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

International Nuclear Information System (INIS)

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

1986-01-01

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

Regulation of protein synthesis during sea urchin early development

International Nuclear Information System (INIS)

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. [ 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

Injury-induced inhibition of small intestinal protein and nucleic acid synthesis

International Nuclear Information System (INIS)

Carter, E.A.; Hatz, R.A.; Yarmush, M.L.; Tompkins, R.G.

1990-01-01

Small intestinal mucosal weight and nutrient absorption are significantly diminished early after cutaneous thermal injuries. Because these intestinal properties are highly dependent on rates of nucleic acid and protein synthesis, in vivo incorporation of thymidine, uridine, and leucine into small intestinal deoxyribonucleic acid, ribonucleic acid, and proteins were measured. Deoxyribonucleic acid synthesis was markedly decreased with the lowest thymidine incorporation in the jejunum (p less than 0.01); these findings were confirmed by autoradiographic identification of radiolabeled nuclei in the intestinal crypts. Protein synthesis was decreased by 6 h postinjury (p less than 0.01) but had returned to normal by 48 h. Consistent with a decreased rate of protein synthesis, ribonucleic acid synthesis was also decreased 18 h postinjury (p less than 0.01). These decreased deoxyribonucleic acid, ribonucleic acid, and protein synthesis rates are not likely a result of ischemia because in other studies of this injury model, intestinal blood flow was not significantly changed by the burn injury. Potentially, factors initiating the acute inflammatory reaction may directly inhibit nucleic acid and protein synthesis and lead to alterations in nutrient absorption and intestinal barrier function after injury

Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits

International Nuclear Information System (INIS)

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

1987-01-01

To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [ 3 H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting

Deducing the kinetics of protein synthesis in vivo from the transition rates measured in vitro.

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Sophia Rudorf

2014-10-01

Full Text Available The molecular machinery of life relies on complex multistep processes that involve numerous individual transitions, such as molecular association and dissociation steps, chemical reactions, and mechanical movements. The corresponding transition rates can be typically measured in vitro but not in vivo. Here, we develop a general method to deduce the in-vivo rates from their in-vitro values. The method has two basic components. First, we introduce the kinetic distance, a new concept by which we can quantitatively compare the kinetics of a multistep process in different environments. The kinetic distance depends logarithmically on the transition rates and can be interpreted in terms of the underlying free energy barriers. Second, we minimize the kinetic distance between the in-vitro and the in-vivo process, imposing the constraint that the deduced rates reproduce a known global property such as the overall in-vivo speed. In order to demonstrate the predictive power of our method, we apply it to protein synthesis by ribosomes, a key process of gene expression. We describe the latter process by a codon-specific Markov model with three reaction pathways, corresponding to the initial binding of cognate, near-cognate, and non-cognate tRNA, for which we determine all individual transition rates in vitro. We then predict the in-vivo rates by the constrained minimization procedure and validate these rates by three independent sets of in-vivo data, obtained for codon-dependent translation speeds, codon-specific translation dynamics, and missense error frequencies. In all cases, we find good agreement between theory and experiment without adjusting any fit parameter. The deduced in-vivo rates lead to smaller error frequencies than the known in-vitro rates, primarily by an improved initial selection of tRNA. The method introduced here is relatively simple from a computational point of view and can be applied to any biomolecular process, for which we have

Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

International Nuclear Information System (INIS)

Sampson, D.A.; Jansen, G.R.

1985-01-01

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- 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

Adeno-associated virus rep protein synthesis during productive infection

International Nuclear Information System (INIS)

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

1989-01-01

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 [ 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

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

Science.gov (United States)

Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

2014-05-20

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

Two transcription products of the vesicular stomatitis virus genome may control L-cell protein synthesis

International Nuclear Information System (INIS)

Dunigan, D.D.; Lucas-Lenard, J.M.

1983-01-01

When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate of protein synthesis ranging from 20 to 85% of that in mock-infected cells. Vesicular stomatitis virus, irradiated with increasing doses of UV light, eventually loses this capacity to inhibit protein synthesis. The UV inactivation curve was biphasic, suggesting that transcription of two regions of the viral genome is necessary for the virus to become inactivated in this capacity. The first transcription produced corresponded to about 373 nucleotides, and the second corresponded to about 42 nucleotides. Inhibition of transcription of the larger product by irradiating the virus with low doses of UV light left a residual inhibition of protein synthesis consisting of approximately 60 to 65% of the total inhibition. This residual inhibition could be obviated by irradiating the virus with a UV dose of greater than 20,000 ergs/mm 2 and was thus considered to represent the effect of the smaller transcription product. In the R1 mutant of another author, the inhibition of transcription of the larger product sufficed to restore protein synthesis to the mock-infected level, suggesting that the smaller transcription product is nonfunctional with respect to protein synthesis inhibition. Extracts from cells infected with virus irradiated with low doses of UV light showed a protein synthesis capacity quite similar to that of their in vivo counterparts, indicating that these extracts closely reflect the in vivo effects of virus infection

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

Science.gov (United States)

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.

Response of rat brain protein synthesis to ethanol and sodium barbital

International Nuclear Information System (INIS)

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 14 C) leucine into protein by all three groups of ribosomes. Under these conditions, the Ethanol Withdrawn group displayed the largest inhibition of the 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 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

Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis

International Nuclear Information System (INIS)

Ortiz, W.

1990-01-01

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 [ 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

Inhibition of skeletal muscle protein synthesis in septic intra-abdominal abscess

International Nuclear Information System (INIS)

Vary, T.C.; Siegel, J.H.; Tall, B.D.; Morris, J.G.; Smith, J.A.

1988-01-01

Chronic sepsis is always associated with profound wasting leading to increased release of amino acids from skeletal muscle. Net protein catabolism may be due to decreased rate of synthesis, increased rate of degradation, or both. To determine whether protein synthesis is altered in chronic sepsis, the rate of protein synthesis in vivo was estimated by measuring the incorporation of [ 3 H]-phenylalanine in skeletal muscle protein in a chronic (5-day) septic rat model induced by creation of a stable intra-abdominal abscess using an E. coli + B. fragilis-infected sterile fecal-agar pellet as foreign body nidus. Septic rats failed to gain weight at rates similar to control animals, therefore control animals were weight matched to the septic animals. The skeletal muscle protein content in septic animals was significantly reduced relative to control animals (0.18 +/- 0.01 vs. 0.21 +/- 0.01 mg protein/gm wet wt; p less than 0.02). The rate of incorporation of [ 3 H]-phenylalanine into skeletal muscle protein from control animals was 39 +/- 4 nmole/gm wet wt/hr or a fractional synthetic rate of 5.2 +/- 0.5%/day. In contrast to control animals, the fractional synthetic rate in septic animals (2.6 +/- 0.2%/day) was reduced by 50% compared to control animals (p less than 0.005). The decreased rate of protein synthesis in sepsis was not due to an energy deficit, as high-energy phosphates and ATP/ADP ratio were not altered. This decrease in protein synthesis occurred even though septic animals consumed as much food as control animals

Simplified analytical methods for the measurement of the synthesis rate of plasma proteins in vivo by the (/sup 14/C)carbonate method

Energy Technology Data Exchange (ETDEWEB)

Caine, S [Glasgow Royal Infirmary (UK); Fleck, A [Charing Cross Hospital, London (UK). Medical School

1984-09-01

A method is described for obtaining the specific activity of /sup 14/C in urea, essential in the measurement of the synthesis rate of a plasma protein in vivo, which is simpler than the original procedure. The principle is the measurement of /sup 14/CO/sub 2/ and NH/sub 4//sup +/ separately, after incubation with urease. A simple alteration gives samples of /sup 13/CO/sub 2/ for mass spectrometry. The 'recoveries' of /sup 14/C and /sup 13/C in urea were invariably between 90 and 96% and the CV was 3%.

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

Science.gov (United States)

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

2017-05-01

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

In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues

International Nuclear Information System (INIS)

Thompson, W.L.; Wannemacher, R.W. Jr.

1990-01-01

Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with [14C]leucine and [3H]thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies

Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review

NARCIS (Netherlands)

Trommelen, J.; Groen, B.; Hamer, H.M.; Groot, de C.P.G.M.; Loon, van L.J.C.

2015-01-01

Background Though it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis rates in vivo in humans. Objective To

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

Science.gov (United States)

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

2016-04-26

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

Measuring protein breakdown rate in individual proteins in vivo

DEFF Research Database (Denmark)

Holm, Lars; Kjaer, Michael

2010-01-01

To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo.......To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo....

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

Science.gov (United States)

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

2017-08-01

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

Protein degradation and protein synthesis in long-term memory formation

Directory of Open Access Journals (Sweden)

Timothy J Jarome

2014-06-01

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

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Science.gov (United States)

Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

1978-09-01

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

Hepatic protein synthetic activity in vivo after ethanol administration

International Nuclear Information System (INIS)

Donohue, T.M. Jr.; Sorrell, M.F.; Tuma, D.J.

1987-01-01

Hepatic protein synthetic activity in vivo was measured by the incorporation of [ 3 H]puromycin into elongating nascent polypeptides of rat liver to form peptidyl-[ 3 H]puromycin. Our initial experiments showed that saturating doses of [ 3 H]puromycin were achieved at 3-6 mumol/100 g body weight, and that maximum labeling of nascent polypeptides was obtained 30 min after injection of the labeled precursor. Labeled puromycin was found to be suitable for measuring changes in the status of protein synthesis, since the formation of the peptidyl-[ 3 H]puromycin was decreased in fasted animals and was increased in rats pretreated with L-tryptophan. [ 3 H]Puromycin incorporation into polypeptides was then measured after acute ethanol administration as well as after prolonged consumption of ethanol which was administered as part of a liquid diet for 31 days. Acute alcohol treatment caused no significant change in [ 3 H]puromycin incorporation into liver polypeptides. In rats exposed to chronic ethanol feeding, peptidyl-[3H]puromycin formation, when expressed per mg of protein, was slightly lower compared to pair-fed controls, but was unchanged compared to chow-fed animals. When the data were expressed per mg of DNA or per 100 g body wt, no differences in protein synthetic activity were observed among the three groups. These findings indicate that neither acute nor chronic alcohol administration significantly affects protein synthetic activity in rat liver. They further suggest that accumulation of protein in the liver, usually seen after prolonged ethanol consumption, is apparently not reflected by an alteration of hepatic protein synthesis

In vivo phosphorylation of axonal proteins in goldfish optic nerve during regeneration

Energy Technology Data Exchange (ETDEWEB)

Larrivee, D.C.; Grafstein, B.

1987-01-01

In vivo phosphorylation of axonal proteins was investigated in normal and regenerating optic nerves of goldfish by two-dimensional gel electrophoresis. By 6-24 h after intraocular injection of H/sub 3/(32)PO/sub 4/, approximately 20 optic nerve proteins ranging in size from 19 to 180 kilodaltons and in pI from 4.4 to 6.8 were seen to have incorporated radiolabel. Five of these proteins showed a robust increase in incorporation of phosphate during regeneration. Among the latter was an acidic (pI 4.5) 45-kilodalton protein, which has previously been shown to be conveyed by fast axonal transport and to increase dramatically in its rate of synthesis during regeneration of goldfish optic axons.

Control of protein synthesis in cell-free extracts of sea urchin embryos

International Nuclear Information System (INIS)

Hansen, L.J.; Huang, W.I.; Jagus, R.

1986-01-01

Although the increase in protein synthesis that occurs after fertilization of sea urchin eggs results from increased utilization of stored maternal mRNA, the underlying mechanism is unknown. The authors have prepared cell-free extracts from S.purpuratus and A.puctulata unfertilized eggs and 2-cell embryos that retain the protein synthetic differences observed in vivo. The method is based on that of Dr. Alina Lopo. 35 S methionine incorporation is linear during a 30 min incubation and is 10-20 fold higher in extracts from 2-cell embryos than unfertilized eggs. Addition of purified mRNA does not stimulate these systems, suggesting a regulatory mechanism other than mRNA masking. Addition of rabbit reticulocyte ribosomal salt wash stimulated protein synthesis in extracts from eggs but not embryos, suggesting deficiencies in translational components in unfertilized eggs. Mixing of egg and embryo lysates indicated the presence of a weak protein synthesis inhibitor in eggs. Translational control in developing sea urchin embryos thus appears to be complex, involving both stimulatory and inhibitory factors

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

Science.gov (United States)

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.

In vivo measurement of myocardial protein turnover using an indicator dilution technique

International Nuclear Information System (INIS)

Revkin, J.H.; Young, L.H.; Stirewalt, W.S.; Dahl, D.M.; Gelfand, R.A.; Zaret, B.L.; Barrett, E.J.

1990-01-01

We applied a nondestructive tracer technique, previously developed for measuring skeletal muscle protein turnover, to the measurement of myocardial protein turnover in vivo. During a continuous infusion of L-[ring-2,6-3H]phenylalanine to anesthetized, overnight-fasted dogs, we measured the uptake of radiolabeled phenylalanine from plasma and the release of unlabeled phenylalanine from myocardial proteolysis using arterial and coronary sinus catheterization and analytic methods previously applied to skeletal muscle. Using these measurements, together with a model of myocardial protein synthesis that assumes rapid equilibration of tracer specific activity between myocardial phenylalanyl-tRNA and circulating phenylalanine, we estimated the rates of heart protein synthesis and degradation. The rate of heart protein synthesis was also estimated directly from the incorporation of labeled phenylalanine into tissue protein. The use of [3H]phenylalanine was compared with L-[1-14C]leucine in the measurement of heart protein turnover in dogs given simultaneous infusion of both tracers. Leucine uptake and release by the myocardium exceeded that of phenylalanine by 3.1 +/- 0.4- and 1.7 +/- 0.3-fold, respectively, consistent with leucine's 2.4-fold greater abundance in heart protein and its metabolism via other pathways. Phenylalanine is the preferred tracer for use with this method because of its limited metabolic fate in muscle. One theoretical limitation to the method, slow equilibration of circulating labeled phenylalanine with myocardial phenylalanyl-tRNA, was resolved by comparison of these specific activities after a 30-minute infusion of labeled phenylalanine in the rat. A second, empirical limitation involves precision in the measurement of the small decrements in phenylalanine specific activity that occur with each pass of blood through the coronary circulation

Protein synthesis evaluation in brain and other organs in human by PET

International Nuclear Information System (INIS)

Bustany, P.; Comar, D.

1985-01-01

The choice of treatment in diseases of the nervous system cannot be based only on symptomatology, but on a presumed underlying pathological state. These pathological states often involve direct modifications of neuronal metabolism. Two areas of cellular biochemistry can be studied in vivo in humans: 1) glucose or oxygen consumption which is mainly responsible for energy and lipid metabolism. 2) amino acid metabolism, which is involved in protein and neurotransmitter synthesis. Here the authors examine protein synthesis, which is the basis of cellular integrity and tissue structure. Study of protein synthesis (PS) by positron emission tomography (PET) is governed by specific requirements dictated by 1) the metabolic pathways we want to explore (the fate of the tracer directly influences the analysis of the results); 2) The construction and validation of a mathematical model to be applied to the computerized images; and 3) the human pathology being studied. The timing of scanning and the experimental protocol must include in their conception some physiological constraints such as volume of organs, rapidity of biological phenomena, etc. All these steps are detailed in the following paragraphs

Modulation of protein synthesis by polyamines.

Science.gov (United States)

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.

Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

Science.gov (United States)

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;Pprotein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;Psynthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;Pprotein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620). In the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and

Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity In Vivo

Science.gov (United States)

Gnanadhas, Divya Prakash; Ben Thomas, Midhun; Thomas, Rony; Raichur, Ashok M.

2013-01-01

The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy in vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity in vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs [with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the in vivo relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities in vivo against Salmonella infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes. PMID:23877702

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

Hiroyuki Kato

2016-06-01

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

MECHANISMS IN ENDOCRINOLOGY: Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review.

Science.gov (United States)

Trommelen, Jorn; Groen, Bart B L; Hamer, Henrike M; de Groot, Lisette C P G M; van Loon, Luc J C

2015-07-01

Though it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis rates in vivo in humans. To assess whether exogenous insulin administration increases muscle protein synthesis rates in young and older adults. A systematic review of clinical trials was performed and the presence or absence of an increase in muscle protein synthesis rate was reported for each individual study arm. In a stepwise manner, multiple models were constructed that excluded study arms based on the following conditions: model 1, concurrent hyperaminoacidemia; model 2, insulin-induced hypoaminoacidemia; model 3, supraphysiological insulin concentrations; and model 4, older, more insulin resistant, subjects. From the presented data in the current systematic review, we conclude that: i) exogenous insulin and amino acid administration effectively increase muscle protein synthesis, but this effect is attributed to the hyperaminoacidemia; ii) exogenous insulin administered systemically induces hypoaminoacidemia which obviates any insulin-stimulatory effect on muscle protein synthesis; iii) exogenous insulin resulting in supraphysiological insulin levels exceeding 50, 000  pmol/l may effectively augment muscle protein synthesis; iv) exogenous insulin may have a diminished effect on muscle protein synthesis in older adults due to age-related anabolic resistance; and v) exogenous insulin administered systemically does not increase muscle protein synthesis in healthy, young adults. © 2015 European Society of Endocrinology.

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

Science.gov (United States)

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

2016-02-01

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

Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

OpenAIRE

Demangeat, Gerard; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, Coralie

1992-01-01

The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a sta...

In vivo somatostatin, vasopressin, and oxytocin synthesis in diabetic rat hypothalamus

International Nuclear Information System (INIS)

Fernstrom, J.D.; Fernstrom, M.H.; Kwok, R.P.

1990-01-01

The in vivo labeling of somatostatin-14, somatostatin-28, arginine vasopressin, and oxytocin was studied in rat hypothalamus after third ventricular administration of [35S]cysteine to streptozotocin-diabetic and normal rats. Immunoreactive somatostatin levels in hypothalamus were unaffected by diabetes, as was the incorporation of [35S]cysteine into hypothalamic somatostatin-14 and somatostatin-28. In contrast, immunoreactive vasopressin levels in hypothalamus and posterior pituitary (and oxytocin levels in posterior pituitary) were below normal in diabetic rats. Moreover, [35S]cysteine incorporation into hypothalamic vasopressin and oxytocin (probably mainly in the paraventricular nucleus because of its proximity to the third ventricular site of label injection) was significantly above normal. The increments in vasopressin and oxytocin labeling were reversed by insulin administration. In vivo cysteine specific activity and the labeling of acid-precipitable protein did not differ between normal and diabetic animals; effects of diabetes on vasopressin and oxytocin labeling were therefore not caused by simple differences in cysteine specific activity. These results suggest that diabetes (1) does not influence the production of somatostatin peptides in hypothalamus but (2) stimulates the synthesis of vasopressin and oxytocin. For vasopressin at least, the increase in synthesis may be a compensatory response to the known increase in its secretion that occurs in uncontrolled diabetes

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

DEFF Research Database (Denmark)

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

2011-01-01

PURPOSE: This study aimed to investigate the effect of protein ingestion on leg protein turnover and vastus lateralis muscle protein synthesis during bicycle exercise and recovery. METHODS: Eight healthy males participated in two experiments in which they ingested either a carbohydrate solution...... sampling, and blood flow measurements. Muscle protein synthesis was calculated from the incorporation of l-[ring-C6]phenylalanine into protein. RESULTS: Consuming protein during exercise increased leg protein synthesis and decreased net leg protein breakdown; however, protein ingestion did not increase...... protein synthesis within the highly active vastus lateralis muscle (0.029%·h(-1), ± 0.004%·h(-1), and 0.030%·h(-1), ± 0.003%·h(-1), in CHO and CHO + P, respectively; P = 0.88). In contrast, consuming protein, during exercise and recovery, increased postexercise vastus lateralis muscle protein synthesis...

Noncovalent synthesis of protein dendrimers

NARCIS (Netherlands)

Lempens, E.H.M.; Baal, van I.; Dongen, van J.L.J.; Hackeng, T.M.; Merkx, M.; Meijer, E.W.

2009-01-01

The covalent synthesis of complex biomolecular systems such as multivalent protein dendrimers often proceeds with low efficiency, thereby making alternative strategies based on noncovalent chemistry of high interest. Here, the synthesis of protein dendrimers using a strong but noncovalent

Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis

International Nuclear Information System (INIS)

Heidenreich, K.A.; Toledo, S.P.

1989-01-01

In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of [3H]leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of [3H]aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that (1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; (2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and (3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons

Suppression of matrix protein synthesis in endothelial cells by herpes simplex virus is not dependent on viral protein synthesis

International Nuclear Information System (INIS)

Kefalides, N.A.

1986-01-01

The synthesis of matrix proteins by human endothelial cells (EC) in vitro was studied before and at various times after infection with Herpes Simplex virus Type 1 (HSV-1) or 2 (HSV-2). Monolayers of EC were either mock-infected or infected with virus for 1 hr at a multiplicity infection (MOI) of 5 to 20 at 37 0 C. Control and infected cultures were pulse-labeled for 1 or 2 hrs with either [ 14 C]proline or [ 35 S]methionine. Synthesis of labeled matrix proteins was determined by SDS-gel electrophoresis. Suppression of synthesis of fibronectin, Type IV collagen and thrombospondin began as early as 2 hrs and became almost complete by 10 hrs post-infection. The degree of suppression varied with the protein and the virus dose. Suppression of Type IV collagen occurred first followed by that of fibronectin and then thrombospondin. Infection of EC with UV irradiated HSV-1 or HSV-2 resulted in suppression of host-cell protein synthesis as well as viral protein synthesis. Infection with intact virus in the presence of actinomycin-D resulted in suppression of both host-cell and viral protein synthesis. The data indicate that infection of EC with HSV leads to suppression of matrix protein synthesis which does not depend on viral protein synthesis

Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

Directory of Open Access Journals (Sweden)

Rick Hursel

Full Text Available 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�

Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta)

Science.gov (United States)

Tischler, M. E.; Wu, M.; Cook, P.; Hodsden, S.

1990-01-01

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.

Protein synthesis in the growing rat lung

International Nuclear Information System (INIS)

Kelley, J.; Chrin, L.

1986-01-01

Developmental control of protein synthesis in the postnatal growth of the lung has not been systematically studied. In male Fischer 344 rats, lung growth continues linearly as a function of body weight (from 75 to 450 g body weight). To study total protein synthesis in lungs of growing rats, we used the technique of constant intravenous infusion of tritiated leucine, an essential amino acid. Lungs of sacrificed animals were used to determine the leucine incorporation rate into newly synthesized protein. The specific radioactivity of the leucine associated with tRNA extracted from the same lungs served as an absolute index of the precursor leucine pool used for lung protein synthesis. On the basis of these measurements, we were able to calculate the fractional synthesis rate (the proportion of total protein destroyed and replaced each day) of pulmonary proteins for each rat. Under the conditions of isotope infusion, leucyl-tRNA very rapidly equilibrates with free leucine of the plasma and of the extracellular space of the lung. Infusions lasting 30 minutes or less yielded linear rates of protein synthesis without evidence of contamination of lung proteins by newly labeled intravascular albumin. The fractional synthesis rate is considerably higher in juvenile animals (55% per day) than in adult rats (20% per day). After approximately 12 weeks of age, the fractional synthesis rate remains extremely constant in spite of continued slow growth of the lung. It is apparent from these data that in both young and adult rats the bulk of total protein synthesis is devoted to rapidly turning over proteins and that less than 4 percent of newly made protein is committed to tissue growth

Influence of nutrition on protein synthesis and 15N tracer data in man

International Nuclear Information System (INIS)

Faust, H.

1984-01-01

Quantitative studies and measurements of parameters of the protein metabolism in vivo require the isotope methodology. Different 15 N tracer methods with special modifications are available which can be used depending on clinical problems. The oral single pulse application of [ 15 N]glycine is equal to other isotope tracer techniques provided that the basic assumptions of the application are fullfilled. The protein metabolism is clearly influenced by the nutritional status whereby the protein synthesis is more sensitive than the breakdown to altered dietary intakes of protein and energy. The importance of standardized experimental conditions is emphasized for studies with 15 N and the interpretation of tracer data. (author)

Increase in tendon protein synthesis in response to insulin-like growth factor-I is preserved in elderly men

DEFF Research Database (Denmark)

Nielsen, Rie Harboe; Holm, Lars; Malmgaard-Clausen, Nikolaj Mølkjær

2014-01-01

the in vivo IGF-I stimulation of tendon protein synthesis in elderly compared with young men. We injected IGF-I in the patellar tendons of young (n = 11, 20-30 yr of age) and old (n = 11, 66-75 yr of age) men, and the acute fractional synthesis rate (FSR) of tendon protein was measured with the stable isotope.......01). This increase in protein synthesis was seen in both young and old men, with no differences between age groups. The old group had markedly lower serum IGF-I levels compared with young (165 ± 17 vs. 281 ± 27 ng/ml, P protein synthesis in both young and old men...... technique and compared with the contralateral side (injected with saline as control). We found that tendons injected with IGF-I had significantly higher protein FSR compared with controls (old group: 0.018 ± 0.015 vs. 0.008 ± 0.008, young group: 0.016 ± 0.009 vs. 0.009 ± 0.006%/h, mean ± SE, P

In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

International Nuclear Information System (INIS)

Mack, John; Vermeiren, Christie; Heinrichs, David E.; Stillman, Martin J.

2004-01-01

We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins

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

OpenAIRE

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 a...

Protein synthesis in geostimulated root caps

Science.gov (United States)

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.

Protein synthesis controls phosphate homeostasis.

Science.gov (United States)

Pontes, Mauricio H; Groisman, Eduardo A

2018-01-01

Phosphorus is an essential element assimilated largely as orthophosphate (Pi). Cells respond to Pi starvation by importing Pi from their surroundings. We now report that impaired protein synthesis alone triggers a Pi starvation response even when Pi is plentiful in the extracellular milieu. In the bacterium Salmonella enterica serovar Typhimurium , this response entails phosphorylation of the regulatory protein PhoB and transcription of PhoB-dependent Pi transporter genes and is eliminated upon stimulation of adenosine triphosphate (ATP) hydrolysis. When protein synthesis is impaired due to low cytoplasmic magnesium (Mg 2+ ), Salmonella triggers the Pi starvation response because ribosomes are destabilized, which reduces ATP consumption and thus free cytoplasmic Pi. This response is transient because low cytoplasmic Mg 2+ promotes an uptake in Mg 2+ and a decrease in ATP levels, which stabilizes ribosomes, resulting in ATP consumption and Pi increase, thus ending the response. Notably, pharmacological inhibition of protein synthesis also elicited a Pi starvation response in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae Our findings identify a regulatory connection between protein synthesis and Pi homeostasis that is widespread in nature. © 2018 Pontes and Groisman; Published by Cold Spring Harbor Laboratory Press.

Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

International Nuclear Information System (INIS)

Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J.

1996-01-01

The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6- 3 H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

Albumin synthesis in protein energy malnutrition

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Duggan, C; Hardy, S; Kleinman, R E [Harvard Medical School, Boston, MA (United States); Lembcke, J [Instituto de Investigacion Nutricional, La Molina, Lima (Peru); Young, V E [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Lab. of Human Nutrition

1994-12-31

The dietary treatment of protein-energy malnutrition (PEM) has been designed on an empirical basis, with outcomes for successful management including body weight gain and resolution of apathy. We propose using the measurements of protein synthesis as a more objective measure of renourishment. We will therefore randomize a group of malnourished children (weigh-for-height Z score <-2.0) to receive either a standard (10% of calories as protein) or increased (15%) amount of dietary protein early in their recovery phase. We will calculate albumin synthesis rates via the flooding dose technique, using {sup 13}C-leucine and serial measurements of {sup 13}C-enrichment of albumin. Isotope infusions will be performed on days one and three, following a standard three hour fast. Since albumin synthesis is reduced under the influence of cytokines which mediate the inflammatory response, results will be stratified according to the presence or absence of clinically apparent infections. We hypothesize that the provision of added dietary protein will optimize albumin synthesis rates in PEM as well as attenuate the reduction in albumin synthesis seen in the presence of infections. (author). 20 refs.

Translational step inhibited in vivo by aflatoxin B1 in rat-liver polysomes

International Nuclear Information System (INIS)

Sarasin, A.; Moule, Y.

1975-01-01

Aflatoxin B 1 strongly inhibits protein synthesis in rat liver cells. This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B 1 . We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451-462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis. Up to 5 h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2 h after dosing, each step of protein synthesis is blocked. Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin-treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin-treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures. Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed. (orig./BSC) [de

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

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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.

Brain protein synthesis in normal and demented patients. A study by P.E.T. with 11C-L methionine

International Nuclear Information System (INIS)

Bustany, P.; Soussaline, F.; Comar, D.; Henry, J.F.

1982-09-01

A compartmental model representing protein synthesis in the brain was validated experimentally in 9 baboons. After sequential injections of 11 C, 3 H and 14 C methionines on the same animal, followed by P.E.T. recording of the γ activity in a chosen brain section with time, the distribution of methionine injected into the different compartments of the model after a bolus was measured by crushing and precipitation with T.C.A. The agreement between direct in vitro findings and computed results is excellent. This method of studying brain protein synthesis in vivo was applied to 28 Alzheimer dementia cases and 20 normal subjects of the same age. The correlation between the results of clinical and psychometric tests and the brain protein synthesis activity confirms an anomaly in this biochemical synthesis process during the illness. A 65% fall in activity may be found in the frontal lobes of certain patients

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

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Lena Thoring

Full Text Available 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.

Synthesis of Lipidated Proteins.

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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.

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

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

2015-09-15

Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

Theiler's virus RNA and protein synthesis in the central nervous system of demyelinating mice

International Nuclear Information System (INIS)

Cash, E.; Chamorro, M.; Brahic, M.

1985-01-01

The authors studied Theiler's virus RNA and capsid protein synthesis in sections of mouse spinal cord using in situ hybridization coupled to immunoperoxidase. They found that the majority of infected cells contain 100 to 500 viral genomes and no detectable capsid antigens. Similarly, baby hamster kidney (BHK) cells, which are permissive to Theiler's virus, do not synthesize capsid if they contain less than 1000 viral genomes. The results demonstrate that virus multiplication is restricted in vivo at the level of RNA replication. They suggest that RNA restriction is sufficient to explain the lack of capsid antigen synthesis

4-(Phenylsulfanylbutan-2-One Suppresses Melanin Synthesis and Melanosome Maturation In Vitro and In Vivo

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Shing-Yi Sean Wu

2015-08-01

Full Text Available In this study, we screened compounds with skin whitening properties and favorable safety profiles from a series of marine related natural products, which were isolated from Formosan soft coral Cladiella australis. Our results indicated that 4-(phenylsulfanylbutan-2-one could successfully inhibit pigment generation processes in mushroom tyrosinase platform assay, probably through the suppression of tyrosinase activity to be a non-competitive inhibitor of tyrosinase. In cell-based viability examinations, it demonstrated low cytotoxicity on melanoma cells and other normal human cells. It exhibited stronger inhibitions of melanin production and tyrosinase activity than arbutin or 1-phenyl-2-thiourea (PTU. Also, we discovered that 4-(phenylsulfanylbutan-2-one reduces the protein expressions of melanin synthesis-related proteins, including the microphthalmia-associated transcription factor (MITF, tyrosinase-related protein-1 (Trp-1, dopachrome tautomerase (DCT, Trp-2, and glycoprotein 100 (GP100. In an in vivo zebrafish model, it presented a remarkable suppression in melanogenesis after 48 h. In summary, our in vitro and in vivo biological assays showed that 4-(phenylsulfanylbutan-2-one possesses anti-melanogenic properties that are significant in medical cosmetology.

Heterotopic cardiac transplantation decreases the capacity for rat myocardial protein synthesis

International Nuclear Information System (INIS)

Klein, I.; Samarel, A.M.; Welikson, R.; Hong, C.

1991-01-01

Heterotopic cardiac isografts are vascularly perfused hearts that maintain structural and functional integrity for prolonged periods of time. When placed in an infrarenal location, the heart is hemodynamically unloaded and undergoes negative growth, leading to cardiac atrophy. At 7 and 14 days after transplantation, the transplanted heart was decreased in size compared with the in situ heart (p less than 0.001). To assess the possible mechanism(s) to account for this reduction in size we studied in vivo rates of total left ventricular (LV) protein synthesis, total LV RNA content, and 18S ribosomal RNA content by nucleic acid hybridization. The LV protein synthetic rate was 4.7 and 5.3 mg/day in the in situ heart and was significantly decreased to 2.9 and 2.7 mg/day in the transplanted hearts at 7 and 14 days, respectively. LV RNA content of the transplant declined to 53% and 48% of the in situ value at 7 and 14 days, respectively. Hybridization studies revealed that LV 18S ribosomal subunit content was reduced proportionately to total RNA in the heterotopic hearts. As a result of these changes, there was no significant difference in the efficiency of total LV protein synthesis between the in situ and transplanted hearts. The present studies demonstrate that the hemodynamic unloading and cardiac atrophy that is characteristic of heterotopic cardiac transplantation is accompanied by a decrease in LV total RNA content and 18S RNA, resulting in a decreased capacity for myocardial protein synthesis

Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes

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Takehiro Nishikawa

2012-01-01

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

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

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Sørensen, Brita Singers; Busk, Morten; Overgaard, Jens; Horsman, Michael R; Alsner, Jan

2015-01-01

protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.

Arraying proteins by cell-free synthesis.

Science.gov (United States)

He, Mingyue; Wang, Ming-Wei

2007-10-01

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

SHORT-TERM MEMORY IS INDEPENDENT OF BRAIN PROTEIN SYNTHESIS

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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.

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

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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.

Cell-free protein synthesis for structure determination by X-ray crystallography.

Science.gov (United States)

Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

2010-01-01

Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. Pt. 1

International Nuclear Information System (INIS)

Skog, S.; Tribukait, B.; Sundius, G.

1985-01-01

The effects of roentgen irradiation on the incorporation of 3 H-uridine and 14 C-leucine into RNA and protein and the RNA and protein contents of in vivo growing Ehrlich ascites tumour cells were studied. The results were related to changes in the composition of cells in cell cycle and compared with the synthesis of RNA and protein in cell material from various parts of the cell cycle obtained by means of elutriator centrifuging. The incorporation expressed by the ratio between acid insoluble/acid soluble activity was unchanged for RNA during the observation period up to 24 hours after a dose of 5.0 Gy. The ratio for protein was markedly decreased between 4 and 24 hours. This decrease was partly due to a decrease of the pool size of leucine as studied by changing the amounts of 14 C leucine used. From these studies, the existence of at least two pools, an expandable and a non-expandable fixed pool can be concluded. There were no differences in the decrease of protein-synthesis between cells from the various parts of the cell cycle. The RNA and protein contents of the irradiated cells from various parts of the cell cycle corresponded to those of non-irradiated cells except for G 1 /early S-phase cells at 15 and 24 hours after irradiation. Possible reasons for this discrepancy are discussed. (orig.)

Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

Energy Technology Data Exchange (ETDEWEB)

Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J. [Queensland Univ., St. Lucia, QLD (Australia). Dept. of Chemistry

1996-12-31

The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6-{sup 3}H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

Protein synthesis in x-irradiated rabbit lens

International Nuclear Information System (INIS)

Garadi, R.; Foltyn, A.R.; Giblin, F.J.; Reddy, V.N.

1984-01-01

The present study deals with the incorporation of 35 S methionine into lens crystallins as a function of time after x-irradiation. Crystallin synthesis is first affected approximately 4 weeks following x-irradiation. This coincides with the time period at which the ratio of the two cations in the lens is affected, as shown in earlier studies. A greater decrease in 35 S-methionine incorporation into crystallins is observed between 5-7 weeks following x-irradiation in good agreement with a cation imbalance at these time intervals. These studies also revealed for the first time that the change in cation distribution can affect not only crystallin synthesis, but also the synthesis of certain polypeptides of lens membranes. No alteration in protein synthesis could be detected in lens epithelium even after 7 weeks following irradiation. In addition to the effect of Na+ and K+ levels on protein synthesis, an impaired transport of amino acids into the x-rayed lens was also found to be a factor in the observed reduction in synthesis of the crystallin, cytoskeletal and membrane proteins of the fiber cells. It is concluded that Na+/K+ ratio as well as the availability of amino acids in the lens are important factors in protein synthesis of x-ray cataracts

Albumin synthesis in protein energy malnutrition

International Nuclear Information System (INIS)

Duggan, C.; Hardy, S.; Kleinman, R.E.; Lembcke, J.; Young, V.E.

1994-01-01

The dietary treatment of protein-energy malnutrition (PEM) has been designed on an empirical basis, with outcomes for successful management including body weight gain and resolution of apathy. We propose using the measurements of protein synthesis as a more objective measure of renourishment. We will therefore randomize a group of malnourished children (weigh-for-height Z score 13 C-leucine and serial measurements of 13 C-enrichment of albumin. Isotope infusions will be performed on days one and three, following a standard three hour fast. Since albumin synthesis is reduced under the influence of cytokines which mediate the inflammatory response, results will be stratified according to the presence or absence of clinically apparent infections. We hypothesize that the provision of added dietary protein will optimize albumin synthesis rates in PEM as well as attenuate the reduction in albumin synthesis seen in the presence of infections. (author). 20 refs

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

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

Scalise, F.W.

1988-01-01

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

Vivo-morpholinos induced transient knockdown of physical activity related proteins.

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David P Ferguson

Full Text Available Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60-97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28. However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001 decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016 increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

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

Science.gov (United States)

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.

Imaging of Protein Synthesis: In Vitro and In Vivo Evaluation of Sc-44-DOTA-Puromycin

Czech Academy of Sciences Publication Activity Database

Eigner, Sebastian; Beckford, Denis R.; Fellner, M.; Loktionova, N.; Piel, M.; Lebeda, Ondřej; Rosch, F.; Ross, T. L.; Eigner-Henke, Kateřina

2013-01-01

Roč. 15, č. 1 (2013), s. 79-86 ISSN 1536-1632 R&D Projects: GA MŠk 2B06165 Institutional support: RVO:61389005 Keywords : protein synthesis * Scandium-44 * DOTA-Pur * therapy control * mu PET * preclinical imaging Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 2.869, year: 2013 http://link.springer.com/content/pdf/10.1007%2Fs11307-012-0561-3

Biochemical investigations of the effect of NaF on mammalian cells. 2. Influence on biosynthesis of nucleic acids and proteins in mouse spleen cells ''in vivo''

Energy Technology Data Exchange (ETDEWEB)

Klein, W; Kocsis, F; Altmann, H

1974-10-01

The influence of NaF on the biosynthesis of nucleic acids and proteins was studied ''in vivo'' with ''Swiss mice''. Using a fluoride concentration of 0.4 ..mu..g/g no effect on DNA-repair appeared within 12 weeks, while DNA-, RNA- and protein-synthesis were suppressed after 10 weeks. Fluoride in a concentration of 3.5 ..mu..g/g gives a nearly complete inhibition of DNA-repair after 10 weeks, while DNA-, RNA- and protein-synthesis were suppressed to various degrees from week 2 until week 12. The phosphorylation of DNA- and RNA-precursors indicated results comparable to both synthesis, but investigating the particular kinase-steps of the phosphorylation, no specific effect on one of them can be localized significantly. (auth)

Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

Science.gov (United States)

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.

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

Science.gov (United States)

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 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.

Targeting tumor-initiating cells: Eliminating anabolic cancer stem cells with inhibitors of protein synthesis or by mimicking caloric restriction

Science.gov (United States)

Lamb, Rebecca; Harrison, Hannah; Smith, Duncan L.; Townsend, Paul A.; Jackson, Thomas; Ozsvari, Bela; Martinez-Outschoorn, Ubaldo E.; Pestell, Richard G.; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

2015-01-01

We have used an unbiased proteomic profiling strategy to identify new potential therapeutic targets in tumor-initiating cells (TICs), a.k.a., cancer stem cells (CSCs). Towards this end, the proteomes of mammospheres from two breast cancer cell lines were directly compared to attached monolayer cells. This allowed us to identify proteins that were highly over-expressed in CSCs and/or progenitor cells. We focused on ribosomal proteins and protein folding chaperones, since they were markedly over-expressed in mammospheres. Overall, we identified >80 molecules specifically associated with protein synthesis that were commonly upregulated in mammospheres. Most of these proteins were also transcriptionally upregulated in human breast cancer cells in vivo, providing evidence for their potential clinical relevance. As such, increased mRNA translation could provide a novel mechanism for enhancing the proliferative clonal expansion of TICs. The proteomic findings were functionally validated using known inhibitors of protein synthesis, via three independent approaches. For example, puromycin (which mimics the structure of tRNAs and competitively inhibits protein synthesis) preferentially targeted CSCs in both mammospheres and monolayer cultures, and was ~10-fold more potent for eradicating TICs, than “bulk” cancer cells. In addition, rapamycin, which inhibits mTOR and hence protein synthesis, was very effective at reducing mammosphere formation, at nanomolar concentrations. Finally, mammosphere formation was also markedly inhibited by methionine restriction, which mimics the positive effects of caloric restriction in cultured cells. Remarkably, mammosphere formation was >18-fold more sensitive to methionine restriction and replacement, as directly compared to monolayer cell proliferation. Methionine is absolutely required for protein synthesis, since every protein sequence starts with a methionine residue. Thus, the proliferation and survival of CSCs is very sensitive to

Predictors of muscle protein synthesis after severe pediatric burns.

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

2015-04-01

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

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

Science.gov (United States)

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.

Induction of hepatic protein synthesis by a peptide in blood plasma of patients with sepsis and trauma.

Science.gov (United States)

Loda, M; Clowes, G H; Dinarello, C A; George, B C; Lane, B; Richardson, W

1984-08-01

Accelerated release of amino acids from muscle and their uptake for protein synthesis by liver and other visceral tissues are characteristic of trauma or sepsis. Experimentally, this response is induced by interleukin-1 (IL-1) generated by activated macrophages in vitro. However, IL-1 has not been demonstrated in human blood. A small 4000-dalton peptide recently isolated from plasma of patients with sepsis and trauma induces muscle proteolysis and is called "proteolysis-inducing factor" (PIF). To test whether this agent has the ability also to induce hepatic protein synthesis, a series of animal experiments and clinical observations were undertaken. The structural and secretory (acute-phase reactants) in vitro protein synthesis in livers of normal rats injected intraperitoneally with IL-1 or PIF was significantly greater than that of normal rats or those injected with Ringer's lactate (p less than 0.01). In patients with sepsis and trauma the central plasma clearance rate of amino acids, a measure of visceral (principally hepatic) amino acid uptake, was elevated and correlated with the rates of protein synthesis in incubated liver slices obtained by biopsy at operation from the same patients (p less than 0.05). Both in vivo measured central plasma clearance rate of amino acids and in vitro measured hepatic protein synthesis correlated with plasma levels of PIF in the same patients (p less than 0.01 and p less than 0.05, respectively). We conclude that since PIF, and not IL-1, is present in human plasma and both are produced by activated macrophages, PIF seems to be the stable circulating cleavage product of IL-1, which induces not only muscle proteolysis but also hepatic protein synthesis, principally in the form of acute-phase reactants during infection and other states in which inflammation is present.

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

Directory of Open Access Journals (Sweden)

Brita Singers Sørensen

novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.

Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

International Nuclear Information System (INIS)

McClure, P.R.; Omholt, T.E.; Pace, G.M.; Bouthyette, P.Y.

1987-01-01

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [ 35 S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [ 35 S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vecinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots

The single-biopsy approach in determining protein synthesis in human slow-turning-over tissue: use of flood-primed, continuous infusion of amino acid tracer

DEFF Research Database (Denmark)

Holm, Lars; Reitelseder, Søren; Dideriksen, Kasper

2014-01-01

Muscle protein synthesis (MPS) rate is determined conventionally by obtaining two or more tissue biopsies during a primed, continuous infusion of a stable isotopically labeled amino acid. The purpose of the present study was to test whether tracer priming given as a flooding dose, thereby securing....... In conclusion, the flood-primed, continuous infusion protocol using phenylalanine as tracer can validly be used to measure the protein synthesis rate in human in vivo experiments by obtaining only a single tissue biopsy after a prolonged infusion period....

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

Science.gov (United States)

Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A

2008-10-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

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

Science.gov (United States)

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.

Liver protein synthesis stays elevated after chemotherapy in tumour-bearing mice.

Science.gov (United States)

Samuels, Sue E; McLaren, Teresa A; Knowles, Andrew L; Stewart, Sarah A; Madelmont, Jean-Claude; Attaix, Didier

2006-07-28

We studied the effect of chemotherapy on liver protein synthesis in mice bearing colon 26 adenocarcinoma (C26). Liver protein mass decreased (-32%; Psynthesis increased (20-35%; Psynthesis. Increased protein synthesis in tumour-bearing mice was primarily mediated by increasing ( approximately 15%; Psynthesis (Cs; mg RNA/g protein). Cystemustine, a nitrosourea chemotherapy that cures C26 with 100% efficacy, rapidly restored liver protein mass; protein synthesis however stayed higher than in healthy mice ( approximately 15%) throughout the initial and later stages of recovery. Chemotherapy had no significant effect on liver protein mass and synthesis in healthy mice. Reduced food intake was not a factor in this model. These data suggest a high priority for liver protein synthesis during cancer cachexia and recovery.

Age-related changes in the synthesis and phosphorylation of proteins

International Nuclear Information System (INIS)

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

1986-01-01

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 [ 3 H]-valine or [ 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

A low-protein diet restricts albumin synthesis in nephrotic rats.

OpenAIRE

Kaysen, G A; Jones, H; Martin, V; Hutchison, F N

1989-01-01

High-protein diets increase albumin synthesis in rats with Heymann nephritis but albuminuria increases also, causing serum albumin concentration to be suppressed further than in nephrotic animals eating a low-protein diet. Experiments were designed to determine whether dietary protein augmentation directly stimulates albumin synthesis, or whether instead increased albumin synthesis is triggered by the decrease in serum albumin concentration. Evidence is presented that dietary protein augmenta...

Inhibition of chloroplast protein synthesis following light chilling of tomato

International Nuclear Information System (INIS)

Kent, J.; Ort, D.

1989-01-01

In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

International Nuclear Information System (INIS)

Panaviene, Zivile; Nagy, Peter D.

2003-01-01

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

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

Science.gov (United States)

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.

Protein synthesis in the presence of carbamoyl-amino acids

International Nuclear Information System (INIS)

Kraus, L.M.; Stephens, M.C.

1987-01-01

The role of exogenous carbamoyl-amino acids in protein biosynthesis has been examined in vitro using a mixture of 14 C amino acids to label newly synthesized protein in human reticulocyte rich (8-18%) peripheral blood. Aliquots of the radiolabeled newly synthesized protein were acid precipitated, washed and the radioactivity measured. Control samples which measured the synthetic capacity of the blood were aliquots of the same blood- 14 C amino acid mixture without added carbamoyl-amino acids or cyanate. N-carbamoyl leucine alone or a 3 N-carbamoyl amino acid mixture of leucine, aspartic acid and tyrosine were used to test inhibition of protein synthesis. Also carbamoyl-amino acids were synthesized using cyanate and Pierce hydrolyzate amino acid calibration standards or the mixture of 14 C amino acids. In this system the carbamoylation of endogenous amino acids by cyanate up to 8 μmol/100μl showed a linear decrease in protein synthesis with time which is inversely related to the cyanate concentration. At greater cyanate levels the inhibition of protein synthesis reaches a plateau. When N-carbamoyl-amino acids only are present there is about a 50% decrease in the 14 C protein at 30 minutes as compared to the synthesis of 14 C protein without N-carbamoyl-amino acids. These results indicate that the presence of carbamoyl-amino acids interferes with protein synthesis

Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion.

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Benjamin Toby Wall

Full Text Available Progressive loss of skeletal muscle mass with aging (sarcopenia forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men.We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y and 72 older (75±1 y men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y and 40 older (74±1 y men.Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion.Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia.

Dendritic protein synthesis in the normal and diseased brain

Science.gov (United States)

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

In vivo cellular imaging using fluorescent proteins - Methods and Protocols

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M. Monti

2012-12-01

Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

A Ca2+-calmodulin-eEF2K-eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions

DEFF Research Database (Denmark)

Rose, Adam John; Alsted, Thomas Junker; Jensen, Thomas Elbenhardt

2009-01-01

Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesised that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1......) phosphorylation by signalling cascades downstream of rises in intracellular [Ca(2+)] and decreased energy charge via AMP activated protein kinase (AMPK) in contracting skeletal muscle. When fast-twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis...... correlated more closely with changes in eEF2 rather than 4EBP1 phosphorylation. Using a combination of Ca(2+) release agents and ATPase inhibitors it was shown that the 60-70% suppression of fast-twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca(2+) and energy...

Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts.

Science.gov (United States)

Xu, Wen-Xiao; Liu, Sheng-Zhi; Wu, Di; Qiao, Guo-Fen; Yan, Jinglong

2015-01-01

Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel.

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

Science.gov (United States)

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.

Understanding Protein Synthesis: An Interactive Card Game Discussion

Science.gov (United States)

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…

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

Science.gov (United States)

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

2017-11-01

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the

The origin of polynucleotide-directed protein synthesis

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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.

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

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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.

Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

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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.

Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

International Nuclear Information System (INIS)

Minor, P.D.; Dimmock, N.J.

1977-01-01

Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

Low-protein, high-carbohydrate diet increases glucose uptake and fatty acid synthesis in brown adipose tissue of rats.

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Aparecida de França, Suélem; Pavani Dos Santos, Maísa; Nunes Queiroz da Costa, Roger Vinícius; Froelich, Mendalli; Buzelle, Samyra Lopes; Chaves, Valéria Ernestânia; Giordani, Morenna Alana; Pereira, Mayara Peron; Colodel, Edson Moleta; Marlise Balbinotti Andrade, Cláudia; Kawashita, Nair Honda

2014-04-01

The aim of this study was to evaluate glucose uptake and the contribution of glucose to fatty acid (FA) synthesis and the glycerol-3-phosphate (G3P) of triacylglycerol synthesis by interscapular brown adipose tissue (IBAT) of low-protein, high-carbohydrate (LPHC) diet-fed rats. LPHC (6% protein; 74% carbohydrate) or control (17% protein; 63% carbohydrate) diets were administered to rats (∼ 100 g) for 15 d. Total FA and G3P synthesis and the synthesis of FA and G3P from glucose were evaluated in vivo by (3)H2O and (14)C-glucose. Sympathetic neural contribution for FA synthesis was evaluated by comparing the synthesis in denervated (7 d before) IBAT with that of the contralateral innervated side. The insulin signaling and β3 adrenergic receptor (β3-AR) contents, as well as others, were determined by Western blot (Student's t test or analysis of variance; P ≤ 0.05). Total FA synthesis in IBAT was 133% higher in the LPHC group and was reduced 85% and 70% by denervation for the LPHC and control groups, respectively. Glucose uptake was 3.5-fold higher in the IBAT of LPHC rats than in that of the control rats, and the contribution of glucose to the total FA synthesis increased by 12% in control rats compared with 18% in LPHC rats. The LPHC diet increased the G3P generation from glucose by 270% and the insulin receptor content and the p-AKT insulin stimulation in IBAT by 120% and reduced the β3-AR content by 50%. The LPHC diet stimulated glucose uptake, both the total rates and the rates derived from glucose-dependent FA and G3P synthesis, by increasing the insulin sensitivity and the sympathetic flux, despite a reduction in the β3-AR content. Copyright © 2014 Elsevier Inc. All rights reserved.

Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

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Demangeat, G; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, C

1992-07-01

The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

Roles of Fe-S proteins: from cofactor synthesis to iron homeostasis to protein synthesis.

Science.gov (United States)

Pain, Debkumar; Dancis, Andrew

2016-06-01

Fe-S cluster assembly is an essential process for all cells. Impairment of Fe-S cluster assembly creates diseases in diverse and surprising ways. In one scenario, the loss of function of lipoic acid synthase, an enzyme with Fe-S cluster cofactor in mitochondria, impairs activity of various lipoamide-dependent enzymes with drastic consequences for metabolism. In a second scenario, the heme biosynthetic pathway in red cell precursors is specifically targeted, and iron homeostasis is perturbed, but lipoic acid synthesis is unaffected. In a third scenario, tRNA modifications arising from action of the cysteine desulfurase and/or Fe-S cluster proteins are lost, which may lead to impaired protein synthesis. These defects can then result in cancer, neurologic dysfunction or type 2 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

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Boyce Mark

2012-08-01

Full Text Available Abstract Background Bluetongue virus (BTV is a double-stranded RNA (dsRNA virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1 as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis.

Science.gov (United States)

Boyce, Mark; Celma, Cristina C P; Roy, Polly

2012-08-29

Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3' poly(A) sequence identifying the 3' end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

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Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

DEFF Research Database (Denmark)

Doessing, Simon; Heinemeier, Katja M; Holm, Lars

2010-01-01

young individuals. rhGH administration caused an increase in serum GH, serum IGF-I, and IGF-I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9-fold and 1.3-fold, respectively (P ...RNA expression and muscle collagen protein synthesis increased by 2.3-fold and 5.8-fold, respectively (P protein synthesis was unaffected by elevation of GH and IGF-I. Moderate exercise did not enhance the effects of GH manipulation. Thus, increased GH availability stimulates...... matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue....

Marginal dietary zinc deprivation augments sepsis-induced alterations in skeletal muscle TNF-α but not protein synthesis.

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Crowell, Kristen T; Kelleher, Shannon L; Soybel, David I; Lang, Charles H

2016-11-01

Severe zinc deficiency is associated with an increased systemic inflammatory response and mortality after sepsis. However, the impact of mild zinc deficiency, which is more common in populations with chronic illnesses and sepsis, is unknown. In this study, we hypothesized that marginal dietary Zn deprivation (ZM) would amplify tissue inflammation and exacerbate the sepsis-induced decrease in muscle protein synthesis. Adult male C57BL/6 mice were fed a zinc-adequate (ZA) or ZM diet (30 or 10 mg Zn/kg, respectively) over 4 weeks, peritonitis was induced by cecal ligation and puncture (CLP), and mice were examined at either 24 h (acute) or 5 days (chronic) post-CLP Acute sepsis decreased the in vivo rate of skeletal muscle protein synthesis and the phosphorylation of the mTOR substrate 4E-BP1. Acutely, sepsis increased TNF-α and IL-6 mRNA in muscle, and the increase in TNF-α was significantly greater in ZM mice. However, muscle protein synthesis and 4E-BP1 phosphorylation returned to baseline 5 days post-CLP in both ZA and ZM mice. Protein degradation via markers of the ubiquitin proteasome pathway was increased in acute sepsis, yet only MuRF1 mRNA was increased in chronic sepsis and ZM amplified this elevation. Our data suggest that mild zinc deficiency increases TNF-α in muscle acutely after sepsis but does not significantly modulate the rate of muscle protein synthesis. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

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

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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.

Synthesis of stress proteins in winter wheat seedlings under gamma-radiation

International Nuclear Information System (INIS)

Gudkova, N.V.; Kosakovskaya, I.V.; Major, P.S.

2001-01-01

A universal cellular response to a number of diverse stresses is the synthesis of a set of stress proteins. Most of them are heat shock proteins (HSP). We show that both heat shock and gamma-radiation enhance the synthesis of HSP70 in the total protein fractions of winter wheat seedlings. It is found that a dose of 15 Gy induced the synthesis of 35 and 45 kD proteins after 5 h of irradiation in both total and mitochondrial protein fractions. On the second day after exposure, both 35 and 45 kD proteins were not observed, but new total proteins with a molecular weight of 90 and 92 kD appeared. The synthesis of 35 and 45 kD proteins after gamma-irradiation is revealed for the first time, their function being now unknown

Protein synthesis and sublethal damage repair in synchronized CHO cells

International Nuclear Information System (INIS)

Yezzi, M.J.; Tobias, C.A.; Blakely, E.A.

1984-01-01

The authors have previously reported that the split dose survival response to x-rays of asynchronous CHO-TSH1 cells is reduced if the cells are held at 40 0 C,a temperature that inhibits protein synthesis, for 2 hours before the first dose and during a 2-hour interval between doses. In conjunction with the survival experiments on asynchronous cells, the authors also examined the DNA rejoining ability in split dose studies with and without inhibition of protein synthesis. The results of these experiments suggest that inhibition of protein synthesis affects a pool of proteins that are necessary for the correct expression of the DNA, although they do not appear to be involved in rejoining DNA breaks. They have extended this work to the study of cells synchronized in G1 phase (2 hour post-mitosis) and S phase (10 hour post-mitosis). Autoradiographic analyses, using 3H-TdR pulse labeling, demonstrated that a delay in the progression of each synchronized cell population occurs after inhibition of protein synthesis. Data are reported on the effects of inhibition of protein synthesis on the ability of G1 and S phase cells to repair sublethal damage

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

NARCIS (Netherlands)

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

2015-01-01

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

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo.

Science.gov (United States)

Wölfle, Ute; Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M

2017-08-22

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema.

Measurement of protein synthesis in vivo in the growing lamb liver using 14C lysyl-tRNAlys

International Nuclear Information System (INIS)

Ferrara, M.; Arnal, M.; Fauconneau, G.

1977-01-01

The distribution of intravenously administered 14 C lysine was followed in the extracellular and intracellular pools of the lamb liver and in the crude aminoacyl-tRNA extracted from them at the same time. The evolution of the specific radioactivity of lysine in these three pools suggested that lysyl-tRNAlys was acrylated with amino acid derived from the extracellular and intracellular pools. The rate of protein synthesis calculated with specific radioactivity of lysine released from aminoacyl-tRNA was intermediate between those obtained with the extracellular and intracellular pools and indicated that determination of specific radioactivity of amino acid released from aminoacyl-tRNA was important for correct assessment of protein turnover [fr

PROSPECTIVE TEACHERS’ COGNITIVE STRUCTURES CONCERNING PROTEIN SYNTHESIS AND THEIR DEGREE OF UNDERSTANDING

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Cem Gerçek

2018-02-01

Full Text Available The purpose of education is to actualise meaningful learning. Therefore, researching the issues on how students process information and how they configure it is important for meaningful learning. The issue of protein synthesis contains a number of abstract topics and concepts. Hence, it is important in biology teaching to be informed of students’ cognitive structures concerning protein synthesis. This research aims to analyse prospective teachers’ cognitive structures about protein synthesis and their degree of understanding the subject. The research group was composed of 17 volunteering prospective teachers who had been chosen through purposeful sampling. The data were collected via semi-structured interviews. Flow maps and content analysis were used in analysing the data. The results demonstrated that prospective teachers had too many misconceptions about protein synthesis and that their knowledge extent and rich connection are inadequate. The prospective teachers’ degree of understanding protein synthesis was divided into three categories. The results obtained in this research suggested that teachers should be careful in teaching the subject of protein synthesis. Students’ prior knowledge and their misconceptions should be determined and content or contexts to facilitate them to learn an abstract subject such as protein synthesis should be presented.

Synthesis and In Vitro AMPK Activation of Cycloalkyl/Alkarylbiguanides with Robust In Vivo Antihyperglycemic Action

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Erika Gutierrez-Lara

2017-01-01

Full Text Available This work describes the design, synthesis in one step, and the in vitro, in vivo, and in silico antidiabetic evaluation of a series of ten alicyclic and aromatic (alkyl +aryl: alkarylbiguanides, analogues of metformin and phenformin. The design was conceived using isosteric replacement, chain-ring transformation, and lower and higher homologation strategies. All compounds were obtained as crystals and their structure was confirmed on the basis of their spectral data (NMR and mass spectra, and their purity was ascertained by microanalysis. Compounds were in vitro evaluated as activators of AMP-Activated Protein Kinase (AMPK. The results indicated that compounds 4, 5, and 6 showed similar or even better effect compared to metformin. Docking analysis was performed with regulatory subunit γ of AMPK, showing several interactions with nucleotide binding pocket. The in vivo evaluation of compounds 4–6 at a single dose of 50 mg/kg was performed in a murine experimental model of diabetes. The results showed an important and robust decrease of plasmatic glucose levels (−40%. Compound 6 was selected for an oral glucose tolerance test, showing an antihyperglycemic effect similar to metformin. The in vivo results indicated that compounds 4–6 may be effective in treating experimental T2DM.

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

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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.

Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies.

Science.gov (United States)

Le Plénier, Servane; Goron, Arthur; Sotiropoulos, Athanassia; Archambault, Eliane; Guihenneuc, Chantal; Walrand, Stéphane; Salles, Jérome; Jourdan, Marion; Neveux, Nathalie; Cynober, Luc; Moinard, Christophe

2017-01-01

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control. Copyright © 2017 the American Physiological Society.

Ethylene-induced senescence-related gene expression requires protein synthesis

International Nuclear Information System (INIS)

Lawton, K.A.; Raghothama, K.G.; Woodson, W.R.

1990-01-01

We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

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Bukowska-Faniband, Ewa; Hederstedt, Lars

2017-07-01

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

Science.gov (United States)

Kent, Stephen Bh

2018-04-04

A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1987-01-01

The effect of salt stress on the incorporation of [ 3 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

Ethylene and protein synthesis

Energy Technology Data Exchange (ETDEWEB)

Osborne, D J

1973-01-01

Ethylene reduces the rate of expansion growth of cells and it is suggestive that the rate of expansion is controlled at least in part by the synthesis of hydroxyproline rich glycopeptides that are secreted with other polysaccharide material through the plasmalemma into the cell wall, thereby enhancing the thickness of the cell wall and also rendering it poorly extensible. In combination, auxin would appear to counteract the effect of ethylene in this respect, for although auxin enhances the synthesis of protein and the content in the cell walls, as well as causing some increase in wall thickness, it reduces the amount of hydroxyproline reaching the wall. Such effects may be instrumental in enhancing wall plasticity, the rate of expansion and the final cell size. These results indicate that ethylene and auxin together afford a dual regulatory system exerted through a control of a specific part of the protein synthetic pathway, the products of which regulate the rate of expansion, and the potential for expansion, of the plant cell wall. 38 references, 3 figures, 8 tables.

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

Science.gov (United States)

Kent, Stephen

2017-09-15

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

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

Science.gov (United States)

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

2011-01-01

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

Late protein synthesis-dependent phases in CTA long-term memory: BDNF requirement

Directory of Open Access Journals (Sweden)

Araceli eMartínez-Moreno

2011-09-01

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

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

Science.gov (United States)

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

2011-01-01

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

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

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Richa Tyagi

2015-02-01

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

Rewiring protein synthesis: From natural to synthetic amino acids.

Science.gov (United States)

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

2017-11-01

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

Photo-synthesis of protein-based nanoparticles and the application in drug delivery

International Nuclear Information System (INIS)

Xie, Jinbing; Wang, Hongyang; Cao, Yi; Qin, Meng; Wang, Wei

2015-01-01

Recently, protein-based nanoparticles as drug delivery systems have attracted great interests due to the excellent behavior of high biocompatibility and biodegradability, and low toxicity. However, the synthesis techniques are generally costly, chemical reagents introduced, and especially present difficulties in producing homogeneous monodispersed nanoparticles. Here, we introduce a novel physical method to synthesize protein nanoparticles which can be accomplished under physiological condition only through ultraviolet (UV) illumination. By accurately adjusting the intensity and illumination time of UV light, disulfide bonds in proteins can be selectively reduced and the subsequent self-assembly process can be well controlled. Importantly, the co-assembly can also be dominated when the proteins mixed with either anti-cancer drugs, siRNA, or active targeting molecules. Both in vitro and in vivo experiments indicate that our synthesized protein–drug nanoparticles (drug-loading content and encapsulation efficiency being ca. 8.2% and 70%, respectively) not only possess the capability of traditional drug delivery systems (DDS), but also have a greater drug delivery efficiency to the tumor sites and a better inhibition of tumor growth (only 35% of volume comparing to the natural growing state), indicating it being a novel drug delivery system in tumor therapy

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

DEFF Research Database (Denmark)

Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe

2017-01-01

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and ......, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms....... to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity...

Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

Science.gov (United States)

Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

2017-01-01

Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1- 13 C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1- 13 C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1- 13 C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P protein-bound l-[1- 13 C]-phenylalanine enrichments (MPE) increased to a greater extent in the stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P protein-derived amino acids in the NMES compared with CON leg. In conclusion, application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older men after a day of bed rest, the application of NMES prior to presleep protein feeding stimulates the use of

Phosphorylation of protein synthesis initiation factor 2 (elF-2) in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Romero, D.P.

1986-01-01

Initiation Factor 2 (elF-2) in the yeast Saccharomyces cerevisiae is comprised of 3 subunits. The control of protein synthesis in mammalian cells have been shown to involve the phosphorylation of the small (alpha) subunit by a specific protein kinase. Phosphorylation results in an inhibition of protein synthesis. In order to determine whether or not an analogous system is operative in yeast, the phosphorylation state of the alpha subunit of elF-2 in Saccharomyces was determined during various growth and nongrowth conditions. Cells were radiolabelled with 32 P and 35 S, and the whole cell lysates were analyzed by two dimensional gel electrophoresis. These experiments revealed that the smallest subunit (alpha, M/sub r/ = 31,000) is a phosphoprotein in vivo under a variety of growth and nongrowth conditions. This is in direct contrast to the pattern exhibited in mammalian cells. The fact that the small subunit of elF-2 in yeast is phosphorylated under a variety of physiological conditions indicates that such a covalent modification is important for some aspects of elF-2 function. In order to investigate this problem further, a protein kinase that specifically labels the alpha subunit of elF-2 in vitro was isolated. The kinase is not autophosphorylating, utilizes ATP as a phosphate donor, phosphorylates an exogenous protein, casein, modifies serine residues in elF-2, is cyclic nucleotide-independent, and is strongly inhibited by heparin

Effects of nonstructural carbohydrates and protein sources on intake, apparent total tract digestibility, and ruminal metabolism in vivo and in vitro with high-concentrate beef cattle diets.

Science.gov (United States)

Rotger, A; Ferret, A; Calsamiglia, S; Manteca, X

2006-05-01

To investigate the effects of synchronizing nonstructural carbohydrate (NSC) and protein degradation on intake and rumen microbial fermentation, four ruminally fistulated Holstein heifers (BW = 132.3 +/- 1.61 kg) fed high-concentrate diets were assigned to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments studied in vivo and in vitro with a dual-flow continuous culture system. Two NSC sources (barley and corn) and 2 protein sources [soybean meal (SBM) and sunflower meal (SFM)] differing in their rate and extent of ruminal degradation were combined resulting in a synchronized rapid fermentation diet (barley-SFM), a synchronized slow fermentation diet (corn-SBM), and 2 unsynchronized diets with a rapidly and a slowly fermenting component (barley-SBM, and corn-SFM). In vitro, the fermentation profile was studied at a constant pH of 6.2, and at a variable pH with 12 h at pH 6.4 and 12 h at pH 5.8. Synchronization tended to result in greater true OM digestion (P = 0.072), VFA concentration (P = 0.067), and microbial N flow (P = 0.092) in vitro, but had no effects on in vivo fermentation pattern or on apparent total tract digestibility. The NSC source affected the efficiency of microbial protein synthesis in vitro, tending to be greater (P = 0.07) for barley-based diets, and in vivo, the NSC source tended to affect intake. Dry matter and OM intake tended to be greater (P > or = 0.06) for corn- than barley-based diets. Ammonia N concentration was lower in vitro (P = 0.006) and tended to be lower in vivo (P = 0.07) for corn- than barley-based diets. In vitro, pH could be reduced from 6.4 to 5.8 for 12 h/d without any effect on ruminal fermentation or microbial protein synthesis. In summary, ruminal synchronization seemed to have positive effects on in vitro fermentation, but in vivo recycling of endogenous N or intake differences could compensate for these effects.

Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

International Nuclear Information System (INIS)

Nosek, T.M.; Adams, R.J.

1986-01-01

Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7μM compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7μM ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100μM CYC or LEU did not affect NKA activity or 3 H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA

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

Directory of Open Access Journals (Sweden)

Je H

2011-01-01

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

Alterations in membrane protein-profile during cold treatment of alfalfa

International Nuclear Information System (INIS)

Mohapatra, S.S.; Poole, R.J.; Dhindsa, R.S.

1988-01-01

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [ 35 S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo

Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

Directory of Open Access Journals (Sweden)

Robert W Tilghman

Full Text Available Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa, cells on soft substrates (150-300 Pa exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins and glycolysis (e.g., phosphofructokinase-1, whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

Science.gov (United States)

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

NARCIS (Netherlands)

Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

2001-01-01

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

Science.gov (United States)

Heppert, Jennifer K; Dickinson, Daniel J; Pani, Ariel M; Higgins, Christopher D; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R; Goldstein, Bob

2016-11-07

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. © 2016 Heppert et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet - A pilot study.

Science.gov (United States)

Jourdan, Marion; Nair, K Sreekumaran; Carter, Rickey E; Schimke, Jill; Ford, G Charles; Marc, Julie; Aussel, Christian; Cynober, Luc

2015-06-01

Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake. Copyright © 2014. Published by Elsevier Ltd.

Ketone-body utilization and lipid synthesis by developing rat brain—a comparison between in vivo and in vitro experiments

NARCIS (Netherlands)

Klein, W.; Lopes-Cardozo, M.

1984-01-01

The distribution of ketone bodies between oxidation and lipid synthesis was analysed in homogenates of developing rat brain. The capacity for lipid synthesis of homogenized or minced brain preparations was compared with rates of lipid synthesis in vivo, assessed by incorporation of ³H from

Ribulose 1,5-bisphosphate carboxylase synthesis during heat shock

International Nuclear Information System (INIS)

Vierling, E.; Key, J.L.

1985-01-01

Ribulose 1,5-bisphosphate carboxylase (RuBPCase) was chosen as a model protein to study how heat shock (HS) affects both chloroplast protein synthesis and the nuclear-chloroplast interaction in production of chloroplast proteins. Experiments were performed using highly chlorophyllous, soybean (Glycine max L. Merr. var Corsoy) cell suspension cultures active in chloroplast protein synthesis. Synthesis of RuBPCase large (L) and small (S) subunits was followed by in vivo labeling, and corresponding mRNA levels were examined by Northern and dot hybridization analyses. Results demonstrate that L and S synthesis declines with increasing HS temperatures (33-40 0 C) and reaches minimum levels (20-30% of control) at temperatures of maximum HS protein synthesis (39-40 0 C). Recovery of L and S synthesis following a 2-hour HS at 38 or 40 0 C was also studied. The changes in S synthesis during HS and recovery correlate with the steady state levels of S mRNA. In contrast, changes in L synthesis show little relationship to the corresponding mRNA levels; levels of L mRNA remain relatively unchanged by HS. The authors conclude that chloroplast protein synthesis shows no greater sensitivity to HS than is observed for cytoplasmic protein synthesis and that transport of proteins into the chloroplast (e.g.,S subunit) continues during HS. Furthermore, there is no apparent coordination of L and S subunit mNRA levels under the conditions examined

Effect of heat stress on the pattern of protein synthesis in wheat endosperm

International Nuclear Information System (INIS)

Inwood, W.; Bernardin, J.

1990-01-01

The exposure of detached wheat heads (T. aestivum L. cv Cheyenne) to elevated temperatures resulted not only in the induction of a typical set of high and low molecular weight heat shock proteins (hsps), but also in a differential effect on the synthesis of wheat storage proteins in endosperm tissue when monitored by SDS PAGE of 35 S-labeled polypeptides. The synthesis of hsps in the endosperm had a rapid onset, reached a maximum rate within the first 2 hours at 40 degree C, and then steadily decreased during the next four hours. When heads were returned to 25 degree C after 3 hours at 40 degree C, hsp synthesis did not cease abruptly, but gradually declined over the next several hours. High molecular weight glutenin protein synthesis was drastically reduced with the same time course as heat shock protein synthesis was induced at 40 degree C. Conversely, the synthesis of gliadin proteins remained at a high level at 40 degree C. The synthesis rates for glutenin and gliadin proteins remained at low and high levels, respectively, for as long as the elevated temperature was maintained up to 7 hours

Longevity in vivo of primary cell wall cellulose synthases.

Science.gov (United States)

Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

2018-02-01

Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

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

Science.gov (United States)

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.

Quantifying drug-protein binding in vivo

International Nuclear Information System (INIS)

Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

2004-01-01

Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

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Barwal Indu

2011-12-01

Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light

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Griffiths, T.D.; Dahle, D.B.; Meechan, P.J.; Carpenter, J.G.

1981-01-01

Chinese hamster V-79 cells were treated with metabolic inhibitors of DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m -2 . After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [ 14 C]thymidine into acid-insoluble material. A 2.5 or 5.0h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of the cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process. (Auth.)

Protein synthesis by isolated type II pneumocytes in suspension and in primary culture

International Nuclear Information System (INIS)

Brandes, M.E.; Finkelstein, J.N.

1987-01-01

Protein synthesis in rabbit type II pneumocytes immediately after isolation or during the first 7 days in culture was examined by incorporation of [ 3 H] leucine or [ 35 S]methionine. After a 1h incubation with label, total cellular protein was analyzed by 1 or 2-D PAGE and fluorography. Following isolation, incorporation was limited to a small number of proteins of apparent molecular weight 70kD, 55-60kD, 25kD and 20+22kD which appear to lack cognates in cultured cells. At 3h, these isolation proteins (IPs) account for ∼ 50% of the labeled protein. Pretreatment with actinomycin D abolished synthesis of the IPs suggesting a requirement for active mRNA production. These proteins are actively synthesized during the first 10h following cell isolation. Loss of active synthesis is accompanied by a gradual enhancement in synthesis of other proteins. Actin synthesis, 125 I-EGF binding to cultured type II cells indicate changing receptor number and binding affinity with time in culture

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

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Steiner, Jennifer L; Pruznak, Anne M; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R; Lang, Charles H

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

International Nuclear Information System (INIS)

Feldherr, C.M.; Hodges, P.; Paine, P.L.

1988-01-01

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

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

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Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

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Jourdan, Marion; Nair, K. Sreekumaran; Carter, Rickey E.; Schimke, Jill; Ford, G. Charles; Marc, Julie; Aussel, Christian; Cynober, Luc

2015-01-01

Background and Aims Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. Methods To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 hours. [ring-13C6] phenylalanine and [15N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. Results FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; p=0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Conclusions Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake. PMID:24972455

Alcohol impairs skeletal muscle protein synthesis and mTOR signaling in a time-dependent manner following electrically stimulated muscle contraction.

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Steiner, Jennifer L; Lang, Charles H

2014-11-15

Alcohol (EtOH) decreases protein synthesis and mammalian target of rapamycin (mTOR)-mediated signaling and blunts the anabolic response to growth factors in skeletal muscle. The purpose of the current investigation was to determine whether acute EtOH intoxication antagonizes the contraction-induced increase in protein synthesis and mTOR signaling in skeletal muscle. Fasted male mice were injected intraperitoneally with 3 g/kg EtOH or saline (control), and the right hindlimb was electrically stimulated (10 sets of 6 contractions). The gastrocnemius muscle complex was collected 30 min, 4 h, or 12 h after stimulation. EtOH decreased in vivo basal protein synthesis (PS) in the nonstimulated muscle compared with time-matched Controls at 30 min, 4 h, and 12 h. In Control, but not EtOH, PS was decreased 15% after 30 min. In contrast, PS was increased in Control 4 h poststimulation but remained unchanged in EtOH. Last, stimulation increased PS 10% in Control and EtOH at 12 h, even though the absolute rate remained reduced by EtOH. The stimulation-induced increase in the phosphorylation of S6K1 Thr(421)/Ser(424) (20-52%), S6K1 Thr(389) (45-57%), and its substrate rpS6 Ser(240/244) (37-72%) was blunted by EtOH at 30 min, 4 h, and 12 h. Phosphorylation of 4E-BP1 Ser(65) was also attenuated by EtOH (61%) at 4 h. Conversely, phosphorylation of extracellular signal-regulated kinase Thr(202)/Tyr(204) was increased by stimulation in Control and EtOH mice at 30 min but only in Control at 4 h. Our data indicate that acute EtOH intoxication suppresses muscle protein synthesis for at least 12 h and greatly impairs contraction-induced changes in synthesis and mTOR signaling. Copyright © 2014 the American Physiological Society.

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

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Lee, Kyung-Ho; Kim, Dong-Myung

2013-11-01

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

Origins of the protein synthesis cycle

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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.

Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

International Nuclear Information System (INIS)

Hodge, B.O.; Kream, B.E.

1988-01-01

We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of [ 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

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

DEFF Research Database (Denmark)

Dikopoltsev, Elena; Foltyn, Veronika N; Zehl, Martin

2014-01-01

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little......, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association...... is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex...

Protective effect of a non specific inflammation on bone marrow protein synthesis in irradiated mice

International Nuclear Information System (INIS)

Herodin, F.; Roques, P.; Court, L.

1988-01-01

Gamma radiations exert a decrease in mouse bone marrow total protein synthesis. A non-specific inflammatory process induced with polyacrylamide microbeads stimulates spleen and marrow protein synthesis and protects the medullar protein synthesis in irradiated mice [fr

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

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

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

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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 H 5 ]-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 0.05). 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 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 protein-derived amino acids in the circulation and does not lower basal muscle protein synthesis rates or increase postprandial muscle protein synthesis rates after ingestion of 25 g protein in older men. This trial was registered at clinicaltrials.gov as NCT

Glucocorticoid effects on hippocampal protein synthesis

International Nuclear Information System (INIS)

Schlatter, L.K.

1988-01-01

Following subcutaneous injection of rats with 5 mg corticosterone, hippocampal slices in vitro show increased [ 35 S]-methionine labeling of a cytosolic protein with an apparent molecular weight (M 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 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 r . A second hippocampal protein with an M 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 r of 45,000 (IEP of 8.7-7.8) are increased in the liver following glucocorticoid administration

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

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

2017-12-09

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

Chemical synthesis of membrane proteins by the removable backbone modification method.

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Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

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Ana Ferreras

2002-04-01

Full Text Available A cell-free system from Plasmodium falciparum able to translate endogenous mRNA was used to determine the effect of artemisinin, chloroquine and primaquine on the protein synthesis mechanism of the parasite. The antimalarial drugs did not inhibit the incorporation of [³H] methionine into parasite proteins even at concentrations higher than the ones found to strongly inhibit the parasite growth. Results clearly indicate that these compounds do not have a direct effect on protein synthesis activity of P. falciparum coded by endogenous mRNA.

Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

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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…

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

International Nuclear Information System (INIS)

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

1991-01-01

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

Inhibition of host cell protein synthesis by UV-inactivated poliovirus

International Nuclear Information System (INIS)

Helentjaris, T.; Ehrenfeld, E.

1977-01-01

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

Chlorophyll biosynthesis and assembly into chlorophyll-protein complexes in isolated developing chloroplasts

International Nuclear Information System (INIS)

Bhaya, D.; Castelfranco, P.A.

1985-01-01

Isolated developing plastids from greening cucumber cotyledons or from photoperiodically grown pea seedlings incorporated 14 C-labeled 5-aminolevulinic acid (ALA) into chlorophyll (Chl). Incorporation was light dependent, enhanced by S-adenosylmethionine, and linear for 1 hr. The in vitro rate of Chl synthesis from ALA was comparable to the in vivo rate of Chl accumulation. Levulinic acid and dioxoheptanoic acid strongly inhibited Chl synthesis but not plastid protein synthesis. Neither chloramphenicol nor spectinomycin affected Chl synthesis, although protein synthesis was strongly inhibited. Components of thylakoid membranes from plastids incubated with [ 14 C]ALA were resolved by electrophoresis and then subjected to autoradiography. This work showed that (i) newly synthesized Chl was assembled into Chl-protein complexes and (ii) the inhibition of protein synthesis during the incubation did not alter the labeling pattern. Thus, there was no observable short-term coregulation between Chl synthesis (from ALA) and the synthesis of membrane proteins in isolated plastids

Effect of diet protein quality on growth and protein synthesis in rats

International Nuclear Information System (INIS)

Chinchalkar, D.V.; Mehta, S.L.

1978-01-01

The effect of diet protein quality on albino rats was studied by feeding normal and opaque-2 maize. The weight gain in rats was 60 percent higher on opaque-2 maize as compared to those fed on normal maize. Rats converted 1.0 g of dietary opaque-2 maize to 0.226 g weight gain as compared to 0.131 g for normal maize. The protein content per liver was higher with opaque-2 maize diet suggesting a higher net protein synthesis in opaque-2 maize fed rat livers. In vitro 14 C-phenylalanine incorporation showed that polysomes from opaque-2 maize fed rat livers were more efficient in protein synthesis than those from normal maize fed rat livers. Addition of poly-U resulted in more enhanced amino acid incorporation with polysomes from normal maize fed rats as compared to other group indicating greater limitation of mRNA in polysomes from normal maize fed rats. The total yield of liver polysomes from opaque-2 maize fed rats was substantially higher. (author)

Evidence for the involvement of a labile protein in stimulation of adrenal steroidogenesis under conditions not inhibitory to protein synthesis

International Nuclear Information System (INIS)

Krueger, R.J.; Orme-Johnson, N.R.

1988-01-01

Evidence is presented to support the hypothesis that synthesis of a labile protein is required for stimulation of steroidogenesis in rat adrenocortical cells. Amino acids L-canavanine and L-S-aminoethylcysteine, at concentrations as high as 5 mM, each inhibited steroidogenesis to a much greater extent than they inhibited protein synthesis. S-Aminoethylcysteine caused a 50% decrease in the stimulated rate of corticosterone production under conditions where incorporation of [35S]methionine into protein was unchanged. Both amino acids block stimulation of steroid synthesis at a step subsequent to the formation of cAMP and before the synthesis of progesterone. The onset of this effect, after the addition of the amino acids, on corticosterone production is quite rapid. These results provide support, that is not dependent on inhibition of protein synthesis, for the hypothesis that a labile protein mediates stimulation of steroidogenesis. Reversal of canavanine and S-aminoethylcysteine inhibition of steroidogenesis by arginine and lysine, respectively, suggests that the inhibitors are functioning as amino acid analogs. S-Aminoethylcysteine inhibits the incorporation of [3H]lysine into protein as well as inhibits steroidogenesis; further, [3H]S-aminoethylcysteine is incorporated into protein that is nonstimulatory. These results suggest that lysine residues play an essential role in the function of the labile protein or that the labile protein contains a large number of lysine residues

Existence of life-time stable proteins in mature rats-Dating of proteins' age by repeated short-term exposure to labeled amino acids throughout age

DEFF Research Database (Denmark)

Bechshøft, Cecilie Leidesdorff; Schjerling, Peter; Bornø, Andreas

2017-01-01

In vivo turnover rates of proteins covering the processes of protein synthesis and breakdown rates have been measured in many tissues and protein pools using various techniques. Connective tissue and collagen protein turnover is of specific interest since existing results are rather diverging. Th...... living days, indicating very slow turnover. The data support the hypothesis that some proteins synthesized during the early development and growth still exist much later in life of animals and hence has a very slow turnover rate.......In vivo turnover rates of proteins covering the processes of protein synthesis and breakdown rates have been measured in many tissues and protein pools using various techniques. Connective tissue and collagen protein turnover is of specific interest since existing results are rather diverging....... The aim of this study is to investigate whether we can verify the presence of protein pools within the same tissue with very distinct turnover rates over the life-span of rats with special focus on connective tissue. Male and female Lewis rats (n = 35) were injected with five different isotopically...

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

DEFF Research Database (Denmark)

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

1995-01-01

Rates of growth, protein synthesis and oxygen consumption were measured in herring larvae, Clupea harengus, in order to estimate the contribution that protein synthesis makes to oxygen consumption during rapid growth at 8°C. Protein synthesis rates were determined in larvae 9 to 17 d after hatching....... Larvae were bathed in (3)H phenylalanine for several hours and the free pool and protein-bound phenylalanine specific radioactivities were determined.Fractional rates of protein synthesis increased 5 to 11 fold with feeding after a period of fasting. Efficiencies of retention of synthesized protein were...... approximately 50% during rapid growth. Rapid growth in herring larvae thus appears to be characterized by moderate levels of protein turnover similar to those obtained for larger fish. Increases in growth rate occurred without changes in RNA concentration, i.e., the larvae increased the efficiency of RNA...

Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis.

Science.gov (United States)

Heude, M; Chanet, R

1975-04-01

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.

Direct determination of the redox status of cysteine residues in proteins in vivo

Energy Technology Data Exchange (ETDEWEB)

Hara, Satoshi [Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503 (Japan); Tatenaka, Yuki; Ohuchi, Yuya [Dojindo Laboratories, 2025-5 Tabaru, Mashiki-machi, Kumamoto 861-2202 (Japan); Hisabori, Toru, E-mail: thisabor@res.titech.ac.jp [Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075 (Japan)

2015-01-02

Highlights: • A new DNA-maleimide which is cleaved by UV irradiation, DNA-PCMal, was developed. • DNA-PCMal can be used like DNA-Mal to analyze the redox state of cysteine residues. • It is useful for detecting the thiol redox status of a protein in vivo by Western blotting method. • Thus, DNA-PCMal can be a powerful tool for redox proteomics analysis. - Abstract: The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells

International Nuclear Information System (INIS)

Seki, Shuji; Hosogi, Nobuo; Oda, Takuzo

1984-01-01

In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97 % by aphidicolin at 10 μg/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30 % and 90 % depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90 % at 100 μg/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 μg/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase α and a non-α DNA polymerase (possibly DNA polymerase β), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase α in UDS favored DNA synthesis in the intranucleosomal region. (author)

Solid-phase synthesis of protein-polymers on reversible immobilization supports.

Science.gov (United States)

Murata, Hironobu; Carmali, Sheiliza; Baker, Stefanie L; Matyjaszewski, Krzysztof; Russell, Alan J

2018-02-27

Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

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

Science.gov (United States)

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

2017-09-15

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

Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

Directory of Open Access Journals (Sweden)

Leila M Luheshi

2010-03-01

Full Text Available Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta peptide by using a small engineered binding protein (Z(Abeta3 that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3 in the brains of Drosophila melanogaster expressing either Abeta(42 or the aggressive familial associated E22G variant of Abeta(42 abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3 not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

Synthesis and thermotolerance of heat shock proteins in Campylobacter jejuni

International Nuclear Information System (INIS)

Kim, C.K.; Kim, H.O.; Lee, K.J.

1991-01-01

The heat shock responses of Campylobacter jejuni were studied by examination of their survival rates and synthesis of heat shock proteins. When C. jejuni cells were treated at the sublethal temperatures of 48C° for 30 minutes, most of the cells maintained their viabilities and synthesized the heat shock proteins of 90, 73, and 66 kD in molecular weight. By the method of two-dimensional electrophoresis, the heat shock proteins of C. jejuni were identified to be Hsp90, Hsp73, and Hsp66. During the heat shock at 48C°, the heat shock proteins were induced from about 5 minutes after the heat shock treatment. Their synthesis was continued upto 30 minutes, but remarkably retarded after 50 minutes. When C. jejune cells were heat shocked at 51C° for 30 minutes, the survival rates of the cells were decreased by about 10 3 fold and synthesis of heat shock proteins and normal proteins was also generally retarded. The cells exposed to 55C° for 30 minutes died off by more than 10 5 cells and the new protein synthesis was not observed. But when C. jejuni cells were heat-shocked at the sublethal temperature of 48C° for 15 to 20 minutes and then were exposed at the lethal temperature of 55C° for 30 minutes, their viabilities were higher than those exposed at 55C° for 30 minutes without pre-heat shock at 48C°. Therefore, the heat shock proteins synthesized at the sublethal temperature of 48C° in C. jejuni were thought to be responsible for thermotolerance. However, when C. jejuni cells heat-shocked at various ranges of sublethal and lethal temperatures were placed back to the optimum temperature of 42C°, the multiplication patterns of the cells pretreated at different temperatures were not much different each other

Regioselective synthesis of isoxazole-mercaptobenzimidazole hybrids and their in vivo analgesic and anti-inflammatory activity studies

DEFF Research Database (Denmark)

Kankala, Shravankumar; Kankala, Ranjith Kumar; Gundepaka, Prasad

2013-01-01

Regioselective synthesis of isoxazole-mercaptobenzimidazole hybrids and their efficiency in in vivo analgesic and anti-inflammatory activity was described. A comparison of structure-activity relationship for there compounds was also emphasized....

Prolonged bed rest decreases skeletal muscle and whole body protein synthesis

Science.gov (United States)

Ferrando, A. A.; Lane, H. W.; Stuart, C. A.; Davis-Street, J.; Wolfe, R. R.

1996-01-01

We sought to determine the extent to which the loss of lean body mass and nitrogen during inactivity was due to alterations in skeletal muscle protein metabolism. Six male subjects were studied during 7 days of diet stabilization and after 14 days of stimulated microgravity (-6 degrees bed rest). Nitrogen balance became more negative (P protein synthesis (PS; P protein also decreased by 46% (P protein breakdown and inward transport. Whole body protein synthesis determined by [15N]alanine ingestion on six subjects also revealed a 14% decrease (P protein breakdown change significantly. These results indicate that the loss of body protein with inactivity is predominantly due to a decrease in muscle PS and that this decrease is reflected in both whole body and skeletal muscle measures.

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.

Science.gov (United States)

Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; De Mey, Marjan; Ajikumar, Parayil Kumaran

2016-03-22

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

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

Science.gov (United States)

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

Improved synthesis of (S)-N-Boc-5-oxaproline for protein synthesis with the α-ketoacid-hydroxylamine (KAHA) ligation.

Science.gov (United States)

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

2017-09-15

We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA) ligation. The new synthetic pathway to the enantiopure oxaproline is based on a chiral amine mediated enantioselective conjugate addition of a hydroxylamine to trans-4-oxo-2-butenoate. This route is practical, scalable and economical and provides decagram amounts of material for protein synthesis and conversion to other protected forms of (S)-oxaproline. Copyright © 2017 Elsevier Ltd. All rights reserved.

Control of protein synthesis in the female pupa of Bombyx mori

International Nuclear Information System (INIS)

Yamao, Masami; Koga, Katsumi

1975-01-01

For the purpose of understanding the mechanisms of insect metamorphosis, protein synthesis by silkmoth pupae has been studied. Synthetic rate and contents of total RNA and protein changed markedly in the female pupae of Bombyx mori. Attempt was made to find what the limiting step for the synthesis of the bulk of proteins during the adult development of female pupae is. Several female pupae of hydridstrain were homogenized at each of stated periods in buffer. The ribosomal fraction prepared from the homogenates was incubated in the buffer containing 3 H-leucine or 3 H-phenylalanine. The incorporation of leucine depending on endogenous mRNA and that of phenylalanine directed by added poly U were the largest in 9--10 days and 7th day, respectively. From the results, the synthesis of protein during the late adult development of female silkworms is controlled at the level of mRNA. The increase of ribosomes, which were active to bind mRNA, preceded the appearance of available endogenous mRNA, and it may be attributed to neogenesis and ''run-off'' of previous ribosomes. It is conceivable that such neogenesis or run-off serves as less direct control for the protein synthesis during the metamorphosis of Bombix mori. (Kobatake, H.)

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

Science.gov (United States)

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

2013-01-01

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

Selective memory generalization by spatial patterning of protein synthesis.

Science.gov (United States)

O'Donnell, Cian; Sejnowski, Terrence J

2014-04-16

Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings, we proposed a two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock

International Nuclear Information System (INIS)

Gailit, James

1990-01-01

Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins. The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m 2 . The induced proteins differed in minimum dose necessary for induction, maximum dose at which induction still occurred and constitutive level present in unirradiated cells. A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins. Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair. Synthesis of five of the proteins was also induced by brief heat shock treatment. These five may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress. (author)

Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey

Energy Technology Data Exchange (ETDEWEB)

Olsson, J E; Sandberg, M [Department of Neurology, University Hospital, S-221 85 Lund, Sweden

1975-01-01

The sites of synthesis of the low molwculat weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissue were cultured in Eagle's minium essential medium in the presence of /sup 14/C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed.

Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey

International Nuclear Information System (INIS)

Olsson, J.-E.; Sandberg, M.

1975-01-01

The sites of synthesis of the low molwculat weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissue were cultured in Eagle's minium essential medium in the presence of 14 C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed. (author)

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

Science.gov (United States)

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

2016-02-18

Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

OpenAIRE

Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

2011-01-01

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

Science.gov (United States)

Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

2015-09-01

Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Activation of protein kinase C inhibits synthesis and release of decidual prolactin

International Nuclear Information System (INIS)

Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

1986-01-01

Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

Synthesis of [18F]-5-fluorouridine (F-18-5-FUR) as a probe for measuring RNA synthesis and tumor growth rates in vivo

International Nuclear Information System (INIS)

Shiue, C.Y.; Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

1979-01-01

A method for the rapid synthesis of high specific activity of [ 18 F]-5-fluorouridine is described. The 20 Ne(d,α) 18 F nuclear reaction is used to produce high specific activity, anhydrous [ 18 F]-F 2 at the Brookhaven National Laboratory 60'' cyclotron. Fluorination of 2',3',5'-tri-0-acetyluridine with [ 18 F]-F 2 in glacial acetic acid at room temperature followed by hydrolysis with sodium methoxide in methanol gives [ 18 F]-5-fluorouridine with a radiochemical yield of 5 to 7% in a synthesis time of 90 minutes from EOB. The compound is required for the study of RNA synthesis and tumor growth rates in vivo

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

Science.gov (United States)

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

2014-09-01

Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Studies on protein synthesis by protoplasts of Saccharomyces carlsbergensis I. The effect of ribonuclease on protein synthesis

NARCIS (Netherlands)

Kloet, S.R. de; Wermeskerken, R.K.A. van; Koningsberger, V.V.

1961-01-01

Ribonuclease was found to inhibit the protein synthesis in the naked yeast protoplast for nearly 100%. Even small concentrations (5 μg/ml) were found inhibitory. The cause of this inhibition can be attributed at least in part to a 90% inhibition of the respiration. Amino acid uptake was found to

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

Science.gov (United States)

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

Key Markers of mTORC1-Dependent and mTORC1-Independent Signaling Pathways Regulating Protein Synthesis in Rat Soleus Muscle During Early Stages of Hindlimb Unloading.

Science.gov (United States)

Mirzoev, Timur; Tyganov, Sergey; Vilchinskaya, Natalia; Lomonosova, Yulia; Shenkman, Boris

2016-01-01

The purpose of the study was to assess the amount of rRNA and phosphorylation status of the key markers of mTORC1-dependent (70s6k, 4E-BP1) and mTORC1-independent (GSK-3β, AMPK) signaling pathways controlling protein synthesis in rat soleus during early stages of mechanical unloading (hindlimb suspension (HS) for 1-, 3- and 7 days). The content of the key signaling molecules of various anabolic signaling pathways was determined by Western-blotting. The amount of 28S rRNA was evaluated by RT-PCR. The rate of protein synthesis was assessed using in-vivo SUnSET technique. HS for 3 and 7 days induced a significant (pprotein synthesis in soleus muscle in comparison with control. HS within 24 hours resulted in a significant (pprotein synthesis in rat soleus during early stages of simulated microgravity is associated with impaired ribosome biogenesis as well as reduced activity of mTORC1-independent signaling pathways. © 2016 The Author(s) Published by S. Karger AG, Basel.

Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

International Nuclear Information System (INIS)

Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

1989-01-01

The effects of three different concentrations (about 20, 100 and 1000 μM) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, 14 C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively. (author)

Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

Energy Technology Data Exchange (ETDEWEB)

Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

1989-01-01

The effects of three different concentrations (about 20, 100 and 1000 ..mu..M) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, /sup 14/C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively.

High Whey Protein Intake Delayed the Loss of Lean Body Mass in Healthy Old Rats, whereas Protein Type and Polyphenol/Antioxidant Supplementation Had No Effects

Science.gov (United States)

Mosoni, Laurent; Gatineau, Eva; Gatellier, Philippe; Migné, Carole; Savary-Auzeloux, Isabelle; Rémond, Didier; Rocher, Emilie; Dardevet, Dominique

2014-01-01

Our aim was to compare and combine 3 nutritional strategies to slow down the age-related loss of muscle mass in healthy old rats: 1) increase protein intake, which is likely to stimulate muscle protein anabolism; 2) use leucine rich, rapidly digested whey proteins as protein source (whey proteins are recognized as the most effective proteins to stimulate muscle protein anabolism). 3) Supplement animals with a mixture of chamomile extract, vitamin E, vitamin D (reducing inflammation and oxidative stress is also effective to improve muscle anabolism). Such comparisons and combinations were never tested before. Nutritional groups were: casein 12% protein, whey 12% protein, whey 18% protein and each of these groups were supplemented or not with polyphenols/antioxidants. During 6 months, we followed changes of weight, food intake, inflammation (plasma fibrinogen and alpha-2-macroglobulin) and body composition (DXA). After 6 months, we measured muscle mass, in vivo and ex-vivo fed and post-absorptive muscle protein synthesis, ex-vivo muscle proteolysis, and oxidative stress parameters (liver and muscle glutathione, SOD and total antioxidant activities, muscle carbonyls and TBARS). We showed that although micronutrient supplementation reduced inflammation and oxidative stress, the only factor that significantly reduced the loss of lean body mass was the increase in whey protein intake, with no detectable effect on muscle protein synthesis, and a tendency to reduce muscle proteolysis. We conclude that in healthy rats, increasing protein intake is an effective way to delay sarcopenia. PMID:25268515

In vivo synthesized 34S enriched amino acid standards for species specific isotope dilution of proteins

DEFF Research Database (Denmark)

Hermann, Gerrit; Moller, Laura Hyrup; Gammelgaard, Bente

2016-01-01

(ICP-MS) combined to anion exchange showed that very high concentrated spike material could be produced with [small mu ]mol amounts of proteinogenic sulfur containing amino acids per g cell dry weight. An enrichment of 34S to 96.3 +/- 0.4% (n = 3) and 98.5 +/- 0.4% (n = 3) for cysteic acid...... with the concept of species specific isotope dilution analysis (IDA). The method relies on the determination of the two sulfur containing amino acids, cysteine and methionine by sulfur speciation analysis and is hence applicable to any protein containing sulfur. In vivo synthesis using 34S as sulfur source...... and methionine sulfone, respectively, was assessed. The established IDA method was validated for the absolute quantification of commercially available lysozyme and ceruloplasmin standards including the calculation of a total combined uncertainty budget....

The limits of adaptation of functional protein synthesis to sever undernutrition

International Nuclear Information System (INIS)

Jahoor, F.; Bhattiprolu, S.; Reeds, P.; Forrester, T.; Boyne, M.

1994-01-01

Our goal is to determine how the stress of infections alters the adaptation to reduced food intake in children. We think that an important element is the need for hepatic synthesis of rapidly turning over acute-phase proteins, a critical factor in overall maintenance of host defenses. When the child's prior intake has been adequate, even though growth may temporarily cease, the presence of adequate amino acid stores in tissues allows the hepatic response to stress to be maintained at the same time as an adequate rate of synthesis of nutrient transport proteins. However, when the immune system is activated in a children whose nutrition is already suboptimal the ability of the liver to synthesize nutrient transport proteins is compromised thereby further impeding nutrient utilization. We will use stable isotope tracer methodology to determine the effects of severe protein energy malnutrition, with and without infection, on the rates of synthesis of nutrient transport proteins and acute-phase proteins in undernourished children at three time points during treatment; in the early resuscitative period, after appetite has returned, and at the end of the catch-up growth phase when normal growth has resumed. (author). 12 refs, 1 fig., 1 tab

The limits of adaptation of functional protein synthesis to sever undernutrition

Energy Technology Data Exchange (ETDEWEB)

Jahoor, F; Bhattiprolu, S; Reeds, P [Baylor Coll. of Medicine, Houston, TX (United States). Children` s Nutrition Research Centre; Forrester, T; Boyne, M [West Indies Univ., Mona (Jamaica). Tropical Metabolism Research Unit

1994-12-31

Our goal is to determine how the stress of infections alters the adaptation to reduced food intake in children. We think that an important element is the need for hepatic synthesis of rapidly turning over acute-phase proteins, a critical factor in overall maintenance of host defenses. When the child`s prior intake has been adequate, even though growth may temporarily cease, the presence of adequate amino acid stores in tissues allows the hepatic response to stress to be maintained at the same time as an adequate rate of synthesis of nutrient transport proteins. However, when the immune system is activated in a children whose nutrition is already suboptimal the ability of the liver to synthesize nutrient transport proteins is compromised thereby further impeding nutrient utilization. We will use stable isotope tracer methodology to determine the effects of severe protein energy malnutrition, with and without infection, on the rates of synthesis of nutrient transport proteins and acute-phase proteins in undernourished children at three time points during treatment; in the early resuscitative period, after appetite has returned, and at the end of the catch-up growth phase when normal growth has resumed. (author). 12 refs, 1 fig., 1 tab.

Synthesis of erythrocyte membrane proteins in dispersed cells from fetal rat liver

International Nuclear Information System (INIS)

Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

1984-01-01

Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [ 35 S] methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispered cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells. (author)

Heat shock proteins of higher plants

International Nuclear Information System (INIS)

Key, J.L.; Lin, C.Y.; Chen, Y.M.

1981-01-01

The pattern of protein synthesis changes rapidly and dramatically when the growth temperture of soybean seedling tissue is increased from 28 0 C (normal) to about 40 0 C (heat shock). The synthesis of normal proteins is greatly decreased and a new set of proteins, heat shock proteins, is induced. The heat shock proteins of soybean consist of 10 new bands on one-dimensional NaDodSO 4 gels; a more complex pattern is observed on two-dimensional gels. when the tissue is returned to 28 0 C after 4 hr at 40 0 C, there is progressive decline in the synthesis of heat shock proteins and reappearance of a normal pattern of synthesis by 3 or 4 hr. In vitro translation of poly(A) + RNAs isolated from tissued grown at 28 and 40 0 C shows that the heat shock proteins are translated from a ndw set of mRNAs induced at 40 0 C; furthermore, the abundant class mRNAs for many of the normal proteins persist even though they are translated weakly (or not at all) in vivo at 40 or 42.5 0 C. The heat shock response in soybean appears similar to the much-studied heat shock phenomenon in Drosophila

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

International Nuclear Information System (INIS)

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

1986-01-01

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-/ 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

Synthesis and processing of structural and intracellular proteins of two enteric coronaviruses

International Nuclear Information System (INIS)

Sardinia, L.M.

1985-01-01

The synthesis and processing of virus-specific proteins of two economically important enteric coronaviruses, bovine enteric coronavirus (BCV) and transmissible gastroenteritis virus (TGEV), were studied at the molecular level. To determine the time of appearance of virus-specific proteins, virus-infected cells were labeled with 35 S-methionine at various times during infection, immunoprecipitated with specific hyperimmune ascitic fluid, and analyzed by SDS-polyacrylamide gel electrophoresis. The peak of BCV protein synthesis was found to be at 12 hours postinfection (hpi). The appearance of all virus-specific protein was coordinated. In contrast, the peak of TGEV protein synthesis was at 8 hpi, but the nucleocapsid proteins was present as early as 4 hpi. Virus-infected cells were treated with tunicamycin to ascertain the types of glycosidic linkages of the glycoproteins. The peplomer proteins of both viruses were sensitive to inhibition by tunicamycin indicating that they possessed N-linked carbohydrates. The matrix protein of TGEV was similarly affected. The matrix protein of BCV, however, was resistant to tunicamycin treatment and, therefore, has O-linked carbohydrates. Only the nucleocapsid protein of both viruses is phosphorylated as detected by radiolabeling with 32 P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships

Predictors of muscle protein synthesis after severe pediatric burns

Science.gov (United States)

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

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

Science.gov (United States)

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

2005-02-23

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

Synthesis of total protein (TP) and myosin heavy chain (HC) isozymes in pressure overloaded rabbit hearts

International Nuclear Information System (INIS)

Nagai, R.; Martin, B.J.; Pritzl, N.; Zak, R.; Low, R.B.; Stirewalt, W.S.; Alpert, N.R.; Litten, R.Z.

1986-01-01

Pulmonary artery banding (PO) leads to a rapid increase in right ventricular (RV) weight as well as a shift toward β myosin isozyme. They determined: (1) the contributions of changes in the capacity (RNA content) and efficiency of total protein synthesis to the increase in RV weight; and (2) the relative contributions of translational and pretranslational mechanisms to the shift in myosin HC isotypes. The rates of synthesis in vivo of TP, α- and β-HC were measured by a constant infusion technique using 3 H-leucine. TP synthesis was 7 +/- 2(SD) mg/day in control (RV:367 +/- 70 mg) and was increased by 2.6 fold at day 2 and 2.9 fold at day 4 following PO (p < 0.01). RV RNA content was increased by 83% at day 2 and 103% at day 4 PO (p < 0.05). The efficiency of synthesis (rate/RNA) was also significantly higher at these time points (1.4- and 1.3-fold). β-HC synthesis was 0.6 +/- 0.2 mg/day in control and increased by 2.6 fold at day 2 and 3.5 fold at day 4 following PO. In contrast, the rate of synthesis of α-HC was unchanged. The relative rates of β-HC to total HC synthesis was correlated linearly with the relative levels of β-myosin mRNA as measured by S1 nuclease mapping. They conclude that increases in the proportion of β-HC myosin following PO is due to increases in the relative amount of β-myosin mRNA and therefore involves modulation of a pretranslational mechanism

Dynamic changes of the early protein synthesis in murine immune cells after low dose radiation

International Nuclear Information System (INIS)

Chen Shali; Liu Shuzheng

1997-01-01

It was shown that there was a marked increase in protein synthesis of thymocytes that were metabolically labelled with 3 H-Leu for 4,6,8 and 12 hours in low dose irradiated mice showing 33.26%, 51.48%, 51.54% and 34.98% increase respectively at different time intervals of incubation when the thymic and splenic cells were sampled 4 hours after whole body irradiation (WBI) with 75 mGy X-rays. The results suggest that there is an increase in protein synthesis with its peak at 6∼8 hours after radiation. Changes in protein synthesis of immune cells in mice 4 hours after radiation and incubated for 4∼12 h were observed with SDS-PAGE followed by densitometrical scanning. It is revealed that 28 kD protein synthesis was increased gradually within 12 hours of incubation and 43 kD protein synthesis was increased in the thymocytes rapidly reaching a maximum 2 hours after incubation. It was also exhibited that the synthesis of 43 kD protein and 32 kD protein was increased in the splenocytes 2 hours after incubation. These findings may have implications in the mechanism of immunoenhancement and adaptive response induced by low dose radiation

Engineering Designed Proteins for Light Capture, Energy Transfer, and Emissive Sensing In Vivo

Science.gov (United States)

Mancini, Joshua A.

Proteins that are used for photosynthetic light harvesting and biological signaling are critical to life. These types of proteins act as scaffolds that hold small, sometimes metal-containing organic molecules in precise locations for light absorption and successive use. For signaling proteins, this energy can be used to induce a photoisomerization of the small molecule that can turn on or off a signaling cascade that controls the physiology of an organism. Alternatively, photosynthetic light-harvesting proteins funnel this energy in a directional manner towards a charge separating catalytic component that can change this light energy into chemical energy. The protein environment also serves to tune the photophysical properties of the small molecules. This is seen extensively with the linear tetrapyrroles that are used in both photosynthetic and signaling proteins. Many efforts have been made to harness these natural proteins for societal use, including improving photophysical properties and interfacing capabilities with manmade catalytic components. Several methods of achieving improvement have entailed structurally guided mutation and directed evolution. However, these methods all have their limitations due to the inherent complexity and fragility of the natural proteins. This work presents an alternative more robust method to natural proteins. My thesis states: that man-made proteins, known as maquettes, employing basic rules of protein folding, can be designed to become light harvesting and signaling proteins that can be assembled fully in vivo providing an alternative, robust, and versatile platform for meeting the diverse array of societal "green chemistry" and biomedical needs. This in vivo assembly is carried out by interacting with cyanobacterial protein and pigment machinery, both as stand-alone units and as protein fusions with natural antenna complexes. Additionally, this work offers insight for fast and tight binding of circular and linear tetrapyrroles

Towards single-molecule observation of protein synthesis

International Nuclear Information System (INIS)

Dulin, David; Le Gall, Antoine; Bouyer, Philippe; Perronet, Karen; Westbrook, Nathalie; Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

2009-01-01

The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, a study at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the development of an advanced surface chemistry to attach an active ribosome to the microscope coverslip and follow the amino-acid incorporation by fluorescence microscopy. The ribosome is labeled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. It should then be possible to observe the protein synthesis in real time as the labeled amino acids are incorporated into the polypeptide chain. (Author)

Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

Science.gov (United States)

Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

2015-09-11

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

On the mechanism of arginine requirement for adenovirus synthesis

International Nuclear Information System (INIS)

Plaat, D.; Weber, J.

1979-01-01

The effects of arginine deprivation on the synthesis and processing of viral proteins and the assembly of incomplete and complete virions were studied during infection with human adenovirus type 2. Arginine deprivation greatly reduced the synthesis of all viral proteins, particularly the precursor to core protein VII. The inhibition was completely reversible by the addition of arginine to the medium. Arginine deprivation between 7 and 20 hours post-infection inhibited the processing of PVII to VII, suggesting that PVII is not cleaved autocatalytically. The assembly of incomplete virions was sensitive to arginine deprivation only prior to 20 hours, while the assembly of complete virions was dependent on the continuous presence of arginine. This observation supports the hypothesis that incomplete virions are precursors of complete virions. The experiments on the PVII-specific endoprotease activity showed that arginine deprivation caused only slight reduction in the in vitro activity, although no activity was observed in vivo. The present results lead to the hypothesis that arginine deficiency inhibits the synthesis of a functional protein essential for virion maturation, other than the synthesis of processing of PVII. (author)

Skeletal muscle morphology, protein synthesis and gene expression in Ehlers Danlos Syndrome

DEFF Research Database (Denmark)

Nygaard, Rie H; Jensen, Jacob K; Voermans, Nicol C

2017-01-01

skeletal muscle biopsies in patients with classic EDS (cEDS, n=5 (Denmark)+ 8 (The Netherlands)) and vascular EDS (vEDS, n=3) and analyzed muscle fiber morphology and content (Western blotting and muscle fiber type/area distributions) and muscle mRNA expression and protein synthesis rate (RT-PCR and stable...... isotope technique). RESULTS: The cEDS patients did not differ from healthy controls (n = 7-11) with regard to muscle fiber type/area, myosin/α-actin ratio, muscle protein synthesis rate or mRNA expression. In contrast, the vEDS patients demonstrated higher expression of matrix proteins compared to c......EDS patients (fibronectin and MMP-2). DISCUSSION: The cEDS patients had surprisingly normal muscle morphology and protein synthesis, whereas vEDS patients demonstrated higher mRNA expression for extracellular matrix remodeling in skeletal musculature compared to cEDS patients....

Fluoride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion.

Science.gov (United States)

Sharma, Ramaswamy; Tsuchiya, Masahiro; Bartlett, John D

2008-09-01

Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER. We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation. We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo. These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.

Effect of whey protein on the In Vivo Release of Aldehydes.

NARCIS (Netherlands)

Weel, K.G.C.; Boelrijk, A.E.M.; Burger, J.J.; Claassen, N.E.; Gruppen, H.; Voragen, A.G.J.

2003-01-01

Retention of aldehydes by whey proteins in solutions buffered at a range of pH values was studied under static and dynamic headspace conditions and in vivo in exhaled air. Static headspace measurements showed a clear increase in retention in the presence of whey proteins for aldehydes with longer

Ribosomal history reveals origins of modern protein synthesis.

Directory of Open Access Journals (Sweden)

Ajith Harish

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

In vivo protein quality of selected cereal-based staple foods enriched with soybean proteins

Directory of Open Access Journals (Sweden)

Laura Acevedo-Pacheco

2016-10-01

Full Text Available Background: One way to diminish protein malnutrition in children is by enriching cereal-based flours for the manufacturing of maize tortillas, wheat flour tortillas, and yeast-leavened breads, which are widely consumed among low socio-economic groups. Objective: The aim was to determine and compare the essential amino acid (EAA scores, protein digestibility corrected amino acid scores (PDCAAS, and in vivo protein quality (protein digestibility, protein efficiency ratio (PER, biological values (BV, and net protein utilization (NPU values of regular versus soybean-fortified maize tortillas, yeast-leavened bread, and wheat flour tortillas. Design: To comparatively assess differences in protein quality among maize tortillas, wheat flour tortillas, and yeast-leavened breads, EAA compositions and in vivo studies with weanling rats were performed. The experimental diets based on regular or soybean-fortified food products were compared with a casein-based diet. Food intake, weight gains, PER, dry matter and protein digestibility, BV, NPU, and PDCAAS were assessed. The soybean-fortified tortillas contained 6% of defatted soybean flour, whereas the yeast-leavened bread flour contained 4.5% of soybean concentrate. Results: The soybean-fortified tortillas and bread contained higher amounts of lysine and tryptophan, which improved their EAA scores and PDCAAS. Rats fed diets based on soybean-fortified maize or wheat tortillas gained considerably more weight and had better BV and NPU values compared with counterparts fed with respective regular products. As a result, fortified maize tortillas and wheat flour tortillas improved PER from 0.73 to 1.64 and 0.69 to 1.77, respectively. The PER improvement was not as evident in rats fed the enriched yeast-leavened bread because the formulation contained sugar that decreased lysine availability possibly to Maillard reactions. Conclusions: The proposed enrichment of cereal-based foods with soybean proteins greatly

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

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Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

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Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe; Del Monaco, Giovanni; She, Qunxin; Rossi, Mosè; Valenti, Anna; Ciaramella, Maria

2017-01-01

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells

International Nuclear Information System (INIS)

Santoro, M.G.; Garaci, E.; Amici, C.

1989-01-01

Prostaglandins (PGs)A 1 and J 2 were found to potently suppress the proliferation of human K562 erythroleukemia cells and to induce the synthesis of a 74-kDa protein (p74) that was identified as a heat shock protein related to the major 70-kDa heat shock protein group. p74 synthesis was stimulated at doses of PGA 1 and PGJ 2 that inhibited cell replication, and its accumulation ceased upon removal of the PG-induced proliferation block. PGs that did not affect K562 cell replication did not induce p74 synthesis. p74 was found to be localized mainly in the cytoplasm of PG-treated cells, but moderate amounts were found also in dense areas of the nucleus after PGJ 2 treatment. p74 was not necessarily associated with cytotoxicity or with inhibition of cell protein synthesis. The results described support the hypothesis that synthesis of the 70-kDa heat shock proteins is associated with changes in cell proliferation. The observation that PGs can induce the synthesis of heat shock proteins expands our understanding of the mechanism of action of these compounds whose regulatory role is well known in many physiological phenomena, including the control of fever production

Pyridine induction of cytochrome P450IIE1: Evidence for enhanced protein synthesis

International Nuclear Information System (INIS)

Kim, S.G.; Novak, R.F.

1990-01-01

The dose-, and time-dependent induction of P450IIE1 in the rat by pyridine (PY) has been characterized. A single injection of PY (100 mg/kg, i.p.) increased as the levels of IIE1 2-, 3- and 4-fold at 6, 10 and 24 hr, respectively, relative to controls based on p-nitrophenol hydroxylase activity and Western blot analysis. Induction of IIE1 was dose-dependent over the range 10 to 200 mg/kg. Cycloheximide administration completely prevented the induction of IIE1 by PY, while actinomycin D failed to affect PY induction of IIE1. The rate of IIE1 synthesis was examined by labelling of proteins with [ 14 C] leucine in vivo, followed by SDS-PAGE and autoradiographic analysis of isolated microsomes. Enhanced intensity of the IIE1 band was observed in microsomes isolated from rats treated with either PY or acetone relative to untreated rats. Slot and Northern blot analyses were employed to assess IIE1 mRNA levels in total RNA and poly(A + ) mRNA isolated from livers of rats at 1, 5 and 12 hr following a single dose of PY. No increase in IIE1 mRNA in total RNA was monitored. A time-dependent decrease in IIE1 poly(A + ) mRNA however, was observed with the maximal decrease occurring at ∼12 hr. These results suggest that induction of IIE1 by PY does not involve transcriptional activation but occurs by protein synthesis possibly through increased translational efficiency

Synthesis and in-vivo detection of boronated compounds for use in BNCT

International Nuclear Information System (INIS)

Kabalka, G.W.

1990-04-01

The primary objective of the DOE Program at the University of Tennessee Biomedical Imaging Center is the development of new technology to detect boron compounds in-vivo. The research focuses on the development of multinuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) techniques for verifying and measuring BNCT agents in-vivo. A small but significant portion of the effort is directed toward the design of boron-containing neutron-capture-therapy agents. The UT -- DOE program is unique in that it has access to two state-of-the-art multinuclear magnetic resonance imaging units housed in the Biomedical Imaging Center at the University of Tennessee Medical Center at Knoxville. Included in this report are two sections describing research accomplishments in multinuclear magnetic resonance imaging and synthesis of potential BNCT agents

Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

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Muhammad Ibrahim

Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

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Ibrahim, Muhammad; Shi, Yu; Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Blondel, Carlos; Santiviago, Carlos A; Contreras, Ines; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

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

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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.

Studying RNA-protein interactions in vivo by RNA immunoprecipitation

DEFF Research Database (Denmark)

Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

2011-01-01

and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

Science.gov (United States)

Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

2017-07-01

The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

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Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

2016-01-01

Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session.

A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

Science.gov (United States)

Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

2017-01-01

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

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

Science.gov (United States)

Hudson, Matthew B; Smuder, Ashley J; Nelson, W Bradley; Wiggs, Michael P; Shimkus, Kevin L; Fluckey, James D; Szeto, Hazel H; Powers, Scott K

2015-01-01

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

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

Directory of Open Access Journals (Sweden)

Matthew B Hudson

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1978-10-01

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

Astatine-211 labelled proteins and their stability in vivo

International Nuclear Information System (INIS)

Yi Changhou; Jin Jannan; Zhang Shuyuan; Wang Ketai; Zhang Dayuan; Zhou Maolun

1989-01-01

211 At or 131 I labelled proteins, e.g. 211 At-IgG or 211 At-BSA (bovine serum albumin) were prepared by 211 At reaction with the diazo-compound of para-aminobenzoic acid, which is then conjugated with IgG or BSA via an acylation reaction. The 211 At-carbon bond was found metabolically stable under in vivo conditions. For the labelling of proteins with 211 At or 131 I, other methods of direct oxidation are also described. The results show that for the labelling of proteins with 211 At, high rate of incorporation can be obtained with hydrogen peroxide as oxidant, but the labelling of proteins with 131 I is more favourable with the strong oxidant Chloramine-T. (author) 12 refs.; 6 figs

In vivo protein-DNA interactions at the β-globin gene locus

International Nuclear Information System (INIS)

Tohru Ikuta; Yuet Wai Kan

1991-01-01

The authors have investigated in vivo protein-DNA interactions in the β-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express var-epsilon- and γ-globin but not β-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The β promoter was also not footprinted, while extensive footprints were observed in the promoter of the active γ-globin gene. No footprints were seen in the A γ and β3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells

Protein synthesis levels are increased in a subset of individuals with Fragile X syndrome

DEFF Research Database (Denmark)

Jacquemont, Sébastien; Pacini, Laura; Jønch, Aia E

2018-01-01

architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical...... severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS...... and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those...

Clofibrate-induced increases in peroxisomal proteins: effect on synthesis, degradation, and mRNA activity

International Nuclear Information System (INIS)

Mortensen, R.M.

1983-01-01

The effect of clofibrate on the polypeptide composition of peroxisomes was determined. A simple method was developed for the isolation of peroxisomes with a purity of 90-95% using sedimentation in a metrizamide gradient. The specific activities of HD did not change with clofibrate treatment so that the increases in enzyme activities are solely due to increases in protein amounts. The hepatic concentration of HD increased 63 times. The HD synthesis rate, as measured by the incorporation of [ 3 H]leucine, increased 74 times, so that the increase in the synthesis was sufficient to account for the increase in protein. Clofibrate caused no discernible change in the degradation rate of HD labeled with [ 14 C]bicarbonate. The half-life of HD was approximately 2 days. The translatable mRBA coding for HD increased 55 times. This value is not significantly different from the increase in HD protein or in HD synthesis. This observation was also true for several other peroxisomal proteins. Therefore, clofibrate causes an increase in the mRNA activity, which increases the synthesis of HD leading to an accumulation of protein and enzyme activity. The kinetics of the clofibrate-induced changes in HD synthesis rate, protein level, and enzymatic activity was analyzed using a simple model which included the half-lives of the drug, mRNA, and protein. The best fit of the model to the data gave an mRNA half-life of 10 hours and a protein half-life of 1.8 days, with no significant change by clofibrate

Safe taste memory consolidation is disrupted by a protein synthesis inhibitor in the nucleus accumbens shell.

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Pedroza-Llinás, R; Ramírez-Lugo, L; Guzmán-Ramos, K; Zavala-Vega, S; Bermúdez-Rattoni, F

2009-07-01

Consolidation is the process by which a new memory is stabilized over time, and is dependent on de novo protein synthesis. A useful model for studying memory formation is gustatory memory, a type of memory in which a novel taste may become either safe by not being followed by negative consequences (attenuation of neophobia, AN), or aversive by being followed by post-digestive malaise (conditioned taste aversion, CTA). Here we evaluated the effects of the administration of a protein synthesis inhibitor in the nucleus accumbens (NAc) shell for either safe or aversive taste memory trace consolidation. To test the effects on CTA and AN of protein synthesis inhibition, anisomycin (100microg/microl) was bilaterally infused into the NAc shell of Wistar rats' brains. We found that post-trial protein synthesis blockade impaired the long-term safe taste memory. However, protein synthesis inhibition failed to disrupt the long-term memory of CTA. In addition, we infused anisomycin in the NAc shell after the pre-exposure to saccharin in a latent inhibition of aversive taste. We found that the protein synthesis inhibition impaired the consolidation of safe taste memory, allowing the aversive taste memory to form and consolidate. Our results suggest that protein synthesis is required in the NAc shell for consolidation of safe but not aversive taste memories, supporting the notion that consolidation of taste memory is processed in several brain regions in parallel, and implying that inhibitory interactions between both taste memory traces do occur.

Alcohol-induced decrease in muscle protein synthesis associated with increased binding of mTOR and raptor: Comparable effects in young and mature rats

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Vary Thomas C

2009-01-01

Full Text Available Abstract Background Acute alcohol (EtOH intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication. Methods Male F344 rats were studied at approximately 3 (young or 12 (mature months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [3H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation. Results Blood alcohol levels (BALs were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL. However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL. EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E·eIF4G and inactive eIF4E·4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6

Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm.

Science.gov (United States)

Kasumov, Takhar; Ilchenko, Serguey; Li, Ling; Rachdaoui, Nadia; Sadygov, Rovshan G; Willard, Belinda; McCullough, Arthur J; Previs, Stephen

2011-05-01

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O. Copyright © 2011 Elsevier Inc. All rights reserved.

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

DEFF Research Database (Denmark)

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

2017-01-01

The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL......) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at –14, +4, +15, and +29 DRTC by measuring [13C]Phe isotopic...... enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[13C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [125I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [13C...

Protein synthesis in muscle cultures from patients with duchenne muscular dystrophy

International Nuclear Information System (INIS)

Ionasescu, V.; Zellweger, H.; Ionasescu, R.; Lara-Braud, C.; Cancilla, P.A.

1976-01-01

Muscle samples for cultures were obtained from the quadriceps by open biopsy under local anesthesia in five patients with early stage of Duchenne muscular dystrophy (DMD) and 10 controls. Primary cultures were grown in Eagle's Minimum Essential Medium (MEM) with 20 per cent fetal calf serum. After 4 weeks, cells were trypsinized, counted, subcultured for 5 days in MEM with 5 per cent horse serum and finally incubated for 4 h with ( 3 H) leucine. Total protein synthesis showed a significant decrease (ALF OF CONTROL VALUES) only in muscle cultures from patients with DMD. Addition of calcium chloride alone or with A23187 ionophore normalized this defect in protein synthesis. By contrast, myosin heavy chain synthesis was measured and found normal in all patients. (author)

Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

Science.gov (United States)

Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

2015-05-01

Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis

International Nuclear Information System (INIS)

Drillien, R.; Spehner, D.; Kirn, A.

1978-01-01

Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by uv irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective

Conversion of leucine to β‐hydroxy‐β‐methylbutyrate by α‐keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes

Science.gov (United States)

Vílchez, José D.; Salto, Rafael; Manzano, Manuel; Sevillano, Natalia; Campos, Nefertiti; Argilés, Josep M.; Rueda, Ricardo; López‐Pedrosa, José M.

2015-01-01

Abstract Background L‐Leu and its metabolite β‐hydroxy‐β‐methylbutyrate (HMB) stimulate muscle protein synthesis enhancing the phosphorylation of proteins that regulate anabolic signalling pathways. Alterations in these pathways are observed in many catabolic diseases, and HMB and L‐Leu have proven their anabolic effects in in vivo and in vitro models. The aim of this study was to compare the anabolic effects of L‐Leu and HMB in myotubes grown in the absence of any catabolic stimuli. Methods Studies were conducted in vitro using rat L6 myotubes under normal growth conditions (non‐involving L‐Leu‐deprived conditions). Protein synthesis and mechanistic target of rapamycin signalling pathway were determined. Results Only HMB was able to increase protein synthesis through a mechanism that involves the phosphorylation of the mechanistic target of rapamycin as well as its downstream elements, pS6 kinase, 4E binding protein‐1, and eIF4E. HMB was significantly more effective than L‐Leu in promoting these effects through an activation of protein kinase B/Akt. Because the conversion of L‐Leu to HMB is limited in muscle, L6 cells were transfected with a plasmid that codes for α‐keto isocaproate dioxygenase, the key enzyme involved in the catabolic conversion of α‐keto isocaproate into HMB. In these transfected cells, L‐Leu was able to promote protein synthesis and mechanistic target of rapamycin regulated pathway activation equally to HMB. Additionally, these effects of leucine were reverted to a normal state by mesotrione, a specific inhibitor of α‐keto isocaproate dioxygenase. Conclusion Our results suggest that HMB is an active L‐Leu metabolite able to maximize protein synthesis in skeletal muscle under conditions, in which no amino acid deprivation occurred. It may be proposed that supplementation with HMB may be very useful to stimulate protein synthesis in wasting conditions associated with chronic diseases, such as cancer or

In vitro versus in vivo protein digestibility techniques for calculating PDCAAS (protein digestibility-corrected amino acid score) applied to chickpea fractions.

Science.gov (United States)

Tavano, Olga Luisa; Neves, Valdir Augusto; da Silva Júnior, Sinézio Inácio

2016-11-01

Seven different in vitro methods to determine the protein digestibility for chickpea proteins were considered and also the application of these methodologies for calculating PDCAAS (protein digestibility-corrected amino acid score), seeking their correlations with the in vivo methodology. In vitro digestibility of raw and heated samples were determined using pepsin-pancreatin hydrolysis, considering soluble nitrogen via Kjeldahl (ppKJ) and hydrolysed peptide linkages using trinitrobenzenesulfonic acid and o-phthaldialdehyde. In vitro digestibility was also determined using trypsin, chymotrypsin and peptidase (3-Enz) or trypsin, chymotrypsin, peptidase and pronase solution (4-Enz). None of the correlations between in vitro and in vivo digestibilities were significant (at p<0.0500), but, strong correlations were observed between PDCAAS calculated by in vitro and in vivo results. PDCAAS-ppKJ, PDCAAS-3-Enz and PDCAAS-4-Enz presented the highest correlations with in vivo method, r=0.9316, 0.9442 and 0.9649 (p<0.0500), respectively. The use of in vitro methods for calculating PDCAAS may be promising and deserves more discussions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

Science.gov (United States)

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

Glucose Synthesis in a Protein-Based Artificial Photosynthesis System.

Science.gov (United States)

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

2015-09-01

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

Equine interferon gamma synthesis in lymphocytes after in vivo infection and in vitro stimulation with EHV-1.

Science.gov (United States)

Paillot, R; Daly, J M; Juillard, V; Minke, J M; Hannant, D; Kydd, J H

2005-08-22

Equine cytotoxic T lymphocyte (CTL) responses to equine herpesvirus-1 (EHV-1) are well characterised but little is known about the cytokine response after infection or vaccination. EHV-1 is common in horses and infects lymphocytes in vivo. This virus was used as a model to measure the synthesis of interferon gamma (IFN-gamma) by equine peripheral blood mononuclear cells (PBMC) after in vivo infection and/or in vitro stimulation with EHV-1. Both flow cytometry and ELISPOT assays were used to quantify equine IFN-gamma using a mouse anti-bovine IFN-gamma monoclonal antibody (clone CC302; shown to cross-react with recombinant equine IFN-gamma) and a rabbit anti-canine IFN-gamma polyclonal antibody. The percentage of PBMC synthesising IFN-gamma after in vitro stimulation with EHV-1 increased with age. In yearlings infected experimentally with EHV-1, PBMC showed two peaks of IFN-gamma synthesis, 11 and 56 days after infection. The IFN-gamma synthesis was principally associated with CD8(+) cells. The patterns of IFN-gamma synthesis detected by intracellular IFN-gamma staining or ELISPOT were compared with CTL data and shown to be similar. These methods were also applied successfully to frozen samples of PBMC. Measurement of equine IFN-gamma using these simple techniques can now be applied to future studies on protective cellular immune responses following virus infection and/or vaccination of horses.

[Effect of metalaxyl on the synthesis of RNA, DNA and protein in Phytophthora nicotianae].

Science.gov (United States)

Wollgiehn, R; Bräutigam, E; Schumann, B; Erge, D

1984-01-01

Metalaxyl is used to control diseases caused by fungi of the order of the Perenosporales. We investigated the action of this fungicid eon nucleic acid and protein synthesis in liquid cultures of Phytophthora nicotianae. The uptake of 32P, 3H-uridine, 3H-thymidine and 14C-leucine as precursors of nuclei acid and protein synthesis by the mycelium was not inhibited by metalaxyl. RNA synthesis as indicated by 3H-uridine incorporation was strongly inhibited (about 80%) by 0.5 micrograms/ml of metalaxyl. The inhibition was visible already few minutes after addition of the toxicant. Since the inhibition of incorporation of 3H-thymidine into DNA and of 14C-leucine into protein became significant 2-3 hours later, we conclude that metalaxyl primarily interfers with RNA synthesis. Synthesis of ribosomal RNA is more affected (more than 90%) than that of tRNA (about 55%) and poly(A)-containing RNA. Since in the presence of actinomycin, in contrast to metalaxyl, protein synthesis is inhibited immediately as a consequence of complete inhibition of RNA synthesis and of the short life-time of mRNA, it is also evident that mRNA synthesis is less strongly inhibited, at least during the early period of metalaxyl action. The molecular mechanism of metalaxyl inhibition of the transcription process remains open. The fungicide did not inhibit the activity of a partially purified RNA polymerase isolated from the fungus. On the other hand, the RNA synthesis (14C-UTP-incorporation) by a cell homogenate and by isolated nuclear fractions was inhibited significantly. Possibilities of the molecular action of metalaxyl are discussed. The RNA synthesis of some plant systems (cell cultures of Lycopersicon peruvianum, isolated nuclei from the same cell cultures, purified RNA polymerase from Spinacia oleracea chloroplasts) was not inhibited by metalaxyl, not even at high concentrations.

Mitochondrial Protein Synthesis, Import, and Assembly

Science.gov (United States)

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

DCB-3503, a tylophorine analog, inhibits protein synthesis through a novel mechanism.

Directory of Open Access Journals (Sweden)

Ying Wang

Full Text Available BACKGROUND: DCB-3503, a tylophorine analog, inhibits the growth of PANC-1 (human pancreatic ductal cancer cell line and HepG2 (human hepatocellular cancer cell line tumor xenografts in nude mice. The inhibition of growth leads to cancer cell differentiation instead of cell death. However, the mechanisms of action of tylophorine analogs is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that DCB-3503 suppresses the expression of pro-oncogenic or pro-survival proteins with short half-lives, including cyclin D1, survivin, beta-catenin, p53, and p21, without decreasing their mRNA levels. Proteasome inhibitor reversed the inhibitory effect of DCB-3503 on expression of these proteins. DCB-3503 inhibited the incorporation of radiolabeled amino acid and thymidine, and to a much lesser degree of uridine, in a panel of cell lines. The mechanism of inhibition of protein synthesis is different from that of cycloheximide (CHX as assayed in cell culture and HeLa in vitro translation system. Furthermore, in contrast to rapamycin, DCB-3503 does not affect protein synthesis through the mTOR pathway. DCB-3503 treatment shifts the sedimentation profiles of ribosomes and mRNAs towards the polysomal fractions while diminishing monosome abundance, indicative of the inhibition of the elongation step of protein synthesis. Preferential down regulation of several studied proteins under these conditions is likely due to the relative short half-lives of these proteins. CONCLUSION/SIGNIFICANCE: The inhibitory effect of DCB-3503 on translation is apparently distinct from any of the current anticancer compounds targeting protein synthesis. Translation inhibitors with novel mechanism could complement current chemotherapeutic agents for the treatment of human cancers and suppress the occurrence of drug resistance.

Studies on protein synthesis by protoplasts of Saccharomyces carlsbergensis II. Reversal of the RNase effect of protein synthesis by polymethacrylic acid

NARCIS (Netherlands)

Kloet, S.R. de; Wermeskerken, R.K.A. van; Koningsberger, V.V.

1961-01-01

The ribonuclease inhibited protein synthesis and respiration of yeast protoplasts can be restored by the addition of several polyanionic compounds, among which polymethacrylic acid proved to be the most effective one. The results of preliminary experiments with the ultracentrifuge indicate a

Synthesis and In Vitro and In Vivo Evaluation of Iodine-131-Labeled Folates: Potential Molecular Diagnostic and Therapeutic Radiopharmaceuticals

International Nuclear Information System (INIS)

Al Jammaz, I.

2009-01-01

Molecular targeting imaging has a great potential to be able to image molecular changes that are currently defined as predisease states which facilitate earlier detection of cancer and consequently, the greatest chance of cure. Advancement of scintigraphic imaging and radiotherapy is highly determined by development of more specific radiotracers. The Membrane-associated-folic acid receptor is a glycosylphospstidylinositol protein that overexpressed in approximately 100% of serious ovarian adenocarcinomas and various epithelial cancers including cervical, colorectal and renal cancers. Meanwhile, this receptor is highly restricted in most normal tissues which make these tumors as an excellent candidates for molecular targeting imaging and therapy through the folate receptor system. As part of our on-going research effort to develop prosthetic precursors for radiohalogenation of bioactive molecules, we have previously reported the synthesis and biological characterization of [ 18 F]- fluorobenzene and pyridine carbohydrazide-folate conjugates ([ 18 F]-SFB and [ 18 F]-SFP-folates). We here report the synthesis and biological characterization of [ 131 I]-iodobenzenecarbohydrazide-folate conjugate ([ 131 I]-SIB-folate) as a potential therapeutic radiopharmaceutical. The synthetic approaches for preparation of [ 131 I]iodobenzene carbohydrazide-folates ([ 131 I]-SIB-folate) entailed sequence of reactions. Hydrazide-folate was reacted with N-succinimidyl-m-[131I]-iodobenzoate-carboxylate ([ 131 I]-SIB) to give [ 131 I]-SIB-folate conjugate. Radiochemical yield was greater than 80% and synthesis times were ranging between 40-45 min. Radiochemical purity was also greater than 97% without HPLC purification. These synthetic approaches hold considerable promise as rapid and simple method for the radiohalogenation of folate in high radiochemical yield in short time. In vitro tests on KB cell line has shown that significant amount of the radioconjugate associated with cell

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

International Nuclear Information System (INIS)

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

2010-01-01

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

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

Science.gov (United States)

Gan, Qinglei; Fan, Chenguang

2017-11-01

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

In vivo stability and inertness of various direct labelled and chelate-tagged protein

International Nuclear Information System (INIS)

Janoki, A.; Korosi, L.; Klivenyi, G.; Spett, B.

1987-01-01

There were looking for methods giving precise information about composition and activity distribution of protein components, both in the initial samples and serum samples after intravenous administration. It was tested the applicability of electroimmunoassay, polyacrilamide gel electrophoresis and high performance liquid chromatography for the assessment of in vivo stability and labelled proteins. The model compound was human serum albumin (HSA) labelled with 99m Tc and 125 I, respectively. Bifunctional chelate labelling was done with desferrioxamine, in this case protein was labelled with 67 Ga. Biodistribution of the labelled compounds and their elimination from the blood were studied in rabbits. Experience with various labelling proteins, especially with Tc-Sn-HSA system indicate that in vivo stability of this compounds are generally low. Following intravenous injection of proteins labelled with metal isotopes, due to dilution and to the presence of considerable amount of compatitive protein in the serum, part of the label is being detached from the carrier protein. Distribution of the detached metal is different from the original distribution of the protein. This problem arises also with radiopharmaceuticals based on monoclonal antibodies. (M.E.L.) [es

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

Science.gov (United States)

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

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

OpenAIRE

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

2011-01-01

It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memo...

Protein synthesis and the recovery of both survival and cytoplasmic ''petite'' mutation in ultraviolet-treated yeast cells

International Nuclear Information System (INIS)

Heude, M.; Chanet, R.; Moustacchi, E.

1975-01-01

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phase haploid yeast cells was examined. This was carried out using cycloheximide, 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 protein synthesis

Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

Science.gov (United States)

Murphy, Caoileann H; Shankaran, Mahalakshmi; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Burke, Louise M; Hawley, John A; Kassis, Amira; Karagounis, Leonidas G; Li, Kelvin; King, Chelsea; Hellerstein, Marc; Phillips, Stuart M

2018-06-01

Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24�

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

Science.gov (United States)

Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

2017-11-01

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Regulation of urea synthesis during the acute phase response in rats

DEFF Research Database (Denmark)

Thomsen, Karen Louise; Jessen, Niels; Buch Møller, Andreas

2013-01-01

The acute-phase response is a catabolic event involving increased waste of amino-nitrogen (N) via hepatic urea synthesis, despite an increased need for amino-N incorporation into acute-phase proteins. This study aimed to clarify the regulation of N elimination via urea during different phases...... of the tumor necrosis factor-α (TNF-α)-induced acute-phase response in rats. We used four methods to study the regulation of urea synthesis: We examined urea cycle enzyme mRNA levels in liver tissue, the hepatocyte urea cycle enzyme proteins, the in vivo capacity of urea-N synthesis (CUNS), and known humoral...... regulators of CUNS at 1, 3, 24, and 72 h after TNF-α injection (25 μg/kg iv rrTNF-α) in rats. Serum acute-phase proteins and their liver mRNA levels were also measured. The urea cycle enzyme mRNA levels acutely decreased and then gradually normalized, whereas the urea cycle enzyme proteins remained...

T-Stimulator effect on cotton protein composition and synthesis in salinization stress

International Nuclear Information System (INIS)

Ibragimova, E.A.; Nazirova, E.R.; Samarkhodjaeva, N.R.; Nalbandyan, A.A.; Babaev, T.A.

2004-01-01

Full text: T-stimulator was established to possess a wide spectrum of physiological effects, to enhance plant adaptation to thermal stress and to increase plant resistance to pathogens. Plant adaptation to unfavorable conditions manifests in changes in many links of metabolism, that of proteins included. We studied effect of cottonseed treatment with T-stimulator on composition and synthesis of plasma membrane proteins upon chloride salinization by means of the radioisotope method. Electrophoretic fractionation of cottonseed plasma membrane proteins showed absence of more than 40 polypeptides with molecular mass from 10 to more than 100 kDa in the cotton root membranes. Major fractions-polypeptides with molecular mass of 61, 53, 46, 25, 21, 20 and 18 kDa constitute about 50% of the total polypeptide composition. The salinization significantly affects the total membrane protein output, proportion of some polypeptides and their synthesis rate. Analysis of phoreogram radioautographs showed that 2-hour exposition of cotton roots to 35 S methionine suppresses synthesis of major polypeptides with molecular mass of 63, 61 and 53 kDa, that of low molecular polypeptides (46, 20, 18 kDa) increasing. Changes in the proportion of major polypeptides in cotton plasma membranes, reduction in rate of biosynthesis of high molecular fractions with the general suppression of label inclusion in the membrane fraction are the evidence for a disturbance in biosynthesis of some membrane proteins in cotton tissue cells upon salinization. The inhibiting effect of salinization on the protein-synthesizing system was observed in plants treated with T-stimulator, but the rate of synthesis in plasma membranes of the treated plants was found significantly higher. The activation of some plasma membrane proteins under T-stimulator effect suggests an association with the increase in adaptation of the treated plants to the disturbing effect of salinization

The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

Directory of Open Access Journals (Sweden)

Proud Christopher G

2002-05-01

Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

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Donohue-Rolfe Arthur

2011-06-01

Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

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

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Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

2008-10-01

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein1[OPEN

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Fernie, Alisdair R.; Stitt, Mark

2015-01-01

Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d−1), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. PMID:25810096

A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

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Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

2017-05-13

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Determination of human muscle protein fractional synthesis rate

DEFF Research Database (Denmark)

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

2014-01-01

In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically...

Sulfur in human nutrition - effects beyond protein synthesis

NARCIS (Netherlands)

Gertjan Schaafsma

2008-01-01

That sulfur is essential to humans is based on the requirement of S-animo acids for normal growth and maintenance of nitrogen balance and not on the optimization of metabolic proccesses involving the synthesis of non-protein sulphur containing compounds. This paper reviews the significance of sulfur

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

DEFF Research Database (Denmark)

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

2011-01-01

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

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

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Yazmín Ramiro-Cortés

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

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

International Nuclear Information System (INIS)

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

1984-01-01

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

A human endogenous protein exerts multi-role biomimetic chemistry in synthesis of paramagnetic gold nanostructures for tumor bimodal imaging.

Science.gov (United States)

Yang, Weitao; Wu, Xiaoli; Dou, Yan; Chang, Jin; Xiang, Chenyang; Yu, Jiani; Wang, Jun; Wang, Xiuli; Zhang, Bingbo

2018-04-01

Protein-mediated biomimetic nanoparticles because of simplicity of their synthesis, subdued nonspecific adsorption, improved pharmacokinetics, and biocompatibility have been receiving increasing attention recently. Nevertheless, only a handful of proteins have been developed for biomimetic synthesis. Worse still, most of them are constrained on single-function usages in chemistry. Exploring new functional proteins, especially those with multi-dentate moieties for multi-role biomimetic chemistry, still remains a substantial challenge. Here, we report on a human endogenous protein, glutathione S-transferase (GST), with favorable amino acid motifs, that has innate talents in incubating high quality gold nanoparticles without adding reducing agents at physiological temperature, and particularly can further anchor gadolinium ions without adding extra chelators. The resultant paramagnetic AuNPs@GST Gd exhibits highly crystallization and uniform size of ca. 10 nm. Compared with clinical contrast agents (Iopamidol, Magnevist), AuNPs@GST Gd shows better imaging performance (e.g. enhanced relaxivity and larger X-ray attenuation efficiency) with clear evidence from Monte Carlo simulation and in vitro experimental results. Further in vivo imaging demonstrates good tumor targeting and clearance of AuNPs@GST Gd without obvious systemic toxicity. Particularly, low immunogenicity of AuNPs@GST Gd is certified by immunological status evaluation of T cells after stimulated with them. This study for the first time demonstrates the manipulation of a human protein for multi-role biomimetic chemistry depending on its unique amino acid motifs and its incorporation into a synthetic agent for potentially addressing some critical issues in cancer nanotheranostics such as synthetic methodology, biocompatibility, function integration, targeting, and immunogenicity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

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Matej Zábrady

Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

Radioautographic study of protein synthesis during early embryogenesis of Leptimotarsa decemlineata Say (Coleoptera)

International Nuclear Information System (INIS)

Maisonhaute, Claude

1976-01-01

Protein synthesis in early embryonic stages of the Colorado beetle has been investigated by radioautography. Radioactive precursor (L. Leucine-3 H) was injected in eggs. At the stage of blastoderm formation amino-acid incorporation decreases sharply: at late blastula stage, incorporation reaches the same levels as during early cleavage, and at gastrula stage becomes higher. Nuclear protein synthesis is first detected during blastoderm formation and increases at gastrula stage [fr

Aberrant regulation of synthesis and degradation of viral proteins in coliphage lambda-infected UV-irradiated cells and in minicells

International Nuclear Information System (INIS)

Shaw, J.E.; Epp, C.; Pearson, M.L.; Reeve, J.N.

1987-01-01

The patterns of bacteriophage lambda proteins synthesized in UV-irradiated Escherichia coli cells and in anucleate minicells are significantly different; both systems exhibit aberrations of regulation in lambda gene expression. In unirradiated cells or cells irradiated with low UV doses (less than 600 J/m2), regulation of lambda protein synthesis is controlled by the regulatory proteins CI, N, CII, CIII, Cro, and Q. As the UV dose increases, activation of transcription of the cI, rexA, and int genes by CII and CIII proteins fails to occur and early protein synthesis, normally inhibited by the action of Cro, continues. After high UV doses (greater than 2000 J/m2), late lambda protein synthesis does not occur. Progression through the sequence of regulatory steps in lambda gene expression is slower in infected minicells. In minicells, there is no detectable cII- and cIII-dependent synthesis of CI, RexA, or Int proteins and inhibition of early protein synthesis by Cro activity is always incomplete. The synthesis of early b region proteins is not subject to control by CI, N, or Cro proteins, and evidence is presented suggesting that, in minicells, transcription of the early b region is initiated at a promoter(s) within the b region. Proteolytic cleavage of the regulatory proteins O and N and of the capsid proteins C, B, and Nu3 is much reduced in infected minicells. Exposure of minicells to very high UV doses before infection does not completely inhibit late lambda protein synthesis

Molecular characterization of the Borrelia burgdorferi in vivo-essential protein PncA.

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Jewett, Mollie W; Jain, Sunny; Linowski, Angelika K; Sarkar, Amit; Rosa, Patricia A

2011-10-01

The conversion of nicotinamide to nicotinic acid by nicotinamidase enzymes is a critical step in maintaining NAD(+) homeostasis and contributes to numerous important biological processes in diverse organisms. In Borrelia burgdorferi, the nicotinamidase enzyme, PncA, is required for spirochaete survival throughout the infectious cycle. Mammals lack nicotinamidases and therefore PncA may serve as a therapeutic target for Lyme disease. Contrary to the in vivo importance of PncA, the current annotation for the pncA ORF suggests that the encoded protein may be inactive due to the absence of an N-terminal aspartic acid residue that is a conserved member of the catalytic triad of characterized PncA proteins. Herein, we have used genetic and biochemical strategies to determine the N-terminal sequence of B. burgdorferi PncA. Our data demonstrate that the PncA protein is 24 aa longer than the currently annotated sequence and that pncA translation is initiated from the rare, non-canonical initiation codon AUU. These findings are an important first step in understanding the catalytic function of this in vivo-essential protein.

Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

DEFF Research Database (Denmark)

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

2005-01-01

We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied...... collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage...... of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise...

Nuclear transport factor directs localization of protein synthesis during mitosis

NARCIS (Netherlands)

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

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

Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

DEFF Research Database (Denmark)

Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek

2004-01-01

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo...... proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear...... was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first...

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

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

2016-05-01

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

Whey and casein labelled with L-[1-13C]-leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

DEFF Research Database (Denmark)

Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

2011-01-01

to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass......), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments......, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after...

Acylation of proteins with myristic acid occurs cotranslationally

International Nuclear Information System (INIS)

Wilcox, C.; Hu, J.S.; Olson, E.N.

1987-01-01

Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC 3 H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [ 3 H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [ 3 H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [ 3 H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

International Nuclear Information System (INIS)

Sudhakaran, P.R.; Stamatoglou, S.C.; Hughes, R.C.

1986-01-01

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and section of α-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as α-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured

Identification of microRNAs linked to regulators of muscle protein synthesis and regeneration in young and old skeletal muscle.

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Evelyn Zacharewicz

Full Text Available BACKGROUND: Over the course of ageing there is a natural and progressive loss of skeletal muscle mass. The onset and progression of age-related muscle wasting is associated with an attenuated activation of Akt-mTOR signalling and muscle protein synthesis in response to anabolic stimuli such as resistance exercise. MicroRNAs (miRNAs are novel and important post-transcriptional regulators of numerous cellular processes. The role of miRNAs in the regulation of muscle protein synthesis following resistance exercise is poorly understood. This study investigated the changes in skeletal muscle miRNA expression following an acute bout of resistance exercise in young and old subjects with a focus on the miRNA species predicted to target Akt-mTOR signalling. RESULTS: Ten young (24.2±0.9 years and 10 old (66.6±1.1 years males completed an acute resistance exercise bout known to maximise muscle protein synthesis, with muscle biopsies collected before and 2 hours after exercise. We screened the expression of 754 miRNAs in the muscle biopsies and found 26 miRNAs to be regulated with age, exercise or a combination of both factors. Nine of these miRNAs are highly predicted to regulate targets within the Akt-mTOR signalling pathway and 5 miRNAs have validated binding sites within the 3' UTRs of several members of the Akt-mTOR signalling pathway. The miR-99/100 family of miRNAs notably emerged as potentially important regulators of skeletal muscle mass in young and old subjects. CONCLUSION: This study has identified several miRNAs that were regulated with age or with a single bout of resistance exercise. Some of these miRNAs were predicted to influence Akt-mTOR signalling, and therefore potentially skeletal muscle mass. These miRNAs should be considered as candidate targets for in vivo modulation.

Problem-Solving Test: The Mechanism of Protein Synthesis

Science.gov (United States)

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…

Viral protein synthesis in cowpea mosaic virus infected protoplasts

International Nuclear Information System (INIS)

Rottier, P.

1980-01-01

Some aspects of cowpea mosaic virus (CPMV) multiplication in cowpea mesophyll protoplasts were studied. The detection and characterization of proteins whose synthesis is induced or is stimulated upon virus infection was performed with the aid of radioactive labelling. (Auth.)

Neurofilament protein synthesis in DRG neurons decreases more after peripheral axotomy than after central axotomy

International Nuclear Information System (INIS)

Greenberg, S.G.; Lasek, R.J.

1988-01-01

Cytoskeletal protein synthesis was studied in DRG neurons after transecting either their peripheral or their central branch axons. Specifically, the axons were transected 5-10 mm from the lumbar-5 ganglion on one side of the animal; the DRGs from the transected side and contralateral control side were labeled with radiolabeled amino acids in vitro; radiolabeled proteins were separated by 2-dimensional (2D) PAGE; and the amounts of radiolabel in certain proteins of the experimental and control ganglia were quantified and compared. We focused on the neurofilament proteins because they are neuron-specific. If either the peripheral or central axons were cut, the amounts of radiolabeled neurofilament protein synthesized by the DRG neurons decreased between 1 and 10 d after transection. Neurofilament protein labeling decreased more after transection of the peripheral axons than after transection of the central axons. In contrast to axonal transections, sham operations or heat shock did not decrease the radiolabeling of the neurofilament proteins, and these procedures also affected the labeling of actin, tubulin, and the heat-shock proteins differently from transection. These results and others indicate that axonal transection leads to specific changes in the synthesis of cytoskeletal proteins of DRG neurons, and that these changes differ from those produced by stress to the animal or ganglia. Studies of the changes in neurofilament protein synthesis from 1 to 40 d after axonal transection indicate that the amounts of radiolabeled neurofilament protein synthesis were decreased during axonal elongation, but that they returned toward control levels when the axons reached cells that stopped elongation

Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies

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Tjandrawinata RR

2014-09-01

Full Text Available Raymond R Tjandrawinata,1 Jessica Trisina,1 Puji Rahayu,1 Lorentius Agung Prasetya,1 Aang Hanafiah,2 Heni Rachmawati3 1Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, Indonesia; 2National Nuclear Energy Agency, Bandung, Indonesia; 3School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia Abstract: DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The “enteric coating” formulation showed no leakage in gastric fluid–like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration–versus-time curve, 99mTc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent. Keywords: bioactive protein fraction, enteric coated tablet, pharmacodynamic

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

OpenAIRE

E. Ruchti; P.J. Roach; A.A. DePaoli-Roach; P.J. Magistretti; I. Allaman

2016-01-01

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the ...

Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

Energy Technology Data Exchange (ETDEWEB)

DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua; Mullen, Daniel; Beese, Lorena S.; Barany, George; Distefano, Mark D. (Duke); (UMM)

2010-11-15

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.

The limits of adaptation of functional protein synthesis to severe undernutrition

International Nuclear Information System (INIS)

Forrester, T.; Jahoor, F.; Reeds, P.

1996-01-01

This project was designed to investigate the limits of adaptation of protein metabolism in the stree of severe childhood malnutrition, representing as it does chronic dietary insufficiency of macronutrients and superimposed infection. The tasks included measurement of concentrations and rates of synthesis of nutrient transport proteins and hepatic acute phase proteins inseverely malnourished children during their acute illness and a recovery

Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process.

Science.gov (United States)

Jeyapalan, Asumthia S; Orellana, Renan A; Suryawan, Agus; O'Connor, Pamela M J; Nguyen, Hanh V; Escobar, Jeffery; Frank, Jason W; Davis, Teresa A

2007-08-01

Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.