated mainly into supernatant and succinate into microsomal fatty acids. Mitochondria only could intensively decarboxylate pyruvate. Avidine inhibited fatty acid synthesis from succinate mainly in the supernatant. It is suggested that succinate is an important physiological precursor of fatty acids in the liver and that an active succino-dehydrogenase is present in microsomes.

  5. A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

    PubMed Central

    Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

    2014-01-01

    Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins. PMID:25486605

  6. The effect of insulin infusion and food intake on muscle protein synthesis in postabsorptive rats.

    PubMed Central

    Garlick, P J; Fern, M; Preedy, V R

    1983-01-01

    1. Insulin was infused into young male rats in the postabsorptive state. Rates of protein synthesis in skeletal muscle were determined during the final 10 min of infusion from the incorporation of label into protein after intravenous injection of a massive dose of [3H]phenylalanine. Rates of synthesis were not altered during the first 10 min of insulin infusion, but were increased significantly between 10 and 60 min. 2. Rats were infused with different amounts of insulin for 30 min. When concentrations were increased from 10 to 40 microunits/ml of plasma there was no change in muscle protein synthesis, but concentrations higher than 70 microunits/ml caused a significant stimulation. Concentrations below 10 microunits/ml, obtained by infusion of anti-insulin serum, did not depress synthesis below that found in the postabsorptive rat. 3. Infusion of glucose for 30 or 60 min led to an increase in plasma insulin to 40 microunits/ml, but this also failed to stimulate muscle protein synthesis. 4. Rates of synthesis in postabsorptive rats, even when stimulated maximally by insulin, were not so high as those in fed rats or in postabsorptive rats refed for 60 min. However, in fed and refed rats insulin concentrations were below that required to stimulate synthesis in postabsorptive animals. Despite this, infusion of large amounts of insulin into fed rats did not increase synthesis further. 5. The sensitivity of plasma glucose to insulin infusion was different from that of protein synthesis. A decrease in glucose concentration preceded the increase in synthesis and occurred at lower insulin concentrations. 6. It is concluded that changes in circulating insulin may have been partly responsible for the increase in muscle protein synthesis brought about by feeding, but that other factors must also play a part. PMID:6347182

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

    PubMed

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

    2009-06-01

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

  8. The diurnal response of muscle and liver protein synthesis in vivo in meal-fed rats

    PubMed Central

    Garlick, P. J.; Millward, D. J.; James, W. P. T.

    1973-01-01

    1. The rate of protein synthesis in rat tissues was measured by constant intravenous infusion of [14C]tyrosine. A modification has been developed for the method of calculating the rate of protein synthesis in individual tissues from the specific radioactivity of the free and protein-bound amino acid in tissue at the end of the infusion. This technique gives greater accuracy and allows a greater choice of labelled amino acids. The specific radioactivity of free tyrosine in plasma was used to calculate the plasma tyrosine flux, an index of the rate of protein synthesis in the whole body. 2. Young male Wistar rats were allowed access to food for only 4h in every 24h. The tyrosine flux and the rate of protein synthesis in liver and muscle at different periods of time after a single feed were estimated. 3. The tyrosine flux did not alter after feeding nor even after starvation for 48h. 4. The average fractional rate of protein synthesis in muscle was 7.2%/day, i.e. the proportion of the protein mass which is replaced each day. The rate rose after eating and declined during starvation for 48h. In addition the rate of muscle protein synthesis correlated with the growth rate of the rat. 5. In liver the average fractional rate of protein synthesis was 50%/day. There was no change in the rate after eating nor after starvation for 48h. In contrast with muscle this suggests that the changes in protein mass were accompanied by changes in the rate of protein breakdown rather than synthesis. PMID:4786539

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

    PubMed

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Kouw, Imre Wk; Wall, Benjamin T; Burd, Nicholas A; de Groot, Lisette Cpgm; van Loon, Luc Jc

    2017-02-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis rates and the ability to increase muscle protein synthesis after protein ingestion. To our knowledge, no previous studies have evaluated the impact of habituation to either low protein intake (LOW PRO) or high protein intake (HIGH PRO) on the postprandial muscle protein synthetic response. We assessed the impact of LOW PRO compared with HIGH PRO on basal and postprandial muscle protein synthesis rates after the ingestion of 25 g whey protein. Twenty-four healthy, older men [age: 62 ± 1 y; body mass index (in kg/m(2)): 25.9 ± 0.4 (mean ± SEM)] participated in a parallel-group randomized trial in which they adapted to either a LOW PRO diet (0.7 g · kg(-1) · d(-1); n = 12) or a HIGH PRO diet (1.5 g · kg(-1) · d(-1); n = 12) for 14 d. On day 15, participants received primed continuous l-[ring-(2)H5]-phenylalanine and l-[1-(13)C]-leucine infusions and ingested 25 g intrinsically l-[1-(13)C]-phenylalanine- and l-[1-(13)C]-leucine-labeled whey protein. Muscle biopsies and blood samples were collected to assess muscle protein synthesis rates as well as dietary protein digestion and absorption kinetics. Plasma leucine concentrations and exogenous phenylalanine appearance rates increased after protein ingestion (P < 0.01) with no differences between treatments (P > 0.05). Plasma exogenous phenylalanine availability over the 5-h postprandial period was greater after LOW PRO than after HIGH PRO (61% ± 1% compared with 56% ± 2%, respectively; P < 0.05). Muscle protein synthesis rates increased from 0.031% ± 0.004% compared with 0.039% ± 0.007%/h in the fasted state to 0.062% ± 0.005% compared with 0.057% ± 0.005%/h in the postprandial state after LOW PRO compared with HIGH PRO, respectively (P < 0.01), with no differences between treatments (P = 0.25). Habituation to LOW PRO (0.7 g · kg(-1) · d(-1)) compared with HIGH PRO (1.5 g · kg(-1) · d(-1)) augments the postprandial

  10. The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells.

    PubMed

    Monreal-Flores, Jessica; Espinosa-García, María Teresa; García-Regalado, Alejandro; Arechavaleta-Velasco, Fabian; Martínez, Federico

    2017-06-01

    Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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

    PubMed Central

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

    2013-01-01

    Summary Many neuropsychiatric symptoms of fragile X syndrome (FXS) are believed to be a consequence of altered regulation of protein synthesis at synapses. We discovered that lovastatin, a drug that is widely prescribed for treatment of high cholesterol, can correct excess hippocampal protein synthesis in themouse model of FXS and can prevent one of the robust functional consequences of increased protein synthesis in FXS, epileptogenesis. These data suggest that lovastatin is potentially disease modifying, and could be a viable prophylactic treatment for epileptogenesis in FXS. PMID:23352161

  12. 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.

  13. 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.

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

    PubMed Central

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

    1994-01-01

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

  15. Basal lamina inhibition suppresses synthesis of calcium-dependent proteins associated with mammary epithelial cell spreading.

    PubMed

    Rocha, V; Hom, Y K; Marinkovich, M P

    1986-08-01

    Spreading of mouse mammary epithelial cells on collagen gels is closely correlated with the synthesis of a group of putative calcium-binding proteins (CBP) (Braslau et al., Exp cell res 155 (1984) 213). Collagen synthesis was shown to occur during cell spreading, while omission of serum prevented cell spreading and the synthesis of collagen. The proline analogues cis-hydroxyproline and L-azetidine-2-carboxylic acid were shown to inhibit epithelial cell spreading and to suppress the collagen synthesis that occurs during serum-supported cell spreading. Inhibition of collagen synthesis resulted in the inhibition of CBP synthesis associated with cell spreading. In contrast, the collagen cross-linking inhibitor B-aminopropionitrile did not inhibit cell spreading nor did it suppress collagen synthesis; CBP synthesis was also normal during treatment with this inhibitor. Thus, mammary epithelial cell spreading on collagen gels and CBP synthesis can both be suppressed by inhibition of collagen synthesis indicating that they may be integrated in some manner. It is suggested that inhibition of cell spreading during inhibition of collagen synthesis results from failure to assemble a normal basal lamina; this may in turn signal suppression of CBP synthesis.

  16. 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.

  17. Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics.

    PubMed

    Xiao, Lin; Xian, Hongxu; Lee, Kit Yee; Xiao, Bin; Wang, Hongyan; Yu, Fengwei; Shen, Han-Ming; Liou, Yih-Cherng

    2015-10-09

    Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Metabolism of 4'-(9-acridinylamino)methanesulfon-m-anisidide by rat liver microsomes

    SciTech Connect

    Shoemaker, D.D.; Cysyk, R.L.; Gormley, P.E.; DeSouza, J.J.; Malspeis, L.

    1984-05-01

    4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) is metabolized by a hepatic microsomal enzyme system composed of rat liver microsomes, a reduced nicotinamide adenine dinucleotide phosphate-generating system, cytosolic protein (or glutathione), and oxygen. Omission of any one of the components, or incubation under an atmosphere of CO or N/sub 2/, results in inhibition of the reaction. Also, the addition of inhibitors of microsomal metabolism (alpha-naphthoflavone, metyrapone, or SKF 525-A) decreases m-AMSA metabolism. Metabolism of m-AMSA is more rapid with microsomes prepared from rats pretreated with phenobarbital or 3-methylcholanthrene. Two microsomal oxidation products of m-AMSA were isolated and identified as N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohex adiene-1', 4'-dimine (m-AQDI) and 3'-methoxy-4'-(9-acridinylamino-2',5'-cyclohexadien-1'-one (m-AQI). m-AQDI reacts with glutathione to form a product previously identified in in vivo studies as the principal rat biliary metabolite and which is not cytotoxic to cultured L1210 cells. Thus, the end result of the microsomal metabolism of m-AMSA is detoxification. However, the two primary oxidation products (m-AQDI and m-AQI) are considerably more cytotoxic to L1210 cells in vitro than is m-AMSA. The concentration of m-AMSA required to produce a 5-log kill is 1.0 microgram/ml compared to 0.01 microgram/ml for m-AQDI and m-AQI. These results indicate that m-AMSA might undergo bioactivation to form the active cytotoxic species of the drug.

  19. Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation

    PubMed Central

    Wills, E. D.

    1969-01-01

    1. Aminopyrine strongly inhibits NADPH-induced lipid peroxide formation in rat liver microsomes, but ascorbate-induced peroxidation is inhibited to a smaller extent. 2. Aminopyrine oxidation is stimulated by Mg2+ but inhibited by Ca2+. Concentrated solutions (10mm) of iron-chelating agents inhibit aminopyrine oxidation, but the more dilute solutions (0·5mm) of chelators that block lipid peroxide formation do not inhibit aminopyrine oxidation. Microsomes prepared from sucrose–EDTA homogenates rapidly oxidize aminopyrine, but do not form lipid peroxide when incubated with ascorbate or NADPH. 3. Aminopyrine oxidation is strongly inhibited by p-chloromercuribenzoate, less by iodoacetamide and weakly by N-ethylmaleimide. The site of action of these compounds is considered to be a ferredoxin-type protein. GSH and cysteine also inhibit. 4. Other drugs oxidized by microsomes such as caffeine, phenobarbitone and hexobarbitone had either no or little effect on lipid peroxide formation, but codeine inhibited. 5. Most aliphatic hydrocarbons, alcohols, ketones and aldehydes did not affect lipid peroxide formation, but chloroform and carbon tetrachloride inhibited. 6. Many aromatic compounds inhibited lipid peroxide formation. Only aromatic acids were without any effect and phenols and amines were very strong inhibitors. 7. Induction of lipid peroxide formation in microsomes by incubation with ascorbate or NADPH or by treatment with ionizing radiation leads to a sharp decline in the ability of microsomes to oxidize aminopyrine or hydroxylate aniline. 8. It is considered that the two processes of hydroxylation and lipid peroxide formation are closely linked in microsomes. They probably depend on the same electron-transport chain, and peroxide formation, which involves membrane disintegration, may be part of the normal membrane remodelling process. PMID:4390103

  20. Induction of heat-shock protein synthesis in chondrocytes at physiological temperatures.

    PubMed

    Madreperla, S A; Louwerenburg, B; Mann, R W; Towle, C A; Mankin, H J; Treadwell, B V

    1985-01-01

    Induction of heat-shock protein (HSP) synthesis is demonstrated in cultured calf-chondrocytes at temperatures shown to occur in normal human cartilage during experiments subjecting intact cadaverous hip joints to the parameters of level walking. A 70,000 MW heat-shock protein (HSP-70) is synthesized by chondrocytes at temperatures above 39 degrees C, while induction of synthesis of a 110,000 MW HSP only occurs at temperatures of 45 degrees C or greater. These differences in critical temperatures for induction, and data showing differences in kinetics of induction and repression of synthesis, suggest that there are differences in the mechanism of induction of the two HSPs. The duration of HSP synthesis and inhibition of synthesis of normal cellular proteins is directly proportional to the duration and magnitude of the temperature rise. Possible relationships between these new findings and the initiation and progression of degenerative joint disease are discussed.

  1. Interferons modulate mitogen-induced protein synthesis in airway smooth muscle

    PubMed Central

    Goncharova, Elena A.; Lim, Poay N.; Chisolm, Amelia; Fogle, Homer W.; Taylor, Jerome H.; Goncharov, Dmitry A.; Eszterhas, Andrew; Panettieri, Reynold A.

    2010-01-01

    Severe asthma is characterized by increased airway smooth muscle (ASM) mass due, in part, to ASM cell growth and contractile protein expression associated with increased protein synthesis. Little is known regarding the combined effects of mitogens and interferons on ASM cytosolic protein synthesis. We demonstrate that human ASM mitogens including PDGF, EGF, and thrombin stimulate protein synthesis. Surprisingly, pleiotropic cytokines IFN-β and IFN-γ, which inhibit ASM proliferation, also increased cytosolic protein content in ASM cells. Thus IFN-β alone significantly increased protein synthesis by 1.62 ± 0.09-fold that was further enhanced by EGF to 2.52 ± 0.17-fold. IFN-γ alone also stimulated protein synthesis by 1.91 ± 0.15-fold; treatment of cells with PDGF, EGF, and thrombin in the presence of IFN-γ stimulated protein synthesis by 2.24 ± 0.3-, 1.25 ± 0.17-, and 2.67 ± 0.34-fold, respectively, compared with growth factors alone. The mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) inhibition with rapamycin inhibited IFN- and EGF-induced protein synthesis, suggesting that IFN-induced protein synthesis is modulated by mTOR/S6K1 activation. Furthermore, overexpression of tumor suppressor protein tuberous sclerosis complex 2 (TSC2), which is an upstream negative regulator of mTOR/S6K1 signaling, also inhibited mitogen-induced protein synthesis in ASM cells. IFN-β and IFN-γ stimulated miR143/145 microRNA expression and increased SM α-actin accumulation but had little effect on ASM cell size. In contrast, EGF increased ASM cell size but had little effect on miR143/145 expression. Our data demonstrate that both IFNs and mitogens stimulate protein synthesis but have differential effects on cell size and contractile protein expression and suggest that combined effects of IFNs and mitogens may contribute to ASM cell growth, contractile protein expression, and ASM remodeling in asthma. PMID:20382746

  2. Effect of ethanol on CHCl{sub 3} metabolism in hepatic microsomes from Osborne-Mendel rats

    SciTech Connect

    Testai, E.; Gemma, S.; Vittozzi, L.

    1994-11-01

    The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to {sup 14}CHCl{sub 3} reactive intermediates in ethanol-pretreated microsomes were identical to those measured in microsomes from untreated rats at any pO{sub 2}. The distribution of radioactivity obtained after transmethylation of the adducts of {sup 14}CHCl{sub 3} intermediates with microsomal phospholipids (PL) indicated that binding to fatty acyl chains (due to {center_dot}CHCl{sub 2} radicals) increased with decreased pO{sub 2}. On the contrary, the binding to polar heads due to phosgene decreased. The ethanol treatment did not affect binding to either PL moieties. These results indicated that, in our experimental conditions, the in vitro production of both oxidative and reductive intermediates of CHCl{sub 3} in the liver of Osborne-Mendel rats were not influenced by ethanol consumption. 44 refs., 4 figs., 2 tabs.

  3. Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein

    PubMed Central

    Casperson, Shanon L.; Sheffield-Moore, Melinda; Hewlings, Susan J.; Paddon-Jones, Douglas

    2013-01-01

    SUMMARY Background & aim Protein-energy supplementation is routinely employed to combat muscle loss. However, success is often compromised by increased satiety, poor palatability, high costs and low compliance. Methods For 2-weeks we supplemented meals of older individuals with leucine (4 g/meal; 3 meals/day; days 2–14). Metabolic studies were performed prior to (Day 1) and following (Day 15) supplementation. Leucine was not provided on metabolic study days. Venous blood and vastus lateralis muscle biopsies were obtained during a primed constant infusion of L-[ring-13C6] phenylalanine. Mixed muscle fractional synthesis rate (FSR), body composition and markers of nutrient signaling (mTOR, 4E-BP1 and p70S6K1 phosphorylation) were measured before and after a low protein/carbohydrate simulated meal. Results The meal modestly increased FSR on Day 1 (postabsorptive: 0.063 ± 0.004 vs. postprandial: 0.075 ± 0.006%/h; p = 0.03), however, two weeks of leucine supplementation increased postabsorptive FSR (p = 0.004) and the response to the meal (p = 0.01) (postabsorptive: 0.074 ± 0.007 vs. postprandial: 0.10 ± 0.007%/h). Changes in FSR were mirrored by increased phosphorylation of mTOR, 4E-BP1 and p70S6K1 (p ≤ 0.1). No change in fat free mass was observed (p > 0.05). Conclusions In older adults, leucine supplementation may improve muscle protein synthesis in response to lower protein meals. PMID:22357161

  4. 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.

  5. Characterisation of theophylline metabolism in human liver microsomes.

    PubMed Central

    Robson, R A; Matthews, A P; Miners, J O; McManus, M E; Meyer, U A; Hall, P M; Birkett, D J

    1987-01-01

    1. A radiometric high performance liquid chromatographic method is described for the assay of theophylline metabolism in vitro by the microsomal fraction of human liver. 2. Formation of the three metabolites of theophylline (3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid) were linear with protein concentrations to 4 mg ml-1 and with incubation times up to 180 min. 3. The coefficients of variation for the formation of 3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid were 1.2%, 1% and 1.6%, respectively. 4. Theophylline is metabolised by microsomal enzymes with a requirement for NADPH. 5. The mean (n = 7) Km values for 1-demethylation, 3-demethylation and 8-hydroxylation were 545, 630 and 788 microM, respectively, and the mean Vmax values were 2.65, 2.84 and 11.23 pmol min-1 mg-1, respectively. 6. There was a high correlation between the Km and Vmax values for the two demethylation pathways suggesting that the demethylations are performed by the same enzyme. 7. Overall the in vitro studies are consistent with the in vivo results which suggest the involvement of two cytochrome P-450 isozymes in the metabolism of theophylline. PMID:3663445

  6. 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…

  7. 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…

  8. Response of rat brain protein synthesis to ethanol and sodium barbital

    SciTech Connect

    Tewari, S.; Greenberg, S.A.; Do, K.; Grey, P.A.

    1987-01-01

    Central nervous system (CNS) depressants such as ethanol and barbiturates under acute or chronic conditions can induce changes in rat brain protein synthesis. While these data demonstrate the individual effects of drugs on protein synthesis, the response of brain protein synthesis to alcohol-drug interactions is not known. The goal of the present study was to determine the individual and combined effects of ethanol and sodium barbital on brain protein synthesis and gain an understanding of the mechanisms by which these alterations in protein synthesis are produced. Specifically, the in vivo and in vitro effects of sodium barbital (one class of barbiturates which is not metabolized by the hepatic tissue) were examined on brain protein synthesis in rats made physically dependent upon ethanol. Using cell free brain polysomal systems isolated from Control, Ethanol and 24 h Ethanol Withdrawn rats, data show that sodium barbital, when intubated intragastrically, inhibited the time dependent incorporation of /sup 14/C) leucine into protein by all three groups of ribosomes. Under these conditions, the Ethanol Withdrawn group displayed the largest inhibition of the /sup 14/C) leucine incorporation into protein when compared to the Control and Ethanol groups. In addition, sodium barbital when added at various concentrations in vitro to the incubation medium inhibited the incorporation of /sup 14/C) leucine into protein by Control and Ethanol polysomes. The inhibitory effects were also obtained following preincubation of ribosomes in the presence of barbital but not cycloheximide. Data suggest that brain protein synthesis, specifically brain polysomes, through interaction with ethanol or barbital are involved in the functional development of tolerance. These interactions may occur through proteins or polypeptide chains or alterations in messenger RNA components associated with the ribosomal units.

  9. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

    PubMed

    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

    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. 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. 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. 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 vs high protein diet (0.042±0.01 vs 0

  10. 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

  11. All about that Amide Bond: The Sixth Chemical Protein Synthesis (CPS) Meeting.

    PubMed

    Weller, Caroline E; Chatterjee, Champak

    2015-11-01

    Endless potential: The sixth Chemical Protein Synthesis Meeting, held recently in St. Augustine, Florida, showed the potential of peptide and protein chemistry when applied toward understanding and controlling complex biological processes. This report highlights the diverse and cutting-edge protein chemistry presented at the meeting.

  12. Characterization of microsomal and cytosolic alpha-1,2-mannosidases from mung bean hypocotyls.

    PubMed

    Forsee, W T

    1985-10-01

    Microsomal and cytosolic alpha-mannosidase activities, which hydrolyze alpha-1,2-mannosyl-mannose linkages in the Man5GlcNAc2 oligosaccharide, have been isolated from homogenates of mung bean hypocotyls. The alpha-1,2-mannosidase activities were readily distinguished from previously described aryl alpha-mannosidases by several criteria. They were optimally active in the presence of Ca2+ between pH 5.5 and 6, they were inhibited by Zn2+, and they had essentially no activity with p-nitrophenyl-alpha-mannoside. The microsomal and cytosolic alpha-1,2-mannosidases demonstrated specificity for oligosaccharides with terminal nonreducing alpha-1,2-mannosyl linkages, and they were inhibited by mannosyl-mannose disaccharides, with the inhibition decreasing in the order of alpha-1,2-greater than alpha-1,3-greater than alpha-1,6-mannosyl-mannose. The cytosolic alpha-1,2-mannosidase activity, which was present in the 100,000 g supernatant, was separated from the aryl alpha-mannosidase by ammonium sulfate precipitation. The microsomal alpha-1,2-mannosidase, which was tightly associated with the particulate fraction, was solubilized with Triton X-100 and 0.2 M KCl. The two alpha-1,2-mannosidase activities were readily differentiated by gel-filtration chromatography. The solubilized microsomal enzyme chromatographed in approximately the same position as a Mr 460,000 globular protein whereas the cytosolic enzyme was eluted in a retarded position, indicating a much smaller protein.

  13. Orally administered lactoferrin increases hepatic protein synthesis in formula-fed newborn pigs.

    PubMed

    Burrin, D G; Wang, H; Heath, J; Dudley, M A

    1996-07-01

    Lactoferrin is a polypeptide which is abundant in colostrum; however, its biologic effect in the neonate is unknown. The objective was to determine the potentially anabolic effect of orally administered lactoferrin on visceral organ growth and protein synthesis in newborn pigs. We studied a total of 18 unsuckled newborn pigs from six litters. Three pigs from each litter were randomly assigned to one of three dietary treatment groups (n = 6) and bottle-fed (10 mL/h) formula, formula containing physiologic levels (1 mg/mL) of added bovine lactoferrin (bLF), or colostrum. After 24 h of feeding, we measured visceral organ protein synthesis in vivo using a flooding dose of [3H]phenylalanine. We also measured visceral organ protein and DNA mass, as well as intestinal hydrolase activities and villus morphology. Hepatic protein synthesis in pigs fed either formula containing bLF or colostrum was similar and in both groups was significantly higher than in pigs fed formula. Splenic protein synthesis was not significantly different in pigs fed either formula or formula containing bLF, but was significantly higher in colostrum-fed animals. There were no significant differences in small intestinal growth, protein synthesis, or hydrolase activities between newborn pigs fed formula, formula containing bLF, or colostrum. Our results demonstrate that feeding formula containing physiologic concentrations of added bLF increased hepatic protein synthesis in newborn pigs, suggesting that colostrumborne lactoferrin serves an anabolic function in neonates.

  14. Effect of chronic protein ingestion on tyrosine and tryptophan levels and catecholamine and serotonin synthesis in rat brain.

    PubMed

    Choi, SuJean; DiSilvio, Briana; Fernstrom, Madelyn H; Fernstrom, John D

    2011-11-01

    Previous studies have shown that brain tyrosine (TYR) levels and catecholamine synthesis rate increase in rats as chronic dietary protein content increases from 2 to 10% (% weight). A single protein, casein, was examined. The present study explores how TYR levels and catecholamine synthesis (and tryptophan (TRP) levels and serotonin synthesis) change when different proteins are ingested chronically over the same range of dietary protein contents. Male rats ingested for 8 days diets contain 2 or 10% protein (zein, gluten, casein, soy protein, or alpha-lactalbumin). On the last day, they were killed 2.5 hours into the dark period, 30 minutes after receiving an injection of m-hydroxybenzylhydrazine, an inhibitor of aromatic l-amino acid decarboxylase. Brain samples were analyzed for amino acids, including 5-hydroxytryptophan (index of serotonin synthesis rate) and dihydroxyphenylalanine (index of catecholamine synthesis rate), by HPLC-electrochemical detection. TYR levels and catecholamine synthesis rate in brain were unaffected by the particular protein ingested. However, TRP levels and serotonin synthesis rate varied markedly, depending on the protein ingested, with effects being most prominent in the 10% protein groups. The effect of dietary protein on brain TRP correlated very highly with its effect on serotonin synthesis. The results indicate that the protein ingested can chronically modify TRP levels and serotonin synthesis in brain, but not TYR levels or catecholamine synthesis, with effects most distinct at an adequate level of protein intake (10%).

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

    PubMed

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

    2014-10-03

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

  16. Protein kinase D activity controls endothelial nitric oxide synthesis.

    PubMed

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. © 2014. Published by The Company of Biologists Ltd.

  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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

  19. Muscle protein synthesis in response to nutrition and exercise.

    PubMed

    Atherton, P J; Smith, K

    2012-03-01

    Muscle protein synthesis (MPS) is the driving force behind adaptive responses to exercise and represents a widely adopted proxy for gauging chronic efficacy of acute interventions, (i.e. exercise/nutrition). Recent findings in this arena have been progressive. Nutrient-driven increases in MPS are of finite duration (∼1.5 h), switching off thereafter despite sustained amino acid availability and intramuscular anabolic signalling. Intriguingly, this 'muscle-full set-point' is delayed by resistance exercise (RE) (i.e. the feeding × exercise combination is 'more anabolic' than nutrition alone) even 24 h beyond a single exercise bout, casting doubt on the importance of nutrient timing vs. sufficiency per se. Studies manipulating exercise intensity/workload have shown that increases in MPS are negligible with RE at 20-40% but maximal at 70-90% of one-repetition maximum when workload is matched (according to load × repetition number). However, low-intensity exercise performed to failure equalises this response. Analysing distinct subcellular fractions (e.g. myofibrillar, sarcoplasmic, mitochondrial) may provide a readout of chronic exercise efficacy in addition to effect size in MPS per se, i.e. while 'mixed' MPS increases similarly with endurance and RE, increases in myofibrillar MPS are specific to RE, prophetic of adaptation (i.e. hypertrophy). Finally, the molecular regulation of MPS by exercise and its regulation via 'anabolic' hormones (e.g. IGF-1) has been questioned, leading to discovery of alternative mechanosensing-signalling to MPS.

  20. A simple construction of electrochemical liver microsomal bioreactor for rapid drug metabolism and inhibition assays.

    PubMed

    Walgama, Charuksha; Nerimetla, Rajasekhara; Materer, Nicholas F; Schildkraut, Deniz; Elman, James F; Krishnan, Sadagopan

    2015-01-01

    In order to design a green microsomal bioreactor on suitably identified carbon electrodes, it is important to understand the direct electrochemical properties at the interfaces between various carbon electrode materials and human liver microsomes (HLM). The novelty of this work is on the investigation of directly adsorbed HLM on different carbon electrodes with the goal to develop a simple, rapid, and new bioanalytical platform of HLM useful for drug metabolism and inhibition assays. These novel biointerfaces are designed in this study by a one step adsorption of HLM directly onto polished basal plane pyrolytic graphite (BPG), edge plane pyrolytic graphite (EPG), glassy carbon (GC), or high-purity graphite (HPG) electrodes. The estimated direct electron transfer (ET) rate constant of HLM on the smooth GC surface was significantly greater than that of the other electrodes. On the other hand, the electroactive surface coverage and stability of microsomal films were greater on highly surface defective, rough EPG and HPG electrodes compared to the smooth GC and less defective hydrophobic BPG surfaces. The presence of significantly higher oxygen functionalities and flatness of the GC surface is attributed to favoring faster ET rates of the coated layer of thin HLM film compared to other electrodes. The cytochrome P450 (CYP)-specific bioactivity of the liver microsomal film on the catalytically superior, stable HPG surface was confirmed by monitoring the electrocatalytic conversion of testosterone to 6β-hydroxytestosterone and its inhibition by the CYP-specific ketoconazole inhibitor. The identification of optimal HPG and EPG electrodes to design biologically active interfaces with liver microsomes is suggested to have immense significance in the design of one-step, green bioreactors for stereoselective drug metabolite synthesis and drug metabolism and inhibition assays.

  1. Protein Synthesis in Isolated Castor Bean Mitochondria Is Stimulated by Cyanide 1

    PubMed Central

    Kaderbhai, Mustak A.; Beechey, R. Brian; Kaderbhai, Naheed

    1989-01-01

    Cyanide added to isolated castor bean (Ricinus communis L.) mitochondria supplemented with ATP and succinate (or NADH) significantly enhanced the rate and extent of organellar protein synthesis. Cyanide stimulated mitochondrial protein synthesis in a dose-dependent manner with an optimum stimulation of over twofold at 1 millimolar cyanide. At this concentration of cyanide, the mitochondrial respiratory activity, in the presence of succinate (or NADH) and ADP was inhibited by 90%. The stimulatory effect of cyanide on mitochondrial translation was reflected in the increased synthesis of all the proteins synthesized within the organelle. Preliminary evidence indicates a role for the alternative, salicylhydroxamic acid-sensitive, oxidase in the cyanide stimulation of protein synthesis. Images Figure 4 PMID:16666599

  2. 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.

  3. Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

    PubMed Central

    Miller, Jennifer L.; Cimen, Huseyin; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially-encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCδ, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications. PMID:19702336

  4. Role of RNA Synthesis in the Estrogen Induction of a Specific Uterine Protein*

    PubMed Central

    DeAngelo, Anthony B.; Gorski, Jack

    1970-01-01

    The rate of amino acid incorporation into a specific uterine protein (induced protein band) isolated by gel electrophoresis has been shown to be markedly stimulated within an hour after estrogen administration. Injection of actinomycin D (8 mg/kg) prior to estrogen blocks the synthesis of induced protein. The accumulation of the product of the actinomycin D-sensitive step (induced protein band RNA) is significant 15 minutes after estrogen, and its synthesis would appear to be initiated as soon as the estrogen-receptor complex reaches the nucleus. Blocking protein synthesis with puromycin or cycloheximide did not affect the accumulation of induced protein band RNA, indicating that this is one of the earliest macromolecular synthetic events to occur after estrogen administration. PMID:5269235

  5. In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes.

    PubMed

    Maul, Ronald; Warth, Benedikt; Schebb, Nils Helge; Krska, Rudolf; Koch, Matthias; Sulyok, Michael

    2015-06-01

    The mycotoxin deoxynivalenol (DON), formed by Fusarium species, is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DON phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DON glucuronidation in human are lacking. In the present study, the DON metabolism was investigated using human microsomal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal species. Only two of the twelve tested human recombinant UGTs led to the formation of DON glucuronides with a different regiospecificity. UGT2B4 predominantly catalyzed the formation of DON-15-O-glucuronide (DON-15GlcA), while for UGT2B7 the DON-3-O-glucuronide (DON-3GlcA) metabolite prevailed. For human UGTs, liver