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Sample records for microbial protein synthesis

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

    SciTech Connect

    Kent, Stephen

    2012-07-20

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

  2. Effects of synchronicity of carbohydrate and protein degradation on rumen fermentation characteristics and microbial protein synthesis.

    PubMed

    Seo, J K; Kim, M H; Yang, J Y; Kim, H J; Lee, C H; Kim, K H; Ha, Jong K

    2013-03-01

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

  3. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    PubMed

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  4. Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyanophycin is a poly(arginyl-aspartate) biopolymer produced and stored intracellularly by bacteria. Cyanophycin has been proposed as a renewable replacement for petrochemical-based industrial products. An abundant source of amino acids and nitrogen such as in the form of protein hydrolysates is n...

  5. Microbial transglutaminase-mediated synthesis of hapten-protein conjugates for immunoassays.

    PubMed

    Josten, A; Meusel, M; Spener, F

    1998-05-01

    Hapten-protein conjugates are essential in many immunochemical assays, in particular, in assays employing titration or competitive assay formats. By exploitation of the catalytic properties of the microbial transglutaminase from Streptoverticillium mobarense sp. (MTGase), i.e., acyl transfer between gamma-carboxamide groups and various primary amines, new techniques for the synthesis of hapten-protein conjugates were developed. This is demonstrated by two examples. The feasibility of MTGase for hapten-protein conjugate synthesis was studied by coupling the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) to casein. Different procedures for the synthesis and the immobilization of these 2,4-D-casein conjugates were evaluated, comprising (i) a batch procedure, (ii) coupling of 2,4-D to an already immobilized layer of casein, and (iii) a method for simultaneous immobilization and conjugation. Kinetic studies revealed that conjugate formation in the batch procedure was almost complete after approx 2 h. By employing the conjugates in a competitive ELISA, detection limits as low as 0.05 microgram/L 2,4-D were reached. Using the approach with simultaneous immobilization and conjugation, the time for the whole assay could be reduced to only 2 h. Finally, to demonstrate the versatility of the enzymatic synthesis of hapten-protein conjugates, an ELISA for 2,4,6-trinitrotoluene (TNT) determination based on transglutaminase-synthesized conjugates was developed. In this assay, a detection limit as low as 0.04 microgram/l TNT was obtained.

  6. Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro.

    PubMed

    Wischer, G; Boguhn, J; Steingaß, H; Schollenberger, M; Rodehutscord, M

    2013-11-01

    Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia

  7. Effects of Different Protein Supplements on Omasal Nutrient Flow and Microbial Protein Synthesis in Lactating Dairy Cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eight ruminally cannulated Holstein cows that were part of a larger lactation trial were used in 2 replicated 4 x 4 Latin squares to quantify effects of supplementing protein as urea, solvent soybean meal (SSBM), cottonseed meal (CSM), or canola meal (CM) on omasal nutrient flows and microbial prote...

  8. Rumen microorganisms, methane production, and microbial protein synthesis affected by mangosteen peel powder supplement in lactating dairy cows.

    PubMed

    Polyorach, Sineenart; Wanapat, Metha; Cherdthong, Anusorn; Kang, Sungchhang

    2016-03-01

    Four crossbred dairy cows (50 % Holstein-Friesian × 50 % Thai native), 404 ± 50.0 kg of body weight (4 years old) and 90 ± 5 day in milk with daily milk production of 9 ± 2.0 kg/day, were randomly assigned according to a 4 × 4 Latin square design to study the effect of mangosteen (Garcinia mangostana) peel powder (MSP) supplementation on rumen microorganisms, methane production, and microbial protein synthesis fed concentrate containing yeast fermented cassava chip protein (YEFECAP). The treatments were different levels of MSP supplementation at 0, 100, 200, and 300 g/head/day. Rice straw was used as a roughage source fed ad libitum, and concentrate containing YEFECAP at 200 g/kg concentrate was offered corresponding to concentrate-to-milk-yield ratio at 1:2. A quantitative real-time PCR approach was used to determine the population densities of ruminal microorganisms. The results revealed that supplementation of MSP did not affect on Fibrobactor succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus (P > 0.05). However, total bacteria was linearly increased (P < 0.01) while methanogens and protozoal population were linearly decreased (P < 0.01) with increasing level of MSP supplementation. Increasing level of MSP supplement could decrease rumen methane production from 27.5 to 23.7 mmol/100 ml(3). Furthermore, cows that received MSP at 300 g/head/day had the highest microbial crude protein and efficiency of rumen microbial N synthesis (416.8 g/day and 16.2 g/kg organic matter truly digested in the rumen (OMDR), respectively). In conclusion, supplementation of MSP at 300 g/head/day with YEFECAP as a protein source in the concentrate mixture revealed an enhancement of rumen fermentation and methane reduction in lactating dairy cows.

  9. Effect of donor animals and their diet on in vitro nutrient degradation and microbial protein synthesis using grass and corn silages.

    PubMed

    Boguhn, J; Zuber, T; Rodehutscord, M

    2013-06-01

    Two nonlactating cows and two wether sheep, all fitted with a permanent cannula into the rumen, were fed either hay plus concentrate, grass silage or corn silage to study the effect of the donor animal and its diet on in vitro fermentation and microbial protein synthesis. Rumen inoculum was obtained before the morning feeding. Grass silage or corn silage was incubated in a semi-continuous rumen simulation system for 14 days. Four replicated vessels were used per treatment. Degradation of crude nutrients and detergent fibre fractions as well as microbial protein synthesis and the production of volatile fatty acids were studied. Additionally, total gas and methane production was measured with a standard in vitro gas test. Gas production and methane concentration was higher when the inoculum used was from sheep than that from cows. The donor animal also affected the degradation of organic matter and ether extract as well as the amount of propionate and butyrate, and the acetate-to-propionate ratio. The effect of the diet fed to the donor animal on fermentation was much greater than the effect of the donor animal itself. Feeding hay plus concentrate resulted in higher gas production and degradation of acid detergent fibre, but in lower degradation of ether extract and reduced microbial protein synthesis. Additionally, the pattern of volatile fatty acids changed significantly when the diet of the donor animals was hay plus concentrate or one of the silages. These results show that in vitro fermentation and microbial protein synthesis is different when based on inoculum from either cattle or sheep. The diet fed to the donor animal is more important than the animal species and is probably mediated by an adjusted microbial activity. With regard to standardized feed evaluations, these results further support the need to harmonize in vitro approaches used in different laboratories.

  10. Effect of dietary energy source and level on nutrient digestibility, rumen microbial protein synthesis, and milk performance in lactating dairy cows.

    PubMed

    Zhou, X Q; Zhang, Y D; Zhao, M; Zhang, T; Zhu, D; Bu, D P; Wang, J Q

    2015-10-01

    This study was conducted to examine the effects of dietary energy source and level on intake, digestion, rumen microbial protein synthesis, and milk production in lactating dairy cows, using corn stover as a forage source. Eight multiparous Holstein cows, 4 of which were fitted with rumen cannulas, were evaluated in a replicated 4 × 4 Latin square design, with each period lasting 21 d. The cows were randomly assigned into 4 treatment groups: low-energy (LE) ground corn (GC), LE steam-flaked corn (SFC), high-energy (HE) GC, and HE SFC. Changes to ruminal energy degradation rates were induced by feeding the cows diets of either finely ground corn or SFC as components of diets with the same total energy level. Milk yield, milk protein content and yield, and milk lactose yield all increased in response to higher levels of dietary energy, whereas contents of milk fat and lactose were unaffected. Cows fed HE diets had a higher crude microbial protein yield and total-tract apparent digestibility than those receiving LE diets. Milk yield, milk protein yield, and microbial protein yield were also higher when SFC replaced GC as the main energy source for lactating cows fed LE diets. These results suggest that an increased dietary energy level and ruminal degradation rate are beneficial to milk protein production, which we suggest is due to increased yields of microbial proteins, when cows are fed corn stover as a dietary forage source.

  11. Effect of feeding garlic leaf on microbial nitrogen supply, kinetics of plasma phenylalanine, tyrosine and protein synthesis in sheep.

    PubMed

    Kamruzzaman, Md; Liang, Xi; Sekiguchi, Natsumi; Sano, Hiroaki

    2014-05-01

    The objective of the present study was to assess the feeding effects of garlic leaf on microbial N supply (MNS), turnover rates of plasma phenylalanine (PheTR) and tyrosine (TyrTR) and whole body protein synthesis (WBPS) in sheep. The sheep were fed either mixed hay (Hay-diet, as control) or hay plus garlic leaf diet (GL-diet, at a ratio of 9:1) in a crossover design each for a 21 day period. The isotope dilution method using [(2) H5 ]Phe and [(2) H2 ]Tyr was performed on the 21st day of each dietary treatment. Nitrogen intake remained similar between the diets and N absorption and N digestibility were higher (P<0.05) in the GL-diet than Hay-diet. Total purine derivatives excretion and MNS were greater (P<0.05) in the GL-diet than the Hay-diet. Plasma PheTR tended to be higher (P=0.06) during GL feeding and TyrTR did not differ between the diets. Further, WBPS tended to be greater (P=0.05) for the GL-diet compared with the Hay-diet. Hence, the present results suggest that garlic leaf may have positive effects on N metabolism by influencing MNS in sheep and could be used as a potential ruminant feed in the future.

  12. Improving the quality of rice straw by urea and calcium hydroxide on rumen ecology, microbial protein synthesis in beef cattle.

    PubMed

    Polyorach, S; Wanapat, M

    2015-06-01

    Four rumen-fistulated beef cattle were randomly assigned to four treatments according to a 4 × 4 Latin square design to study the influence of urea and calcium hydroxide [Ca(OH)2 ] treatment of rice straw to improve the nutritive value of rice straw. Four dietary treatments were as follows: untreated rice straw, 50 g/kg urea-treated rice straw, 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw and 30 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw. All animals were kept in individual pens and fed with concentrate at 0.5 g/kg of BW (DM), rice straw was fed ad libitum. The experiment was conducted for four periods, and each period lasted for 21 days. During the first 14 days, DM feed intake measurements were made while during the last 7 days, all cattle were moved to metabolism crates for total faeces and urine collections. The results revealed that 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw improved the nutritive value of rice straw, in terms of dry matter intake, digestibility, ruminal volatile fatty acids, population of bacteria and fungi, nitrogen retention and microbial protein synthesis. Based on this study, it could be concluded that using urea plus calcium hydroxide was one alternative method to improve the nutritive value of rice straw, rumen ecology and fermentation and thus a reduction of treatment cost.

  13. Steers grazing blue grama rangeland throughout the growing season. II. Site and extent of digestion and microbial protein synthesis.

    PubMed

    Funk, M A; Galyean, M L; Branine, M E

    1987-11-01

    Effects of advancing forage maturity and drought-induced summer dormancy on site and extent of digestion and microbial protein synthesis in beef steers grazing native blue grama rangeland were evaluated in four sampling periods. Five steers (avg initial wt 227 kg) fitted with ruminal, duodenal and ileal cannulae and three steers cannulated at the esophagus freely grazed a 12-ha study pasture. Sampling periods lasted 11 d and started June 2, which was during the early growing season (EGS); June 22, during early summer dormancy (ESD); July 21, during late summer dormancy (LSD); and August 25, 1985, during the late growing season (LGS). Dietary N content was lower (P less than .05) in ESD and LSD than in EGS and LGS. Neutral detergent fiber (NDF) content was lower (P less than .05) in EGS than in other sampling periods. Ruminal organic matter (OM) digestion was lower (P less than .05) in ESD than in EGS, probably because of increased dietary NDF and lower N content. Ruminal OM digestion was greater (P less than .05) in LSD and LGS than in ESD because of increased fiber digestion. Neutral detergent fiber and acid detergent fiber (ADF) digestion occurring in the rumen was greater (P less than .05) in LSD and LGS than in EGS and ESD. Organic matter digestion in the small intestine and OM, NDF and ADF digestion in the hindgut were similar for all sampling periods. Over 90% of the fiber digestion occurred ruminally.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Microbial Engineering for Aldehyde Synthesis

    PubMed Central

    Kunjapur, Aditya M.

    2015-01-01

    Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity. PMID:25576610

  15. Effect of diet and absence of protozoa on the rumen microbial community and on the representativeness of bacterial fractions used in the determination of microbial protein synthesis.

    PubMed

    Belanche, A; de la Fuente, G; Pinloche, E; Newbold, C J; Balcells, J

    2012-11-01

    Accurate estimates of microbial synthesis in the rumen are vital to optimize ruminant nutrition. Liquid- (LAB) and solid-associated bacterial fractions (SAB) harvested from the rumen are generally considered as microbial references when microbial yield is calculated; however, factors that determine their composition are not completely understood. The aim of this study was to evaluate the effect of diet and absence or presence of rumen protozoa on the rumen microbial community. It was hypothesized that these treatments could modify the composition and representativeness of LAB and SAB. Twenty twin lambs (Ovis aries) were used; one-half of the twins were kept protozoa-free, and each respective twin sibling was faunated. At 6 mo of age, 5 animals from each group were randomly allocated to the experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain. After 15 d of adaptation to the diet, animals were euthanized, rumen and abomasum contents were sampled, and LAB and SAB isolated. The presence of protozoa buffered the effect of diet on the rumen bacterial population. Faunated animals fed alfalfa hay had a greater abundance of F. succinogenes, anaerobic fungi and methanogens, as well as an enhanced rumen bacterial diversity. Cellulolytic bacteria were more abundant in SAB, whereas the abomasal abundance of most of the microorganisms studied was closer to those values observed in LAB. Rumen and abomasal samples showed similar bacterial DNA concentrations, but the fungal and protozoal DNA concentration in the abomasum was only 69% and 13% of that observed in the rumen, respectively, suggesting fungal and protozoal sequestration in the rumen or possible preferential degradation of fungal and protozoal DNA in the abomasum, or both. In conclusion, absence of protozoa and type of diet extensively modified the chemical composition of LAB and SAB as a consequence of changes in the microbial composition of these fractions.

  16. Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage.

    PubMed

    Ramos, S; Tejido, M L; Martínez, M E; Ranilla, M J; Carro, M D

    2009-09-01

    Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square to evaluate the effects of 4 diets on microbial synthesis, microbial populations, and ruminal digestion. The experimental diets had forage to concentrate ratios (F:C; DM basis) of 70:30 (HF) or 30:70 (HC) with alfalfa hay (A) or grass hay (G) as forage and were designated as HFA, HCA, HFG, and HCG. The concentrate was based on barley, gluten feed, wheat middlings, soybean meal, palmkern meal, wheat, corn, and mineral-vitamin premix in the proportions of 22, 20, 20, 13, 12, 5, 5, and 3%, respectively (as-is basis). Sheep were fed the diets at a daily rate of 56 g/kg of BW(0.75) to minimize feed selection. High-concentrate diets resulted in greater (P < 0.001) total tract apparent OM digestibility compared with HF diets, but no differences were detected in NDF digestibility. Ruminal digestibility of OM, NDF, and ADF was decreased by increasing the proportion of concentrate, but no differences between forages were detected. Compared with sheep fed HF diets, sheep receiving HC diets had less ruminal pH values and acetate proportions, but greater butyrate proportions. No differences among diets were detected in numbers of cellulolytic bacteria, but protozoa numbers were less (P = 0.004) and total bacteria numbers tended (P = 0.08) to be less for HC diets. Carboxymethylcellulase, xylanase, and amylase activities were greater for HC compared with HF diets, with A diets showing greater (P = 0.008) carboxymethylcellulase activities than G diets. Retained N ranged from 28.7 to 37.9% of N intake and was not affected by F:C (P = 0.62) or the type of forage (P = 0.31). Microbial N synthesis and its efficiency was greater (P < 0.001) for HC diets compared with HF diets. The results indicate that concentrates with low cereal content can be included in the diet of sheep up to 70% of the diet without detrimental effects on ruminal activity, microbial synthesis efficiency, and N

  17. Supplementation with non-fibrous carbohydrates reduced fiber digestibility and did not improve microbial protein synthesis in sheep fed fresh forage of two nutritive values.

    PubMed

    Tebot, I; Cajarville, C; Repetto, J L; Cirio, A

    2012-04-01

    To determine whether non-fibrous carbohydrate (NFC) supplementation improves fiber digestibility and microbial protein synthesis, 18 Corriedale ewes with a fixed intake level (40 g dry matter (DM)/kg BW0.75) were assigned to three (n = 6) diets: F = 100% fresh temperate forage, FG = 70% forage + 30% barley grain and FGM = 70% forage + 15% barley grain + 15% molasses-based product (MBP, Kalori 3000). Two experimental periods were carried out, with late (P1) and early (P2) vegetative stage forage. For P2, ewes were fitted with ruminal catheters. Forage was distributed at 0900 h, 1300 h, 1800 h and 2300 h, and supplement added at 0900 h and 1800 h meals. Digestibility of the different components of the diets, retained N and rumen microbial protein synthesis were determined. At the end of P2, ruminal pH and N-NH3 concentration were determined hourly for 24 h. Supplementation increased digestibility of DM (P < 0.001) and organic matter (OM; P < 0.001) and reduced NDF digestibility (P = 0.043) in both periods, with greater values in P2 (P = 0.008) for the three diets. Daily mean ruminal pH differed (P < 0.05) among treatments: 6.33 (F), 6.15 (FG) and 6.51 (FGM). The high pH in FGM was attributed to Ca(OH)2 in MBP. Therefore, the decreased fiber digestibility in supplemented diets could not be attributed to pH changes. The mean ruminal concentration of N-NH3 was 18.0 mg/dl, without differences among treatments or sampling hours. Microbial protein synthesis was greater in P2 (8.0 g/day) than in P1 (6.1 g/day; P = 0.006), but treatments did not enhance this parameter. The efficiency of protein synthesis tended to be lower in supplemented groups (16.4, 13.9 and 13.4 in P1, and 20.8, 16.7 and 16.2 g N/kg digestible OM ingested in P2, for F, FG and FGM, respectively; P = 0.07) without differences between supplements. The same tendency was observed for retained N: 2.55, 1.38 and 1.98 in P1, and 2.28, 1.23 and 1.10 g/day in P2, for F, FG and FGM, respectively; P = 0.05). The

  18. Microbial production of spider silk proteins.

    PubMed

    Fahnestock, S R; Yao, Z; Bedzyk, L A

    2000-08-01

    The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene. PMID:11763501

  19. Microbial synthesis of pinene.

    PubMed

    Sarria, Stephen; Wong, Betty; García Martín, Hector; Keasling, Jay D; Peralta-Yahya, Pamela

    2014-07-18

    The volumetric heating values of today's biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ~28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio. PMID:24679043

  20. Microbial synthesis of pinene.

    PubMed

    Sarria, Stephen; Wong, Betty; García Martín, Hector; Keasling, Jay D; Peralta-Yahya, Pamela

    2014-07-18

    The volumetric heating values of today's biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ~28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.

  1. Effect of Grape Pomace Powder, Mangosteen Peel Powder and Monensin on Nutrient Digestibility, Rumen Fermentation, Nitrogen Balance and Microbial Protein Synthesis in Dairy Steers.

    PubMed

    Foiklang, S; Wanapat, M; Norrapoke, T

    2016-10-01

    This study was designed to investigate the effect of grape pomace powder (GPP), mangosteen peel powder (MPP) and monensin on feed intake, nutrients digestibility, microorganisms, rumen fermentation characteristic, microbial protein synthesis and nitrogen balance in dairy steers. Four, rumen fistulated dairy steers with initial body weight (BW) of 220±15 kg were randomly assigned according to a 4×4 Latin square design to receive four treatments. The treatments were as follows: T1 = control, T2 = supplementation with monensin at 33 mg/kg diet, T3 = supplementation with GPP at 2% of dry matter intake, and T4 = supplementation with MPP at 30 g/kg diet. The steers were offered the concentrate diet at 0.2% BW and 3% urea treated rice straw (UTRS) was fed ad libitum. It was found that GPP supplemented group had higher UTRS intake and nutrient digestibility in terms of neutral detergent fiber and acid detergent fiber than those in control group (p<0.05). Ammonia nitrogen (NH3-N) and blood urea-nitrogen concentration were higher in monensin, GPP and MPP supplemented groups (p<0.05). Total volatile fatty acids and propionate in the GPP group were higher than those in the control group (p<0.05) while acetate concentration, and acetate to propionate ratio were decreased (p<0.01) when steers were supplemented with GPP, monensin, and MPP, respectively. Moreover, protozoal populations in GPP, MPP, and monensin supplementation were significantly lower than those in the control group (p<0.05), while cellulolytic bacterial population was significantly higher in the control group (p<0.05). Nitrogen retention, microbial crude protein and efficiency of microbial nitrogen synthesis were found significantly higher in steers that received GPP (p<0.05). Based on this study it could be concluded that the GPP has potential as an alternative feed supplement in concentrate diets which can result in improved rumen fermentation efficiency, digestibility and microbial protein synthesis in

  2. Effect of Grape Pomace Powder, Mangosteen Peel Powder and Monensin on Nutrient Digestibility, Rumen Fermentation, Nitrogen Balance and Microbial Protein Synthesis in Dairy Steers

    PubMed Central

    Foiklang, S.; Wanapat, M.; Norrapoke, T.

    2016-01-01

    This study was designed to investigate the effect of grape pomace powder (GPP), mangosteen peel powder (MPP) and monensin on feed intake, nutrients digestibility, microorganisms, rumen fermentation characteristic, microbial protein synthesis and nitrogen balance in dairy steers. Four, rumen fistulated dairy steers with initial body weight (BW) of 220±15 kg were randomly assigned according to a 4×4 Latin square design to receive four treatments. The treatments were as follows: T1 = control, T2 = supplementation with monensin at 33 mg/kg diet, T3 = supplementation with GPP at 2% of dry matter intake, and T4 = supplementation with MPP at 30 g/kg diet. The steers were offered the concentrate diet at 0.2% BW and 3% urea treated rice straw (UTRS) was fed ad libitum. It was found that GPP supplemented group had higher UTRS intake and nutrient digestibility in terms of neutral detergent fiber and acid detergent fiber than those in control group (p<0.05). Ammonia nitrogen (NH3-N) and blood urea-nitrogen concentration were higher in monensin, GPP and MPP supplemented groups (p<0.05). Total volatile fatty acids and propionate in the GPP group were higher than those in the control group (p<0.05) while acetate concentration, and acetate to propionate ratio were decreased (p<0.01) when steers were supplemented with GPP, monensin, and MPP, respectively. Moreover, protozoal populations in GPP, MPP, and monensin supplementation were significantly lower than those in the control group (p<0.05), while cellulolytic bacterial population was significantly higher in the control group (p<0.05). Nitrogen retention, microbial crude protein and efficiency of microbial nitrogen synthesis were found significantly higher in steers that received GPP (p<0.05). Based on this study it could be concluded that the GPP has potential as an alternative feed supplement in concentrate diets which can result in improved rumen fermentation efficiency, digestibility and microbial protein synthesis in

  3. Effect of time of cutting and maceration on nutrient flow, microbial protein synthesis, and digestibility in dual-flow continuous culture.

    PubMed

    Kokko, C; Soder, K J; Brito, A F; Hovey, R C; Berthiaume, R

    2013-04-01

    Maceration and evening-cutting are 2 forage management techniques that have independently improved forage quality and nutrient utilization in ruminants, but have not been evaluated in combination. Using a dual-flow continuous culture fermenter system, this preliminary study was designed to evaluate the individual and combined effects of time of cutting and maceration on in vitro ruminal digestion, nutrient flows, and microbial protein synthesis. Forages were harvested as hay from a timothy (Phleum pratense L.)-birdsfoot trefoil (Lotus corniculatus L.) stand in the morning (AM) or evening (PM). Half of each morning- and evening-cut treatment was macerated (AM-M, PM-M). The chemical composition (DM, OM, CP, NDF, ADF), including nonstructural carbohydrates (NSC) and water-soluble carbohydrates (WSC), was determined for each of the 4 treatments (AM, AM-M, PM, PM-M). Forages were ground to 2 mm and allocated to separate fermenters at 60 g of DM/d in a 4 × 4 Latin square design. Fermenters were operated over four 10-d periods with the first 7 d for adaptation followed by 3 d of sampling. Evening-cutting enhanced the apparent digestibility of NDF (P = 0.02) and ADF (P = 0.006), with a tendency (P < 0.10) for improved true DM digestibility and microbial protein synthesis. Molar proportions of individual VFA were not affected (P > 0.10) by time of cutting, though evening-cutting increased (P = 0.02) total concentration of VFA. Maceration had no effect (P > 0.10) on true nutrient digestibility or microbial protein synthesis. An interaction of time of cutting and maceration (P < 0.05) was observed whereby maceration decreased true DM and OM digestibilities in evening-cut treatments, but had no effect in morning-cut treatments. Similarly, maceration reduced total N supply (P < 0.001) and molar proportions of acetate (P = 0.04) and increased molar proportions of propionate (P = 0.01) in evening-cut treatments with no effect on morning-cut treatments. These results indicate

  4. Microbial Protein-tyrosine Kinases*

    PubMed Central

    Chao, Joseph D.; Wong, Dennis; Av-Gay, Yossef

    2014-01-01

    Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins. PMID:24554699

  5. Total replacement of corn by mesquite pod meal considering nutritional value, performance, feeding behavior, nitrogen balance, and microbial protein synthesis of Holstein-Zebu crossbred dairy steers.

    PubMed

    de Oliveira Moraes, Gláucia Sabrine; de Souza, Evaristo Jorge Oliveira; Véras, Antonia Sherlânea Chaves; de Paula Almeida, Marina; da Cunha, Márcio Vieira; Torres, Thaysa Rodrigues; da Silva, Camila Sousa; Pereira, Gerfesson Felipe Cavalcanti

    2016-10-01

    The objective of the present study to assess the effects of mesquite pod addition replacing corn (0, 250, 500, 750, and 1000 g/kg in the dry matter basis) on nutrient intake, animal performance, feeding behavior, nutrient digestibility, nitrogen balance, and microbial protein synthesis. Twenty-five Holstein-Zebu crossbred dairy steers at 219 ± 22 kg initial body weight and 18 months of age were used. The experiment lasted 84 days, divided into three periods of 28 days. A completely randomized design was used, and data were submitted to analysis using PROC GLM for analysis of variance and PROC REG for regression analysis using the software Statistical Analysis Systems version 9.1. Experimental diets were composed of Tifton 85 hay, soybean meal, ground corn, mesquite pod meal, and mineral salt. Samples of food offered were collected during the last 3 days of each period, and the leftovers were collected daily, with samples bulked per week. At the end of each 28-day period, the remaining animals were weighed to determine total weight gain and average daily gain. The assessment of behavioral patterns was performed through instantaneous scans in 5-min intervals for three consecutive 12-h days. A single urine sample from each animal was collected on the last day of each collection period at about 4 h after the first feeding. The replacement of corn by mesquite pod meal did not significantly influence treatments regarding nutrients intake, animal performance, and feeding behavior. Retained and consumed nitrogen ratio did not statistically differ between replacement levels. Likewise, there were no statistical differences regarding microbial protein synthesis and efficiency between replacement levels. Mesquite pod meal can be used in Holstein-Zebu crossbred dairy steers' diet with total corn replacement. PMID:27387896

  6. Engineering microbial factories for synthesis of value-added products

    PubMed Central

    Du, Jing; Shao, Zengyi; Zhao, Huimin

    2011-01-01

    Microorganisms have become an increasingly important platform for the production of drugs, chemicals, and biofuels from renewable resources. Advances in protein engineering, metabolic engineering, and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for the production of natural and unnatural value-added compounds. In this review, we describe the recent progress on engineering microbial factories for synthesis of valued-added products including alkaloids, terpenoids, flavonoids, polyketides, non-ribosomal peptides, biofuels, and chemicals. Related topics on lignocellulose degradation, sugar utilization, and microbial tolerance improvement will also be discussed. PMID:21526386

  7. Maximizing efficiency of rumen microbial protein production

    PubMed Central

    Hackmann, Timothy J.; Firkins, Jeffrey L.

    2015-01-01

    Rumen microbes produce cellular protein inefficiently partly because they do not direct all ATP toward growth. They direct some ATP toward maintenance functions, as long-recognized, but they also direct ATP toward reserve carbohydrate synthesis and energy spilling (futile cycles that dissipate heat). Rumen microbes expend ATP by vacillating between (1) accumulation of reserve carbohydrate after feeding (during carbohydrate excess) and (2) mobilization of that carbohydrate thereafter (during carbohydrate limitation). Protozoa account for most accumulation of reserve carbohydrate, and in competition experiments, protozoa accumulated nearly 35-fold more reserve carbohydrate than bacteria. Some pure cultures of bacteria spill energy, but only recently have mixed rumen communities been recognized as capable of the same. When these communities were dosed glucose in vitro, energy spilling could account for nearly 40% of heat production. We suspect that cycling of glycogen (a major reserve carbohydrate) is a major mechanism of spilling; such cycling has already been observed in single-species cultures of protozoa and bacteria. Interconversions of short-chain fatty acids (SCFA) may also expend ATP and depress efficiency of microbial protein production. These interconversions may involve extensive cycling of intermediates, such as cycling of acetate during butyrate production in certain butyrivibrios. We speculate this cycling may expend ATP directly or indirectly. By further quantifying the impact of reserve carbohydrate accumulation, energy spilling, and SCFA interconversions on growth efficiency, we can improve prediction of microbial protein production and guide efforts to improve efficiency of microbial protein production in the rumen. PMID:26029197

  8. Effects of Synchronization of Carbohydrate and Protein Supply in Total Mixed Ration with Korean Rice Wine Residue on Ruminal Fermentation, Nitrogen Metabolism and Microbial Protein Synthesis in Holstein Steers

    PubMed Central

    Piao, Min Yu; Kim, Hyun J.; Seo, J. K.; Park, T. S.; Yoon, J. S.; Kim, K. H.; Ha, Jong K.

    2012-01-01

    Three Holstein steers in the growing phase, each with a ruminal cannula, were used to test the hypothesis that the synchronization of the hourly rate of carbohydrate and nitrogen (N) released in the rumen would increase the amount of retained nitrogen for growth and thus improve the efficiency of microbial protein synthesis (EMPS). In Experiment 1, in situ degradability coefficients of carbohydrate and N in feeds including Korean rice wine residue (RWR) were determined. In Experiment 2, three total mixed ration (TMR) diets having different rates of carbohydrate and N release in the rumen were formulated using the in situ degradability of the feeds. All diets were made to contain similar contents of crude protein (CP) and neutral detergent fiber (NDF) but varied in their hourly pattern of nutrient release. The synchrony index of the three TMRs was 0.51 (LS), 0.77 (MS) and 0.95 (HS), respectively. The diets were fed at a restricted level (2% of the animal’s body weight) in a 3×3 Latin-square design. Synchronizing the hourly supply of energy and N in the rumen did not significantly alter the digestibility of dry matter, organic matter, crude protein, NDF or acid detergent fiber (ADF) (p>0.05). The ruminal NH3-N content of the LS group at three hours after feeding was significantly higher (p<0.05) than that of the other groups; however, the mean values of ruminal NH3-N, pH and VFA concentration among the three groups were not significantly different (p>0.05). In addition, the purine derivative (PD) excretion in urine and microbial-N production (MN) among the three groups were not significantly different (p>0.05). In conclusion, synchronizing dietary energy and N supply to the rumen did not have a major effect on nutrient digestion or microbial protein synthesis (MPS) in Holstein steers. PMID:25049518

  9. Chemical Synthesis of Proteins

    PubMed Central

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

    2010-01-01

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

  10. Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. I. Fermentation, biohydrogenation, and microbial protein synthesis.

    PubMed

    Karnati, S K R; Sylvester, J T; Ribeiro, C V D M; Gilligan, L E; Firkins, J L

    2009-08-01

    Methane is an end product of ruminal fermentation that is energetically wasteful and contributes to global climate change. Bromoethanesulfonate, animal-vegetable fat, and monensin were compared with a control treatment to suppress different functional groups of ruminal prokaryotes in the presence or absence of protozoa to evaluate changes in fermentation, digestibility, and microbial N outflow. Four dual-flow continuous culture fermenter systems were used in 4 periods in a 4 x 4 Latin square design split into 2 subperiods. In subperiod 1, a multistage filter system (50-microm smallest pore size) retained most protozoa. At the start of subperiod 2, conventional filters (300-microm pore size) were substituted to efflux protozoa via filtrate pumps over 3 d; after a further 7 d of adaptation, the fermenters were sampled for 3 d. Treatments were retained during both subperiods. Flow of total N and digestibilities of NDF and OM were 18, 16, and 9% higher, respectively, for the defaunated subperiod but were not different among treatments. Ammonia concentration was 33% higher in the faunated fermenters but was not affected by treatment. Defaunation increased the flow of nonammonia N and bacterial N from the fermenters. Protozoal counts were not different among treatments, but bromoethanesulfonate increased the generation time from 43.2 to 55.6 h. Methanogenesis was unaffected by defaunation but tended to be increased by unsaturated fat. Defaunation did not affect total volatile fatty acid production but decreased the acetate:propionate ratio; monensin increased production of isovalerate and valerate. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because effluent flows of oleic, linoleic, and linolenic acids were 60, 77, and 69% higher, and the ratio of vaccenic acid:unsaturated FA ratio was decreased by 34% in the effluent. This ratio was increased in both subperiods with the added fat diet, indicating an accumulation of

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-01

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

  13. Screening of protease producing fungi for microbial digestion of seed proteins and synthesis of amino acids-metalnutrient chelates.

    PubMed

    Deore, G B; Limaye, A S; Dushing, Y A; Dhobale, S B; Kale, S; Laware, S L

    2013-01-15

    The problem of metalnutrient deficiency is becoming more serious with the introduction of modern agricultural practices. As a result, metalnutrient deficiency is recognized as one of the critical yield limiting factors. Metalnutrients are generally offered in their sulphate or oxide forms. However, it is reported that organically bound minerals generally have a higher bioavailability than inorganic minerals. Chelation makes otherwise unavailable metalnutrients plant available. Amino acids are well known among various chelating agents. In present investigation the fungus Paecilomyces variotii PR-4 was isolated from soil and was used for production of protease and determination of its activity. Proteins from germinating seeds of chick pea, mung bean, soybean and cowpea were hydrolyzed for the production of amino acids. Amino acids were recovered, estimated and utilized for chelation of metalnutrients viz., Zn, Cu, Fe, Mn, Mg, B and Mo. The resultant chelates were employed to detect with Fourier Transform Infra-Red Spectrophotometer (FTIR) analysis. The peaks of most intensive bands in the IR spectra of ligands recorded were present in the intervals of the wave numbers 3500-3300 and 1720-1700 cm(-1). Chelation of metalnutrients led to the broadening of peak and changes of the peak position of hydroxyl groups, which indicated the binding of the carboxylic groups and primary amine groups of amino acids to the metalnutrients. The resultant amino acids-metalnutrient chelates can be utilized as organic fertilizer.

  14. Tuning microbial hosts for membrane protein production

    PubMed Central

    2009-01-01

    The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months. This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production. PMID:20040113

  15. The importance of the form of nitrogen on microbial protein synthesis in the rumen of cattle receiving grass silage and continuous intrarumen infusions of sucrose.

    PubMed

    Rooke, J A; Armstrong, D G

    1989-01-01

    1. In a 4 x 4 Latin square design experiment, four cattle were given grass silage in two meals per d to satisfy maintenance energy requirements. In addition, sucrose (170 g/kg silage dry matter (DM] was infused intraruminally at a constant rate with no nitrogen supplementation; with the infusion intraruminally of either casein (23 g/kg silage DM) or urea (8 g/kg silage DM); or with soya-bean meal (64 g/kg silage DM) fed in two equal portions. 2. Samples of duodenal digesta representative of a 24 h period were obtained using chromium-EDTA and ytterbium acetate for flow estimation and 35S as a marker of microbial N entering the small intestine. Samples of rumen fluid were also taken for estimation of rumen pH and concentrations of ammonia-N and volatile fatty acids. Estimates of apparent organic matter (OM) and N digestibility and of the rates of silage DM and N disappearance from porous synthetic-fibre bags incubated in the rumen were also made. 3. The N supplements had no significant effects on rumen pH, concentrations of volatile fatty acids, their molar proportions or the disappearance of DM or N from porous synthetic-fibre bags. N supplementation increased rumen ammonia-N concentrations (urea, P less than 0.05; casein, soya-bean meal, not significant). 4. N supplementation had no significant effects on the digestion of OM, acid-detergent fibre or soluble carbohydrate. 5. Infusion of casein increased the quantities of total non-ammonia-N (not significant) and microbial N (P less than 0.05) entering the small intestine daily and the efficiency of rumen microbial N synthesis (not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Water Stress and Protein Synthesis

    PubMed Central

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

    1975-01-01

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

  17. Evaluation of isoquinoline alkaloid supplementation levels on ruminal fermentation, characteristics of digestion, and microbial protein synthesis in steers fed a high-energy diet.

    PubMed

    Aguilar-Hernández, J A; Urías-Estrada, J D; López-Soto, M A; Barreras, A; Plascencia, A; Montaño, M; González-Vizcarra, V M; Estrada-Angulo, A; Castro-Pérez, B I; Barajas, R; Rogge, H I; Zinn, R A

    2016-01-01

    Four Holstein steers with ruminal and duodenal cannulas were used in a 4 × 4 Latin square design to examine the effect of daily intake of 0, 2, 4 or 6 g/steer of standardized plant extract containing a mixture of quaternary benzophenanthridine alkaloids and protopine alkaloids (QBA+PA) on the characteristics of ruminal fermentation and characteristics of digestion. The basal diet consisted of a steam-flaked corn-based finishing diet that contained 62% corn and 12% sudangrass hay and the rest of diet was composed of mainly dried distillers grains, molasses, fat, and minerals. The source of QBA+PA used was Sangrovit-RS (Phytobiotics Futterzusatzstoffe GmbH, Eltville, Germany) and supplementation levels of 2, 4, and 6 g Sangrovit-RS∙steer∙d, which represented a net daily ingestion of approximately 6, 12, and 18 mg of QBA+PA compounds, respectively. Inclusion of QBA+PA linearly increased ( = 0.04) flow to the duodenum of nonammonia N and linearly decreased ( < 0.01) duodenal flows of ammonia N. Ruminal microbial efficiency (duodenal microbial N; g/kg OM fermented in the rumen) and protein efficiency (duodenal nonammonia N; g/g N intake) were increased ( < 0.05) as the level of QBA+PA increased. There were no effects of QBA+PA supplementation on ruminal, postruminal, and total tract digestion of OM, starch, and NDF, but postruminal and total tract digestion of N increased ( < 0.01) as the level of QBA+PA increased. Digestible energy of the diet tended to increase (linear affect, = 0.09) with QBA+PA supplementation. Ruminal pH and total VFA molar concentrations were not different between treatments. Ruminal NH-N concentration linearly decreased ( = 0.02) with QBA+PA supplementation. Ruminal molar proportion of acetate increased ( = 0.04) as the supplementation level of QBA+PA increased. It is concluded that QBA+PA supplementation enhances efficiency of N utilization in feedlot steers fed a steam-flaked corn-based finishing diet. This effect was due, in part, to

  18. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres.

    PubMed

    Gonzalez-Gil, Graciela; Lens, Piet N L; Saikaly, Pascal E

    2016-01-01

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se(0)), insights into the microbial community structure and synthesis of Se(0) within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se(0) (nano)spheres, with some cells having high numbers of intracellular Se(0) spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se(0) spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se(0) spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se(0) spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se(0) spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se(0) spheres. In view of these and previous findings, a schematic model for the synthesis of Se(0) spheres by the microorganisms inhabiting the granular sludge is proposed. PMID:27199909

  19. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres

    PubMed Central

    Gonzalez-Gil, Graciela; Lens, Piet N. L.; Saikaly, Pascal E.

    2016-01-01

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0), insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano)spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed. PMID:27199909

  20. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres.

    PubMed

    Gonzalez-Gil, Graciela; Lens, Piet N L; Saikaly, Pascal E

    2016-01-01

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se(0)), insights into the microbial community structure and synthesis of Se(0) within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se(0) (nano)spheres, with some cells having high numbers of intracellular Se(0) spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se(0) spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se(0) spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se(0) spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se(0) spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se(0) spheres. In view of these and previous findings, a schematic model for the synthesis of Se(0) spheres by the microorganisms inhabiting the granular sludge is proposed.

  1. The Modern Synthesis in the Light of Microbial Genomics.

    PubMed

    Booth, Austin; Mariscal, Carlos; Doolittle, W Ford

    2016-09-01

    We review the theoretical implications of findings in genomics for evolutionary biology since the Modern Synthesis. We examine the ways in which microbial genomics has influenced our understanding of the last universal common ancestor, the tree of life, species, lineages, and evolutionary transitions. We conclude by advocating a piecemeal toolkit approach to evolutionary biology, in lieu of any grand unified theory updated to include microbial genomics. PMID:27482743

  2. Chloroplast ribosomes and protein synthesis.

    PubMed Central

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

    1994-01-01

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

  3. Melatonin modifies the rhythm of protein synthesis.

    PubMed

    Brodsky, V Y; Dubovaya, N D; Zvezdina, T K; Fateeva, V I; Mal'chenko, L A

    2010-07-01

    Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.

  4. Microbial synthesis of n-butanol, isobutanol, and other higher alcohols from diverse resources.

    PubMed

    Lan, Ethan I; Liao, James C

    2013-05-01

    Microbial production of fuel and chemical feedstock is a promising approach to solving energy and environmental problems. n-Butanol, isobutanol and other higher alcohols are of particular interest because they can serve as both fuel and chemical feedstock. Alternative resources such as CO2, syngas, waste protein, and lignocellulose are currently being investigated for their potential to produce these compounds. Except for lignocellulose, utilization of such alternative resource has not been examined extensively. This review aims to summarize the development of metabolic pathways for efficient synthesis of these higher alcohols and the current status of microbial strain development for the conversion of diverse resources into higher alcohols.

  5. Total Synthesis of Glycosylated Proteins

    PubMed Central

    Brailsford, John; Zhang, Qiang; Shieh, Jae-Hung; Moore, Malcolm A.S.

    2016-01-01

    Glycoproteins are an important class of naturally occurring biomolecules which play a pivotal role in many biological processes. They are biosynthesized as complex mixtures of glycoforms through post-translational protein glycosylation. This fact, together with the challenges associated with producing them in homogeneous form, has hampered detailed structure-function studies of glycoproteins as well as their full exploitation as potential therapeutic agents. By contrast, chemical synthesis offers the unique opportunity to gain access to homogeneous glycoprotein samples for rigorous biological evaluation. Herein, we review recent methods for the assembly of complex glycopeptides and glycoproteins and present several examples from our laboratory towards the total chemical synthesis of clinically relevant glycosylated proteins that have enabled synthetic access to full-length homogeneous glycoproteins. PMID:25805144

  6. Microbial Synthesis of Alka(e)nes

    PubMed Central

    Wang, Weihua; Lu, Xuefeng

    2013-01-01

    Alka(e)nes are the predominant constituents of gasoline, diesel, and jet fuels. They can be produced naturally by a wide range of microorganisms. Bio-alka(e)nes can be used as drop-in biofuels. To date, five microbial pathways that convert free fatty acids or fatty acid derivatives into alka(e)nes have been identified or reconstituted. The discoveries open a door to achieve microbial production of alka(e)nes with high efficiency. The modules derived from these alka(e)ne biosynthetic pathways can be assembled as biological parts and synthetic biology strategies can be employed to optimize the metabolic pathways and improve alka(e)ne production. PMID:25023719

  7. Microbial protein production: maximizing protein production efficiency in Space habitats

    NASA Astrophysics Data System (ADS)

    Clauwaert, Peter; Alloul, Abbas; Muys, Maarten; Sui, Yixing; Boon, Nico; Luther, Amanda; Christiaens, Marlies E. R.; Ilgrande, Chiara; Lindeboom, Ralph E. F.; Rabaey, Korneel; Vlaeminck, Siegfried

    2016-07-01

    On top of the goal of a closed material cycle for Space habitats or deep Space missions with food production, extreme requirements apply to such Life Support Systems (LSS) in terms of mass, volume, crew time, energy consumption and controllability. Although relatively high water recovery efficiencies (~70-90%) can be achieved, all Space missions until now have relied on terrestrial food resupply and thus no nutrient recovery has been achieved so far. Researchers and Space agencies have typically been focussing on the cultivation of higher plants to produce food for crew members for future Space LSS. It can be assumed that the required surface area (50-500 m2 per crew member), plant evaporation rates (~200 kg per crew member per day), power consumption (~65 kW per crew member) and the degree of controllability of a higher plant compartment will have a great impact on the feasibility of realizing a future closed loop LSS in Space for the first time. As the food production density is so critical in a LSS, a combination of higher plant cultivation and microbial protein production might increase the chances of success of future Space LSS's since the production densities are significantly higher. Higher plants in Space LSS's would typically have an average specific protein production rate in the order of 0-4 kg protein m-3 year-1 (calculated from Do, Owens et al. (2016)), whereas bacterial biomass can be produced continuously at a rate up to ~1000 kg protein m-3 year-1. Several routes for microbial food production will be discussed in this presentation, ranging from aerobic heterotrophic production with for instance Candida ingens (Strayer, Finger et al. 1997), photoheterotrophic production with PNSB such as Rhodospirillum rubrum (Hendrickx, De Wever et al. 2006) and hydrogenotrophic production with HOB such as Cupriavidus necator (Matassa, Boon et al. 2015)) and photoautotrophic production of oxygen and microbial food (e.g. Arthrospira sp. (Hendrickx, De Wever et al

  8. Effects of replacing canola meal as the major protein source with wheat dried distillers grains with solubles on ruminal function, microbial protein synthesis, omasal flow, and milk production in cows.

    PubMed

    Chibisa, G E; Christensen, D A; Mutsvangwa, T

    2012-02-01

    A study was conducted to determine the effects of replacing canola meal (CM) as the major protein source with wheat-based dried distillers grains with solubles (W-DDGS) on ruminal fermentation, microbial protein production, omasal nutrient flow and animal performance. Eight lactating dairy cows were fed in a replicated 4 × 4 Latin square design with 28-d periods (20 d of dietary adaptation and 8 d of measurements). Four cows in one Latin square were ruminally cannulated for measurements of ruminal fermentation characteristics and flow of nutrients at the omasal canal. Cows were fed either a standard barley silage-based total mixed ration containing CM as the major protein supplement (0% W-DDGS, control) or diets formulated to contain 10, 15, and 20% W-DDGS (dry matter basis), with W-DDGS replacing primarily CM. Diets were isonitrogenous (18.9% crude protein) and contained 3.0, 3.2, 3.5, and 3.7% ether extract for 0, 10, 15, and 20% W-DDGS, respectively. Diets contained 50% forage and 50% concentrate. Inclusion of W-DDGS linearly increased dry matter intake (29.5, 31.2, 30.2, and 31.9 kg/d for 0, 10, 15, and 20% W-DDGS, respectively). The addition of W-DDGS in place of CM resulted in a 1.2- to 1.8-kg increase in milk yield (42.9, 44.7, 44.1, and 44.5 kg/d for 0, 10, 15, and 20% W-DDGS); however, a quadratic change in feed efficiency (i.e., milk yield/DM intake) occurred as the dietary level of W-DDGS increased. Treatments did not differ for milk fat, protein, and lactose concentrations; however, quadratic changes were observed in milk yields of fat (1.48, 1.56, 1.62, and 1.55 kg/d for 0, 10, 15, and 20% W-DDGS, respectively), protein (1.44, 1.46, 1.49, and 1.42 kg/d) and lactose (1.96, 2.02, 2.09, and 1.93 kg/d). Ruminal fermentation characteristics did not change except that the inclusion of 20% W-DDGS resulted in a decrease and a tendency for a decrease in molar concentrations of isobutyrate and total volatile fatty acids, respectively. Omasal flow of total

  9. Effects of alfalfa silage storage structure and roasting corn on ruminal digestion and microbial CP synthesis in lactating dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this experiment was to determine the effects of unroasted ground shelled corn (GSC) or roasted GSC (RGSC), when fed with alfalfa, ensiled in bag, bunker, or O2-limiting tower silos on ruminal digestion and microbial protein synthesis in lactating dairy cows. The roasted corn was hea...

  10. Effects of extruding wheat dried distillers grains with solubles with peas or canola meal on ruminal fermentation, microbial protein synthesis, nutrient digestion, and milk production in dairy cows.

    PubMed

    Claassen, R M; Christensen, D A; Mutsvangwa, T

    2016-09-01

    Our objective was to examine the effects of feeding coextruded and nonextruded supplements consisting of wheat dried distillers grains with solubles with peas (WDDGS-peas) or canola meal (WDDGS-CM) on ruminal fermentation, omasal flow, and production performance in Holstein cows. Eight cows (4 ruminally cannulated) were used in a replicated 4×4 Latin square with 28-d periods and a 2×2 factorial arrangement of dietary treatments. Dietary treatments were coextruded or nonextruded mixtures of WDDGS-peas and WDDGS-CM that were included in total mixed rations at 15.1% [dry matter (DM) basis]. Diet had no effect on DM intake. Milk yield was greater in cows fed coextruded diets compared with those fed nonextruded diets. Milk fat content was greater in cows fed nonextruded diets compared with those fed coextruded diets, but milk fat yield was greater in cows fed coextruded diets compared with those fed nonextruded diets. Milk yield tended to be greater and milk protein yield was greater in cows fed WDDGS-peas compared with those fed WDDGS-CM. Cows fed nonextruded diets had a greater milk urea-N concentration compared with those fed coextruded diets. Cows fed coextruded diets had greater ruminal digestion of DM and tended to have greater ruminal digestion of organic matter compared with those fed nonextruded diets. Total-tract digestibilities of organic matter, crude protein, ether extract, and starch were greater, whereas that of acid detergent fiber and neutral detergent fiber tended to be greater in cows fed coextruded compared with those fed nonextruded diets. Total-tract digestibility of ether extract was lower whereas that of starch was greater and that of crude protein tended to be greater in cows fed WDDGS-peas compared with those fed WDDGS-CM. Total N excretion and milk N efficiency were unaffected by diet. Ruminal NH3-N concentration tended to be greater in cows fed WDDGS-CM compared with those fed WDDGS-peas. Ruminal propionate concentration was greater whereas

  11. Effects of extruding wheat dried distillers grains with solubles with peas or canola meal on ruminal fermentation, microbial protein synthesis, nutrient digestion, and milk production in dairy cows.

    PubMed

    Claassen, R M; Christensen, D A; Mutsvangwa, T

    2016-09-01

    Our objective was to examine the effects of feeding coextruded and nonextruded supplements consisting of wheat dried distillers grains with solubles with peas (WDDGS-peas) or canola meal (WDDGS-CM) on ruminal fermentation, omasal flow, and production performance in Holstein cows. Eight cows (4 ruminally cannulated) were used in a replicated 4×4 Latin square with 28-d periods and a 2×2 factorial arrangement of dietary treatments. Dietary treatments were coextruded or nonextruded mixtures of WDDGS-peas and WDDGS-CM that were included in total mixed rations at 15.1% [dry matter (DM) basis]. Diet had no effect on DM intake. Milk yield was greater in cows fed coextruded diets compared with those fed nonextruded diets. Milk fat content was greater in cows fed nonextruded diets compared with those fed coextruded diets, but milk fat yield was greater in cows fed coextruded diets compared with those fed nonextruded diets. Milk yield tended to be greater and milk protein yield was greater in cows fed WDDGS-peas compared with those fed WDDGS-CM. Cows fed nonextruded diets had a greater milk urea-N concentration compared with those fed coextruded diets. Cows fed coextruded diets had greater ruminal digestion of DM and tended to have greater ruminal digestion of organic matter compared with those fed nonextruded diets. Total-tract digestibilities of organic matter, crude protein, ether extract, and starch were greater, whereas that of acid detergent fiber and neutral detergent fiber tended to be greater in cows fed coextruded compared with those fed nonextruded diets. Total-tract digestibility of ether extract was lower whereas that of starch was greater and that of crude protein tended to be greater in cows fed WDDGS-peas compared with those fed WDDGS-CM. Total N excretion and milk N efficiency were unaffected by diet. Ruminal NH3-N concentration tended to be greater in cows fed WDDGS-CM compared with those fed WDDGS-peas. Ruminal propionate concentration was greater whereas

  12. Microbial synthesis of multishaped gold nanostructures.

    PubMed

    Das, Sujoy K; Das, Akhil R; Guha, Arun K

    2010-05-01

    The development of methodologies for the synthesis of nanoparticles of well-defined size and shape is a challenging one and constitutes an important area of research in nanotechnology. This Full Paper describes the controlled synthesis of multishaped gold nanoparticles at room temperature utilizing a simple, green chemical method by the interaction of chloroauric acid (HAuCl4 x 3H20) and cell-free extract of the fungal strain Rhizopus oryzae. The cell-free extract functions as a reducing, shape-directing, as well as stabilizing, agent. Different shapes of gold nanocrystals, for example, triangular, hexagonal, pentagonal, spherical, spheroidal, urchinlike, two-dimensional nanowires, and nanorods, are generated by manipulating key growth parameters, such as gold ion concentration, solution pH, and reaction time. The synthesized nanostructures are characterized by UV/Vis and Fourier-transform infrared spectroscopy, transmission electron microscopy, and energy dispersive X-ray analysis studies. Electron diffraction patterns reveal the crystalline nature of the nanoparticles and a probable mechanism is proposed for the formation of the different structural entities. PMID:20376859

  13. Efficient total synthesis of novel bioactive microbial metabolites.

    PubMed

    Sunazuka, Toshiaki; Hirose, Tomoyasu; Omura, Satoshi

    2008-02-01

    Bioactive natural products produced by microbes have almost limitless potential in pharmaceutical applications, and the organic synthesis of such products as lead compounds will result in the creation of new and widely useful pharmaceutical products. A program of discovery of naturally occurring bioactive microbial metabolites has been ongoing at the Kitasato Institute. We have also developed efficient, rational, and highly flexible production methods for generation of target compounds, synthesis of related compounds, elucidation of their structure-activity relationships, and the possible creation of improved bioactive compounds. In this Account, the isolation and total synthesis of naturally occurring bioactive microbial metabolites in order to create novel medicines for specific illnesses is described. This covers diseases and conditions such as atherosclerosis, Alzheimer's disease, cancer, inflammation, and osteoporosis, among others, and focuses on six specific compounds. Pyripyropenes were discovered from Aspergillus fumigatus FO-1289 through our screening of microbial metabolites that strongly inhibit acyl-CoA cholesterol acyltransferase (ACAT) in order to develop a new class of cholesterol-lowering agents. These novel polyoxygenated mixed polyketide-terpenoid (meroterpenoid) metabolites contain a fused pyridyl alpha-pyrone moiety. We carried out the first total synthesis of (+)-pyripyropene A via a flexible, concise, and highly efficient route and also clarified the structure-activity relationships. Arisugacins were discovered from Penicillium sp. FO-4259 by our screening of microbial metabolites that strongly inhibit acetylcholinesterase (AChE) in order to create novel medicines for Alzheimer's disease (AD). Arisugacins are also meroterpenoids. We have achieved the first convergent total synthesis of arisugacins A and B. Lactacystin was isolated from Streptomyces sp. OM-6519 via our screening of microbial metabolites that promote the differentiation of the

  14. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

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

    1998-01-01

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

  15. T-2 mycotoxin inhibits mitochondrial protein synthesis

    SciTech Connect

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

    1988-01-01

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

  16. Local Protein Synthesis in Axonal Growth Cones

    PubMed Central

    Šatkauskas, Saulius

    2007-01-01

    While initially thought to be essentially a developmental characteristic observed in artificial in vitro models, local protein synthesis in growth cones has been described in the adult, and more interestingly, during nerve regeneration. This emerging field is under intense investigation, revealing new functions of localized protein synthesis that include axon guidance, growth cone adaptation and sensitivity modulation at intermediate targets or axon regeneration. Here, we will review these functions and provide a short survey of the current knowledge on mechanisms of mRNA transport and regulation of localized protein synthesis. In addition, we will consider what lessons can be learned from localized protein synthesis in dendrites and what developments can be expected next in the field. This latter question relates to the crucial point of which technical strategy to adopt for an ideal and pertinent analysis of the phenomenon. PMID:19262143

  17. A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors

    PubMed Central

    Henrich, Curtis J.

    2016-01-01

    Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis. PMID:27768779

  18. Protein synthesis in geostimulated root caps

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.

    1982-01-01

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

  19. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

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

  20. Microbial growth and macromolecular synthesis in the northwestern Atlantic Ocean

    SciTech Connect

    Cuhel, R.L.; Jannasch, H.W.; Taylor, C.D.

    1983-01-01

    Simultaneous time-course measurements of /sup 35/SO/sub 4//sup 2 -/, /sup 32/PO/sup 43 -/, /sup 15/NH/sub 4//sup +/, and (/sup 14/C)acetate, glucose, and glutamate uptake were made at three stations in the northwestern Atlantic Ocean, using water samples taken from well below the euphotic zone. Marked deviations from linearity were observed in 14 of the 15 cases. At the two most inshore stations uptake of /sup 15/NH/sub 4//sup +/ or incorporation of /sup 35/SO/sub 4//sup 2 -/ into protein was undetectable for 16-30 h, followed by very rapid increases in the rates of activity. The sudden burst of SO/sub 4//sup 2 -/and NH/sub 4//sup +/ uptake was accompanied by a major increase in the incorporation of /sup 32/P into RNA and lipid fractions of the microbial population at a continental slope station. At a station in Sargasso Sea, all substrates were taken up without lag. Extended incubations led to a growth plateau which may be a measure of the total biologically labile organic nutrient supply. In all cases tested, chloramphenicol severely restricted uptake. One of the inshore stations was revisited a year later with similar results. The combined data demonstrate the utility of using inorganic nutrient uptake and subcellular incorporation patterns to measure microbial growth and metabolism and stress the necessity of time-course rather than end-point incubations.

  1. Origins of the protein synthesis cycle

    NASA Technical Reports Server (NTRS)

    Fox, S. W.

    1981-01-01

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

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

    PubMed

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

    2014-05-01

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

  3. Antibiotics that target protein synthesis.

    PubMed

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

    2011-01-01

    The key role of the bacterial ribosome makes it an important target for antibacterial agents. Indeed, a large number of clinically useful antibiotics target this complex translational ribonucleoprotein machinery. The majority of these compounds, mostly of natural origin, bind to one of the three key ribosomal sites: the decoding (or A-site) on the 30S, the peptidyl transferase center (PTC) on the 50S, and the peptide exit tunnel on the 50S. Antibiotics that bind the A-site, such as the aminoglycosides, interfere with codon recognition and translocation. Peptide bond formation is inhibited when small molecules like oxazolidinones bind at the PTC. Finally, macrolides tend to block the growth of the amino acid chain at the peptide exit tunnel. In this article, the major classes of antibiotics that target the bacterial ribosome are discussed and classified according to their respective target. Notably, most antibiotics solely interact with the RNA components of the bacterial ribosome. The surge seen in the appearance of resistant bacteria has not been met by a parallel development of effective and broad-spectrum new antibiotics, as evident by the introduction of only two novel classes of antibiotics, the oxazolidinones and lipopeptides, in the past decades. Nevertheless, this significant health threat has revitalized the search for new antibacterial agents and novel targets. High resolution structural data of many ribosome-bound antibiotics provide unprecedented insight into their molecular contacts and mode of action and inspire the design and synthesis of new candidate drugs that target this fascinating molecular machine. PMID:21957007

  4. Inhibition of Toxoplasma gondii protein synthesis by azithromycin.

    PubMed Central

    Blais, J; Garneau, V; Chamberland, S

    1993-01-01

    Azithromycin was shown to specifically inhibit the protein synthesis of Toxoplasma gondii in experimental systems by using free tachyzoites and T. gondii-infected mouse macrophages. RNA synthesis of the parasite was not affected by azithromycin. Inhibition of protein synthesis was also proportional to the relative anti-Toxoplasma activity of three macrolides. PMID:8215287

  5. Protein synthesis inhibitor from potato tuber

    SciTech Connect

    Romaen, R. )

    1989-04-01

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

  6. Protein synthesis by ribosomes with tethered subunits.

    PubMed

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

    2015-08-01

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

  7. Microbial chemical factories: recent advances in pathway engineering for synthesis of value added chemicals.

    PubMed

    Dhamankar, Himanshu; Prather, Kristala L J

    2011-08-01

    The dwindling nature of petroleum and other fossil reserves has provided impetus towards microbial synthesis of fuels and value added chemicals from biomass-derived sugars as a renewable resource. Microbes have naturally evolved enzymes and pathways that can convert biomass into hundreds of unique chemical structures, a property that can be effectively exploited for their engineering into Microbial Chemical Factories (MCFs). De novo pathway engineering facilitates expansion of the repertoire of microbially synthesized compounds beyond natural products. In this review, we visit some recent successes in such novel pathway engineering and optimization, with particular emphasis on the selection and engineering of pathway enzymes and balancing of their accessory cofactors.

  8. Glucocorticoid effects on hippocampal protein synthesis

    SciTech Connect

    Schlatter, L.K.

    1988-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Mizutani, H.; Ponnamperuma, C.

    1977-01-01

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

  10. Measurement of Microbial Activity and Growth in the Ocean by Rates of Stable Ribonucleic Acid Synthesis

    PubMed Central

    Karl, David M.

    1979-01-01

    A relatively simple and extremely sensitive technique for measuring rates of stable ribonucleic acid (RNA) synthesis was devised and applied to bacterial cultures and seawater samples. The procedure is based upon the uptake and incorporation of exogenous radiolabeled adenine into cellular RNA. To calculate absolute rates of synthesis, measurements of the specific radioactivity of the intracellular adenosine 5′-triphosphate pools (precursor to RNA) and of the total amount of radioactivity incorporated into stable cellular RNA per unit time are required. Since the rate of RNA synthesis is positively correlated with growth rate, measurements of RNA synthesis should be extremely useful for estimating and comparing the productivities of microbial assemblages in nature. Adenosine 5′-triphosphate, adenylate energy charge, and rates of stable RNA synthesis have been measured at a station located in the Columbian Basin of the Caribbean Sea. A subsurface peak in RNA synthesis (and therefore growth) was located within the dissolved oxygen minimum zone (450 m), suggesting in situ microbiological utilization of dissolved molecular oxygen. Calculations of the specific rates of RNA synthesis (i.e., RNA synthesis per unit of biomass) revealed that the middepth maximum corresponded to the highest specific rate of growth (420 pmol of adenine incorporated into RNA·day−1) of all depths sampled, including the euphotic zone. The existence of an intermediate depth zone of active microbial growth may be an important site for nutrient regeneration and may serve as a source of reduced carbon for mesopelagic and deep sea environments. PMID:16345461

  11. Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas

    PubMed Central

    1983-01-01

    Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga. PMID:6841455

  12. Mitochondrial Protein Synthesis, Import, and Assembly

    PubMed Central

    Fox, Thomas D.

    2012-01-01

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

  13. Characterization and optimization of cathodic conditions for H2O2 synthesis in microbial electrochemical cells

    EPA Science Inventory

    Cathode potential and O2 supply methods were investigated to improve H2O2 synthesis in an electrochemical cell, and optimal cathode conditions were applied for microbial electrochemical cells (MECs). Using aqueous O2 for the cathode significantly improved current density, but H2...

  14. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

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

    2013-01-01

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

  15. Celecoxib transiently inhibits cellular protein synthesis.

    PubMed

    Pyrko, Peter; Kardosh, Adel; Schönthal, Axel H

    2008-01-15

    To uncover the full spectrum of its pharmacological activities, the selective COX-2 inhibitor celecoxib is routinely being used at concentrations of up to 100 microM in cell culture. At these elevated concentrations, several COX-2-independent effects were identified, although many details of these events have remained unclear. Here, we report a COX-2-independent effect of celecoxib that might have profound consequences for the interpretation of previous results obtained at elevated concentrations of this drug in vitro. We found that celecoxib rapidly inhibits general protein translation at concentrations as low as 30 microM. This appears to be a consequence of endoplasmic reticulum (ER) stress and entails the phosphorylation and inactivation of eukaryotic translation initiation factor 2 alpha (eIF2alpha). These effects were not achieved by other coxibs (rofecoxib, valdecoxib) or traditional NSAIDs (indomethacin, flurbiprofen), but were mimicked by the COX-2-inactive celecoxib analog, 2,5-dimethyl-celecoxib (DMC), indicating COX-2 independence. Considering the obvious impact of blocked translation on cellular function, we provide evidence that this severe inhibition of protein synthesis might suffice to explain some of the previously reported COX-2-independent effects of celecoxib, such as the down-regulation of the essential cell cycle regulatory protein cyclin D, which is a short-lived protein that rapidly disappears in response to the inhibition of protein synthesis. Taken together, our findings establish ER stress-induced inhibition of general translation as a critical outcome of celecoxib treatment in vitro, and suggest that this effect needs to be considered when interpreting observations from the use of this drug in cell culture. PMID:17920040

  16. The Involvement of Microbially Derived Extracellular Proteins in Nanoparticle Formation and Aggregation

    NASA Astrophysics Data System (ADS)

    Pearce, C. I.; Moreau, J. W.

    2007-12-01

    While humans are newcomers to the field of nanoscience, microbes have been synthesizing functional nanoscale structures for billions of years. Bacteria have evolved the capability to produce proteins that can unite cellular processes with inorganic substrates, transfer electrons, template biomineralization, and facilitate adhesion. Biominerals are commonly nano-composite materials in which biomolecules such as proteins and/or polysaccharides act as a template to direct nanoparticle nucleation and growth. Understanding the capability of microbes to form nanoparticles and influence their reactive transport properties offers potential for bioremediation and materials synthesis applications. The identification of biomolecules and functional groups associated with biogenic nanoparticle formation in both environmental and laboratory systems is the objective of our research. Two such systems in which protein-nanoparticle interactions were studied are discussed. First, the biogenic reduction of selenium oxyanions to Se0 was studied in pure cultures of Veillonella atypica, Bacillus selenitireducens and Geobacter sulfurreducens. Biogenic Se0 nanostructures were observed as spherical, fibrillar, granular or amorphous aggregates, both in the cytoplasm or periplasmic space and extracellularly. These nanoparticles formed as protein-nanoparticle complexes that could be separated from the cells on the basis of density. A protein of ~39 kDa associated with biogenic nano-Se0 was recovered via polyacrylamide gel electrophoresis for characterization by MALDI-TOF mass spectrometry. Initial results suggest that this protein plays an integral, structural role in Se0 nanosphere formation. Second, the nanoparticulate products of bacterial sulfate reduction in a biofilm growing in minewater were investigated with multiple high- spatial resolution microanalyses. Biogenic zinc-sulfide nanoparticles exhibited evidence for rapid, highly efficient aggregation to form orders-of-magnitude larger

  17. MannDB: A microbial annotation database for protein characterization

    SciTech Connect

    Zhou, C; Lam, M; Smith, J; Zemla, A; Dyer, M; Kuczmarski, T; Vitalis, E; Slezak, T

    2006-05-19

    MannDB was created to meet a need for rapid, comprehensive automated protein sequence analyses to support selection of proteins suitable as targets for driving the development of reagents for pathogen or protein toxin detection. Because a large number of open-source tools were needed, it was necessary to produce a software system to scale the computations for whole-proteome analysis. Thus, we built a fully automated system for executing software tools and for storage, integration, and display of automated protein sequence analysis and annotation data. MannDB is a relational database that organizes data resulting from fully automated, high-throughput protein-sequence analyses using open-source tools. Types of analyses provided include predictions of cleavage, chemical properties, classification, features, functional assignment, post-translational modifications, motifs, antigenicity, and secondary structure. Proteomes (lists of hypothetical and known proteins) are downloaded and parsed from Genbank and then inserted into MannDB, and annotations from SwissProt are downloaded when identifiers are found in the Genbank entry or when identical sequences are identified. Currently 36 open-source tools are run against MannDB protein sequences either on local systems or by means of batch submission to external servers. In addition, BLAST against protein entries in MvirDB, our database of microbial virulence factors, is performed. A web client browser enables viewing of computational results and downloaded annotations, and a query tool enables structured and free-text search capabilities. When available, links to external databases, including MvirDB, are provided. MannDB contains whole-proteome analyses for at least one representative organism from each category of biological threat organism listed by APHIS, CDC, HHS, NIAID, USDA, USFDA, and WHO. MannDB comprises a large number of genomes and comprehensive protein sequence analyses representing organisms listed as high

  18. Control of RNA synthesis by chromatin proteins.

    PubMed Central

    Cedar, H; Solage, A; Zurucki, F

    1976-01-01

    The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA. PMID:787926

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

    SciTech Connect

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

    1981-01-01

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

  20. Subglacial Lake Whillans microbial biogeochemistry: a synthesis of current knowledge.

    PubMed

    Mikucki, J A; Lee, P A; Ghosh, D; Purcell, A M; Mitchell, A C; Mankoff, K D; Fisher, A T; Tulaczyk, S; Carter, S; Siegfried, M R; Fricker, H A; Hodson, T; Coenen, J; Powell, R; Scherer, R; Vick-Majors, T; Achberger, A A; Christner, B C; Tranter, M

    2016-01-28

    Liquid water occurs below glaciers and ice sheets globally, enabling the existence of an array of aquatic microbial ecosystems. In Antarctica, large subglacial lakes are present beneath hundreds to thousands of metres of ice, and scientific interest in exploring these environments has escalated over the past decade. After years of planning, the first team of scientists and engineers cleanly accessed and retrieved pristine samples from a West Antarctic subglacial lake ecosystem in January 2013. This paper reviews the findings to date on Subglacial Lake Whillans and presents new supporting data on the carbon and energy metabolism of resident microbes. The analysis of water and sediments from the lake revealed a diverse microbial community composed of bacteria and archaea that are close relatives of species known to use reduced N, S or Fe and CH4 as energy sources. The water chemistry of Subglacial Lake Whillans was dominated by weathering products from silicate minerals with a minor influence from seawater. Contributions to water chemistry from microbial sulfide oxidation and carbonation reactions were supported by genomic data. Collectively, these results provide unequivocal evidence that subglacial environments in this region of West Antarctica host active microbial ecosystems that participate in subglacial biogeochemical cycling.

  1. Subglacial Lake Whillans microbial biogeochemistry: a synthesis of current knowledge.

    PubMed

    Mikucki, J A; Lee, P A; Ghosh, D; Purcell, A M; Mitchell, A C; Mankoff, K D; Fisher, A T; Tulaczyk, S; Carter, S; Siegfried, M R; Fricker, H A; Hodson, T; Coenen, J; Powell, R; Scherer, R; Vick-Majors, T; Achberger, A A; Christner, B C; Tranter, M

    2016-01-28

    Liquid water occurs below glaciers and ice sheets globally, enabling the existence of an array of aquatic microbial ecosystems. In Antarctica, large subglacial lakes are present beneath hundreds to thousands of metres of ice, and scientific interest in exploring these environments has escalated over the past decade. After years of planning, the first team of scientists and engineers cleanly accessed and retrieved pristine samples from a West Antarctic subglacial lake ecosystem in January 2013. This paper reviews the findings to date on Subglacial Lake Whillans and presents new supporting data on the carbon and energy metabolism of resident microbes. The analysis of water and sediments from the lake revealed a diverse microbial community composed of bacteria and archaea that are close relatives of species known to use reduced N, S or Fe and CH4 as energy sources. The water chemistry of Subglacial Lake Whillans was dominated by weathering products from silicate minerals with a minor influence from seawater. Contributions to water chemistry from microbial sulfide oxidation and carbonation reactions were supported by genomic data. Collectively, these results provide unequivocal evidence that subglacial environments in this region of West Antarctica host active microbial ecosystems that participate in subglacial biogeochemical cycling. PMID:26667908

  2. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

    PubMed Central

    Gasser, Brigitte; Saloheimo, Markku; Rinas, Ursula; Dragosits, Martin; Rodríguez-Carmona, Escarlata; Baumann, Kristin; Giuliani, Maria; Parrilli, Ermenegilda; Branduardi, Paola; Lang, Christine; Porro, Danilo; Ferrer, Pau; Tutino, Maria Luisa; Mattanovich, Diethard; Villaverde, Antonio

    2008-01-01

    Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. PMID:18394160

  3. Influence of branched-chain fatty acid supplementation on voluntary intake, site and extent of digestion, ruminal fermentation, digesta kinetics and microbial protein synthesis in beef heifers consuming grass hay.

    PubMed

    Gunter, S A; Krysl, L J; Judkins, M B; Broesder, J T; Barton, R K

    1990-09-01

    Four heifers (British x British; average BW 372 kg) cannulated at the rumen and duodenum and consuming a grass hay (fescue-orchardgrass) diet were used in a 4 x 4 Latin square and supplemented with four levels (0, 20, 40, and 60 g.head-1.d-1) of supplemental four- and five-carbon VFA (BCFA). Forage OM, ADF, NDF and N intakes and digestibilities were not affected (P greater than .10) by BCFA supplementation. Likewise, duodenal N (microbial, feed and ammonia) flows and microbial efficiency were not altered (P greater than .10) by BCFA supplementation. Neither particulate and fluid passage rate nor in situ rate of NDF digestion was affected (P greater than .10) by treatment. Ruminal pH, ammonia concentrations and total VFA concentrations were similar (P greater than .10) among treatments. Ruminal proportions of acetate and propionate were not affected (P greater than .10) by treatment; however, butyrate responded in a cubic (P less than .05) fashion to BCFA, with the lowest proportion of butyrate at the 40 g BCFA feeding level. A time x treatment interaction (P less than .05) was noted for isobutyrate, isovalerate and valerate proportions; they were increased as a function of BCFA dosage at 2 to 8 h postdosing. Supplemental four- and five-carbon VFA had no effect on digestion and fermentation of grass hay. Supplementation of low-quality roughages with BCFA is not justified.

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

    SciTech Connect

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

    1980-09-01

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

  5. Understanding Protein Synthesis: An Interactive Card Game Discussion

    ERIC Educational Resources Information Center

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

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

  6. Influence of soybean meal and sorghum grain supplementation on intake, digesta kinetics, ruminal fermentation, site and extent of digestion and microbial protein synthesis in beef steers grazing blue grama rangeland.

    PubMed

    Krysl, L J; Branine, M E; Cheema, A U; Funk, M A; Galyean, M L

    1989-11-01

    Six beef steers (British x Brahman) cannulated at the rumen, duodenum and ileum (avg wt 334 kg) and three mature steers (British x British) cannulated at the esophagus were used in a replicated 3 x 3 latin square design and fed no supplement (C), .5 kg soybean meal (SBM) or .5 kg steam-flaked sorghum grain (SFS).head-1.d-1 (DM basis) while grazing blue grama rangeland. Periods of the latin square included a minimum of 14 d for adaptation and 11 d for esophageal masticate collection and digesta sampling. In September, October and November, respectively, forage collected by esophageally cannulated steers averaged 74.5, 88.8 and 71.0% grasses; 2.06, 1.53 and 1.77% N and 68.3, (P greater than .10) by treatment, but total N intake was greater (P less than .05) for SBM vs C and SFS treatments. No differences (P greater than .10) were detected among treatments in OM, NDF, ADF and N digestibilities in the rumen, small intestine or hindgut, but total tract OM digestibility was greater (P less than .10) for SBM and SFS than for C, and total tract N digestibility was greater (P less than .10) for SBM than for C or SFS. Duodenal ammonia N flow was greater (P less than .05) when SBM was fed that when SFS and C were fed, but microbial N and non-ammonia, non-microbial N flows and microbial efficiency were not altered by treatment. Likewise, ileal N flow was not affected (P greater than .10) by treatment. Particulate passage rate, gastrointestinal mean retention time, forage in vitro OM disappearance and in situ rate of forage NDF digestion also were not affected (P greater than .10) by treatments. Ruminal fluid volume was greater (P less than .05) for SFS vs SBM and C treatments, but no differences were noted in fluid dilution rate. Ruminal fluid ammonia concentration was greater (P less than .05) when SBM was fed than when SFS and C were fed (13.5, 9.9 and 8.7 mg/dl, respectively), whereas pH and total VFA concentrations were not different (P greater than .10). Proportion of

  7. A biocompatible alkene hydrogenation merges organic synthesis with microbial metabolism.

    PubMed

    Sirasani, Gopal; Tong, Liuchuan; Balskus, Emily P

    2014-07-21

    Organic chemists and metabolic engineers use orthogonal technologies to construct essential small molecules such as pharmaceuticals and commodity chemicals. While chemists have leveraged the unique capabilities of biological catalysts for small-molecule production, metabolic engineers have not likewise integrated reactions from organic synthesis with the metabolism of living organisms. Reported herein is a method for alkene hydrogenation which utilizes a palladium catalyst and hydrogen gas generated directly by a living microorganism. This biocompatible transformation, which requires both catalyst and microbe, and can be used on a preparative scale, represents a new strategy for chemical synthesis that combines organic chemistry and metabolic engineering.

  8. A Biocompatible Alkene Hydrogenation Merges Organic Synthesis with Microbial Metabolism**

    PubMed Central

    Sirasani, Gopal; Tong, Liuchuan; Balskus, Emily P.

    2014-01-01

    Organic chemists and metabolic engineers use largely orthogonal technologies to construct essential small molecules like pharmaceuticals and commodity chemicals. While chemists have leveraged the unique capabilities of biological catalysts for small molecule production, metabolic engineers have not likewise integrated reactions from organic synthesis with the metabolism of living organisms. Here we report a method for alkene hydrogenation that utilizes a palladium catalyst and hydrogen gas generated directly by a living microorganism. This biocompatible transformation, which requires both catalyst and microbe and can be used on a preparative scale, represents a new strategy for chemical synthesis that combines organic chemistry and metabolic engineering. PMID:24916924

  9. Stream microbial diversity in response to environmental changes: review and synthesis of existing research

    PubMed Central

    Zeglin, Lydia H.

    2015-01-01

    The importance of microbial activity to ecosystem function in aquatic ecosystems is well established, but microbial diversity has been less frequently addressed. This review and synthesis of 100s of published studies on stream microbial diversity shows that factors known to drive ecosystem processes, such as nutrient availability, hydrology, metal contamination, contrasting land-use and temperature, also cause heterogeneity in bacterial diversity. Temporal heterogeneity in stream bacterial diversity was frequently observed, reflecting the dynamic nature of both stream ecosystems and microbial community composition. However, within-stream spatial differences in stream bacterial diversity were more commonly observed, driven specifically by different organic matter (OM) compartments. Bacterial phyla showed similar patterns in relative abundance with regard to compartment type across different streams. For example, surface water contained the highest relative abundance of Actinobacteria, while epilithon contained the highest relative abundance of Cyanobacteria and Bacteroidetes. This suggests that contrasting physical and/or nutritional habitats characterized by different stream OM compartment types may select for certain bacterial lineages. When comparing the prevalence of physicochemical effects on stream bacterial diversity, effects of changing metal concentrations were most, while effects of differences in nutrient concentrations were least frequently observed. This may indicate that although changing nutrient concentrations do tend to affect microbial diversity, other environmental factors are more likely to alter stream microbial diversity and function. The common observation of connections between ecosystem process drivers and microbial diversity suggests that microbial taxonomic turnover could mediate ecosystem-scale responses to changing environmental conditions, including both microbial habitat distribution and physicochemical factors. PMID:26042102

  10. Effects of Replacing Dry-rolled Corn with Increasing Levels of Corn Dried Distillers Grains with Solubles on Characteristics of Digestion, Microbial Protein Synthesis and Digestible Energy of Diet in Hair Lambs Fed High-concentrate Diets

    PubMed Central

    Castro-Pérez, B. I.; Garzón-Proaño, J. S.; López-Soto, M. A.; Barreras, A.; González, V. M.; Plascencia, A.; Estrada-Angulo, A.; Dávila-Ramos, H.; Ríos-Rincón, F. G.; Zinn, R. A.

    2013-01-01

    Four male lambs (Katahdin; average live weight 25.9±2.9 kg) with “T” type cannulas in the rumen and proximal duodenum were used in a 4×4 Latin square experiment to evaluate the influence of supplemental dry distillers grain with solubles (DDGS) levels (0, 10, 20 and 30%, dry matter basis) in substitution for dry-rolled (DR) corn on characteristics of digestive function and digestible energy (DE) of diet. Treatments did not influence ruminal pH. Substitution of DR corn with DDGS increased ruminal neutral detergent fiber (NDF) digestion (quadratic effect, p<0.01), but decreased ruminal organic matter (OM) digestion (linear effect, p<0.01). Replacing corn with DDGS increased (linear, p≤0.02) duodenal flow of lipids, NDF and feed N. But there were no treatment effects on flow to the small intestine of microbial nitrogen (MN) or microbial N efficiency. The estimated UIP value of DDGS was 44%. Postruminal digestion of OM, starch, lipids and nitrogen (N) were not affected by treatments. Total tract digestion of N increased (linear, p = 0.04) as the DDGS level increased, but DDGS substitution tended to decrease total tract digestion of OM (p = 0.06) and digestion of gross energy (p = 0.08). However, it did not affect the dietary digestible energy (DE, MJ/kg), reflecting the greater gross energy content of DDGS versus DR corn in the replacements. The comparative DE value of DDGS may be considered similar to the DE value of the DR corn it replaced up to 30% in the finishing diets fed to lambs. PMID:25049896

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

    PubMed

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

    2016-07-01

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

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

    NASA Astrophysics Data System (ADS)

    Basu, Aakash; Chowdhury, Debashish

    2007-10-01

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

  13. Microbial production of fructosyltransferases for synthesis of pre-biotics.

    PubMed

    Maiorano, Alfredo Eduardo; Piccoli, Rosane Moniz; da Silva, Elda Sabino; de Andrade Rodrigues, Maria Filomena

    2008-11-01

    Fructooligosaccharides (FOS) are prebiotic substances found in several vegetable or natural foods. The main commercial production of FOS comes from enzymatic transformation of sucrose by the microbial enzyme fructosyltransferase. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists with production by several microorganisms being studied. This article reviews and discusses FOS chemical structure, enzyme characteristics, the nomenclature, producer microorganisms and enzyme production both in solid state fermentation and submerged cultivation.

  14. Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis.

    PubMed

    Xing, Mei-Ning; Zhang, Xue-Zhu; Huang, He

    2012-01-01

    Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks. PMID:22306331

  15. Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis.

    PubMed

    Xing, Mei-Ning; Zhang, Xue-Zhu; Huang, He

    2012-01-01

    Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.

  16. Role of RNA and Protein Synthesis in Abscission

    PubMed Central

    Abeles, F. B.

    1968-01-01

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

  17. A short formal synthesis of squalamine from a microbial metabolite.

    PubMed

    Kinney, W A; Zhang, X; Williams, J I; Johnston, S; Michalak, R S; Deshpande, M; Dostal, L; Rosazza, J P

    2000-09-21

    A short formal synthesis of squalamine is described, utilizing the biotransformation product 2, which is available in one step from commercially available 3-keto-23,24-bisnorchol-4-en-22-ol (1). Regioselective C-22 oxidation and C-24 sulfation of the corresponding alcohols in the presence of a free C-7 alcohol make for an efficient preparation of squalamine intermediate 11. PMID:10986072

  18. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During Infant Gut Colonization Case

    SciTech Connect

    Young, Jacque C.; Pan, Chongle; Adams, Rachel M.; Brooks, Brandon; Banfield, Jillian F.; Morowitz, Michael J.; Robert L. Hettich

    2015-01-01

    The microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. Thus, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity and functions increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data indicating three distinct colonization phases. Overall microbial community functions were established relatively early in development and remained stable. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Moreover, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. Our study provides the first snapshot of coordinated human and microbial protein expression in the infant gut during early development.

  19. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During Infant Gut Colonization Case

    DOE PAGESBeta

    Young, Jacque C.; Pan, Chongle; Adams, Rachel M.; Brooks, Brandon; Banfield, Jillian F.; Morowitz, Michael J.; Robert L. Hettich

    2015-01-01

    The microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. Thus, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity and functions increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data indicating three distinct colonization phases. Overall microbial community functions were established relatively early in development andmore » remained stable. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Moreover, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. Our study provides the first snapshot of coordinated human and microbial protein expression in the infant gut during early development.« less

  20. Microbial synthesis of magnetite and Mn-substituted magnetite nanoparticles: influence of bacteria and incubation temperature.

    PubMed

    Roh, Yul; Jang, Hee-Dong; Suh, Yongjae

    2007-11-01

    Microbial synthesis of magnetite and metal (Co, Cr, Ni)-substituted magnetites has only recently been reported. The objective of this study was to examine the influence of Mn ion on the microbial synthesis of magnetite nanoparticles. The reductive biotransformation of an akaganeite (beta-FeOOH) or a Mn-substituted (2-20 mol%) akaganeite (Fe(1-x)Mn(x)OOH) by Shewanella loiha (PV-4, 25 degrees C) and Thermoanaerobacter ethanolicus (TOR-39, 60 degrees C) was investigated under anaerobic conditions at circumneutral pH (pH = 7-8). Both bacteria formed magnetite nanoparticles using akaganeite as a magnetite precursor. By comparison of iron minerals formed by PV-4 and TOR-39 using Mn-mixed akaganeite as the precursor, it was shown that PV-4 formed siderite (FeCO3), green rust [Fe2+Fe3+(OH)16CO3 x 4H2O], and magnetite at 25 degrees C, whereas TOR-39 formed mainly nm-sized magnetite at 60 degrees C. The presence of Mn in the magnetite formed by TOR-39 was revealed by energy dispersive X-ray analysis (EDX) is indicative of Mn substitution into magnetite crystals. EDX analysis of iron minerals formed by PV-4 showed that Mn was preferentially concentrated in the siderite and green rust. These results demonstrate that coprecipitated/sorbed Mn induced microbial formation of siderite and green rust by PV-4 at 25 degrees C, but the synthesis of Mn-substituted magnetite nanoparticles proceeded by TOR-39 at 60 degrees C. These results indicate that the bacteria have the ability to synthesize magnetite and Mn-substituted magnetite nano-crystals. Microbially facilitated synthesis of magnetite and metal-substituted magnetites at near ambient temperatures may expand the possible use of specialized ferromagnetic nano-particles.

  1. Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form

    PubMed Central

    Nielsen, Morten T.; Ranberg, Johan Andersen; Christensen, Ulla; Christensen, Hanne Bjerre; Harrison, Scott J.; Olsen, Carl Erik; Hamberger, Björn; Møller, Birger Lindberg

    2014-01-01

    Forskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices. Hence, substantial efforts and resources have been invested in developing sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial synthesis of (13R)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other medically important labdane-type terpenoids. Process optimization enabled synthesis of enantiomerically pure (13R)-manoyl oxide as the sole metabolite, providing a pure compound in just two steps with a yield of 10 mg/liter. The work presented here demonstrates the value of a standardized bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable synthesis of complex biochemicals. PMID:25239892

  2. Microbial synthesis of the energetic material precursor 1,2,4-butanetriol.

    PubMed

    Niu, Wei; Molefe, Mapitso N; Frost, J W

    2003-10-29

    The lack of a route to precursor 1,2,4-butanetriol that is amenable to large-scale synthesis has impeded substitution of 1,2,4-butanetriol trinitrate for nitroglycerin. To identify an alternative to the current commercial synthesis of racemic d,l-1,2,4-butanetriol involving NaBH4 reduction of esterified d,l-malic acid, microbial syntheses of d- and l-1,2,4-butanetriol have been established. These microbial syntheses rely on the creation of biosynthetic pathways that do not exist in nature. Oxidation of d-xylose by Pseudomonas fragi provides d-xylonic acid in 70% yield. Escherichia coli DH5alpha/pWN6.186A then catalyzes the conversion of d-xylonic acid into d-1,2,4-butanetriol in 25% yield. P. fragi is also used to oxidize l-arabinose to a mixture of l-arabino-1,4-lactone and l-arabinonic acid in 54% overall yield. After hydrolysis of the lactone, l-arabinonic acid is converted to l-1,2,4-butanetriol in 35% yield using E. coli BL21(DE3)/pWN6.222A. As a catalytic route to 1,2,4-butanetriol, microbial synthesis avoids the high H2 pressures and elevated temperatures required by catalytic hydrogenation of malic acid.

  3. Effects of feeding corn silage inoculated with microbial additives on the ruminal fermentation, microbial protein yield, and growth performance of lambs.

    PubMed

    Basso, F C; Adesogan, A T; Lara, E C; Rabelo, C H S; Berchielli, T T; Teixeira, I A M A; Siqueira, G R; Reis, R A

    2014-12-01

    This study aimed to examine the effects of feeding corn silage inoculated without or with either Lactobacillus buchneri (LB) alone or a combination of LB and Lactobacillus plantarum (LBLP) on the apparent digestibility, ruminal fermentation, microbial protein synthesis, and growth performance of lambs. Thirty Santa Inês×Dorper crossbred intact males lambs weighing 20.4±3.8 kg were blocked by weight into 10 groups. Lambs in each group were randomly assigned to 1 of the following 3 dietary treatments: untreated (Control), LB, and LBLP silage. Lambs were fed experimental diets for 61 d. The apparent digestibility was indirectly estimated from indigestible NDF measured on d 57 to 59. Spot urine samples were collected from all animals on d 59 to estimate microbial protein synthesis. Lambs were slaughtered for carcass evaluation on d 61 when they weighed 32.4±5.2 kg. Six additional ruminally cannulated Santa Inês×Dorper crossbred wethers weighing 40.5±1.8 kg were used to examine dietary effects on ruminal fermentation. Average daily gain was increased when lambs were fed LBLP silage (P<0.05) but not LB silage. The LBLP silage had the highest (P<0.05) lactic acid concentration and both inoculated silages had greater acetic acid concentrations than the Control silage (P<0.05). Inoculation of corn silage increased intakes of DM, OM, CP, NDF, total carbohydrate (CHO), and GE by the lambs but decreased digestibility of DM, OM, CP, total and nonstructural carbohydrates, and concentration of GE and ME. (P<0.05). Nevertheless, lambs fed inoculated silages had greater microbial N supply than those on the Control treatment (P<0.05). The acetate to propionate ratio was lower in ruminal fluid of wethers in LBLP treatment than LB and Control treatment (P<0.05) and ruminal pH tended to be greater in LB lambs than in LBLP and Control wethers (P<0.10). Finally, the inoculation with both bacteria combined enhanced the silage fermentation. The intakes of DM, OM, CP, NDF, and GE

  4. [Synthesis of microbial exopolysaccharide ethapolan on ethanol and molasses mix].

    PubMed

    Pyroh, T P; Korzh, Iu V; Lashchuk, N V; Zborovs'ka, B M

    2006-01-01

    A possibility to change glucose, when cultivating exopolysaccharide (EPS) producer etapolan Acinetobacter sp. B-7005 on a mix of C2-C6-compounds, by the inexpensive substrate--molasses has been shown. The highest indices of EPS synthesis were observed under the conditions of preliminary hydrolysis of molasses, availability of growth factors (yeast autholisate and calcium pantothenate) in the medium, lack of the mineral source of nitrogen nutrition and use of inoculation material grown on acetate. In such conditions of cultivation of bacteria on the mix of ethanol (0.75% in volume) and molasses (0.75 wt. % as to carbohydrates) the amount of synthesized EPS reached 10 g/l, EPS-synthesizing capacity--5 g of EPS/g of biomass, the EPS yield from substrate--74% that is 1.3-1.5 times more than in cultivation on molasses. The increase of EPS synthesis on molasses, as well as on etanol and molasses mix with the use of inoculate grown on C2--substrates (compared with the use of inoculate obtained on the medium with molasses) is determined by the induction of gluconeogenesis that was evidenced by the decrease of isocytrate dehydrogenase activity, increase of activity of enzymes of glyoxylate cycle and key enzyme of gluconeogenesis of phosphoenolpyruvate synthetase. PMID:16869140

  5. [Synthesis of microbial exopolysaccharide ethapolan on ethanol and molasses mix].

    PubMed

    Pyroh, T P; Korzh, Iu V; Lashchuk, N V; Zborovs'ka, B M

    2006-01-01

    A possibility to change glucose, when cultivating exopolysaccharide (EPS) producer etapolan Acinetobacter sp. B-7005 on a mix of C2-C6-compounds, by the inexpensive substrate--molasses has been shown. The highest indices of EPS synthesis were observed under the conditions of preliminary hydrolysis of molasses, availability of growth factors (yeast autholisate and calcium pantothenate) in the medium, lack of the mineral source of nitrogen nutrition and use of inoculation material grown on acetate. In such conditions of cultivation of bacteria on the mix of ethanol (0.75% in volume) and molasses (0.75 wt. % as to carbohydrates) the amount of synthesized EPS reached 10 g/l, EPS-synthesizing capacity--5 g of EPS/g of biomass, the EPS yield from substrate--74% that is 1.3-1.5 times more than in cultivation on molasses. The increase of EPS synthesis on molasses, as well as on etanol and molasses mix with the use of inoculate grown on C2--substrates (compared with the use of inoculate obtained on the medium with molasses) is determined by the induction of gluconeogenesis that was evidenced by the decrease of isocytrate dehydrogenase activity, increase of activity of enzymes of glyoxylate cycle and key enzyme of gluconeogenesis of phosphoenolpyruvate synthetase.

  6. Investigating Bacterial Protein Synthesis Using Systems Biology Approaches.

    PubMed

    Gagarinova, Alla; Emili, Andrew

    2015-01-01

    Protein synthesis is essential for bacterial growth and survival. Its study in Escherichia coli helped uncover features conserved among bacteria as well as universally. The pattern of discovery and the identification of some of the longest-known components of the protein synthesis machinery, including the ribosome itself, tRNAs, and translation factors proceeded through many stages of successively more refined biochemical purifications, finally culminating in the isolation to homogeneity, identification, and mapping of the smallest unit required for performing the given function. These early studies produced a wealth of information. However, many unknowns remained. Systems biology approaches provide an opportunity to investigate protein synthesis from a global perspective, overcoming the limitations of earlier ad hoc methods to gain unprecedented insights. This chapter reviews innovative systems biology approaches, with an emphasis on those designed specifically for investigating the protein synthesis machinery in E. coli.

  7. Artificial siderophores. 1. Synthesis and microbial iron transport capabilities.

    PubMed

    Lee, B H; Miller, M J; Prody, C A; Neilands, J B

    1985-03-01

    Several di- and trihydroxamate analogues of natural microbial iron chelators have been prepared. The syntheses involved linkage of core structural units, including pyridinedicarboxylic acid, benzenetricarboxylic acid, nitrilotriacetic acid, and tricarballylic acid, by amide bonds to 1-amino-omega-(hydroxyamino)alkanes to provide the polyhydroxamates 1-5. The required protected (hydroxyamino)alkanes 8, 16, and 21 were prepared by different routes. 1-Amino-3-[(benzyloxy)amino]propane di-p-toluenesulfonate (8) was prepared from the N-protected aminopropanol 6 by oxidation to the aldehyde, formation of the substituted oxime, and reduction with NaBH3CN followed by deprotection of the Boc group. The pentyl derivatives 16 and 21 were made by direct alkylation with either benzyl acetohydroxamate or N-carbobenzoxy-O-benzylhydroxylamine. In Escherichia coli RW193 most of the analogues behaved nutritionally as ferrichrome. However, in E. coli AN193, a mutant lacking the ferrichrome receptor, capacity to use other natural siderophores was retained while response to all analogues was lost.

  8. Microbial utilization and biopolyester synthesis of bagasse hydrolysates.

    PubMed

    Yu, Jian; Stahl, Heiko

    2008-11-01

    Cellulosic biomass is a potentially inexpensive renewable feedstock for the biorefineries of fuels, chemicals and materials. Sugarcane bagasse was pretreated in dilute acid solution under moderately severe conditions, releasing sugars and other hydrolysates including volatile organic acids, furfurals and acid soluble lignin. Utilization of the hydrolysates by an aerobic bacterium, Ralstonia eutropha, was investigated to determine if the organic inhibitors can be removed for potential recycling and reuse of the process water. Simultaneous biosynthesis of polyhydroxyalkanoates (PHAs) for the production of value-added bioplastics was also investigated. An inhibitory effect of hydrolysates on microbial activity was observed, but it could be effectively relieved by using (a) a large inoculum, (b) a diluted hydrolysate solution, and (c) a tolerant strain, or a combination of the three. The major organic inhibitors including formic acid, acetic acid, furfural and acid soluble lignin were effectively utilized and removed to low concentration levels (less than 100ppm) while at the same time, PHA biopolyesters were synthesized and accumulated to 57wt% of cell mass under appropriate C/N ratios. Poly(3-hydroxybutyrate) was the predominant biopolyester formed on the hydrolysates, but the cells could also synthesize co-polyesters that exhibit high ductility. PMID:18474421

  9. Nonfeed application of rendered animal proteins for microbial production of eicosapentaenoic acid by the fungus Pythium irregulare.

    PubMed

    Liang, Yi; Garcia, Rafael A; Piazza, George J; Wen, Zhiyou

    2011-11-23

    Rendered animal proteins are well suited for animal nutrition applications, but the market is maturing, and there is a need to develop new uses for these products. The objective of this study is to explore the possibility of using animal proteins as a nutrient source for microbial production of omega-3 polyunsaturated fatty acids by the microalga Schizochytrium limacinum and the fungus Pythium irregulare. To be absorbed by the microorganisms, the proteins needed to be hydrolyzed into small peptides and free amino acids. The utility of the protein hydrolysates for microorganisms depended on the hydrolysis method used and the type of microorganism. The enzymatic hydrolysates supported better cell growth performance than the alkali hydrolysates did. P. irregulare displayed better overall growth performance on the experimental hydrolysates compared to S. limacinum. When P. irregulare was grown in medium containing 10 g/L enzymatic hydrolysate derived from meat and bone meal or feather meal, the performance of cell growth, lipid synthesis, and omega-3 fatty acid production was comparable to the that of culture using commercial yeast extract. The fungal biomass derived from the animal proteins had 26-29% lipid, 32-34% protein, 34-39% carbohydrate, and <2% ash content. The results show that it is possible to develop a nonfeed application for rendered animal protein by hydrolysis of the protein and feeding to industrial microorganisms which can produce omega-3 fatty acids for making omega-3-fortified foods or feeds.

  10. Predictors of Muscle Protein Synthesis after Severe Pediatric Burns

    PubMed Central

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

    2015-01-01

    Background Following a major burn, skeletal muscle protein synthesis rate increases, but is often insufficient to compensate for massively elevated muscle protein breakdown rates. Given the long-term nature of the pathophysiologic response to burn injury, we hypothesized that muscle protein synthesis rate would be chronically elevated in severely burned children. The objectives of this study were to characterize muscle protein synthesis rate of burned children over a period of 24 months post-injury, and identify predictors that influence this response. Study design 87 children with ≥40% total body surface area (TBSA) burn were included. Patients participated in stable isotope infusion studies at 1, 2 and ~ 4 weeks post-burn, and at 6, 12 and 24 months post-injury to determine skeletal muscle fractional synthesis rate. Generalized estimating equations with log link normal distribution were applied to account for clustering of patients and control for patient characteristics. Results Patients (8±6 years) had large (62, 51–72% TBSA) and deep (47±21% TBSA third degree) burns. Muscle fractional synthesis rate was elevated throughout the first 12 months post-burn compared to established values from healthy young adults. Muscle fractional synthesis rate was lower in boys, children >3 years old, and when burns were >80% TBSA. Conclusions Muscle protein synthesis is elevated for at least one year after injury, suggesting that greater muscle protein turnover is a component of the long-term pathophysiological response to burn trauma. Muscle protein synthesis is highly affected by gender, age and burn size in severely burned children. These findings may explain the divergence in net protein balance and lean body mass in different populations of burn victims. PMID:25807408

  11. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

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

    2015-01-01

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

  12. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening. PMID:24395411

  13. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.

  14. The origin of polynucleotide-directed protein synthesis

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1989-01-01

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

  15. Microbial conversion of synthesis gas components to useful fuels and chemicals

    SciTech Connect

    Madhukar, G.R.; Elmore, B.B.; Huckabay, H.K.

    1996-12-31

    Enriched culture techniques have been used to isolate microbial cultures exhibiting growth on synthesis gas components. Three rod-shaped, gram-positive cultures have been isolated from petroleum-contaminated soil, a cow manure-soil mixture, and sheep rumen fluid. Each culture exhibits growth on carbon monoxide as its primary carbon source, producing alcohols and acids in the fermentation medium. Quantities of up to 7.5, 0.58, and 0.25 g/L of acetate, ethanol, and methanol, respectively, have been produced in batch culture with lesser amounts of acetone, butyric, and propionic acid detected. 15 refs., 5 figs., 3 tabs.

  16. Predictors of muscle protein synthesis after severe pediatric burns

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

    PubMed Central

    Lam, E; Green, P J; Wong, M; Chua, N H

    1989-01-01

    We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes. Images PMID:2583082

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

    PubMed

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

    2015-07-01

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

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

    PubMed

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

    2015-09-01

    The objective of this study was to understand glucose synthesis of a protein-based artificial photosynthesis system affected by operating conditions, including the concentrations of reactants, reaction temperature, and illumination. Results from non-vesicle-based glyceraldehyde-3-phosphate (GAP) and glucose synthesis showed that the initial concentrations of ribulose-1,5-bisphosphate (RuBP) and adenosine triphosphate (ATP), lighting source, and temperature significantly affected glucose synthesis. Higher initial concentrations of RuBP and ATP significantly enhanced GAP synthesis, which was linearly correlated to glucose synthesis, confirming the proper functions of all catalyzing enzymes in the system. White fluorescent light inhibited artificial photosynthesis and reduced glucose synthesis by 79.2 % compared to in the dark. The reaction temperature of 40 °C was optimum, whereas lower or higher temperature reduced glucose synthesis. Glucose synthesis in the vesicle-based artificial photosynthesis system reconstituted with bacteriorhodopsin, F 0 F 1 ATP synthase, and polydimethylsiloxane-methyloxazoline-polydimethylsiloxane triblock copolymer was successfully demonstrated. This system efficiently utilized light-induced ATP to drive glucose synthesis, and 5.2 μg ml(-1) glucose was synthesized in 0.78-ml reaction buffer in 7 h. Light-dependent reactions were found to be the bottleneck of the studied artificial photosynthesis system. PMID:26170084

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

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

    PubMed

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

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

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

    PubMed

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

    1996-12-13

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

  4. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1981-01-01

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

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

    PubMed

    Feduchi, E; Cosín, M; Carrasco, L

    1985-03-01

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

  7. Development of microbial spoilage and lipid and protein oxidation in rabbit meat.

    PubMed

    Nakyinsige, K; Sazili, A Q; Aghwan, Z A; Zulkifli, I; Goh, Y M; Abu Bakar, F; Sarah, S A

    2015-10-01

    This experiment aimed to determine microbial spoilage and lipid and protein oxidation during aerobic refrigerated (4°C) storage of rabbit meat. Forty male New Zealand white rabbits were slaughtered according to the Halal slaughter procedure. The hind limbs were used for microbial analysis while the Longissimus lumborum m. was used for determination of lipid and protein oxidation. Bacterial counts generally increased with aging time and the limit for fresh meat (10(8)cfu/g) was reached at d 7 postmortem. Significant differences in malondialdehyde content were observed after 3d of storage. The thiol concentration significantly decreased with increase in aging time. The band intensities of myosin heavy chain and troponin T significantly reduced with increased refrigerated storage while actin remained relatively stable. This study thus proposes protein oxidation as a potential deteriorative change in refrigerated rabbit meat along with microbial spoilage and lipid oxidation. PMID:26115345

  8. Development of microbial spoilage and lipid and protein oxidation in rabbit meat.

    PubMed

    Nakyinsige, K; Sazili, A Q; Aghwan, Z A; Zulkifli, I; Goh, Y M; Abu Bakar, F; Sarah, S A

    2015-10-01

    This experiment aimed to determine microbial spoilage and lipid and protein oxidation during aerobic refrigerated (4°C) storage of rabbit meat. Forty male New Zealand white rabbits were slaughtered according to the Halal slaughter procedure. The hind limbs were used for microbial analysis while the Longissimus lumborum m. was used for determination of lipid and protein oxidation. Bacterial counts generally increased with aging time and the limit for fresh meat (10(8)cfu/g) was reached at d 7 postmortem. Significant differences in malondialdehyde content were observed after 3d of storage. The thiol concentration significantly decreased with increase in aging time. The band intensities of myosin heavy chain and troponin T significantly reduced with increased refrigerated storage while actin remained relatively stable. This study thus proposes protein oxidation as a potential deteriorative change in refrigerated rabbit meat along with microbial spoilage and lipid oxidation.

  9. Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals

    PubMed Central

    Ruiz, Jimena A.; de Almeida, Alejandra; Godoy, Manuel S.; Mezzina, Mariela P.; Bidart, Gonzalo N.; Méndez, Beatriz S.; Pettinari, M. Julia; Nikel, Pablo I.

    2013-01-01

    Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level. PMID:24688679

  10. Effect of treating alfalfa silage with pistachio by-products extract on Saanen dairy goats performance and microbial nitrogen synthesis.

    PubMed

    Mokhtarpour, A; Naserian, A A; Pourmollae, F; Ghaffari, M H

    2016-08-01

    A lactation experiment was conducted to determine the influence of addition of pistachio by-products extract (PBE) to alfalfa silage (AS) on performance, rumen fermentation, milk yield and composition, and microbial nitrogen synthesis. Eight multiparous dairy goats (1.8 ± 0.25 kg of milk yield) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments to compare two types of AS (supplemented with or without PBE) with two levels of dietary crude protein (14% vs. 16% CP). Dietary treatments were (i) AS with 14% CP of DM diet without PBE (14%CP-PBE), (ii) AS with 14% CP of DM diet with PBE (14%CP + PBE), (iii) AS with 16% CP of DM diet without PBE (16%CP-PBE) and (iv) AS with 16% CP of DM diet with PBE (16%CP + PBE). PBE was sprayed on fresh alfalfa at a ratio of 500 ml/kg alfalfa DM to get the final concentration of 1% tannin as tannic acid equivalent on DM basis. Intake of CP was greater (p < 0.01) in goats fed 16% CP diets than those fed 14% CP diets, regardless of PBE supplementation. Supplementation of PBE tended to decrease (p = 0.09) rumen NH3 -N concentration regardless of the level of CP in the diet. Supplementation of PBE tended (p = 0.09) to decrease total purine derivatives regardless of the level of CP in the diet with no significant change in microbial nitrogen supply. Efficiency of microbial nitrogen synthesis (EMNS) had a tendency (p = 0.07) to decrease in PBE supplemented diets. There was also a tendency (p = 0.10) for more EMNS in 14% CP fed goats than those fed 16% CP diets. Therefore, AS supplemented with PBE may lead to less concentration of ruminal NH3 -N because of decreased degradation of CP by rumen micro-organisms in response to pistachio by-products tannins. PMID:26336063

  11. Effect of treating alfalfa silage with pistachio by-products extract on Saanen dairy goats performance and microbial nitrogen synthesis.

    PubMed

    Mokhtarpour, A; Naserian, A A; Pourmollae, F; Ghaffari, M H

    2016-08-01

    A lactation experiment was conducted to determine the influence of addition of pistachio by-products extract (PBE) to alfalfa silage (AS) on performance, rumen fermentation, milk yield and composition, and microbial nitrogen synthesis. Eight multiparous dairy goats (1.8 ± 0.25 kg of milk yield) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments to compare two types of AS (supplemented with or without PBE) with two levels of dietary crude protein (14% vs. 16% CP). Dietary treatments were (i) AS with 14% CP of DM diet without PBE (14%CP-PBE), (ii) AS with 14% CP of DM diet with PBE (14%CP + PBE), (iii) AS with 16% CP of DM diet without PBE (16%CP-PBE) and (iv) AS with 16% CP of DM diet with PBE (16%CP + PBE). PBE was sprayed on fresh alfalfa at a ratio of 500 ml/kg alfalfa DM to get the final concentration of 1% tannin as tannic acid equivalent on DM basis. Intake of CP was greater (p < 0.01) in goats fed 16% CP diets than those fed 14% CP diets, regardless of PBE supplementation. Supplementation of PBE tended to decrease (p = 0.09) rumen NH3 -N concentration regardless of the level of CP in the diet. Supplementation of PBE tended (p = 0.09) to decrease total purine derivatives regardless of the level of CP in the diet with no significant change in microbial nitrogen supply. Efficiency of microbial nitrogen synthesis (EMNS) had a tendency (p = 0.07) to decrease in PBE supplemented diets. There was also a tendency (p = 0.10) for more EMNS in 14% CP fed goats than those fed 16% CP diets. Therefore, AS supplemented with PBE may lead to less concentration of ruminal NH3 -N because of decreased degradation of CP by rumen micro-organisms in response to pistachio by-products tannins.

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

    PubMed Central

    Martin, Colin; Thimann, Kenneth V.

    1972-01-01

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

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

    PubMed

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

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

  14. DNA Nanoparticles for Improved Protein Synthesis In Vitro.

    PubMed

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

    2016-02-24

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2013-07-31

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

  17. Stress protein synthesis, a potential toxicity marker in Escherichia coli.

    PubMed

    Odberg-Ferragut, C; Espigares, M; Dive, D

    1991-06-01

    Various chemicals were tested in Escherichia coli for the ability to modify the cellular growth rate and to induce the synthesis of heat shock and stress proteins. The toxicity of chemicals as observed by modification of the growth rate depended on concentration and duration of treatment, except for thiram. In this last case, no modification was observed up to a concentration of 10 micrograms.ml-1. In contrast, all toxicants tested enhanced the synthesis of heat shock and stress proteins. The stress response was similar but not identical. Heat shock proteins and stress proteins appear to be a more sensitive toxicity marker than growth inhibition. Suggestions for the use of stress proteins as a practical bioassay are made.

  18. Selective memory generalization by spatial patterning of protein synthesis

    PubMed Central

    O’Donnell, Cian; Sejnowski, Terrence J.

    2014-01-01

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

  19. Microbial protein: future sustainable food supply route with low environmental footprint.

    PubMed

    Matassa, Silvio; Boon, Nico; Pikaar, Ilje; Verstraete, Willy

    2016-09-01

    Microbial biotechnology has a long history of producing feeds and foods. The key feature of today's market economy is that protein production by conventional agriculture based food supply chains is becoming a major issue in terms of global environmental pollution such as diffuse nutrient and greenhouse gas emissions, land use and water footprint. Time has come to re-assess the current potentials of producing protein-rich feed or food additives in the form of algae, yeasts, fungi and plain bacterial cellular biomass, producible with a lower environmental footprint compared with other plant or animal-based alternatives. A major driver is the need to no longer disintegrate but rather upgrade a variety of low-value organic and inorganic side streams in our current non-cyclic economy. In this context, microbial bioconversions of such valuable matters to nutritive microbial cells and cell components are a powerful asset. The worldwide market of animal protein is of the order of several hundred million tons per year, that of plant protein several billion tons of protein per year; hence, the expansion of the production of microbial protein does not pose disruptive challenges towards the process of the latter. Besides protein as nutritive compounds, also other cellular components such as lipids (single cell oil), polyhydroxybuthyrate, exopolymeric saccharides, carotenoids, ectorines, (pro)vitamins and essential amino acids can be of value for the growing domain of novel nutrition. In order for microbial protein as feed or food to become a major and sustainable alternative, addressing the challenges of creating awareness and achieving public and broader regulatory acceptance are real and need to be addressed with care and expedience. PMID:27389856

  20. Microbial Protein-Protein Interactions (MiPPI) Data from the Genomics: GTL Center for Molecular and Cellular Systems (CMCS)

    DOE Data Explorer

    The Genomic Science Center for Molecular and Cellular Systems (CMCS), established in 2002, seeks to identify and characterize the complete set of protein complexes within a cell to provide a mechanistic basis for the understanding of biochemical functions. The CMCS is anchored at ORNL and PNNL. CMCS initially focused on the identification and characterization of protein complexes in two microbial systems,Rhodopseudomonas palustris (R. palustris) and Shewanella oneidensis (S. oneidensis). These two organisms have also been the focus of major DOE Genomic Science/Microbial Cell Program (MCP) projects. To develop an approach for identifying the diverse types of complexes present in microbial organisms, CMCS incorporates a number of molecular biology, microbiology, analytical and computational tools in an integrated pipeline.

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

  2. Determination of Microbial Growth by Protein Assay in an Air-Cathode Single Chamber Microbial Fuel Cell.

    PubMed

    Li, Na; Kakarla, Ramesh; Moon, Jung Mi; Min, Booki

    2015-07-01

    Microbial fuel cells (MFCs) have gathered attention as a novel bioenergy technology to simultaneously treat wastewater with less sludge production than the conventional activated sludge system. In two different operations of the MFC and aerobic process, microbial growth was determined by the protein assay method and their biomass yields using real wastewater were compared. The biomass yield on the anode electrode of the MFC was 0.02 g-COD-cell/g- COD-substrate and the anolyte planktonic biomass was 0.14 g-COD-cell/g-COD-substrate. An MFC without anode electrode resulted in the biomass yield of 0.07 ± 0.03 g-COD-cell/g-COD-substrate, suggesting that oxygen diffusion from the cathode possibly supported the microbial growth. In a comparative test, the biomass yield under aerobic environment was 0.46 ± 0.07 g-COD-cell/g-COD-substrate, which was about 3 times higher than the total biomass value in the MFC operation.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-03

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

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

    PubMed Central

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

    2016-01-01

    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. PMID:26951651

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

    PubMed

    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. PMID:26951651

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

    PubMed Central

    Tyagi, Richa; Shahani, Neelam; Gorgen, Lindsay; Ferretti, Max; Pryor, William; Chen, Po Yu; Swarnkar, Supriya; Worley, Paul F.; Karbstein, Katrin; Snyder, Solomon H.; Subramaniam, Srinivasa

    2015-01-01

    SUMMARY 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 through enhancing the phosphorylation of eIF2α by protein kinase-like endoplasmic reticulum kinase (PERK). Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses. PMID:25660019

  8. Effects of minerals on feed degradation and protein synthesis by rumen micro-organisms in a dual effluent fermenter.

    PubMed

    Broudiscou, L P; Papon, Y; Broudiscou, A F

    1999-01-01

    In dual outflow continuous fermenters on a 75:25 hay/barley diet, feed degradation and protein synthesis by mixed rumen microbes were tested in relation to the concentrations of HPO4(2-), HCO3- and Cl- and Na+/K+ ratio in artificial saliva, by applying a 16-run Franquart design, and by fitting second-order polynomial models. The HPO4(2-), HCO3-, Cl- concentrations and Na+/K+ ratio ranged from 0.1 to 4 g.L-1, from 0.5 to 7 g.L-1, from 0.1 to 0.5 g.L-1 and from 0.5 to 15 g.g-1, respectively. Buffer salts, particularly HPO4(2-), were the major factors while Cl- concentration had negligible effects on microbial metabolism. Maximal neutral detergent fibre, acid detergent fibre and organic matter degradabilities occurred at intermediate values of HPO4(2-) and HCO3- concentrations. The outflow of microbial protein and the efficiency of microbial protein synthesis, which varied from 26.2 to 37.1 g.N.kg-1 of organic matter truly degraded, reached minima at the centre of the experimental domain. PMID:10327453

  9. Effects of minerals on feed degradation and protein synthesis by rumen micro-organisms in a dual effluent fermenter.

    PubMed

    Broudiscou, L P; Papon, Y; Broudiscou, A F

    1999-01-01

    In dual outflow continuous fermenters on a 75:25 hay/barley diet, feed degradation and protein synthesis by mixed rumen microbes were tested in relation to the concentrations of HPO4(2-), HCO3- and Cl- and Na+/K+ ratio in artificial saliva, by applying a 16-run Franquart design, and by fitting second-order polynomial models. The HPO4(2-), HCO3-, Cl- concentrations and Na+/K+ ratio ranged from 0.1 to 4 g.L-1, from 0.5 to 7 g.L-1, from 0.1 to 0.5 g.L-1 and from 0.5 to 15 g.g-1, respectively. Buffer salts, particularly HPO4(2-), were the major factors while Cl- concentration had negligible effects on microbial metabolism. Maximal neutral detergent fibre, acid detergent fibre and organic matter degradabilities occurred at intermediate values of HPO4(2-) and HCO3- concentrations. The outflow of microbial protein and the efficiency of microbial protein synthesis, which varied from 26.2 to 37.1 g.N.kg-1 of organic matter truly degraded, reached minima at the centre of the experimental domain.

  10. Structure and Function of Microbial Metal-Reduction Proteins

    SciTech Connect

    Xu, Ying; Crawford, Oakly H.; Xu, Dong; Larimer, Frank W.; Uberbacher, Edward C.; Zhou, Jizhong

    2009-09-02

    In this project, we proposed (i) identification of metal-reduction genes, (ii) development of new threading techniques and (iii) fold recognition and structure prediction of metal-reduction proteins. However, due to the reduction of the budget, we revised our plan to focus on two specific aims of (i) developing a new threading-based protein structure prediction method, and (ii) developing an expert system for protein structure prediction.

  11. Microwave-irradiation-assisted hybrid chemical approach for titanium dioxide nanoparticle synthesis: microbial and cytotoxicological evaluation.

    PubMed

    Ranjan, Shivendu; Dasgupta, Nandita; Rajendran, Bhavapriya; Avadhani, Ganesh S; Ramalingam, Chidambaram; Kumar, Ashutosh

    2016-06-01

    Titanium dioxide nanoparticles (TNPs) are widely used in the pharmaceutical and cosmetics industries. It is used for protection against UV exposure due to its light-scattering properties and high refractive index. Though TNPs are increasingly used, the synthesis of TNPs is tedious and time consuming; therefore, in the present study, microwave-assisted hybrid chemical approach was used for TNP synthesis. In the present study, we demonstrated that TNPs can be synthesized only in 2.5 h; however, the commonly used chemical approach using muffle furnace takes 5 h. The activity of TNP depends on the synthetic protocol; therefore, the present study also determined the effect of microwave-assisted hybrid chemical approach synthetic protocol on microbial and cytotoxicity. The results showed that TNP has the best antibacterial activity in decreasing order from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The IC50 values of TNP for HCT116 and A549 were found to be 6.43 and 6.04 ppm, respectively. Cell death was also confirmed from trypan blue exclusion assay and membrane integrity loss was observed. Therefore, the study determines that the microwave-assisted hybrid chemical approach is time-saving; hence, this technique can be upgraded from lab scale to industrial scale via pilot plant scale. Moreover, it is necessary to find the mechanism of action at the molecular level to establish the reason for greater bacterial and cytotoxicological toxicity. Graphical abstract A graphical representation of TNP synthesis.

  12. Microwave-irradiation-assisted hybrid chemical approach for titanium dioxide nanoparticle synthesis: microbial and cytotoxicological evaluation.

    PubMed

    Ranjan, Shivendu; Dasgupta, Nandita; Rajendran, Bhavapriya; Avadhani, Ganesh S; Ramalingam, Chidambaram; Kumar, Ashutosh

    2016-06-01

    Titanium dioxide nanoparticles (TNPs) are widely used in the pharmaceutical and cosmetics industries. It is used for protection against UV exposure due to its light-scattering properties and high refractive index. Though TNPs are increasingly used, the synthesis of TNPs is tedious and time consuming; therefore, in the present study, microwave-assisted hybrid chemical approach was used for TNP synthesis. In the present study, we demonstrated that TNPs can be synthesized only in 2.5 h; however, the commonly used chemical approach using muffle furnace takes 5 h. The activity of TNP depends on the synthetic protocol; therefore, the present study also determined the effect of microwave-assisted hybrid chemical approach synthetic protocol on microbial and cytotoxicity. The results showed that TNP has the best antibacterial activity in decreasing order from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The IC50 values of TNP for HCT116 and A549 were found to be 6.43 and 6.04 ppm, respectively. Cell death was also confirmed from trypan blue exclusion assay and membrane integrity loss was observed. Therefore, the study determines that the microwave-assisted hybrid chemical approach is time-saving; hence, this technique can be upgraded from lab scale to industrial scale via pilot plant scale. Moreover, it is necessary to find the mechanism of action at the molecular level to establish the reason for greater bacterial and cytotoxicological toxicity. Graphical abstract A graphical representation of TNP synthesis. PMID:26976013

  13. Synthesis, modification and turnover of proteins during aging.

    PubMed

    Rattan, Suresh I S

    2010-01-01

    Iterations in the rate and extent of protein synthesis, accuracy, post-translational modifications and turnover are among the main molecular characteristics of aging. A decline in the cellular capacity through proteasomal and lysosomal pathways to recognize and preferentially degrade damaged proteins leads to the accumulation of abnormal proteins during aging. The consequent increase in molecular heterogeneity and impaired functioning of proteins is the basis of several age-related pathologies, such as cataracts, sarcopenia and neurodegerative diseases. Understanding the proteomic spectrum and its functional implications during aging can facilitate developing effective means of intervention, prevention and therapy of aging and age-related diseases.

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

    ERIC Educational Resources Information Center

    Templin, Mark A.; Fetters, Marcia K.

    2002-01-01

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

  15. PII Signal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

    PubMed Central

    Arcondéguy, Tania; Jack, Rachael; Merrick, Mike

    2001-01-01

    The PII family of signal transduction proteins are among the most widely distributed signal proteins in the bacterial world. First identified in 1969 as a component of the glutamine synthetase regulatory apparatus, PII proteins have since been recognized as playing a pivotal role in control of prokaryotic nitrogen metabolism. More recently, members of the family have been found in higher plants, where they also potentially play a role in nitrogen control. The PII proteins can function in the regulation of both gene transcription, by modulating the activity of regulatory proteins, and the catalytic activity of enzymes involved in nitrogen metabolism. There is also emerging evidence that they may regulate the activity of proteins required for transport of nitrogen compounds into the cell. In this review we discuss the history of the PII proteins, their structures and biochemistry, and their distribution and functions in prokaryotes. We survey data emerging from bacterial genome sequences and consider other likely or potential targets for control by PII proteins. PMID:11238986

  16. Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens

    PubMed Central

    Rack, Johannes Gregor Matthias; Morra, Rosa; Barkauskaite, Eva; Kraehenbuehl, Rolf; Ariza, Antonio; Qu, Yue; Ortmayer, Mary; Leidecker, Orsolya; Cameron, David R.; Matic, Ivan; Peleg, Anton Y.; Leys, David; Traven, Ana; Ahel, Ivan

    2015-01-01

    Summary Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species. PMID:26166706

  17. Frog Foam Nest Protein Diversity and Synthesis.

    PubMed

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

    2016-08-01

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

  18. Frog Foam Nest Protein Diversity and Synthesis.

    PubMed

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

    2016-08-01

    Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest. PMID:27460953

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    ERIC Educational Resources Information Center

    Goodridge, Frank

    1983-01-01

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

  1. Initiation of protein-primed picornavirus RNA synthesis

    PubMed Central

    Paul, Aniko V.; Wimmer, Eckard

    2015-01-01

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

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

    ERIC Educational Resources Information Center

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

    2004-01-01

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

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

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

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

  4. Structures and functions of autotransporter proteins in microbial pathogens.

    PubMed

    Benz, Inga; Schmidt, M Alexander

    2011-08-01

    Since their discovery more than 20 years ago the autotransporter protein superfamily has been growing continuously and currently represents the largest protein family in (pathogenic) Gram-negative bacteria. Autotransporter proteins (AT) adhere to a common structural principle and are composed of a C-terminal β-barrel-shaped 'translocator' domain and an N-terminal 'passenger' domain. The translocator is anchored in the outer membrane and is indispensable for the N-terminal passenger part to traverse the outer membrane. Most if not all AT harbor a chaperone segment that increases protein stability and may be located in the passenger or translocator domain. The passenger mediates the specific virulence function(s) of the particular AT. Accordingly, passenger domains of AT can be quite variable. Interestingly, AT have been identified as the first glycosylated proteins in Gram-negative bacteria. Despite the considerable efforts invested in the characterization of autotransporter biogenesis, various aspects such as the participation of accessory proteins, the fate of the translocator, or the translocation of glycosylated proteins still remain only poorly understood. In addition, recent evidence indicates that the prefix 'auto' might be slightly exaggerated. Here, we will selectively discuss novel insights at various stages of AT biogenesis. PMID:21616712

  5. Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation 1

    PubMed Central

    Reddy, A. Sathyanarayana; Raina, Anjana; Gunnery, Shobha; Datta, Asis

    1987-01-01

    A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The Mr of the purified enzyme is 95,000 with two nonidentical subunits of Mr 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of Mr 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis. Images Fig. 2 Fig. 6 Fig. 7 PMID:16665377

  6. Metal affinity enrichment increases the range and depth of proteome identification for extracellular microbial proteins

    SciTech Connect

    Wheeler, Korin; Erickson, Brian K; Mueller, Ryan; Singer, Steven; Verberkmoes, Nathan C; Hwang, Mona; Thelen, Michael P.; Hettich, Robert {Bob} L

    2012-01-01

    Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added 20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as hypothetical were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

  7. Microbial response to single-cell protein production and brewery wastewater treatment.

    PubMed

    Lee, Jackson Z; Logan, Andrew; Terry, Seth; Spear, John R

    2015-01-01

    As global fisheries decline, microbial single-cell protein (SCP) produced from brewery process water has been highlighted as a potential source of protein for sustainable animal feed. However, biotechnological investigation of SCP is difficult because of the natural variation and complexity of microbial ecology in wastewater bioreactors. In this study, we investigate microbial response across a full-scale brewery wastewater treatment plant and a parallel pilot bioreactor modified to produce an SCP product. A pyrosequencing survey of the brewery treatment plant showed that each unit process selected for a unique microbial community. Notably, flow equalization basins were dominated by Prevotella, methanogenesis effluent had the highest levels of diversity, and clarifier wet-well samples were sources of sequences for the candidate bacterial phyla of TM7 and BD1-5. Next, the microbial response of a pilot bioreactor producing SCP was tracked over 1 year, showing that two different production trials produced two different communities originating from the same starting influent. However, SCP production resulted generally in enrichment of several clades of rhizospheric diazotrophs of Alphaproteobacteria and Betaproteobacteria in the bioreactor and even more so in the final product. These diazotrophs are potentially useful as the basis of a SCP product for commercial feed production. PMID:24837420

  8. Microbial response to single-cell protein production and brewery wastewater treatment

    PubMed Central

    Lee, Jackson Z; Logan, Andrew; Terry, Seth; Spear, John R

    2015-01-01

    As global fisheries decline, microbial single-cell protein (SCP) produced from brewery process water has been highlighted as a potential source of protein for sustainable animal feed. However, biotechnological investigation of SCP is difficult because of the natural variation and complexity of microbial ecology in wastewater bioreactors. In this study, we investigate microbial response across a full-scale brewery wastewater treatment plant and a parallel pilot bioreactor modified to produce an SCP product. A pyrosequencing survey of the brewery treatment plant showed that each unit process selected for a unique microbial community. Notably, flow equalization basins were dominated by Prevotella, methanogenesis effluent had the highest levels of diversity, and clarifier wet-well samples were sources of sequences for the candidate bacterial phyla of TM7 and BD1-5. Next, the microbial response of a pilot bioreactor producing SCP was tracked over 1 year, showing that two different production trials produced two different communities originating from the same starting influent. However, SCP production resulted generally in enrichment of several clades of rhizospheric diazotrophs of Alphaproteobacteria and Betaproteobacteria in the bioreactor and even more so in the final product. These diazotrophs are potentially useful as the basis of a SCP product for commercial feed production. PMID:24837420

  9. The performance of a thermophilic microbial fuel cell fed with synthesis gas.

    PubMed

    Hussain, A; Mehta, P; Raghavan, V; Wang, H; Guiot, S R; Tartakovsky, B

    2012-08-10

    This study demonstrated electricity generation in a thermophilic microbial fuel cell (MFC) operated on synthesis gas (syngas) as the sole electron donor. At 50°C, a volumetric power output of 30-35 mWL(R)(-1) and a syngas conversion efficiency of 87-98% was achieved. The observed pathway of syngas conversion to electricity primarily consisted of a two-step process, where the carbon monoxide and hydrogen were first converted to acetate, which was then consumed by the anodophilic bacteria to produce electricity. A denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rDNA revealed the presence of Geobacter species, Acetobacter, methanogens and several uncultured bacteria and archaea in the anodic chamber. PMID:22759536

  10. Comparative proteomic analysis in pea treated with microbial consortia of beneficial microbes reveals changes in the protein network to enhance resistance against Sclerotinia sclerotiorum.

    PubMed

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Singh, Vinay; Singh, Harikesh Bahadur

    2015-06-15

    Microbial consortia may provide protection against pathogenic ingress via enhancing plant defense responses. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 were used either singly or in consortia in the pea rhizosphere to observe proteome level changes upon Sclerotinia sclerotiorum challenge. Thirty proteins were found to increase or decrease differentially in 2-DE gels of pea leaves, out of which 25 were identified by MALDI-TOF MS or MS/MS. These proteins were classified into several functional categories including photosynthesis, respiration, phenylpropanoid metabolism, protein synthesis, stress regulation, carbohydrate and nitrogen metabolism and disease/defense-related processes. The respective homologue of each protein identified was trapped in Pisum sativum and a phylogenetic tree was constructed to check the ancestry. The proteomic view of the defense response to S. sclerotiorum in pea, in the presence of beneficial microbes, highlights the enhanced protection that can be provided by these microbes in challenged plants.

  11. Comparative proteomic analysis in pea treated with microbial consortia of beneficial microbes reveals changes in the protein network to enhance resistance against Sclerotinia sclerotiorum.

    PubMed

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Singh, Vinay; Singh, Harikesh Bahadur

    2015-06-15

    Microbial consortia may provide protection against pathogenic ingress via enhancing plant defense responses. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 were used either singly or in consortia in the pea rhizosphere to observe proteome level changes upon Sclerotinia sclerotiorum challenge. Thirty proteins were found to increase or decrease differentially in 2-DE gels of pea leaves, out of which 25 were identified by MALDI-TOF MS or MS/MS. These proteins were classified into several functional categories including photosynthesis, respiration, phenylpropanoid metabolism, protein synthesis, stress regulation, carbohydrate and nitrogen metabolism and disease/defense-related processes. The respective homologue of each protein identified was trapped in Pisum sativum and a phylogenetic tree was constructed to check the ancestry. The proteomic view of the defense response to S. sclerotiorum in pea, in the presence of beneficial microbes, highlights the enhanced protection that can be provided by these microbes in challenged plants. PMID:26067380

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  13. Protein synthesis subserves reconsolidation or extinction depending on reminder duration.

    PubMed

    Pedreira, María Eugenia; Maldonado, Héctor

    2003-06-19

    When learned associations are recalled from long-term memory stores by presentation of an unreinforced conditioned stimulus (CS), two processes are initiated. One, termed reconsolidation, re-activates the association between the conditioned and unconditioned stimuli and transfers it from a stable protein synthesis-independent form of storage to a more labile protein-dependent state. The other is an extinction process in which presentation of the CS alone degrades the association between CS and US. To address the mechanistic relationship between reconsolidation and extinction, we have used an invertebrate model of contextual memory, which involves an association between the learning context and a visual danger stimulus. Here, we show that re-exposure duration to the learning context acts as a switch guiding the memory course toward reconsolidation or extinction, each depending on protein synthesis. Manipulation of this variable allows findings of impaired extinction to be discriminated from those of disrupted reconsolidation.

  14. Effects of a new microbial α-amylase inhibitor protein on Helicoverpa armigera larvae.

    PubMed

    Zeng, Fanrong; Wang, Xiaojing; Cui, Jinjie; Ma, Yan; Li, Qiannan

    2013-03-01

    A new microbial α-amylase inhibitor gene was cloned and characterized. The encoded, recombinant, α-amylase inhibitor protein was induced and expressed by isopropyl β-d-1-thiogalactopyranoside (IPTG) in Escherichia coli M15 cells. The effects of the α-amylase inhibitor protein on Helicoverpa armigera larvae were studied. Compared to the control, the weight of H. armigera larvae fed the diet with recombinant α-amylase inhibitor protein added at a concentration of 20 μg/g was reduced by 49.8%. The total soluble protein of H. armigera larvae fed the diet with the α-amylase inhibitor protein added was also reduced by 36.8% compared to the control. The recombinant α-amylase inhibitor protein showed inhibition activity against α-amylase of H. armigera. These results suggested that this α-amylase inhibitor protein may be a promising bioinsecticide candidate for controlling H. armigera.

  15. Melatonin-induced protein synthesis in the rat parotid gland.

    PubMed

    Cevik-Aras, H; Godoy, T; Ekstrom, J

    2011-02-01

    Melatonin occurs in large amounts in the intestinal mucosa and is released during a meal. Recent studies of ours reveal that exogenous melatonin evokes the in vivo secretion of protein and amylase from the rat parotid gland. The aim of the present study was to investigate the effect of melatonin on the protein synthesis of the parotid gland of pentobarbitone-anaesthetised rats as estimated by the rate of incorporation of [³H]leucine into trichloroacetic acid-insoluble material of the gland. Compared with the parotid protein synthesis (set at 100%) of those rats exposed to an intravenous infusion of melatonin (25 mg/kg during 1 hour), under muscarinic and α- and β-adrenoceptor blockade, the synthesis in the corresponding glands of saline-treated control rats was less (by 25%). The synthesis was also less when the melatonin administration was combined with the melatonin 2-preferring receptor antagonist luzindole (24%), the non-selective nitric oxide synthase inhibitor L-NAME (18%) and the neuronal nitric oxide synthase inhibitor N-PLA (21%). Almost all the melatonin receptor-mediated effect was due to nitric oxide generation via the activity of neuronal type nitric oxide synthase. The present findings lend further weight to the idea that salivary glandular activity associated with food intake is hormonally influenced and they also suggest clinical implications for melatonin in the treatment of xerostomia. Since melatonin is known to exert anti-inflammatory actions in the oral cavity, the stimulatory effect of melatonin may include the synthesis of proteins of importance for the oral defence.

  16. Effect of different levels of phosphorus on rumen microbial fermentation and synthesis determined using a continuous culture technique.

    PubMed

    Komisarczuk, S; Merry, R J; McAllan, A B

    1987-03-01

    A continuous culture technique was used to study the phosphorus requirements of rumen micro-organisms. Solutions of artificial saliva containing 120, 80, 40 and 0 mg inorganic phosphorus (Pi)/l were infused into the reaction vessels previously inoculated with rumen contents, resulting in Pi concentrations in the vessel contents of 48, 28, 4 and less than 1 mg/l respectively. Various fermentative and synthetic characteristics were examined. In the vessel contents, concentrations of protozoa (about 0.9 X 10(5)/ml) were not significantly affected by Pi concentration. Total volatile fatty acids (VFA) produced averaged about 6.83 mmol/h with Pi levels of 48 and 28 mg/l. Reduction in Pi concentrations to 4 and less than 1 mg/l resulted in significant reductions in total VFA to approximately 6.25 and 3.75 mmol/h respectively, accompanied by a rise in pH from 6.5 to 7.3. Ammonia-nitrogen values, which averaged about 131 mg/l at the higher Pi concentrations, also increased with the lowest level of Pi to about 240 mg/l. ATP concentrations averaged about 14 mumol/l at the highest Pi concentration and fell progressively with each reduction in Pi concentration to a final value of 2.5 mumol/l with the Pi level less than 1 mg/l. At Pi concentrations of 48 and 28 mg/l, the digestibilities of xylose, arabinose and cellulose-glucose were maintained at about 0.90, 0.62 and 0.70 g/g input respectively. At lower Pi concentrations these digestibilities fell significantly and corresponding values at Pi less than 1 mg/l were 0.73, 0.41 and 0.31 respectively. Starch digestion was unaffected by Pi concentration and remained at about 0.90 g/g input. The amount of microbial-N synthesized averaged 0.48 g/d and was maintained with Pi concentrations down to 4 mg/l. There was, however, a significant reduction to 0.26 g/d with Pi concentrations of less than 1 mg/l. The efficiency of microbial protein synthesis was variable but averaged approximately 25 g N/kg total carbohydrate fermented. It was

  17. Acute metabolic acidosis decreases muscle protein synthesis but not albumin synthesis in humans.

    PubMed

    Kleger, G R; Turgay, M; Imoberdorf, R; McNurlan, M A; Garlick, P J; Ballmer, P E

    2001-12-01

    Chronic metabolic acidosis induces negative nitrogen balance by either increased protein breakdown or decreased protein synthesis. Few data exist regarding effects of acute metabolic acidosis on protein synthesis. We investigated fractional synthesis rates (FSRs) of muscle protein and albumin, plasma concentrations of insulin-like growth factor-I (IGF-I), thyroid-stimulating hormone (TSH), and thyroid hormones (free thyroxin [fT(4)] and triiodothyronine [fT(3)]) in seven healthy human volunteers after a stable controlled metabolic period of 5 days and again 48 hours later after inducing metabolic acidosis by oral ammonium chloride intake (4.2 mmol/kg/d divided in six daily doses). Muscle and albumin FSRs were obtained by the [(2)H(5)ring]phenylalanine flooding technique. Ammonium chloride induced a significant decrease in pH (7.43 +/- 0.02 versus 7.32 +/- 0.04; P < 0.0001) and bicarbonate concentration (24.6 +/- 1.6 versus 16.0 +/- 2.7 mmol/L; P < 0.0001) within 48 hours. Nitrogen balance decreased significantly on the second day of acidosis. The FSR of muscle protein decreased (1.94 +/- 0.25 versus 1.30 +/- 0.39; P < 0.02), whereas the FSR of albumin remained constant. TSH levels increased significantly (1.1 +/- 0.5 versus 1.9 +/- 1.1 mU/L; P = 0.03), whereas IGF-I, fT(4), and fT(3) levels showed no significant change. We conclude that acute metabolic acidosis for 48 hours in humans induces a decrease in muscle protein synthesis, which contributes substantially to a negative nitrogen balance. In contrast to prolonged metabolic acidosis of 7 days, a short period of acidosis in the present study did not downregulate albumin synthesis.

  18. Accelerated chemical synthesis of peptides and small proteins

    PubMed Central

    Miranda, Les P.; Alewood, Paul F.

    1999-01-01

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

  19. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  20. Ribosomal history reveals origins of modern protein synthesis.

    PubMed

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

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

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

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

  2. Thermally activated charge transport in microbial protein nanowires

    PubMed Central

    Lampa-Pastirk, Sanela; Veazey, Joshua P.; Walsh, Kathleen A.; Feliciano, Gustavo T.; Steidl, Rebecca J.; Tessmer, Stuart H.; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

  3. Thermally activated charge transport in microbial protein nanowires.

    PubMed

    Lampa-Pastirk, Sanela; Veazey, Joshua P; Walsh, Kathleen A; Feliciano, Gustavo T; Steidl, Rebecca J; Tessmer, Stuart H; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

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

    PubMed Central

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

    2015-01-01

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

  5. Impact of Enzymatic and Microbial Bioprocessing on Protein Modification and Nutritional Properties of Wheat Bran.

    PubMed

    Arte, Elisa; Rizzello, Carlo G; Verni, Michela; Nordlund, Emilia; Katina, Kati; Coda, Rossana

    2015-10-01

    Besides providing dietary fiber, wheat bran is a recognized source of protein and is considered a very valuable substitute for other protein-rich sources in the food and feed industry. Nonetheless, several factors affect protein bioavailability, including bran's layered structure. This study showed the influence on the release and protein modification of wheat bran of different bioprocessing methods involving the activation of endogenous enzymes of bran, the addition of an enzyme mixture having carbohydrase activity, and microbial fermentation. Bioprocessing in acidic conditions significantly enhanced the solubilization of protein from wheat bran, reaching the highest value in the treatment where the sole endogenous protease activity was activated. Bioprocessing through controlled fermentation allowed a more intense proteolysis and strongly impacted the in vitro digestibility of proteins. The combined use of starter cultures and cell-wall-degrading enzymes was characterized by the highest increase of phytase activity and total phenols. PMID:26365885

  6. Impact of Enzymatic and Microbial Bioprocessing on Protein Modification and Nutritional Properties of Wheat Bran.

    PubMed

    Arte, Elisa; Rizzello, Carlo G; Verni, Michela; Nordlund, Emilia; Katina, Kati; Coda, Rossana

    2015-10-01

    Besides providing dietary fiber, wheat bran is a recognized source of protein and is considered a very valuable substitute for other protein-rich sources in the food and feed industry. Nonetheless, several factors affect protein bioavailability, including bran's layered structure. This study showed the influence on the release and protein modification of wheat bran of different bioprocessing methods involving the activation of endogenous enzymes of bran, the addition of an enzyme mixture having carbohydrase activity, and microbial fermentation. Bioprocessing in acidic conditions significantly enhanced the solubilization of protein from wheat bran, reaching the highest value in the treatment where the sole endogenous protease activity was activated. Bioprocessing through controlled fermentation allowed a more intense proteolysis and strongly impacted the in vitro digestibility of proteins. The combined use of starter cultures and cell-wall-degrading enzymes was characterized by the highest increase of phytase activity and total phenols.

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

    PubMed

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

    1980-11-01

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

  8. Biorefining of by-product streams from sunflower-based biodiesel production plants for integrated synthesis of microbial oil and value-added co-products.

    PubMed

    Leiva-Candia, D E; Tsakona, S; Kopsahelis, N; García, I L; Papanikolaou, S; Dorado, M P; Koutinas, A A

    2015-08-01

    This study focuses on the valorisation of crude glycerol and sunflower meal (SFM) from conventional biodiesel production plants for the separation of value-added co-products (antioxidant-rich extracts and protein isolate) and for enhancing biodiesel production through microbial oil synthesis. Microbial oil production was evaluated using three oleaginous yeast strains (Rhodosporidium toruloides, Lipomyces starkeyi and Cryptococcus curvatus) cultivated on crude glycerol and nutrient-rich hydrolysates derived from either whole SFM or SFM fractions that remained after separation of value-added co-products. Fed-batch bioreactor cultures with R. toruloides led to the production of 37.4gL(-1) of total dry weight with a microbial oil content of 51.3% (ww(-1)) when a biorefinery concept based on SFM fractionation was employed. The estimated biodiesel properties conformed with the limits set by the EN 14214 and ASTM D 6751 standards. The estimated cold filter plugging point (7.3-8.6°C) of the lipids produced by R. toruloides is closer to that of biodiesel derived from palm oil.

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

    PubMed

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

    2009-05-01

    CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life. PMID:19388845

  10. Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet.

    PubMed

    Dudley, M A; Schoknecht, P A; Dudley, A W; Jiang, L; Ferraris, R P; Rosenberger, J N; Henry, J F; Reeds, P J

    2001-04-01

    The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.

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

    PubMed

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

    2015-08-14

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

  12. Question 7: Optimized Energy Consumption for Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Szaflarski, Witold; Nierhaus, Knud H.

    2007-10-01

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

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

    SciTech Connect

    Horst, M.N. )

    1990-12-01

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

  14. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.

    PubMed

    Atkins, Coleen M; Nozaki, Naohito; Shigeri, Yasushi; Soderling, Thomas R

    2004-06-01

    Phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) regulates protein synthesis in hippocampal dendrites. CPEB binds the 3' untranslated region (UTR) of cytoplasmic mRNAs and, when phosphorylated, initiates mRNA polyadenylation and translation. We report that, of the protein kinases activated in the hippocampus during synaptic plasticity, calcium/calmodulin-dependent protein kinase II (CaMKII) robustly phosphorylated the regulatory site (threonine 171) in CPEB in vitro. In postsynaptic density fractions or hippocampal neurons, CPEB phosphorylation increased when CaMKII was activated. These increases in CPEB phosphorylation were attenuated by a specific peptide inhibitor of CaMKII and by the general CaM-kinase inhibitor KN-93. Inhibitors of protein phosphatase 1 increased basal CPEB phosphorylation in neurons; this was also attenuated by a CaM-kinase inhibitor. To determine whether CaM-kinase activity regulates CPEB-dependent mRNA translation, hippocampal neurons were transfected with luciferase fused to a 3' UTR containing CPE-binding elements. Depolarization of neurons stimulated synthesis of luciferase; this was abrogated by inhibitors of protein synthesis, mRNA polyadenylation, and CaMKII. These results demonstrate that CPEB phosphorylation and translation are regulated by CaMKII activity and provide a possible mechanism for how dendritic protein synthesis in the hippocampus may be stimulated during synaptic plasticity.

  15. Protein chemical synthesis by serine and threonine ligation

    PubMed Central

    Zhang, Yinfeng; Lam, Hiu Yung; Lee, Chi Lung; Li, Xuechen

    2013-01-01

    An efficient method has been developed for the salicylaldehyde ester-mediated ligation of unprotected peptides at serine (Ser) or threonine (Thr) residues. The utility of this peptide ligation approach has been demonstrated through the convergent syntheses of two therapeutic peptides––ovine-corticoliberin and Forteo––and the human erythrocyte acylphosphatase protein (∼11 kDa). The requisite peptide salicylaldehyde ester precursor is prepared in an epimerization-free manner via Fmoc–solid-phase peptide synthesis. PMID:23569249

  16. [Peptide synthesis aiming at elucidation and creation of protein functions].

    PubMed

    Futaki, S

    1998-11-01

    The recent development of molecular biology has been elucidating outlines of the cross-talk of biomolecules. The understanding of the function of these biomolecules from the viewpoint of chemistry is now demanded not only for the understanding of biological systems but also for the creation of novel functional molecules. Here two topics are described about peptide synthesis aiming at the elucidation and the creation of protein functions. The first topic is the development of approaches for the synthesis of Tyr (SO3H)-containing peptides. Tyrosine sulfation is one of the most popular protein post-translational modifications. Synthetic peptides are of great help for the elucidation of the biological significance of tyrosine sulfation. We have developed two approaches for the efficient synthesis of tyrosine sulfate [Tyr (SO3H)]-containing peptides. The first approach employs a dimethylformamide-sulfur trioxide (DMF-SO3) complex as a sulfating agent and safety-catch protecting groups for the selective sulfation of tyrosine in the presence of serine. The second approach employs the direct introduction of Tyr(SO3H) into the peptide chain in the form of Fmoc-Tyr(SO3Na) followed by deprotection at 4 degrees C in trifluoroacetic acid. These approaches were successfully applied for the synthesis of cholecystokinin (CCK)-related peptides. The second topic deals with new approaches for the creation of artificial proteins through assembling alpha-helical peptides via selective disulfide or thioether formation. Approaches to assemble individual peptide segments on a peptide template were also developed. Four peptides corresponding to the transmembrane segments of the sodium channel (S4 in repeat I-IV) were assembled on a peptide template to give a protein having ion channel activity with rectification.

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

    Camarero, J A; Mitchell, A R

    2005-01-20

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

  19. Alcohol myopathy: impairment of protein synthesis and translation initiation.

    PubMed

    Lang, C H; Kimball, S R; Frost, R A; Vary, T C

    2001-05-01

    Alcohol consumption leads to numerous morphological, biochemical and functional changes in skeletal and cardiac muscle. One such change observed in both tissues after either acute alcohol intoxication or chronic alcohol consumption is a characteristic decrease in the rate of protein synthesis. A decrease in translation efficiency appears to be responsible for at least part of the reduction. This review highlights advances in determining the molecular mechanisms by which alcohol impairs protein synthesis and places these observations in context of earlier studies on alcoholic myopathy. Both acute and chronic alcohol administration impairs translational control by modulating various aspects of peptide-chain initiation. Moreover, this alcohol-induced impairment in initiation is associated with a decreased availability of eukaryotic initiation factor (eIF) 4E in striated muscle, as evidenced by an increase in the amount of the inactive eIF4E.4E-BP1 complex and decrease in the active eIF4E.eIF4G complex. In contrast, alcohol does not produce consistent alterations in the control of translation initiation by the eIF2 system. The etiology of these changes remain unresolved. However, defects in the availability or effectiveness of various anabolic hormones, particularly insulin-like growth factor-I, are consistent with the alcohol-induced decrease in protein synthesis and translation initiation.

  20. PATtyFams: Protein Families for the Microbial Genomes in the PATRIC Database

    PubMed Central

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-01-01

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). This new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods. PMID:26903996

  1. Pulse-chase analysis for studying protein synthesis and maturation.

    PubMed

    Fritzsche, Susanne; Springer, Sebastian

    2014-11-03

    Pulse-chase analysis is a well-established and highly adaptable tool for studying the life cycle of endogenous proteins, including their synthesis, folding, subunit assembly, intracellular transport, post-translational processing, and degradation. This unit describes the performance and analysis of a radiolabel pulse-chase experiment for following the folding and cell surface trafficking of a trimeric murine MHC class I glycoprotein. In particular, the unit focuses on the precise timing of pulse-chase experiments to evaluate early/short-time events in protein maturation in both suspended and strictly adherent cell lines. The advantages and limitations of radiolabel pulse-chase experiments are discussed, and a comprehensive section for troubleshooting is provided. Further, ways to quantitatively represent pulse-chase results are described, and feasible interpretations on protein maturation are suggested. The protocols can be adapted to investigate a variety of proteins that may mature in very different ways.

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

    PubMed

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

    2003-03-01

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

  3. A synthesis of the effects of pesticides on microbial persistence in aquatic ecosystems.

    PubMed

    Staley, Zachery R; Harwood, Valerie J; Rohr, Jason R

    2015-01-01

    Pesticides have a pervasive presence in aquatic ecosystems throughout the world. While pesticides are intended to control fungi, insects, and other pests, their mechanisms of action are often not specific enough to prevent unintended effects, such as on non-target microbial populations. Microorganisms, including algae and cyanobacteria, protozoa, aquatic fungi, and bacteria, form the basis of many food webs and are responsible for crucial aspects of biogeochemical cycling; therefore, the potential for pesticides to alter microbial community structures must be understood to preserve ecosystem services. This review examines studies that focused on direct population-level effects and indirect community-level effects of pesticides on microorganisms. Generally, insecticides, herbicides, and fungicides were found to have adverse direct effects on algal and fungal species. Insecticides and fungicides also had deleterious direct effects in the majority of studies examining protozoa species, although herbicides were found to have inconsistent direct effects on protozoans. Our synthesis revealed mixed or no direct effects on bacterial species among all pesticide categories, with results highly dependent on the target species, chemical, and concentration used in the study. Examination of community-level, indirect effects revealed that all pesticide categories had a tendency to reduce higher trophic levels, thereby diminishing top-down pressures and favoring lower trophic levels. Often, indirect effects exerted greater influence than direct effects. However, few studies have been conducted to specifically address community-level effects of pesticides on microorganisms, and further research is necessary to better understand and predict the net effects of pesticides on ecosystem health.

  4. A synthesis of the effects of pesticides on microbial persistence in aquatic ecosystems.

    PubMed

    Staley, Zachery R; Harwood, Valerie J; Rohr, Jason R

    2015-01-01

    Pesticides have a pervasive presence in aquatic ecosystems throughout the world. While pesticides are intended to control fungi, insects, and other pests, their mechanisms of action are often not specific enough to prevent unintended effects, such as on non-target microbial populations. Microorganisms, including algae and cyanobacteria, protozoa, aquatic fungi, and bacteria, form the basis of many food webs and are responsible for crucial aspects of biogeochemical cycling; therefore, the potential for pesticides to alter microbial community structures must be understood to preserve ecosystem services. This review examines studies that focused on direct population-level effects and indirect community-level effects of pesticides on microorganisms. Generally, insecticides, herbicides, and fungicides were found to have adverse direct effects on algal and fungal species. Insecticides and fungicides also had deleterious direct effects in the majority of studies examining protozoa species, although herbicides were found to have inconsistent direct effects on protozoans. Our synthesis revealed mixed or no direct effects on bacterial species among all pesticide categories, with results highly dependent on the target species, chemical, and concentration used in the study. Examination of community-level, indirect effects revealed that all pesticide categories had a tendency to reduce higher trophic levels, thereby diminishing top-down pressures and favoring lower trophic levels. Often, indirect effects exerted greater influence than direct effects. However, few studies have been conducted to specifically address community-level effects of pesticides on microorganisms, and further research is necessary to better understand and predict the net effects of pesticides on ecosystem health. PMID:26565685

  5. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

    SciTech Connect

    Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

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

    PubMed Central

    Siddell, S G; Ellis, R J

    1975-01-01

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

  7. Microbial synthesis of polyhydroxyalkanoate using seaweed-derived crude levulinic acid as co-nutrient.

    PubMed

    Bera, Anupam; Dubey, Sonam; Bhayani, Khushbu; Mondal, Dibyendu; Mishra, Sandhya; Ghosh, Pushpito K

    2015-01-01

    Production of polyhydroxyalkanoates (PHAs) from Jatropha biodiesel residues, namely crude glycerol and oil cake hydrolysate, has been reported previously. Halomonas hydrothermalis (MTCC accession no. 5445; NCBI Genbank accession no. GU938192), a wild marine strain, was used in the bio-synthesis. The present study was initiated to vary the properties of the polymer. Seaweed-derived crude levulinic acid (SDCLA), containing formic acid, residual sugars and dissolved minerals additionally, was proposed as co-feed along with the biodiesel residues. Experiments were conducted at 100mL scale in batch process. Whereas the PHA yield was only 0.40 ± 0.01 g when only biodiesel residues were employed, it rose to 1.07 ± 0.02 g in presence of 0.35% (w/v) of SDCLA. The corresponding carbon utilisation efficiencies were 29.3% and 57.5%, respectively. 3-Hydroxy valerate incorporation in the PHA was pronounced in presence of SDCLA, with associated changes in polymer properties. The microbial synthesis fared poorly when SDCLA was substituted with pure levulinic acid. Thus, Halomonas hydrothermalis had a poor response to levulinic acid, as such, and other constituents present in SDCLA appear to have played a vital role in bacterial cell division and accumulation of PHA. Biodegradability tests in moist soil were also conducted as part of the study. Marine microalgal cultivation for biodiesel and seaweed cultivation for fuels may help generate biodiesel residues and crude levulinic acid in proximity, which would open up the possibility of large scale PHA manufacture in efficient and practical manner in the future through the methodology of the present study.

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

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

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

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

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

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

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

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-04-11

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

  13. Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily

    PubMed Central

    Dunwell, Jim M.; Khuri, Sawsan; Gane, Paul J.

    2000-01-01

    This review summarizes the recent discovery of the cupin superfamily (from the Latin term “cupa,” a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic β-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1,2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications. PMID:10704478

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

    PubMed

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

    2016-06-25

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

  15. Initiation of picornavirus protein synthesis in ascites cell extracts.

    PubMed

    Oberg, B F; Shatkin, A J

    1972-12-01

    The current model of picornavirus protein formation implies that initiation of protein synthesis occurs at a single site on the viral RNA, and that the large polypeptide formed is later cleaved. A direct test of this model was made in vitro by studying the incorporation of [(35)S]methionine from rabbit liver Met-tRNA(M) (Met) and fMet-tRNA(F) (Met) into encephalomyocarditis virus RNA-coded proteins in extracts of Ehrlich ascites cells. The incorporation of N-formylmethionine was complete within 5 min, while utilization of Met-tRNA(M) (Met) continued for 20 min. Tryptic digests of [(35)S]methionine-labeled products from Met-tRNA(M) (Met) analyzed by anion-exchange chromatography yielded more than 30 peptides, as compared to about 15 [(35)S]methionine-labeled peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-tRNA(F) (Met) yielded one major (26)S-labeled tryptic peptide. The N-terminal location of methionine in this peptide was verified by Edman degradation. One predominant N-terminal tryptic peptide was also obtained with fMet-tRNA(F) (Met) when mouse Elberfeld and mengo-virus RNAs were used as messengers. On the basis of N-terminal compared with internal labeling of the products, no evidence for in vitro post-translational cleavage was found. The results are consistent with a single initiation site for synthesis of picornavirus proteins.

  16. Protein synthesis and consolidation of memory-related synaptic changes.

    PubMed

    Lynch, Gary; Kramár, Enikö A; Gall, Christine M

    2015-09-24

    Although sometimes disputed, it has been assumed for several decades that new proteins synthesized following a learning event are required for consolidation of subsequent memory. Published findings and new results described here challenge this idea. Protein synthesis inhibitors did not prevent Theta Bust Stimulation (TBS) from producing extremely stable long-term potentiation (LTP) in experiments using standard hippocampal slice protocols. However, the inhibitors were effective under conditions that likely depleted protein levels prior to attempts to induce the potentiation effect. Experiments showed that induction of LTP at one input, and thus a prior episode of protein synthesis, eliminated the effects of inhibitors on potentiation of a second input even in depleted slices. These observations suggest that a primary role of translation and transcription processes initiated by learning events is to prepare neurons to support future learning. Other work has provided support for an alternative theory of consolidation. Specifically, if the synaptic changes that support memory are to endure, learning events/TBS must engage a complex set of signaling processes that reorganize and re-stabilize the spine actin cytoskeleton. This is accomplished in fast (10 min) and slow (50 min) stages with the first requiring integrin activation and the second a recovery of integrin functioning. These results align with, and provide mechanisms for, the long-held view that memories are established and consolidated over a set of temporally distinct phases. This article is part of a Special Issue entitled SI: Brain and Memory. PMID:25485773

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

    Hurewitz, J.; Jagendorf, A.T.

    1986-04-01

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

  19. Characterization and optimization of cathodic conditions for H2O2 synthesis in microbial electrochemical cells.

    PubMed

    Sim, Junyoung; An, Junyeong; Elbeshbishy, Elsayed; Ryu, Hodon; Lee, Hyung-Sool

    2015-11-01

    Cathode potential and O2 supply methods were investigated to improve H2O2 synthesis in an electrochemical cell, and optimal cathode conditions were applied for microbial electrochemical cells (MECs). Using aqueous O2 for the cathode significantly improved current density, but H2O2 conversion efficiency was negligible at 0.3-12%. Current density decreased for passive O2 diffusion to the cathode, but H2O2 conversion efficiency increased by 65%. An MEC equipped with a gas diffusion cathode was operated with acetate medium and domestic wastewater, which presented relatively high H2O2 conversion efficiency from 36% to 47%, although cathode overpotential was fluctuated. Due to different current densities, the maximum H2O2 production rate was 141 mg H2O2/L-h in the MEC fed with acetate medium, but it became low at 6 mg H2O2/L-h in the MEC fed with the wastewater. Our study clearly indicates that improving anodic current density and mitigating membrane fouling would be key parameters for large-scale H2O2-MECs.

  20. Action of microbial transglutaminase (MTGase) in the modification of food proteins: a review.

    PubMed

    Gaspar, Ana Luisa Camolezi; de Góes-Favoni, Silvana Pedroso

    2015-03-15

    Microbial transglutaminase (MTGase) is an enzyme of the class of transferases widely known to modify protein functional properties in food systems. The main mechanisms of action involved are polymerisations, which result in changes in the molecule's hydrophobicity. Among the functional properties, MTGase affects solubility and hence gelation, emulsification, foaming, viscosity and water-holding capacity, which all depend on protein solubility. Although the enzyme's benefits for protein functionality in a variety of food systems are widely reported in the literature, few studies have focussed on its mechanisms. The purpose of this review is therefore to investigate the mechanisms of action of MTGase and consider its influence on the functional properties with different protein substrates. Understanding these mechanisms is the first step in the development and application, in food production, of new and better functional properties with improved and/or innovative characteristics that can satisfy consumer expectations.

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

    SciTech Connect

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

    1986-03-05

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

  2. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine

    PubMed Central

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L.; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  3. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine.

    PubMed

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  4. Protein synthesis in tomato-fruit locule tissue

    PubMed Central

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

    1967-01-01

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

  5. Effects of influenza A virus NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis.

    PubMed

    Salvatore, Mirella; Basler, Christopher F; Parisien, Jean-Patrick; Horvath, Curt M; Bourmakina, Svetlana; Zheng, Hongyong; Muster, Thomas; Palese, Peter; García-Sastre, Adolfo

    2002-02-01

    The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

  6. Signals, Synapses, and Synthesis: How New Proteins Control Plasticity

    PubMed Central

    Zukin, R. Suzanne; Richter, Joel D.; Bagni, Claudia

    2009-01-01

    Localization of mRNAs to dendrites and local protein synthesis afford spatial and temporal regulation of gene expression and endow synapses with the capacity to autonomously alter their structure and function. Emerging evidence indicates that RNA binding proteins, ribosomes, translation factors and mRNAs encoding proteins critical to synaptic structure and function localize to neuronal processes. RNAs are transported into dendrites in a translationally quiescent state where they are activated by synaptic stimuli. Two RNA binding proteins that regulate dendritic RNA delivery and translational repression are cytoplasmic polyadenylation element binding protein and fragile X mental retardation protein (FMRP). The fragile X syndrome (FXS) is the most common known genetic cause of autism and is characterized by the loss of FMRP. Hallmark features of the FXS include dysregulation of spine morphogenesis and exaggerated metabotropic glutamate receptor-dependent long term depression, a cellular substrate of learning and memory. Current research focuses on mechanisms whereby mRNAs are transported in a translationally repressed state from soma to distal process and are activated at synaptic sites in response to synaptic signals. PMID:19838324

  7. Interferon Production and Protein Synthesis in Chick Cells

    PubMed Central

    Friedman, Robert M.

    1966-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. Diverse and divergent protein post-translational modifications in two growth stages of a natural microbial community

    SciTech Connect

    Li, Zhou; Wang, Yingfeng; Yao, Qiuming; Justice, Nicholas B.; Ahn, Tae-Hyuk; Xu, Dong; Hettich, Robert {Bob} L; Banfield, Jillian F.; Pan, Chongle

    2014-01-01

    Detailed characterization of posttranslational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.

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

    PubMed

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

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples. PMID:27260286

  11. Protein synthesis by synaptosomes from rat brain. Contribution by the intraterminal mitochondria

    PubMed Central

    Hernández, A. G.

    1974-01-01

    (1) The characteristics of protein synthesis in microsomal and synaptosomal fractions from rat brain were examined. A high sensitivity to ribonuclease and to cycloheximide, and the need for the presence of pH5 enzymes distinguished protein synthesis in microsomal fractions from protein synthesis in synaptosomes. (2) Under various conditions of incubation synaptosomal fractions prepared in sucrose showed limited protein synthesis compared with synaptosomal fractions prepared by using Ficoll. Such discrepancies could not be attributed to: (i) animal age, (ii) the metabolic state of the synaptosomal fraction, (iii) the absence of bivalent cations in the incubation medium or (iv) the temperature. (3) Protein synthesis in synaptosomal fractions was inhibited 50–65% by cycloheximide, 38–50% by chloramphenicol, 95% by puromycin, 70% by azide and 40% by deoxyglucose; ribonuclease had only a negligible inhibitory effect. (4) As a first approximation to the localization of the protein-synthetic machinery present in the synaptosomal fraction, the distribution of enzymes and radioactivity in subfractions of prelabelled synaptosomes was determined after osmotic shock with water. Approximately 60% of the total protein synthesis in the synaptosomal fraction occurred in the intraterminal mitochondria. (5) Protein synthesis in the intraterminal mitochondria did not show any fundamental difference from synthesis in somatic mitochondria, with respect to inhibition by cycloheximide and chloramphenicol. (6) It was concluded that if extramitochondrial protein synthesis occurs in synaptosomes, it must be very low. PMID:4441374

  12. The effect of temperature on post-prandial protein synthesis in juvenile barramundi, Lates calcarifer.

    PubMed

    Katersky, Robin S; Carter, Chris G

    2010-08-01

    The experiment aimed to measure post-prandial protein synthesis at three different temperatures. Juvenile barramundi (10.81+/-3.46 g) were held at 21, 27 and 33 degrees C and fed to satiation daily. Samples were taken over a 24h period at 0 (24h after the previous meal) and then at 4, 8, 12 and 24h after feeding to measure protein synthesis in the white muscle, liver and remaining carcass. Protein synthesis at 27 and 33 degrees C peaked 4h after feeding in all tissues and returned to pre-feeding rates by 12h. At 21 degrees C protein synthesis remained constant over 24h in all tissues. While the concentration of RNA remained stable over the 24h cycle and across temperatures, the ribosomal activity increased after feeding. This meant k(RNA), not the absolute amount of RNA, was the driving force underlying the post-prandial increase in protein synthesis. However, relative differences in protein synthesis between tissues were attributed to differences in RNA concentration. There was a significant positive relationship between white muscle and whole body protein synthesis. This was the first study to show an interaction between temperature and the time after feeding on protein synthesis for an ectotherm, and that a post-prandial peak in protein synthesis only occurred under optimum temperature conditions.

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

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

  15. Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.

    PubMed

    Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2014-07-18

    5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.

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

    NASA Astrophysics Data System (ADS)

    Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

    2016-05-01

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

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

    PubMed Central

    Sahraeian, Sayed Mohammad Ebrahim; Yoon, Byung-Jun

    2012-01-01

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

  18. Sildenafil increases muscle protein synthesis and reduces muscle fatigue.

    PubMed

    Sheffield-Moore, Melinda; Wiktorowicz, John E; Soman, Kizhake V; Danesi, Christopher P; Kinsky, Michael P; Dillon, Edgar L; Randolph, Kathleen M; Casperson, Shannon L; Gore, Dennis C; Horstman, Astrid M; Lynch, James P; Doucet, Barbara M; Mettler, Joni A; Ryder, Jeffrey W; Ploutz-Snyder, Lori L; Hsu, Jean W; Jahoor, Farook; Jennings, Kristofer; White, Gregory R; McCammon, Susan D; Durham, William J

    2013-12-01

    Reductions in skeletal muscle function occur during the course of healthy aging as well as with bed rest or diverse diseases such as cancer, muscular dystrophy, and heart failure. However, there are no accepted pharmacologic therapies to improve impaired skeletal muscle function. Nitric oxide may influence skeletal muscle function through effects on excitation-contraction coupling, myofibrillar function, perfusion, and metabolism. Here we show that augmentation of nitric oxide-cyclic guanosine monophosphate signaling by short-term daily administration of the phosphodiesterase 5 inhibitor sildenafil increases protein synthesis, alters protein expression and nitrosylation, and reduces fatigue in human skeletal muscle. These findings suggest that phosphodiesterase 5 inhibitors represent viable pharmacologic interventions to improve muscle function. PMID:24330691

  19. Evolution of Protein Synthesis from an RNA World

    PubMed Central

    Noller, Harry F.

    2012-01-01

    SUMMARY Because of the molecular complexity of the ribosome and protein synthesis, it is a challenge to imagine how translation could have evolved from a primitive RNA World. Two specific suggestions are made here to help to address this, involving separate evolution of the peptidyl transferase and decoding functions. First, it is proposed that translation originally arose not to synthesize functional proteins, but to provide simple (perhaps random) peptides that bound to RNA, increasing its available structure space, and therefore its functional capabilities. Second, it is proposed that the decoding site of the ribosome evolved from a mechanism for duplication of RNA. This process involved homodimeric “duplicator RNAs,” resembling the anticodon arms of tRNAs, which directed ligation of trinucleotides in response to an RNA template. PMID:20610545

  20. [Protein synthesis by the ribosome: a pathway full of pitfalls].

    PubMed

    Macé, Kevin; Giudice, Emmanuel; Gillet, Reynald

    2015-03-01

    Protein synthesis is accomplished through a process known as translation and is carried out by the ribosome, a large macromolecular complex found in every living organism. Given the huge amount of biological data that must be deciphered, it is not uncommon for ribosomes to regularly stall during the process of translation. Any disruption of this finely tuned process will jeopardize the viability of the cell. In bacteria, the main quality-control mechanism for rescuing ribosomes that undergo arrest during translation is trans-translation, which is performed by transfer-messenger RNA (tmRNA) in association with small protein B (SmPB). However, other rescue systems have been discovered recently, revealing a far more complicated network of factors dedicated to ribosome rescue. These discoveries make it possible to consider inhibition of these pathways as a very promising target for the discovery of new antibiotics.

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

    PubMed

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

    2015-10-21

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

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

    PubMed Central

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

    2015-01-01

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

  3. Plastid protein synthesis is required for plant development in tobacco

    PubMed Central

    Ahlert, Daniela; Ruf, Stephanie; Bock, Ralph

    2003-01-01

    Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts of chloroplast translation in a cell-line-specific manner. The resulting plants are chimeric and, in the presence of translational inhibitors, exhibit severe developmental abnormalities. In the absence of active plastid protein synthesis, leaf blade development is abolished because of an apparent arrest of cell division. This effect appears to be cell-autonomous in that adjacent sectors of cells with translating plastids are phenotypically normal but cannot complement for the absence of plastid translation in mutant sectors. Developmental abnormalities also are seen in flower morphology, indicating that the defects are not caused by inhibited expression of plastid photosynthesis genes. Taken together, our data point to an unexpected essential role of plastid genes and gene expression in plant development and cell division. PMID:14660796

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

    PubMed

    Orndorff, P E; Dworkin, M

    1982-01-01

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

  5. Microbial Physiology of the Conversion of Residual Oil to Methane: A Protein Prospective

    NASA Astrophysics Data System (ADS)

    Morris, Brandon E. L.; Bastida-Lopez, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Suflita, Joseph M.

    2010-05-01

    Traditional petroleum recovery techniques are unable to extract the majority of oil in most petroliferous deposits. The recovery of even a fraction of residual hydrocarbon in conventional reserves could represent a substantive energy supply. To this end, the microbial conversion of residual oil to methane has gained increasing relevance in recent years [1,2]. Worldwide demand for methane is expected to increase through 2030 [3], as it is a cleaner-burning alternative to traditional fuels [4]. To investigate the microbial physiology of hydrocarbon-decomposition and ultimate methanogenesis, we initiated a two-pronged approach. First, a model alkane-degrading sulfate-reducing bacterium, Desulfoglaeba alkanexedens, was used to interrogate the predominant metabolic pathway(s) differentially expressed during growth on either n-decane or butyrate. A total of 81 proteins were differentially expressed during bacterial growth on butyrate, while 100 proteins were unique to the alkane-grown condition. Proteins related to alkylsuccinate synthase, or the homologous 1-methyl alkylsuccinate synthase, were identified only in the presence of the hydrocarbon. Secondly, we used a newly developed stable isotope probing technique [5] targeted towards proteins to monitor the flux of carbon through a residual oil-degrading bacterial consortium enriched from a gas-condensate contaminated aquifer [1]. Combined carbon and hydrogen stable isotope fractionation identified acetoclastic methanogenesis as the dominant process in this system. Such findings agree with the previous clone library characterization of the consortium. Furthermore, hydrocarbon activation was determined to be the rate-limiting process during the net conversion of residual oil to methane. References 1. Gieg, L.M., K.E. Duncan, and J.M. Suflita, Bioenegy production via microbial conversion of residual oil to natural gas. Appl Environ Micro, 2008. 74(10): p. 3022-3029. 2. Jones, D.M., et al., Crude-oil biodegradation via

  6. Proteomic profiling of microbial transglutaminase-induced polymerization of milk proteins.

    PubMed

    Hsieh, J F; Pan, P H

    2012-02-01

    Microbial transglutaminase (MTGase)-induced polymerization of individual milk proteins during incubation was investigated using a proteomics-based approach. The addition of MTGase (0.25-2.0 units/mL) caused the milk proteins to polymerize after a 3-h incubation period. Sodium dodecyl sulfate-PAGE analysis showed that the total intensities of the protein bands that corresponded to α(S)-casein, β-casein, and κ-casein decreased from 8,245.6, 6,677.2, and 586.6 arbitrary units to 1,911.7, 0.0, and 66.2 arbitrary units, respectively. Components with higher molecular weights were observed, and the intensity of these proteins increased after 3h of incubation. These results support that inter- or intramolecular crosslinking occurred in the casein proteins of MTGase-treated milk. Two-dimensional electrophoresis analysis indicated that isomers of β-casein, κ-casein, a fraction of serum albumin, α(S1)-casein, α(S2)-casein, β-lactoglobulin, and α-lactalbumin in the milk were polymerized following incubation with MTGase. In addition, MTGase-induced polymerization occurred earlier for β-casein and κ-casein isomers than for other milk proteins.

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

    PubMed

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

    2008-05-01

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

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

    PubMed Central

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

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

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

    SciTech Connect

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

    1985-04-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

  11. Expanded microbial genome coverage and improved protein family annotation in the COG database

    PubMed Central

    Galperin, Michael Y.; Makarova, Kira S.; Wolf, Yuri I.; Koonin, Eugene V.

    2015-01-01

    Microbial genome sequencing projects produce numerous sequences of deduced proteins, only a small fraction of which have been or will ever be studied experimentally. This leaves sequence analysis as the only feasible way to annotate these proteins and assign to them tentative functions. The Clusters of Orthologous Groups of proteins (COGs) database (http://www.ncbi.nlm.nih.gov/COG/), first created in 1997, has been a popular tool for functional annotation. Its success was largely based on (i) its reliance on complete microbial genomes, which allowed reliable assignment of orthologs and paralogs for most genes; (ii) orthology-based approach, which used the function(s) of the characterized member(s) of the protein family (COG) to assign function(s) to the entire set of carefully identified orthologs and describe the range of potential functions when there were more than one; and (iii) careful manual curation of the annotation of the COGs, aimed at detailed prediction of the biological function(s) for each COG while avoiding annotation errors and overprediction. Here we present an update of the COGs, the first since 2003, and a comprehensive revision of the COG annotations and expansion of the genome coverage to include representative complete genomes from all bacterial and archaeal lineages down to the genus level. This re-analysis of the COGs shows that the original COG assignments had an error rate below 0.5% and allows an assessment of the progress in functional genomics in the past 12 years. During this time, functions of many previously uncharacterized COGs have been elucidated and tentative functional assignments of many COGs have been validated, either by targeted experiments or through the use of high-throughput methods. A particularly important development is the assignment of functions to several widespread, conserved proteins many of which turned out to participate in translation, in particular rRNA maturation and tRNA modification. The new version of the

  12. Post-prandial changes in protein synthesis in red drum (Sciaenops ocellatus) larvae.

    PubMed

    McCarthy, Ian D; Fuiman, Lee A

    2011-06-01

    Protein synthesis is one of the major energy-consuming processes in all living organisms. Post-prandial changes in protein synthesis have been studied in a range of animal taxa but have been little studied in fish larvae. Using the flooding-dose method, we measured post-prandial changes in whole-body rates of protein synthesis in regularly fed red drum Sciaenops ocellatus (Linnaeus) larvae for 24-28 h following their daily meal. Fractional rates of protein synthesis increased from a baseline (pre-feeding) rate of 16% day(-1) to a post-prandial peak of 48% day(-1) ca. 8 h after feeding before declining to 12% day(-1) after 24-28 h. The overall mean daily rate of protein synthesis was calculated as 27% day(-1). Although suggested as energetically impossible in larval poikilotherms, our results show that rates in excess of 30% day(-1) can be attained by larval fishes for a few hours but are not sustained. The average daily energetic cost of protein synthesis was estimated as 34% of daily total oxygen consumption, ranging from 19% immediately before feeding to 61% during the post-prandial peak in protein synthesis. This suggests that during the post-prandial peak, protein synthesis will require a large proportion of the hourly energy production, which, given the limited metabolic scope in fish larvae, may limit the energy that could otherwise be allocated to other energy-costly functions, such as foraging and escape responses. PMID:21562168

  13. Recalling an Aversive Experience by Day-Old Chicks Is Not Dependent on Somatic Protein Synthesis

    ERIC Educational Resources Information Center

    Mileusnic, Radmila; Lancashire, Christine L.; Rose, Steven P. R.

    2005-01-01

    Long-term memory is dependent on protein synthesis and inhibiting such synthesis following training results in amnesia for the task. Proteins synthesized during training must be transported to the synapse and disrupting microtubules with Colchicines, and hence, blocking transport, results in transient amnesia. Reactivating memory for a previously…

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. On the Role of Hippocampal Protein Synthesis in the Consolidation and Reconsolidation of Object Recognition Memory

    ERIC Educational Resources Information Center

    Rossato, Janine I.; Bevilaqua, Lia R. M.; Myskiw, Jociane C.; Medina, Jorge H.; Izquierdo, Ivan; Cammarota, Martin

    2007-01-01

    Upon retrieval, consolidated memories are again rendered vulnerable to the action of metabolic blockers, notably protein synthesis inhibitors. This has led to the hypothesis that memories are reconsolidated at the time of retrieval, and that this depends on protein synthesis. Ample evidence indicates that the hippocampus plays a key role both in…

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    ERIC Educational Resources Information Center

    Wolf, Gerald; Engelmann, Mario; Richter, Karin

    2005-01-01

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

  18. PATtyFams: Protein families for the microbial genomes in the PATRIC database

    DOE PAGESBeta

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-02-08

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

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

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

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

  20. Alphavirus RNA synthesis and non-structural protein functions

    PubMed Central

    Rupp, Jonathan C.; Sokoloski, Kevin J.; Gebhart, Natasha N.

    2015-01-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field. PMID:26219641

  1. Microbial population dynamics in an anaerobic CSTR treating a chemical synthesis-based pharmaceutical wastewater.

    PubMed

    Oz, Nilgun Ayman; Ince, Orhan; Ince, Bahar Kasapgil; Akarsubasi, Alper Tunga; Eyice, Ozge

    2003-01-01

    Effects of a chemical synthesis based pharmaceutical wastewater on performance of an anaerobic completely stirred tank reactor (CSTR), activity of acetoclastic methanogens and microbial composition were evaluated under various influent compositions. Initially, the CSTR was fed with glucose up to an organic loading rate (OLR) of 6 kg COD/m3 x d corresponding to an F/M ratio of 0.43 with a hydraulic retention time (HRT) of 2.5 days. A COD removal efficiency of 92% and a methane yield of 0.32 m3 CH4/kg COD(removed) were achieved whilst specific methanogenic activity (SMA) was found to be 336mL CH4/gTVS x d. After the CSTR was fed with pre-aerated wastewater diluted by glucose in different dilution ratios of 10% (w/v), 30% (w/v), 70% (w/v), and 100% (w/v) pre-aerated wastewater, gradual decreases in COD removal efficiency to 71%, methane yield to 0.28 m3CH4/kg COD(removed) and SMA to 166 mL CH4/gTVS d occurred whilst volatile fatty acid concentration reached to 1474 mg/L. After the raw wastewater diluted with the pre-aerated wastewater was fed into the CSTR in increasing ratios of 10% (w/v), 30% (w/v), and 60% (w/v), there was a proportional deterioration in performance in terms of COD removal efficiency, methane yield and acetoclastic methanogenic activity. Epifluorescence microscopy of the seed sludge revealed that Methanococcus-like species, short, and medium rods were found to be equally dominant. The short and medium rod species remained equally dominant groups in the CSTR throughout the feeding regime whilst Methanococcus-like species and long rods were found to be in insignificant numbers at the end of the study. Changes in archael diversity were determined using molecular analyses such as polymerase chain reaction (PCR), and denaturent gradient gel electrophoresis (DGGE). Results showed that overall archeal diversity did not change much whereas changes in composition of eubacterial population occurred.

  2. Competition For Resources in a Model for Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Cook, Larry; Zia, Royce

    2009-03-01

    The Totally Asymmetric Simple Exclusion Process (TASEP) is often used to explore translation during protein synthesis. The particles represent ribosomes that move along mRNA, which is represented by the one-dimensional lattice. Unlike ordinary TASEP where the supply of particles is unlimited, there is a finite number of ribosome in a cell. In addition, there are many genes which compete for this pool of ribosomes. Thus, we are motivated to consider the effects of multiple TASEPs (of varying lengths) coupled to a single, finite reservoir of particles. In particular, the total occupation numbers, the density profiles and the particle currents of individual TASEPs are studied, as the overall reservoir of particles is varied. Both Monte Carlo simulation results and analytic considerations will be presented.

  3. Design and synthesis of a protein. beta. -turn mimetic

    SciTech Connect

    Olson, G.L.; Voss, M.E.; Hill, D.E.; Kahn, M.; Madison, V.S.; Cook, C.M. )

    1990-01-03

    A nine-membered-ring lactam system (1) has been chosen as a framework for the development of non-peptide molecules to mimic structural features of peptide and protein {beta}-turns. The synthesis of model di- and tetrapeptide mimetics starting from 1,5-cyclooctadiene derivatives is reported. In the model dipeptide mimetic (9), the amide linkages is trans (NMR, X-ray) and functional groups at positions adjacent to the lactam amide bond correspond closely to the side-chain positions of residues i + 1 and i + 2 of classical type II{prime} {beta}-turns. In the model tetrapeptide mimetic (30), all four side chains of low-energy trans amide conformers of the mimetic are well matched to their peptide counterparts.

  4. Changes in regulation of ribosomal protein synthesis during vegetative growth and sporulation of Saccharomyces cerevisiae.

    PubMed Central

    Pearson, N J; Haber, J E

    1980-01-01

    When diploid Saccharomyces cerevisiae cells logarithmically growing in acetate medium were placed in sporulation medium, the relative rates of synthesis of 40 or more individual ribosomal proteins (r-proteins) were coordinately depressed to approximately 20% of those of growing cells. These new depressed rates remained constant for at least 10 h into sporulation. If yeast nitrogen base was added 4 yh after the beginning of sporulation to shift the cells back to vegetative growth, the original relative rates of r-protein synthesis were rapidly reestablished. this upshift in the rates occurred even in diploids homozygous for the regulatory mutation rna2 at the restrictive temperature for this mutation (34 degrees C). However, once these mutant cells began to bud and grow at 34 degrees C, the phenotype of rna2 was expressed and the syntheses of r-proteins were again coordinately depressed. At least one protein whose rate of synthesis was not depressed by rna2 in vegetative cells did have a decreased rate of synthesis during sporulation. Another r-protein whose synthesis was depressed by rna2 maintained a high rate of synthesis at the beginning of sporulation. These data suggest that the mechanism responsible for coordinate control of r-protein synthesis during sporulation does not require the gene product of RNA2 and thus defines a separate mechanism by which r-proteins are coordinately controlled in S. cerevisiae. Images PMID:6997272

  5. Linking microbial and ecosystem ecology using ecological stoichiometry: a synthesis of conceptual and empirical approaches

    USGS Publications Warehouse

    Hall, E.K.; Maixner, F.; Franklin, O.; Daims, H.; Richter, A.; Battin, T.

    2011-01-01

    Currently, one of the biggest challenges in microbial and ecosystem ecology is to develop conceptual models that organize the growing body of information on environmental microbiology into a clear mechanistic framework with a direct link to ecosystem processes. Doing so will enable development of testable hypotheses to better direct future research and increase understanding of key constraints on biogeochemical networks. Although the understanding of phenotypic and genotypic diversity of microorganisms in the environment is rapidly accumulating, how controls on microbial physiology ultimately affect biogeochemical fluxes remains poorly understood. We propose that insight into constraints on biogeochemical cycles can be achieved by a more rigorous evaluation of microbial community biomass composition within the context of ecological stoichiometry. Multiple recent studies have pointed to microbial biomass stoichiometry as an important determinant of when microorganisms retain or recycle mineral nutrients. We identify the relevant cellular components that most likely drive changes in microbial biomass stoichiometry by defining a conceptual model rooted in ecological stoichiometry. More importantly, we show how X-ray microanalysis (XRMA), nanoscale secondary ion mass spectroscopy (NanoSIMS), Raman microspectroscopy, and in situ hybridization techniques (for example, FISH) can be applied in concert to allow for direct empirical evaluation of the proposed conceptual framework. This approach links an important piece of the ecological literature, ecological stoichiometry, with the molecular front of the microbial revolution, in an attempt to provide new insight into how microbial physiology could constrain ecosystem processes.

  6. Microbial metaproteomics: Identifying the repertoire of proteins that microorganisms use to compete and cooperate in complex environmental communities

    SciTech Connect

    Hettich, Robert {Bob} L; Sharma, Ritin; Chourey, Karuna; Giannone, Richard J

    2012-01-01

    The availability of genome information for microbial consortia, including unculturable species, from environmental samples has enabled systems-biology interrogation by providing a means to access genomic, transcriptomic, and proteomic information. This provides a unique opportunity to characterize the molecular activities and interactions of these microbial systems at a comprehensive level never before possible. Such information not only provides details about the organizational, functional, and metabolic activities of such systems, but also the untapped reserve of molecular activities that might be invoked and exploited under certain environmental conditions. Since bacteria naturally exist in complex ecosystems, it is imperative to develop and utilize analytical approaches that can provide molecular level details on systems consisting of mixed microbial membership. This is the realm of metaproteomics - the characterization of the complement of proteins expressed by a microbial community in an environmental sample

  7. Short-Term Protein Stable Isotope Probing of Microbial Communities to Associate Functions with Taxa (Invited)

    NASA Astrophysics Data System (ADS)

    Lipton, M. S.; Slysz, G. W.; Steinke, L. A.; Ward, D. M.; Klatt, C. G.; Clauss, T. R.; Purvine, S. O.; Anderson, G. A.; Payne, S. H.; Bryant, D. A.

    2013-12-01

    Determining which taxa in a community perform which functions is essential for understanding metabolite fluxes and metabolic interactions among community members. Specific taxa will alter their metabolism in order to acclimate to changing environmental factors such as light through the diel cycle, changing temperature and other factors. Monitoring which proteins are being expressed, and the quantitative protein expression patterns in the individual taxa as a response to external stimuli is key to understanding these mechanisms. Protein stable isotope probing (Pro-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. In Pro-SIP studies, label incorporation is determined by the extent of the change in the isotopic profile of peptides when measured by mass spectrometry. While most Pro-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), these techniques have not been applied to short term in situ studies due to the small degree of partial labeling of the proteins. We have applied Pro-SIP to study the assimilation of a labeled substrate into proteins to determine which taxa are responsible for sequestration of dissolved inorganic carbon in microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park. This community is fueled by sunlight as it transitions from dark to light; the aim was to understand the light-dependent pathway of inorganic carbon incorporation into different taxa during the early morning hours when the mat was in low light and anoxic. Each mat sample was incubated with 13C-bicarbonate for 3 h. Substrate assimilation was determined through standard proteomic techniques along with the use of SIPPER, a collection of algorithms that sensitively measure small changes in peptide isotopic patterns, allowing the determination of which taxa assimilated the substrate during this period. For the

  8. Application of a Rapid Knowledge Synthesis and Transfer Approach To Assess the Microbial Safety of Low-Moisture Foods.

    PubMed

    Young, Ian; Waddell, Lisa; Cahill, Sarah; Kojima, Mina; Clarke, Renata; Rajić, Andrijana

    2015-12-01

    Low-moisture foods (LMF) are increasingly implicated in outbreaks of foodborne illness, resulting in a significant public health burden. To inform the development of a new Codex Alimentarius code of hygienic practice for LMF, we applied a rapid knowledge synthesis and transfer approach to review global research on the burden of illness, prevalence, and interventions to control nine selected microbial hazards in eight categories of LMF. Knowledge synthesis methods included an integrated scoping review (search strategy, relevance screening and confirmation, and evidence mapping), systematic review (detailed data extraction), and meta-analysis of prevalence data. Knowledge transfer of the results was achieved through multiple reporting formats, including evidence summary cards. We identified 214 unique outbreaks and 204 prevalence and 126 intervention studies. Cereals and grains (n = 142) and Salmonella (n = 278) were the most commonly investigated LMF and microbial hazard categories, respectively. Salmonella was implicated in the most outbreaks (n = 96, 45%), several of which were large and widespread, resulting in the most hospitalizations (n = 895, 89%) and deaths (n = 14, 74%). Salmonella had a consistently low prevalence across all LMF categories (0 to 3%), but the prevalence of other hazards (e.g., Bacillus cereus) was highly variable. A variety of interventions were investigated in small challenge trials. Key knowledge gaps included underreporting of LMF outbreaks, limited reporting of microbial levels in prevalence studies, and a lack of intervention efficacy research under commercial conditions. Summary cards were a useful knowledge transfer format to inform complementary risk ranking activities. This review builds upon previous work in this area by synthesizing a broad range of evidence using a structured, transparent, and integrated approach to provide timely evidence informed inputs into international guidelines.

  9. Application of a Rapid Knowledge Synthesis and Transfer Approach To Assess the Microbial Safety of Low-Moisture Foods.

    PubMed

    Young, Ian; Waddell, Lisa; Cahill, Sarah; Kojima, Mina; Clarke, Renata; Rajić, Andrijana

    2015-12-01

    Low-moisture foods (LMF) are increasingly implicated in outbreaks of foodborne illness, resulting in a significant public health burden. To inform the development of a new Codex Alimentarius code of hygienic practice for LMF, we applied a rapid knowledge synthesis and transfer approach to review global research on the burden of illness, prevalence, and interventions to control nine selected microbial hazards in eight categories of LMF. Knowledge synthesis methods included an integrated scoping review (search strategy, relevance screening and confirmation, and evidence mapping), systematic review (detailed data extraction), and meta-analysis of prevalence data. Knowledge transfer of the results was achieved through multiple reporting formats, including evidence summary cards. We identified 214 unique outbreaks and 204 prevalence and 126 intervention studies. Cereals and grains (n = 142) and Salmonella (n = 278) were the most commonly investigated LMF and microbial hazard categories, respectively. Salmonella was implicated in the most outbreaks (n = 96, 45%), several of which were large and widespread, resulting in the most hospitalizations (n = 895, 89%) and deaths (n = 14, 74%). Salmonella had a consistently low prevalence across all LMF categories (0 to 3%), but the prevalence of other hazards (e.g., Bacillus cereus) was highly variable. A variety of interventions were investigated in small challenge trials. Key knowledge gaps included underreporting of LMF outbreaks, limited reporting of microbial levels in prevalence studies, and a lack of intervention efficacy research under commercial conditions. Summary cards were a useful knowledge transfer format to inform complementary risk ranking activities. This review builds upon previous work in this area by synthesizing a broad range of evidence using a structured, transparent, and integrated approach to provide timely evidence informed inputs into international guidelines. PMID:26613924

  10. Polyribosome Formation and Protein Synthesis in Imbibed but Dormant Lettuce Seeds 1

    PubMed Central

    Fountain, David W.; Bewley, J. Derek

    1973-01-01

    Dormancy is maintained in Grand Rapids lettuce (Lactuca sativa) seeds imbibed on water in darkness at 25 C. Polyribosome formation and protein synthesis occur early in the imbibition phase and considerable polysomal material is also present after 24 and 48 hours, even though the seeds have failed to germinate. Incorporation of labeled leucine into protein following a 24-hour preincubation period shows that these polysomes are active in protein synthesis. PMID:16658614

  11. Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser

    SciTech Connect

    Greco, M.; Guida, G.; Perlino, E.; Marra, E.; Quagliariello, E. )

    1989-09-29

    To gain further insight into the mechanism of cell photostimulation by laser light, both RNA and protein synthesis were measured in mitochondria irradiated with the low power continuous wave He-Ne laser (Energy dose: 5 Joules/cm{sup 2}). Following mitochondrial irradiation, both the rate and amount of incorporation of alpha-({sup 32}P)UTP and L-({sup 35}S)methionine, used to monitor RNA and protein synthesis respectively, proved to increase. Electrophoretic analysis made of the synthesis products clearly shows that He-Ne laser irradiation stimulates the synthesis of all mitochondrial transcription and translation products.

  12. Effects of replacing dietary starch with neutral detergent-soluble fibre on ruminal fermentation, microbial synthesis and populations of ruminal cellulolytic bacteria using the rumen simulation technique (RUSITEC).

    PubMed

    Zhao, X H; Liu, C J; Liu, Y; Li, C Y; Yao, J H

    2013-12-01

    A rumen simulation technique (RUSITEC) apparatus with eight 800 ml fermenters was used to investigate the effects of replacing dietary starch with neutral detergent-soluble fibre (NDSF) by inclusion of sugar beet pulp in diets on ruminal fermentation, microbial synthesis and populations of ruminal cellulolytic bacteria. Experimental diets contained 12.7, 16.4, 20.1 or 23.8% NDSF substituted for starch on a dry matter basis. The experiment was conducted over two independent 15-day incubation periods with the last 8 days used for data collection. There was a tendency that 16.4% NDSF in the diet increased the apparent disappearance of organic matter (OM) and neutral detergent fibre (NDF). Increasing dietary NDSF level increased carboxymethylcellulase and xylanase activity in the solid fraction and apparent disappearance of acid detergent fibre (ADF) but reduced the 16S rDNA copy numbers of Ruminococcus albus in both liquid and solid fractions and R. flavefaciens in the solid fraction. The apparent disappearance of dietary nitrogen (N) was reduced by 29.6% with increased dietary NDSF. Substituting NDSF for starch appeared to increase the ratios of acetate/propionate and methane/volatile fatty acids (VFA) (mol/mol). Replacing dietary starch with NDSF reduced the daily production of ammonia-N and increased the growth of the solid-associated microbial pellets (SAM). Total microbial N flow and efficiency of microbial synthesis (EMS), expressed as g microbial N/kg OM fermented, tended to increase with increased dietary NDSF, but the numerical increase did not continue as dietary NDSF exceeded 20.1% of diet DM. Results suggested that substituting NDSF for starch up to 16.4% of diet DM increased digestion of nutrients (except for N) and microbial synthesis, and further increases (from 16.4% to 23.8%) in dietary NDSF did not repress microbial synthesis but did significantly reduce digestion of dietary N.

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

    SciTech Connect

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

    1986-06-01

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

  14. The role of protein synthesis in memory consolidation: Progress amid decades of debate

    PubMed Central

    Hernandez, Pepe J.; Abel, Ted

    2009-01-01

    A major component of consolidation theory holds that protein synthesis is required to produce the synaptic modification needed for long-term memory storage. Protein synthesis inhibitors have played a pivotal role in the development of this theory. However, these commonly used drugs have unintended effects that have prompted some to reevaluate the role of protein synthesis in memory consolidation. Here we review the role of protein synthesis in memory formation as proposed by consolidation theory calling special attention to the controversy involving the non-specific effects of a group of protein synthesis inhibitors commonly used to study memory formation in vivo. We argue that molecular and genetic approaches that were subsequently applied to the problem of memory formation confirm the results of less selective pharmacological studies. Thus, to a certain extent, the debate over the role of protein synthesis in memory based on interpretational difficulties inherent to the use of protein synthesis inhibitors may be somewhat moot. We conclude by presenting avenues of research we believe will best provide answers to both long-standing and more recent questions facing field of learning and memory. PMID:18053752

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

    PubMed

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

    2008-09-01

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

  16. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility.

    PubMed

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

    2016-01-01

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

  17. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-02-01

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

  19. Tacaribe virus Z protein interacts with the L polymerase protein to inhibit viral RNA synthesis.

    PubMed

    Jácamo, Rodrigo; López, Nora; Wilda, Maximiliano; Franze-Fernández, María T

    2003-10-01

    Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. The TV genome encodes four proteins, the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a small RING finger protein (Z). Using a reverse genetic system, we recently demonstrated that TV N and L are sufficient to drive transcription and full-cycle RNA replication mediated by TV-like RNAs and that Z is a powerful inhibitor of these processes (N. López, R. Jácamo, and M. T. Franze-Fernández, J. Virol. 65:12241-12251, 2001). In the present study we investigated whether Z might interact with either of the proteins, N and L, required for RNA synthesis. To that end, we used coimmunoprecipitation with monospecific antibodies against the viral proteins and coimmunoprecipitation with serum against glutathione S-transferase (GST) and binding to glutathione-Sepharose beads when Z was expressed as a fusion protein with GST. We demonstrated that Z interacted with L but not with N and that Z inhibitory activity was dependent on its ability to bind to L. We also evaluated the contribution of different Z regions to its binding ability and functional activity. We found that integrity of the RING structure is essential for Z binding to L and for Z inhibitory activity. Mutants with deletions at the N and C termini of Z showed that amino acids within the C-terminal region and immediately adjacent to the RING domain N terminus contribute to efficient Z-L interaction and are required for inhibitory activity. The data presented here provide the first evidence of an interaction between Z and L, suggesting that Z interferes with viral RNA synthesis by direct interaction with L. In addition, coimmunoprecipitation studies revealed a previously unreported interaction between N and L.

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

    PubMed

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

    2009-06-01

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-09-01

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

  3. Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.

    PubMed

    Kleppe, Rune; Herfindal, Lars; Døskeland, Stein Ove

    2015-10-01

    Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity. PMID:26506362

  4. Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

    SciTech Connect

    Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; Nandhikonda, Premchendar; Smith, Richard D.; Wright, Aaron T.

    2015-09-25

    Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.

  5. Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

    PubMed Central

    Stephens, Raymond E.

    1997-01-01

    When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [3H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state. PMID:9362062

  6. Production of Microbial Biomass Protein from Potato Processing Wastes by Cephalosporium eichhorniae

    PubMed Central

    Stevens, Coleen A.; Gregory, Kenneth F.

    1987-01-01

    The use of Cephalosporium eichhorniae 152 (ATCC 38255) (reclassified as Acremonium alabamense; see Addendum in Proof), a thermophilic, acidophilic, amylolytic fungus, for the conversion of potato processing wastes into microbial protein for use as animal feed was studied. The fungus was not inhibited by α-solanine or β-2-chaconine, antimicrobial compounds in potatoes, or by morpholine or cyclohexylamine (additives to steam used in the peeling process) at levels likely to be encountered in this substrate. Mixed effluent from holding tanks at a potato-processing plant contained about 109 bacteria per ml and inhibited fungal growth. The fungus grew well on fresh potato wastes containing up to 5% total carbohydrate and utilized both starch and protein at 45°C and pH 3.75. On potato homogenate medium containing 2% carbohydrate (about 14% fresh potato) supplemented with monoammonium phosphate (0.506 g/liter) and ferric iron (0.1 g/liter), with pH control (at 3.75) and additional nitrogen supplied by the automatic addition of ammonium hydroxide, typical yields were 0.61 g (dry weight) of product and 0.3 g of crude protein per g of carbohydrate supplied. An aerobic, spore-forming bacterium, related to Bacillus brevis, commonly contaminated nonsterilized batch cultures but was destroyed by heating for 15 min at 100°C. PMID:16347277

  7. ChemCell : a particle-based model of protein chemistry and diffusion in microbial cells.

    SciTech Connect

    Plimpton, Steven James; Slepoy, Alexander

    2003-12-01

    Prokaryotic single-cell microbes are the simplest of all self-sufficient living organisms. Yet microbes create and use much of the molecular machinery present in more complex organisms, and the macro-molecules in microbial cells interact in regulatory, metabolic, and signaling pathways that are prototypical of the reaction networks present in all cells. We have developed a simple simulation model of a prokaryotic cell that treats proteins, protein complexes, and other organic molecules as particles which diffuse via Brownian motion and react with nearby particles in accord with chemical rate equations. The code models protein motion and chemistry within an idealized cellular geometry. It has been used to simulate several simple reaction networks and compared to more idealized models which do not include spatial effects. In this report we describe an initial version of the simulation code that was developed with FY03 funding. We discuss the motivation for the model, highlight its underlying equations, and describe simulations of a 3-stage kinase cascade and a portion of the carbon fixation pathway in the Synechococcus microbe.

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

    SciTech Connect

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

    1986-03-01

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

  9. Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain.

    PubMed

    Costa, D F A; Quigley, S P; Isherwood, P; McLennan, S R; Poppi, D P

    2016-05-01

    A series of 3 experiments were conducted to evaluate the use of microalgae as supplements for ruminants consuming low-CP tropical grasses. In Exp. 1, the chemical composition and in vitro protein degradability of 9 algae species and 4 protein supplements were determined. In Exp. 2, rumen function and microbial protein (MCP) production were determined in steers fed speargrass hay alone or supplemented with , , , or cottonseed meal (CSM). In Exp. 3, DMI and ADG were determined in steers fed speargrass hay alone or supplemented with increasing amounts of NPN (urea combined with ammonia sulfate), CSM, or . In Exp. 1, the CP content of and (675 and 580 g/kg DM) was highest among the algae species and higher than the other protein supplements evaluated, and sp. had the highest crude lipid (CL) content (198 g/kg DM). In Exp. 2, supplementation increased speargrass hay intake, the efficiency of MCP production, the fractional outflow rate of digesta from the rumen, the concentration of NHN, and the molar proportion of branched-chain fatty acids in the rumen fluid of steers above all other treatments. acceptance by steers was low and this resulted in no significant difference to unsupplemented steers for all parameters measured for this algae supplement. In Exp. 3, ADG linearly increased with increasing supplementary N intake from both and NPN, with no difference between the 2 supplements. In contrast, ADG quadratically increased with increasing supplementary N intake from CSM. It was concluded that and may potentially be used as protein sources for cattle grazing low-CP pastures. PMID:27285702

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

    PubMed

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

    2015-02-01

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

  11. Limiting amino acid for protein synthesis with mammary cells in tissue culture.

    PubMed

    Park, C S; Chandler, P T; Norman, A W

    1976-05-01

    To identify the limiting amino acid in the minimal essential medium as published by Eagle (Science 130:432, 1959) for milk protein synthesis in rat mammary cells in tissue culture, two different experimental approaches were used. The first study involved the reduction of amino acids singly from the total amino acid complement of the medium for milk protein synthesis. The second study was to investigate the effect on milk protein synthesis of single amino acid addition to the basic complement of amino acids. Order of limiting amino acids was lysine (first) and possible methionine, valine, or arginine (second).

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

    SciTech Connect

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

    1991-06-01

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

  13. Induction of heat-shock protein synthesis in chondrocytes at physiological temperatures

    SciTech Connect

    Madreperla, S.A.; Louwerenburg, B.; Mann, R.W.; Towle, C.A.; Mankin, H.J.; Treadwell, B.V.

    1985-01-01

    Induction of heat-shock protein (HSP) synthesis is demonstrated in cultured calf-chondrocytes at temperatures shown to occur in normal human cartilage during experiments subjecting intact cadaverous hip joints to the parameters of level walking. A 70,000 MW heat-shock protein (HSP-70) is synthesized by chondrocytes at temperatures above 39 degrees C, while induction of synthesis of a 110,000 MW HSP only occurs at temperatures of 45 degrees C or greater. These differences in critical temperatures for induction, and data showing differences in kinetics of induction and repression of synthesis, suggest that there are differences in the mechanism of induction of the two HSPs. The duration of HSP synthesis and inhibition of synthesis of normal cellular proteins is directly proportional to the duration and magnitude of the temperature rise. Possible relationships between these new findings and the initiation and progression of degenerative joint disease are discussed.

  14. Cell-free synthesis system suitable for disulfide-containing proteins

    SciTech Connect

    Matsuda, Takayoshi; Watanabe, Satoru; Kigawa, Takanori

    2013-02-08

    Highlights: ► Cell-free synthesis system suitable for disulfide-containing proteins is proposed. ► Disulfide bond formation was facilitated by the use of glutathione buffer. ► DsbC catalyzed the efficient shuffling of incorrectly formed disulfide bonds. ► Milligram quantities of functional {sup 15}N-labeled BPTI and lysozyme C were obtained. ► Synthesized proteins were both catalytically functional and properly folded. -- Abstract: Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional {sup 15}N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.

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

    PubMed

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

    2014-11-01

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

  16. De novo protein synthesis in mature platelets: a consideration for transfusion medicine.

    PubMed

    Schubert, P; Devine, D V

    2010-08-01

    Platelet function in thrombosis and haemostasis is reasonably well understood at the molecular level with respect to the proteins involved in cellular structure, signalling networks and platelet interaction with clotting factors and other cells. However, the natural history of these proteins has only recently garnered the attention of platelet researchers. De novo protein synthesis in platelets was discovered 40 years ago; however, it was generally dismissed as merely an interesting minor phenomenon until studies over the past few years renewed interest in this aspect of platelet proteins. It is now accepted that anucleate platelets not only have the potential to synthesize proteins, but this capacity seems to be required to fulfil their function. With translational control as the primary mode of regulation, platelets are able to express biologically relevant gene products in a timely and signal-dependent manner. Platelet protein synthesis during storage of platelet concentrates is a nascent area of research. Protein synthesis does occur, although not for all proteins found in the platelet protein profile. Furthermore, mRNA appears to be well preserved under standard storage conditions. Although its significance is not yet understood, the ability to replace proteins may form a type of cellular repair mechanism during storage. Disruption by inappropriate storage conditions or processes that block protein synthesis such as pathogen reduction technologies may have direct effects on the ability of platelets to synthesize proteins during storage.

  17. Response of rat brain protein synthesis to ethanol and sodium barbital

    SciTech Connect

    Tewari, S.; Greenberg, S.A.; Do, K.; Grey, P.A.

    1987-01-01

    Central nervous system (CNS) depressants such as ethanol and barbiturates under acute or chronic conditions can induce changes in rat brain protein synthesis. While these data demonstrate the individual effects of drugs on protein synthesis, the response of brain protein synthesis to alcohol-drug interactions is not known. The goal of the present study was to determine the individual and combined effects of ethanol and sodium barbital on brain protein synthesis and gain an understanding of the mechanisms by which these alterations in protein synthesis are produced. Specifically, the in vivo and in vitro effects of sodium barbital (one class of barbiturates which is not metabolized by the hepatic tissue) were examined on brain protein synthesis in rats made physically dependent upon ethanol. Using cell free brain polysomal systems isolated from Control, Ethanol and 24 h Ethanol Withdrawn rats, data show that sodium barbital, when intubated intragastrically, inhibited the time dependent incorporation of /sup 14/C) leucine into protein by all three groups of ribosomes. Under these conditions, the Ethanol Withdrawn group displayed the largest inhibition of the /sup 14/C) leucine incorporation into protein when compared to the Control and Ethanol groups. In addition, sodium barbital when added at various concentrations in vitro to the incubation medium inhibited the incorporation of /sup 14/C) leucine into protein by Control and Ethanol polysomes. The inhibitory effects were also obtained following preincubation of ribosomes in the presence of barbital but not cycloheximide. Data suggest that brain protein synthesis, specifically brain polysomes, through interaction with ethanol or barbital are involved in the functional development of tolerance. These interactions may occur through proteins or polypeptide chains or alterations in messenger RNA components associated with the ribosomal units.

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

    ERIC Educational Resources Information Center

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

    2010-01-01

    Memory consolidation and reconsolidation require kinase activation and protein synthesis. Blocking either process during or shortly after training or recall disrupts memory stabilization, which suggests the existence of a critical time window during which these processes are necessary. Using a computational model of kinase synthesis and…

  19. Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells.

    PubMed

    Lee, Myung-Ja; Feliers, Denis; Sataranatarajan, Kavithalakshmi; Mariappan, Meenalakshmi M; Li, Manli; Barnes, Jeffrey L; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

    2010-01-01

    High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.

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

    PubMed Central

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

    2012-01-01

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

  1. BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis

    PubMed Central

    Grespi, Francesca; Soratroi, Claudia; Krumschnabel, Gerhard; Sohm, Benedicte; Ploner, Christian; Geley, Stephan; Hengst, Ludger; Häcker, Georg; Villunger, Andreas

    2010-01-01

    Tight transcriptional regulation, post-translational modifications and/or alternative splicing of BH3-only proteins fine-tune their pro-apoptotic function. Here, we characterize the gene locus of the BH3-only protein Bmf (Bcl-2 modifying factor) and describe the generation of two major isoforms from a common transcript where initiation of protein synthesis involves leucine-coding CUG. BmfCUG and the originally described isoform, Bmf short (BmfS), display comparable binding affinities to pro-survival Bcl-2 family members, localize preferentially to the outer mitochondrial membrane and induce rapid Bcl-2-blockable apoptosis. Notably, endogenous Bmf expression is induced upon forms of cell stress known to cause the repression of the CAP-dependent translation machinery such as serum-deprivation, hypoxia, inhibition of the PI3K/AKT pathway or mTOR, as well as direct pharmacological inhibition of eukaryotic translation initiation factor eIF-4E. Knock-down or deletion of Bmf reduces apoptosis under some of these conditions demonstrating that Bmf can act as a sentinel for the stress-impaired CAP-dependent protein translation machinery (150). PMID:20706276

  2. Oxidation-induced unfolding facilitates Myosin cross-linking in myofibrillar protein by microbial transglutaminase.

    PubMed

    Li, Chunqiang; Xiong, Youling L; Chen, Jie

    2012-08-15

    Myofibrillar protein from pork Longissimus muscle was oxidatively stressed for 2 and 24 h at 4 °C with mixed 10 μM FeCl(3)/100 μM ascorbic acid/1, 5, or 10 mM H(2)O(2) (which produces hydroxyl radicals) and then treated with microbial transglutaminase (MTG) (E:S = 1:20) for 2 h at 4 °C. Oxidation induced significant protein structural changes (P < 0.05) as evidenced by suppressed K-ATPase activity, elevated Ca-ATPase activity, increased carbonyl and disulfide contents, and reduced conformational stability, all in a H(2)O(2) dose-dependent manner. The structural alterations, notably with mild oxidation, led to stronger MTG catalysis. More substantial amine reductions (19.8-27.6%) at 1 mM H(2)O(2) occurred as compared to 11.6% in nonoxidized samples (P < 0.05) after MTG treatment. This coincided with more pronounced losses of myosin in oxidized samples (up to 33.2%) as compared to 21.1% in nonoxidized (P < 0.05), which was attributed to glutamine-lysine cross-linking as suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID:22809283

  3. Catabolism of tritiated thymidine by aquatic microbial communities and incorporation of tritium into RNA and protein

    SciTech Connect

    Brittain, A.M.; Karl, D.M. )

    1990-05-01

    The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. Nonspecific labeling was greatest in sediment samples, for which {>=}95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. We also evaluated the specificity of (2-{sup 3}H) adenine incorporation into adenylate residues in both RNA and DNA in parallel with the ({sup 3}H) thymidine experiments and compared the degree of nonspecific labeling by ({sup 3}H) adenine with that derived from ({sup 3}H)thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.

  4. Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics.

    PubMed

    Corchero, José Luis; Gasser, Brigitte; Resina, David; Smith, Wesley; Parrilli, Ermenegilda; Vázquez, Felícitas; Abasolo, Ibane; Giuliani, Maria; Jäntti, Jussi; Ferrer, Pau; Saloheimo, Markku; Mattanovich, Diethard; Schwartz, Simó; Tutino, Maria Luisa; Villaverde, Antonio

    2013-01-01

    Both conventional and innovative biomedical approaches require cost-effective protein drugs with high therapeutic potency, improved bioavailability, biocompatibility, stability and pharmacokinetics. The growing longevity of the human population, the increasing incidence and prevalence of age-related diseases and the better comprehension of genetic-linked disorders prompt to develop natural and engineered drugs addressed to fulfill emerging therapeutic demands. Conventional microbial systems have been for long time exploited to produce biotherapeutics, competing with animal cells due to easier operation and lower process costs. However, both biological platforms exhibit important drawbacks (mainly associated to intracellular retention of the product, lack of post-translational modifications and conformational stresses), that cannot be overcome through further strain optimization merely due to physiological constraints. The metabolic diversity among microorganisms offers a spectrum of unconventional hosts, that, being able to bypass some of these weaknesses, are under progressive incorporation into production pipelines. In this review we describe the main biological traits and potentials of emerging bacterial, yeast, fungal and microalgae systems, by comparing selected leading species with well established conventional organisms with a long run in protein drug production.

  5. Targeting deubiquitinases enabled by chemical synthesis of proteins.

    PubMed

    Ohayon, Shimrit; Spasser, Liat; Aharoni, Amir; Brik, Ashraf

    2012-02-15

    Ubiquitination/ubiquitylation is involved in a wide range of cellular processes in eukaryotes, such as protein degradation and DNA repair. Ubiquitination is a reversible post-translational modification, with the removal of the ubiquitin (Ub) protein being catalyzed by a family of enzymes known as deubiquitinases (DUBs). Approximately 100 DUBs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. DUBs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets. Several directions have been pursued in the search for lead anti-DUB compounds. However, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay. In this study, we report on the design and preparation of a FRET-based assay for DUBs based on the application of our recent chemical method for the synthesis of Ub bioconjugates. In the assay, the ubiquitinated peptide was specifically labeled with a pair of FRET labels and used to screen a library comprising 1000 compounds against UCH-L3. Such analysis identified a novel and potent inhibitor able to inhibit this DUB in time-dependent manner with k(inact) = 0.065 min(-1) and K(i) = 0.8 μM. Our assay, which was also found suitable for the UCH-L1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of DUBs in health and disease.

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

    SciTech Connect

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

    1988-05-01

    We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of (/sup 3/H)proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis.

  7. Relief memory consolidation requires protein synthesis within the nucleus accumbens.

    PubMed

    Bruning, Johann E A; Breitfeld, Tino; Kahl, Evelyn; Bergado-Acosta, Jorge R; Fendt, Markus

    2016-06-01

    Relief learning refers to the association of a stimulus with the relief from an aversive event. The thus-learned relief stimulus then can induce, e.g., an attenuation of the startle response or approach behavior, indicating positive valence. Previous studies revealed that the nucleus accumbens is essential for the acquisition and retrieval of relief memory. Here, we ask whether the nucleus accumbens is also the brain site for consolidation of relief memory into a long-term form. In rats, we blocked local protein synthesis within the nucleus accumbens by local infusions of anisomycin at different time points during a relief conditioning experiment. Accumbal anisomycin injections immediately after the relief conditioning session, but not 4 h later, prevented the consolidation into long-term relief memory. The retention of already consolidated relief memory was not affected by anisomycin injections. This identifies a time window and site for relief memory consolidation. These findings should complement our understanding of the full range of effects of adverse experiences, including cases of their distortion in humans such as post-traumatic stress disorder and/or phobias. PMID:26792192

  8. Protein kinase D activity controls endothelial nitric oxide synthesis.

    PubMed

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. PMID:24928905

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

    PubMed Central

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

    2014-01-01

    Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2Akita diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy. PMID:24740573

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

    PubMed

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

    2011-01-01

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

  11. In vitro synthesis of ribosomal proteins directed by Escherichia coli DNA.

    PubMed

    Kaltschmidt, E; Kahan, L; Nomura, M

    1974-02-01

    In vitro synthesis of a number of E. coli 30S ribosomal proteins has been demonstrated in a cell-free system consisting of ribosomes, initiation factors, RNA polymerase, a fraction containing soluble enzymes and factors, and E. coli DNA. DNA-dependent synthesis of the following 30S proteins has been demonstrated: S4, S5, S7, S8, S9, S10, S13, S14, S16, S19, and S20.

  12. Isolation and Characterization of a Protein That Stimulates DNA Synthesis from Avian Myeloblastosis Virus*

    PubMed Central

    Leis, Jonathan P.; Hurwitz, Jerard

    1972-01-01

    A protein has been isolated from avian myeloblastosis virus that stimulates the rate and yield of DNA synthesis primed by viral RNA with purified viral polymerase. It specifically affects the viral polymerase and does not stimulate other DNA polymerases under the conditions tested. The viral polymerase, in conjunction with this protein, transcribes extended single-stranded regions of DNA, and permits the enzyme to initiate synthesis from single-strand breaks in DNA. PMID:4340754

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

    PubMed Central

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

    2011-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1978-01-01

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

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

    PubMed

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

    2011-01-01

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

  16. Protein synthesis of muscle fractions from the small intestine in alcohol fed rats.

    PubMed Central

    Preedy, V R; Peters, T J

    1990-01-01

    The effects of chronic ethanol feeding on the amounts and synthesis rates of cytoplasmic, contractile, and stromal protein fractions were investigated in the small intestine of eight pairs of immature and seven pairs of mature rats. Treated rats were fed ethanol as 36% of total energy in a nutritionally adequate liquid diet. Paired controls were fed isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose. After six weeks the total cytoplasmic and contractile protein content in immature rats was reduced by 18% and 31%, respectively (p less than or equal to 0.007). The decline in the stromal protein content (26%) was not statistically significant (p = 0.130). In mature rats the protein contents were also reduced in the cytoplasmic (25%, p = 0.035) and contractile (27%, p = 0.005) protein fractions, though the stromal protein fraction was unaltered (p = 0.913). In immature rats fractional rates of protein synthesis in cytoplasmic and contractile protein fractions of the small intestine were unaltered by chronic ethanol feeding (p less than or equal to 0.853). In mature rats, the synthesis rates of corresponding fractions declined, by 18% and 31%, respectively, but were also not statistically significant (p less than or equal to 0.369). Absolute rates of protein synthesis in immature rats fell by 6% (p = 0.549) in the cytoplasmic and 31% in the contractile protein fraction (p = 0.045). In mature rats, the corresponding reductions were 38% (p = 0.106) and 48% (p = 0.033), respectively. Virtually no radioactivity could be detected in the stromal fraction, signifying very low synthesis rates. Chronic ethanol feeding reduces the amount of protein in the small intestine of the immature and mature rat with the contractile protein fraction showing the greatest decrease. In the absence of statistically significant reductions in fractional synthesis rates a partial adaptation in turnover rates may have occurred. PMID:2323594

  17. Imidazolium tagged acridines: Synthesis, characterization and applications in DNA binding and anti-microbial activities

    NASA Astrophysics Data System (ADS)

    Raju, Gembali; Vishwanath, S.; Prasad, Archana; Patel, Basant K.; Prabusankar, Ganesan

    2016-03-01

    New water soluble 4,5-bis imidazolium tagged acridines have been synthesized and structurally characterized by multinuclear NMR and single crystal X-ray diffraction techniques. The DNA binding and anti-microbial activities of these acridine derivatives were investigated by fluorescence and far-UV circular dichroism studies.

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

    PubMed

    Chong, Shaorong

    2014-10-01

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

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

    PubMed

    Suryawan, Agus; Davis, Teresa A

    2011-01-01

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

  20. Synthesis of Silver Nanoparticles from Microbial Source-A Green Synthesis Approach, and Evaluation of its Antimicrobial Activity against Escherichia coli

    NASA Astrophysics Data System (ADS)

    Behera, S. S.; Jha, S.; Arakha, M.; Panigrahi, T. K.

    2012-03-01

    TRACT Nanoparticles synthesis by biological methods using various microorganisms, plants, and plant extracts and enzymes have attracted a great attention as these are cost effective, nontoxic, eco-friendly and an alternative to physical and chemical methods. In this research, Silver nanoparticles (Ag-NPs) were synthesized from AgNO3 solution by green synthesis process with the assistance of microbial source only. The detailed characterization of the Ag NPs were carried out using UV-visible spectroscopy, Scanning electron microscopy (SEM), Energy dispersive X-ray Spectroscopy (EDS), Dynamic light scattering (DLS) analysis, and their antimicrobial evaluation was done against Escherichia coli. The UV-visible spectroscopy analysis showed the surface plasmon resonance property of nanoparticles. The DLS analysis showed the particle distribution of synthesized silver nanoparticles in solution, and SEM analysis showed the morphology of nanoparticles. The elemental composition of synthesized sample was confirmed by EDS analysis. Antibacterial assay of synthesized Ag NP was carried out in solid (Nutrient Agar) growth medium against E.coli. The presence of zone of inhibition clearly indicated the antibacterial activity of silver nanoparticles.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

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

  3. Protein synthesis in imaginal disks of Plodia interpunctella during development in vivo and in vitro.

    PubMed

    Oberlander, H; Leach, C E

    1978-08-01

    Wing imaginal disks were dissected from larvae of Plodia interpunctella (Hübner) at various stages during the larval-pupal transformation. The wing-disk proteins separated by electrophoresis and scanned with a densitometer changed quantitatively but not qualitatively during development in vivo. Treatment of wing disks in vitro with beta-ecdysone resulted in a 2-fold increase in synthesis of proteins after only 2 hr incubation. The maximum rate of protein synthesis was reached 16 hr after treatment with hormone. The pattern of proteins separated by electrophoresis of wing disks that were incubated in vitro with beta-ecdysone did not change qualitatively. The major features of protein synthesis during wing-disk development in vivo were similar to those observed during beta-ecdysone-induced development in vitro.

  4. Muscle protein synthesis in response to testosterone administration in wether lambs.

    PubMed

    Lobley, G E; Connell, A; Milne, E; Buchan, V; Calder, A G; Anderson, S E; Vint, H

    1990-11-01

    A method has been developed based on stable isotopes and biopsy procedures which allows the large-dose procedure for measurement of protein synthesis to be applied in serial studies to farm species. Measurements of total nitrogen retention and protein synthesis in m. longissimus dorsi and m. vastus lateralis were made in five wether lambs (40-44 kg) infused intravenously, successively, with vehicle (10 d); testosterone (15 d; 9 mg/d); vehicle (15 d). N retention was improved by testosterone infusion (+2.9 g N/d; a 96% improvement total over control periods). Muscle protein synthesis was not significantly altered by exogenous hormone administration, nor were RNA:protein, RNA:DNA or protein:DNA. The implication of the developed procedure for dynamic studies in accessible tissues of large animals is discussed.

  5. Flexible Programming of Cell-Free Protein Synthesis Using Magnetic Bead-Immobilized Plasmids

    PubMed Central

    Lee, Ka-Young; Lee, Kyung-Ho; Park, Ji-Woong; Kim, Dong-Myung

    2012-01-01

    The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis. PMID:22470570

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

    PubMed

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

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

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

    PubMed Central

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

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

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

    PubMed

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

    2016-01-01

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

  9. Cereal seed storage protein synthesis: fundamental processes for recombinant protein production in cereal grains.

    PubMed

    Kawakatsu, Taiji; Takaiwa, Fumio

    2010-12-01

    Cereal seeds provide an ideal production platform for high-value products such as pharmaceuticals and industrial materials because seeds have ample and stable space for the deposition of recombinant products without loss of activity at room. Seed storage proteins (SSPs) are predominantly synthesized and stably accumulated in maturing endosperm tissue. Therefore, understanding the molecular mechanisms regulating SSP expression and accumulation is expected to provide valuable information for producing higher amounts of recombinant products. SSP levels are regulated by several steps at the transcriptional (promoters, transcription factors), translational and post-translational levels (modification, processing trafficking, and deposition). Our objective is to develop a seed production platform capable of producing very high yields of recombinant product. Towards this goal, we review here the individual regulatory steps controlling SSP synthesis and accumulation.

  10. [Effects of organic fish protein liquid fertilizer on enzyme activities and microbial biomass C and N in a silt soil].

    PubMed

    Wei, Xiu-Li; Lei, Ping; Shi, Wei-Yong

    2010-08-01

    By the method of thermostatic culture, this paper studied the effects of different application rates (0.5, 1.5, and 2.5 ml x kg(-1)) of organic fish protein liquid fertilizer on the enzyme activities and microbial biomass C and N in a silt soil, and the relationships between these parameters and soil nutrient contents. Under the application of the liquid fertilizer, soil pH varied in the range of 7.07-7.31, but had no significant difference from the control. With the increasing application rate of the liquid fertilizer, the activities of soil phosphatase, urease, and protease, as well as the soil biomass C and N, all increased significantly, and the increment was 127, 190 and 196%, 39.81, 78.06 and 173.24%, 56.37, 108.29 and 199.98%, 167, 395 and 474%, and 121, 243 and 406%, respectively, compared with the control. The peak time of the soil urease and protease activities and microbial biomass C and N differed with the fertilization treatments. Soil phosphase, urease, and protease activities and microbial biomass C and N were significantly positively correlated with soil nutrient contents, suggesting that applying organic fish protein liquid fertilizer to silt soil could improve soil microbial growth and enzyme activities, and accordingly, promote the decomposition and transformation of soil organic matter and the release of soil available nutrient elements.

  11. Effects of oral meal feeding on whole body protein breakdown and protein synthesis in cachectic pancreatic cancer patients

    PubMed Central

    van Dijk, David PJ; van de Poll, Marcel CG; Moses, Alastair GW; Preston, Thomas; Olde Damink, Steven WM; Rensen, Sander S; Deutz, Nicolaas EP; Soeters, Peter B; Ross, James A; Fearon, Kenneth CH; Dejong, Cornelis HC

    2015-01-01

    Background Pancreatic cancer is often accompanied by cachexia, a syndrome of severe weight loss and muscle wasting. A suboptimal response to nutritional support may further aggravate cachexia, yet the influence of nutrition on protein kinetics in cachectic patients is poorly understood. Methods Eight cachectic pancreatic cancer patients and seven control patients received a primed continuous intravenous infusion of l-[ring-2H5]phenylalanine and l-[3,3-2H2]tyrosine for 8 h and ingested sips of water with l-[1-13C]phenylalanine every 30 min. After 4 h, oral feeding was started. Whole body protein breakdown, protein synthesis, and net protein balance were calculated. Results are given as median with interquartile range. Results Baseline protein breakdown and protein synthesis were higher in cachectic patients compared with the controls (breakdown: 67.1 (48.1–79.6) vs. 45.8 (42.6–46.3) µmol/kg lean body mass/h, P = 0.049; and synthesis: 63.0 (44.3–75.6) vs. 41.8 (37.6–42.5) µmol/kg lean body mass/h, P = 0.021). During feeding, protein breakdown decreased significantly to 45.5 (26.9–51.1) µmol/kg lean body mass/h (P = 0.012) in the cachexia group and to 33.7 (17.4–37.1) µmol/kg lean body mass/h (P = 0.018) in the control group. Protein synthesis was not affected by feeding in cachectic patients: 58.4 (46.5–76.1) µmol/kg lean body mass/h, but was stimulated in controls: 47.9 (41.8–56.7) µmol/kg lean body mass/h (P = 0.018). Both groups showed a comparable positive net protein balance during feeding: cachexia: 19.7 (13.1–23.7) and control: 16.3 (13.6–25.4) µmol/kg lean body mass/h (P = 0.908). Conclusion Cachectic pancreatic cancer patients have a higher basal protein turnover. Both cachectic patients and controls show a comparable protein anabolism during feeding, albeit through a different pattern of protein kinetics. In cachectic patients, this is primarily related to reduced protein breakdown, whereas in controls, both protein breakdown and

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

    PubMed Central

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

    1979-01-01

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

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

    SciTech Connect

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

    1988-08-01

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

  14. Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

    PubMed Central

    Higgins, S J; Burchell, J M; Mainwaring, W I

    1976-01-01

    1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug. Images PLATE 1(a) PLATES 1(b), 1(c) AND 1(d) PLATE 2 PMID:985427

  15. Epitopes of Microbial and Human Heat Shock Protein 60 and Their Recognition in Myalgic Encephalomyelitis

    PubMed Central

    Elfaitouri, Amal; Herrmann, Björn; Bölin-Wiener, Agnes; Wang, Yilin; Gottfries, Carl-Gerhard; Zachrisson, Olof; Pipkorn, Rϋdiger; Rönnblom, Lars; Blomberg, Jonas

    2013-01-01

    Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001). IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity. PMID:24312270

  16. Epitopes of microbial and human heat shock protein 60 and their recognition in myalgic encephalomyelitis.

    PubMed

    Elfaitouri, Amal; Herrmann, Björn; Bölin-Wiener, Agnes; Wang, Yilin; Gottfries, Carl-Gerhard; Zachrisson, Olof; Pipkorn, Rϋdiger; Rönnblom, Lars; Blomberg, Jonas

    2013-01-01

    Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001). IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity.

  17. Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

    DOE PAGESBeta

    Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; Nandhikonda, Premchendar; Smith, Richard D.; Wright, Aaron T.

    2015-09-25

    Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosicmore » bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.« less

  18. Soy protein hydrolysis with microbial protease to improve antioxidant and functional properties.

    PubMed

    de Oliveira, Cibele Freitas; Corrêa, Ana Paula Folmer; Coletto, Douglas; Daroit, Daniel Joner; Cladera-Olivera, Florencia; Brandelli, Adriano

    2015-05-01

    Soybean proteins are widely used as nutritional and functional food ingredients. This investigation evaluated through a 2(3) central composite design the effect of three variables (pH, temperature and enzyme/substrate (E/S) ratio) on the production of soy protein isolate (SPI) hydrolysates with a microbial protease. Soluble peptides, antioxidant activity, and foaming and emulsifying capabilities of the hydrolysates were analyzed. All variables, as well as their interactions, were significant for the soluble peptides content of SPI hydrolysates. Optimal conditions for obtaining soluble peptides were around 30-35 °C, pH 6.5-9.5, and E/S ratios of 1,650-6,300 U g(-1). SPI hydrolysates produced at 30-45 °C, pH 8.0-9.5, and E/S ratios of 4,000-8,000 U g(-1) showed higher capacity to scavenge the 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical. Models for soluble peptides and ABTS activity of hydrolysates were obtained. In the range studied, the variables had not significant influence on the ability of hydrolysates to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. SPI hydrolysates also presented reducing power and ability to chelate iron. Hydrolysis temperature was significant for the Fe(2+)-chelating ability of hydrolysates. Temperature of hydrolysis was significant for the foaming capacity of hydrolysates, with higher values observed at 45 °C and 8,000 U g(-1). For emulsifying capacity, only E/S ratio presented a significant effect. Temperature and E/S ratio appeared to be more significant variables influencing the properties of the SPI hydrolysates. The results of this study indicate that specific hydrolysis conditions should be selected to obtain SPI hydrolysates with preferred characteristics.

  19. Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community.

    PubMed

    Jeans, Chris; Singer, Steven W; Chan, Clara S; Verberkmoes, Nathan C; Shah, Manesh; Hettich, Robert L; Banfield, Jillian F; Thelen, Michael P

    2008-05-01

    Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5-1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt(572), which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt(572). It readily oxidizes Fe(2+) in the physiologically relevant acidic regime, from pH 0.95-3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt(572). By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt(572). MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe(2+) oxidation activity at very low pH, we propose that Cyt(572) provides a critical function for fitness within the ecological niche of these acidophilic microbial communities.

  20. [Progress of cell-free protein synthesis system and its applications in pharmaceutical engineering - A review].

    PubMed

    Jia, Xiaoge; Deng, Zixin; Liu, Tiangang

    2016-03-01

    Cell-free protein synthesis (CFPS) systems have been widely used for decades as a rapid and efficient tool in fundamental biology. Without the requirements for cell viability and growth, CFPS systems have distinct advantages over in vivo systems for protein production. Recently, great efforts have been made to further optimize CFPS systems to produce proteins at high yields, reduced cost and increased scale, including simplifying extract preparation procedures, developing new energy regeneration systems to protein synthesis, stabilizing substrate supply and promoting protein folding. Nowadays, CFPS systems are emerging as a powerful platform for industrial and high-throughput production of protein therapeutics, providing an alternative solution to solve problems in biopharmaceutical engineering. Moreover, CFPS systems have been successfully applied to high-throughput drug screening, large-scale protein therapeutics production, custom-made anti-cancer vaccines. These achievements highlight that CFPS systems have great potential for a wide range of applications in biopharmaceutical engineering in the future. PMID:27382794

  1. [Progress of cell-free protein synthesis system and its applications in pharmaceutical engineering - A review].

    PubMed

    Jia, Xiaoge; Deng, Zixin; Liu, Tiangang

    2016-03-01

    Cell-free protein synthesis (CFPS) systems have been widely used for decades as a rapid and efficient tool in fundamental biology. Without the requirements for cell viability and growth, CFPS systems have distinct advantages over in vivo systems for protein production. Recently, great efforts have been made to further optimize CFPS systems to produce proteins at high yields, reduced cost and increased scale, including simplifying extract preparation procedures, developing new energy regeneration systems to protein synthesis, stabilizing substrate supply and promoting protein folding. Nowadays, CFPS systems are emerging as a powerful platform for industrial and high-throughput production of protein therapeutics, providing an alternative solution to solve problems in biopharmaceutical engineering. Moreover, CFPS systems have been successfully applied to high-throughput drug screening, large-scale protein therapeutics production, custom-made anti-cancer vaccines. These achievements highlight that CFPS systems have great potential for a wide range of applications in biopharmaceutical engineering in the future.

  2. Dietary crude protein intake influences rates of whole-body protein synthesis in weanling horses.

    PubMed

    Tanner, S L; Wagner, A L; Digianantonio, R N; Harris, P A; Sylvester, J T; Urschel, K L

    2014-11-01

    The objective of this study was to measure whole-body protein kinetics in weanling horses receiving forage and one of two different concentrates: (1) commercial crude protein (CCP) concentrate, which with the forage provided 4.1 g CP/kg bodyweight (BW)/day (189 mg lysine (Lys)/kg BW/day), and (2) recommended crude protein (RCP) concentrate which, with the same forage, provided 3.1 g CP/kg BW/day (194 mg Lys/kg BW/day). Blood samples were taken to determine the response of plasma amino acid concentrations to half the daily concentrate allocation. The next day, a 2 h-primed, constant infusion of [(13)C]sodium bicarbonate and a 4 h-primed, constant infusion of [1-(13)C]phenylalanine were used with breath and blood sampling to measure breath (13)CO2 and blood [(13)C]phenylalanine enrichment. Horses on the CCP diet showed an increase from baseline in plasma isoleucine, leucine, lysine, threonine, valine, alanine, arginine, asparagine, glutamine, ornithine, proline, serine, and tyrosine at 120 min post-feeding. Baseline plasma amino acid concentrations were greater with the CCP diet for histidine, isoleucine, leucine, threonine, valine, asparagine, proline, and serine. Phenylalanine, lysine, and methionine were greater in the plasma of horses receiving the RCP treatment at 0 and 120 min. Phenylalanine intake was standardized between groups; however, horses receiving the RCP diet had greater rates of phenylalanine oxidation (P = 0.02) and lower rates of non-oxidative phenylalanine disposal (P = 0.04). Lower whole-body protein synthesis indicates a limiting amino acid in the RCP diet. PMID:24973006

  3. Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture

    PubMed Central

    1978-01-01

    A simple model system is described for studying synthesis of plasma proteins. The system is based on chick embryo hepatocytes in primary monolayer culture which synthesize a broad spectrum of plasma proteins and secrete them into the culture medium. The secreted proteins are stable and consist almost exclusively of plasma proteins. The cultured cells are nonproliferating hepatic parenchymal cells whose cell mass remains constant in culture. By a modification of Laurell's rocket immunoelectrophoresis, the secreted plasma proteins can be detected in nanogram amounts in 3 microliter of unconcentrated culture medium. Kinetics of secretion are obtained by sequential assay of proteins accumulating in the medium. In this system it is demonstrated that: (a) intracellular plasma protein levels are equivalent to less than 5% of the daily secretion; (b) synthesis and secretion are continuous; and (c) the overall half-time for plasma protein movement along the secretory pathway is less than 10 min. From these results, it follows that the rate at which the plasma proteins are secreted gives a valid estimate of their rate of synthesis. This feature of the culture and the sensitivity of the assay allow routine measurements of plasma protein synthesis without disruption of the cells and without the use of radioisotopes. It is shown, furthermore, that the overall rate of plasma protein synthesis in cultured hepatocytes is constant over a 3- day period and is similar to that of the intact liver. 3,000,000 cells, containing 1 mg cell protein, synthesize 0.2 mg of plasma proteins daily, amounting to one-fifth of hepatocellular protein synthesis. Under the conditions used, albumin synthesis steadily decreases with culture time whereas the synthesis of many other plasma proteins increases. The observed phenotypic changes and reorganization of plasma protein synthesis illustrate how the system may be exploited for studying the regulatory processes governing plasma protein synthesis. PMID

  4. In situ expression of eukaryotic ice-binding proteins in microbial communities of Arctic and Antarctic sea ice

    PubMed Central

    Uhlig, Christiane; Kilpert, Fabian; Frickenhaus, Stephan; Kegel, Jessica U; Krell, Andreas; Mock, Thomas; Valentin, Klaus; Beszteri, Bánk

    2015-01-01

    Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice. PMID:25885562

  5. In situ expression of eukaryotic ice-binding proteins in microbial communities of Arctic and Antarctic sea ice.

    PubMed

    Uhlig, Christiane; Kilpert, Fabian; Frickenhaus, Stephan; Kegel, Jessica U; Krell, Andreas; Mock, Thomas; Valentin, Klaus; Beszteri, Bánk

    2015-11-01

    Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

    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 /sup 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 /sup 32/P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships.

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

    SciTech Connect

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

    2010-06-15

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

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

    SciTech Connect

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

    1986-12-01

    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 ..cap alpha..-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 ..cap alpha..-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.

  10. Complete genome sequence of Klebsiella pneumoniae J1, a protein-based microbial flocculant-producing bacterium.

    PubMed

    Pang, Changlong; Li, Ang; Cui, Di; Yang, Jixian; Ma, Fang; Guo, Haijuan

    2016-02-20

    Klebsiella pneumoniae J1 is a Gram-negative strain, which belongs to a protein-based microbial flocculant-producing bacterium. However, little genetic information is known about this species. Here we carried out a whole-genome sequence analysis of this strain and report the complete genome sequence of this organism and its genetic basis for carbohydrate metabolism, capsule biosynthesis and transport system. PMID:26806487

  11. Triennial growth symposium: Leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial increases in AA and insulin independently stimulate protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to AA. We have shown that the postprandial increase in leucine, but not isoleucine or valine, acutely stimulates muscle protein synth...

  12. Enteral B-hydroxy-B-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite B-hydr...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infants unable to maintain oral feeding can be nourished by orogastric tube. We have shown that orogastric continuous feeding restricts muscle protein synthesis compared with intermittent bolus feeding in neonatal pigs. To determine whether leucine leu infusion can be used to enhance protein synthes...

  15. Heat-induced Accumulation of Chloroplast Protein Synthesis Elongation Factor, EF-TU, in Winter Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays L.). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum L.). In this ...

  16. Insulin and amino acids stimulate whole body protein synthesis in neonates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin and amino acids (AA) stimulate muscle protein synthesis in neonatal pigs. To determine the effects of insulin and AA on whole body protein turnover, hyperinsulinemic (0 and 100 ng/(kg[0.66]/min))-euglycemic-AA clamps were performed during euaminoacidemia or hyperaminoacidemia in fasted 7-d-...

  17. Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

    PubMed Central

    Rawat, A K

    1976-01-01

    Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'. PMID:1016246

  18. PROTEIN SYNTHESIS IN THE VISUAL CELLS OF THE HONEYBEE DRONE AS STUDIED WITH ELECTRON MICROSCOPE RADIOAUTOGRAPHY

    PubMed Central

    Perrelet, Alain

    1972-01-01

    Protein synthesis was studied in the visual cells of an insect (honeybee drone, Apis mellifera) by electron microscope radioautography. After a single injection of tritiated leucine, the radioactivity first appears in the cytoplasm of the visual cell which contains ribosomes. Later, part of this radioactivity migrates to the rhabdome, the visual cell region which is specialized in light absorption. A maximal concentration of radioactivity is reached there 48 hr after the injection of leucine. This pattern of protein synthesis and transport resembles that described in vertebrate visual cells (rods and cones), where newly synthesized proteins have been shown to contribute to the renewal of the photoreceptor membrane. PMID:4656703

  19. Protein synthesis in the visual cells of the honeybee drone as studied with electron microscope radioautography.

    PubMed

    Perrelet, A

    1972-12-01

    Protein synthesis was studied in the visual cells of an insect (honeybee drone, Apis mellifera) by electron microscope radioautography. After a single injection of tritiated leucine, the radioactivity first appears in the cytoplasm of the visual cell which contains ribosomes. Later, part of this radioactivity migrates to the rhabdome, the visual cell region which is specialized in light absorption. A maximal concentration of radioactivity is reached there 48 hr after the injection of leucine. This pattern of protein synthesis and transport resembles that described in vertebrate visual cells (rods and cones), where newly synthesized proteins have been shown to contribute to the renewal of the photoreceptor membrane.

  20. MINLP models for the synthesis of optimal peptide tags and downstream protein processing.

    PubMed

    Simeonidis, Evangelos; Pinto, Jose M; Lienqueo, M Elena; Tsoka, Sophia; Papageorgiou, Lazaros G

    2005-01-01

    The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis. PMID:15932268

  1. Impact of high-pressure processing on microbial shelf-life and protein stability of refrigerated soymilk.

    PubMed

    Smith, K; Mendonca, A; Jung, S

    2009-12-01

    The effects of pressure (400, 500 and 600 MPa), dwell time (1 and 5 min) and temperature (25 and 75 degrees C) on microbial quality and protein stability of soymilk during 28 days of storage (4 degrees C) were evaluated under aerobic and anaerobic conditions. After processing and during storage, there were significant differences in total bacterial count (TBC), numbers of psychrotrophs (PSY) and Enterobacteriaceae (ENT), and protein stability between untreated (control) and pressurized samples (P < 0.05). Pressure applied at an initial temperature of 75 degrees C resulted in a greater suppression in growth of PSY compared to TBC. No ENT was detected in pressurized samples throughout the storage period tested. Dwell time had no significant effect on log reduction of TBC at 25 or 75 degrees C (P > 0.05). Pressure at 400 MPa (5 min), 500 and 600 MPa (1 and 5 min) produced 100% sub-lethal injury in surviving bacterial populations irrespective of temperature. After 28 days of refrigerated storage, both aerobic and anaerobic pressurized samples had better or similar stability as the control on day one of storage. Soymilk control samples were spoiled after 7 days whereas pressurization increased soymilk shelf-life by at least 2 weeks. Pressure (600 MPa) at 75 degrees C for 1 min not only significantly reduced initial microbial populations and increased the microbial shelf-life but also extended the protein stability of soymilk (P < 0.05).

  2. Needles in the blue sea: sub-species specificity in targeted protein biomarker analyses within the vast oceanic microbial metaproteome.

    PubMed

    Saito, Mak A; Dorsk, Alexander; Post, Anton F; McIlvin, Matthew R; Rappé, Michael S; DiTullio, Giacomo R; Moran, Dawn M

    2015-10-01

    Proteomics has great potential for studies of marine microbial biogeochemistry, yet high microbial diversity in many locales presents us with unique challenges. We addressed this challenge with a targeted metaproteomics workflow for NtcA and P-II, two nitrogen regulatory proteins, and demonstrated its application for cyanobacterial taxa within microbial samples from the Central Pacific Ocean. Using METATRYP, an open-source Python toolkit, we examined the number of shared (redundant) tryptic peptides in representative marine microbes, with the number of tryptic peptides shared between different species typically being 1% or less. The related cyanobacteria Prochlorococcus and Synechococcus shared an average of 4.8 ± 1.9% of their tryptic peptides, while shared intraspecies peptides were higher, 13 ± 15% shared peptides between 12 Prochlorococcus genomes. An NtcA peptide was found to target multiple cyanobacteria species, whereas a P-II peptide showed specificity to the high-light Prochlorococcus ecotype. Distributions of NtcA and P-II in the Central Pacific Ocean were similar except at the Equator likely due to differential nitrogen stress responses between Prochlorococcus and Synechococcus. The number of unique tryptic peptides coded for within three combined oceanic microbial metagenomes was estimated to be ∼4 × 10(7) , 1000-fold larger than an individual microbial proteome and 27-fold larger than the human proteome, yet still 20 orders of magnitude lower than the peptide diversity possible in all protein space, implying that peptide mapping algorithms should be able to withstand the added level of complexity in metaproteomic samples.

  3. Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas

    PubMed Central

    1982-01-01

    We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta- tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools. PMID:7118994

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

  5. Microbial synthesis of poly-γ-glutamic acid: current progress, challenges, and future perspectives.

    PubMed

    Luo, Zhiting; Guo, Yuan; Liu, Jidong; Qiu, Hua; Zhao, Mouming; Zou, Wei; Li, Shubo

    2016-01-01

    Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made from repeating units of l-glutamic acid, d-glutamic acid, or both. Since some bacteria are capable of vigorous γ-PGA biosynthesis from renewable biomass, γ-PGA is considered a promising bio-based chemical and is already widely used in the food, medical, and wastewater industries due to its biodegradable, non-toxic, and non-immunogenic properties. In this review, we consider the properties, biosynthetic pathway, production strategies, and applications of γ-PGA. Microbial biosynthesis of γ-PGA and the molecular mechanisms regulating production are covered in particular detail. Genetic engineering and optimization of the growth medium, process control, and downstream processing have proved to be effective strategies for lowering the cost of production, as well as manipulating the molecular mass and conformational/enantiomeric properties that facilitate screening of competitive γ-PGA producers. Finally, future prospects of microbial γ-PGA production are discussed in light of recent progress, challenges, and trends in this field. PMID:27366207

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

    PubMed

    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. PMID:27314672

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

    PubMed Central

    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. PMID:27314672

  8. Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community

    SciTech Connect

    Verberkmoes, Nathan C; Singer, Steven; Shah, Manesh B; Thelen, Michael P.; Hettich, Robert {Bob} L; Banfield, Jillian F.

    2008-01-01

    We have discovered and characterized a novel membrane cytochrome of an iron oxidizing microbial biofilm obtained from the surface of extremely acidic mine water. This protein was initially identified through proteogenomic analysis as one of many novel gene products of Leptospirillum group II, the dominant bacterium of this community (Ram et al, 2005, Science 308, 1915-20). Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. Covalently bound to heme, the purified cytochrome has a unique spectral signature at 572 nm and is thus called Cyt572. It readily oxidizes Fe2+ even in the presence of Fe3+ over a pH range from 0.95 to 3.4. Independent experiments involving 2D blue-native polyacrylamide gel electrophoresis and chemical crosslinking establish a homotetrameric structure for Cyt572. Also, circular dichroism spectroscopy indicates that the protein is largely beta-stranded, consistent with an outer membrane location. Although no significant sequence homology to the full-length cytochrome is detected in protein databases, environmental DNA sequences from both Leptospirillum groups II and III reveal at least 17 strain variants of Cyt572. Due to its abundance, cellular location and Fe2+ oxidation activity, we propose Cyt572 is the iron oxidase of the Leptospirillum bacteria, providing a critical function for fitness within the ecological niche of this acidophilic microbial community.

  9. Autotrophic nitrogen assimilation and carbon capture for microbial protein production by a novel enrichment of hydrogen-oxidizing bacteria.

    PubMed

    Matassa, Silvio; Verstraete, Willy; Pikaar, Ilje; Boon, Nico

    2016-09-15

    Domestic used water treatment systems are currently predominantly based on conventional resource inefficient treatment processes. While resource recovery is gaining momentum it lacks high value end-products which can be efficiently marketed. Microbial protein production offers a valid and promising alternative by upgrading low value recovered resources into high quality feed and also food. In the present study, we evaluated the potential of hydrogen-oxidizing bacteria to upgrade ammonium and carbon dioxide under autotrophic growth conditions. The enrichment of a generic microbial community and the implementation of different culture conditions (sequenced batch resp. continuous reactor) revealed surprising features. At low selection pressure (i.e. under sequenced batch culture at high solid retention time), a very diverse microbiome with an important presence of predatory Bdellovibrio spp. was observed. The microbial culture which evolved under high rate selection pressure (i.e. dilution rate D = 0.1 h(-1)) under continuous reactor conditions was dominated by Sulfuricurvum spp. and a highly stable and efficient process in terms of N and C uptake, biomass yield and volumetric productivity was attained. Under continuous culture conditions the maximum yield obtained was 0.29 g cell dry weight per gram chemical oxygen demand equivalent of hydrogen, whereas the maximum volumetric loading rate peaked 0.41 g cell dry weight per litre per hour at a protein content of 71%. Finally, the microbial protein produced was of high nutritive quality in terms of essential amino acids content and can be a suitable substitute for conventional feed sources such as fishmeal or soybean meal. PMID:27262118

  10. Autotrophic nitrogen assimilation and carbon capture for microbial protein production by a novel enrichment of hydrogen-oxidizing bacteria.

    PubMed

    Matassa, Silvio; Verstraete, Willy; Pikaar, Ilje; Boon, Nico

    2016-09-15

    Domestic used water treatment systems are currently predominantly based on conventional resource inefficient treatment processes. While resource recovery is gaining momentum it lacks high value end-products which can be efficiently marketed. Microbial protein production offers a valid and promising alternative by upgrading low value recovered resources into high quality feed and also food. In the present study, we evaluated the potential of hydrogen-oxidizing bacteria to upgrade ammonium and carbon dioxide under autotrophic growth conditions. The enrichment of a generic microbial community and the implementation of different culture conditions (sequenced batch resp. continuous reactor) revealed surprising features. At low selection pressure (i.e. under sequenced batch culture at high solid retention time), a very diverse microbiome with an important presence of predatory Bdellovibrio spp. was observed. The microbial culture which evolved under high rate selection pressure (i.e. dilution rate D = 0.1 h(-1)) under continuous reactor conditions was dominated by Sulfuricurvum spp. and a highly stable and efficient process in terms of N and C uptake, biomass yield and volumetric productivity was attained. Under continuous culture conditions the maximum yield obtained was 0.29 g cell dry weight per gram chemical oxygen demand equivalent of hydrogen, whereas the maximum volumetric loading rate peaked 0.41 g cell dry weight per litre per hour at a protein content of 71%. Finally, the microbial protein produced was of high nutritive quality in terms of essential amino acids content and can be a suitable substitute for conventional feed sources such as fishmeal or soybean meal.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Drillien, R; Spehner, D; Kirn, A

    1978-12-01

    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.

  13. Hypoxia stimulates prostacyclin synthesis in newborn pulmonary artery endothelium by increasing cyclooxygenase-1 protein.

    PubMed

    North, A J; Brannon, T S; Wells, L B; Campbell, W B; Shaul, P W

    1994-07-01

    In newborn lambs, pulmonary prostacyclin (PGI2) production increases acutely in response to low oxygen. We tested the hypothesis that decreased oxygenation directly stimulates PGI2 synthesis in arterial segments and cultured endothelial cells from newborn lamb intrapulmonary arteries. In segments studied at PO2 of 680 mm Hg, the synthesis of PGI2 exceeded prostaglandin E2 (PGE2) by 73%. Endothelium removal lowered PGI2 by 77% and PGE2 by 66%. At low oxygen tension (PO2, 40 mm Hg), PGI2 and PGE2 synthesis rose by 96% and 102%, respectively. Similarly, in endothelial cells studied at PO2 of 680 mm Hg, the synthesis of PGI2 exceeded PGE2 by 50%, and at low oxygen tension both PGI2 and PGE2 increased (89% and 64%, respectively). Endothelial cell PGI2 synthesis maximally stimulated by bradykinin, A23187, or arachidonic acid was also increased at low PO2 by 50%, 66%, and 48%, respectively. PGE2 synthesis was similarly altered, increasing by 33%, 37%, and 41%, respectively. In contrast, lowering oxygen had minimal effect on PGI2 and PGE2 synthesis with exogenous PGH2, which is the product of cyclooxygenase. Immunoblot analyses revealed that there was a 2.6-fold greater abundance of cyclooxygenase-1 protein at PO2 of 40 versus 680 mm Hg, and the increase at lower oxygen tension was inhibited by cycloheximide. The cyclooxygenase-2 isoform was not detected. Thus, attenuated oxygenation directly stimulates PGI2 and PGE2 synthesis in intrapulmonary arterial segments and endothelial cells from newborn lambs. This process is due to enhanced cyclooxygenase activity related to increased abundance of the cyclooxygenase-1 protein, and this effect may be due to increased synthesis of the enzyme protein.

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

    SciTech Connect

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

    1988-07-01

    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 (/sup 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 (/sup 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.

  15. Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals. [Hibiscus rosa-sinensis

    SciTech Connect

    Woodson, W.R.; Handa, A.K.

    1986-04-01

    Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of /sup 3/H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide.

  16. Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling

    PubMed Central

    Magennis, E. Peter; Fernandez-Trillo, Francisco; Sui, Cheng; Spain, Sebastian G.; Bradshaw, David; Churchley, David; Mantovani, Giuseppe; Winzer, Klaus; Alexander, Cameron

    2014-01-01

    The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms, for diagnostic or anti-infective applications, but which can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerisation of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms which produced them. This ‘bacteria-instructed synthesis’ can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the ‘instructing’ cell types. We further expand on the bacterial redox chemistries to ‘click’ fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualisation of pathogens. PMID:24813421

  17. Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

    PubMed Central

    Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

    2014-01-01

    Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

  18. Synthesis of new microbial pesticide metal complexes derived from coumarin-imine ligand.

    PubMed

    Elhusseiny, Amel F; Aazam, Elham S; Al-Amri, Huda M

    2014-07-15

    A series of metal complexes of zinc(II), cadmium(II), copper(II), nickel(II) and palladium(II) have been synthesized from coumarin-imine ligand, 8-[(1E)-1-(2-aminophenyliminio)ethyl]-2-oxo-2H-chromen-7-olate, [HL]. The structures of the complexes were proposed in the light of their spectroscopic, molar conductance, magnetic and thermal studies. The ligand coordinated in a tridentate manner through the azomethine nitrogen, the phenolic oxygen and the amine nitrogen and all complexes were non-electrolytes with different geometrical arrangements around the central metal ion. Photoluminescence data unambiguously showed remarkable fluorescence enhancement to Zn(2+) over other cations. The antimicrobial screening tests revealed that copper(II) complex exhibited the highest potency and its minimum inhibitory concentration on the enzymatic activities of the tested microbial species was determined. No toxin productivity was detected for all tested toxigenic species upon the exposure of copper complex. PMID:24704603

  19. Synthesis of new microbial pesticide metal complexes derived from coumarin-imine ligand

    NASA Astrophysics Data System (ADS)

    Elhusseiny, Amel F.; Aazam, Elham S.; Al-Amri, Huda M.

    2014-07-01

    A series of metal complexes of zinc(II), cadmium(II), copper(II), nickel(II) and palladium(II) have been synthesized from coumarin-imine ligand, 8-[(1E)-1-(2-aminophenyliminio)ethyl]-2-oxo-2H-chromen-7-olate, [HL]. The structures of the complexes were proposed in the light of their spectroscopic, molar conductance, magnetic and thermal studies. The ligand coordinated in a tridentate manner through the azomethine nitrogen, the phenolic oxygen and the amine nitrogen and all complexes were non-electrolytes with different geometrical arrangements around the central metal ion. Photoluminescence data unambiguously showed remarkable fluorescence enhancement to Zn2+ over other cations. The antimicrobial screening tests revealed that copper(II) complex exhibited the highest potency and its minimum inhibitory concentration on the enzymatic activities of the tested microbial species was determined. No toxin productivity was detected for all tested toxigenic species upon the exposure of copper complex.

  20. Microbial synthesis gas utilization and ways to resolve kinetic and mass-transfer limitations.

    PubMed

    Yasin, Muhammad; Jeong, Yeseul; Park, Shinyoung; Jeong, Jiyeong; Lee, Eun Yeol; Lovitt, Robert W; Kim, Byung Hong; Lee, Jinwon; Chang, In Seop

    2015-02-01

    Microbial conversion of syngas to energy-dense biofuels and valuable chemicals is a potential technology for the efficient utilization of fossils (e.g., coal) and renewable resources (e.g., lignocellulosic biomass) in an environmentally friendly manner. However, gas-liquid mass transfer and kinetic limitations are still major constraints that limit the widespread adoption and successful commercialization of the technology. This review paper provides rationales for syngas bioconversion and summarizes the reaction limited conditions along with the possible strategies to overcome these challenges. Mass transfer and economic performances of various reactor configurations are compared, and an ideal case for optimum bioreactor operation is presented. Overall, the challenges with the bioprocessing steps are highlighted, and potential solutions are suggested. Future research directions are provided and a conceptual design for a membrane-based syngas biorefinery is proposed.

  1. [Recent progress on galacto-oligosaccharides synthesis by microbial beta-galactosidase--a review].

    PubMed

    Lu, Lili; Li, Zhengyi; Xiao, Min

    2008-07-01

    Galacto-oligosaccharides are among the most promising non-digestible oligosaccharides that are recognized as prebiotics. Commercial GOS are synthesized from lactose using the transglycosylation activity of beta-galactosidase from microorganisms. The structure of GOS varies with different enzyme source. The oligosaccharide yields of GOS produced by natural enzymes are generally 20%-45% and they could be improved by artificial enzyme. Reaction conditions also have effect on the oligosaccharide yield. Using enzymes in water-hydrophobic solvent mixtures or reverse micelles may improve the yield to some extent. GOS can be large-scale synthesized by packed bed reactor, plugflow reactor or membrane reactor. The glucose and lactose in the GOS products can be removed by using chromatography, enzyme oxidation, nanofiltration membrane or microbial fermentation.

  2. Microbial synthesis gas utilization and ways to resolve kinetic and mass-transfer limitations.

    PubMed

    Yasin, Muhammad; Jeong, Yeseul; Park, Shinyoung; Jeong, Jiyeong; Lee, Eun Yeol; Lovitt, Robert W; Kim, Byung Hong; Lee, Jinwon; Chang, In Seop

    2015-02-01

    Microbial conversion of syngas to energy-dense biofuels and valuable chemicals is a potential technology for the efficient utilization of fossils (e.g., coal) and renewable resources (e.g., lignocellulosic biomass) in an environmentally friendly manner. However, gas-liquid mass transfer and kinetic limitations are still major constraints that limit the widespread adoption and successful commercialization of the technology. This review paper provides rationales for syngas bioconversion and summarizes the reaction limited conditions along with the possible strategies to overcome these challenges. Mass transfer and economic performances of various reactor configurations are compared, and an ideal case for optimum bioreactor operation is presented. Overall, the challenges with the bioprocessing steps are highlighted, and potential solutions are suggested. Future research directions are provided and a conceptual design for a membrane-based syngas biorefinery is proposed. PMID:25443672

  3. Fluorescent In Situ Folding Control for Rapid Optimization of Cell-Free Membrane Protein Synthesis

    PubMed Central

    Müller-Lucks, Annika; Bock, Sinja; Wu, Binghua; Beitz, Eric

    2012-01-01

    Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality. PMID:22848743

  4. Control of storage-protein synthesis during seed development in pea (Pisum sativum L.).

    PubMed Central

    Gatehouse, J A; Evans, I M; Bown, D; Croy, R R; Boulter, D

    1982-01-01

    The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by 'Northern' hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level. Images Fig. 2. Fig. 3. PMID:6897609

  5. PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility

    PubMed Central

    Zhu, Siying; Henninger, Keely; McGrath, Barbara C.; Cavener, Douglas R.

    2016-01-01

    PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK’s role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility. PMID:27627766

  6. PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility.

    PubMed

    Zhu, Siying; Henninger, Keely; McGrath, Barbara C; Cavener, Douglas R

    2016-01-01

    PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK's role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility. PMID:27627766

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

    NASA Technical Reports Server (NTRS)

    Lin, B. L.; Raghavan, V.

    1991-01-01

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

  8. Soybean meal substitution with a yeast-derived microbial protein source in dairy cow diets.

    PubMed

    Sabbia, J A; Kalscheur, K F; Garcia, A D; Gehman, A M; Tricarico, J M

    2012-10-01

    The objective of this study was to examine the effects substituting soybean meal with a yeast-derived microbial protein (YMP) on rumen and blood metabolites, dry matter intake, and milk production of high-producing dairy cows. Sixteen Holstein cows (12 multiparous and 4 primiparous), 93 ± 37 DIM (mean ± SD) at the beginning of the experiment, were used in a 4×4 Latin square design with four 28-d periods. Cows were blocked by parity and production, with 1 square consisting of 4 animals fitted with rumen cannulas. Basal diets, formulated for 16.1% crude protein and 1.56 Mcal/kg of net energy for lactation, contained 40% corn silage, 20% alfalfa hay, and 40% concentrate mix. During each period, cows were fed 1 of 4 treatment diets corresponding to YMP (DEMP; Alltech Inc., Nicholasville, KY) concentrations of 0, 1.14, 2.28, and 3.41% DM. Soybean meal (44% CP) was replaced by YMP to attain isonitrogenous and isoenergetic diets. Dietary treatments had no effect on pH and on most ruminal volatile fatty acid concentrations, with the exception of isovalerate, which decreased linearly with the addition of YMP. Rumen ammonia concentration decreased linearly, whereas free amino acids, total amino acid nitrogen, and soluble proteins weighing more than 10 kDa showed a cubic response on rumen N fractionation. A quadratic response was observed in oligopeptides that weighed between 3 and 10 kDa and peptides under 3kDa when expressed as percentages of total amino acids and total nitrogen. Although nonesterified fatty acid concentration in blood did not differ between treatments, β-hydroxybutyrate and plasma glucose increased linearly as YMP increased. Dry matter intake showed a cubic effect, where cows fed 1.14, and 3.41% YMP had the highest intake. Milk production was not affected by YMP, whereas a trend was observed for a quadratic increase for 4% fat-corrected milk and energy-corrected milk. Medium- and long-chain fatty acid concentrations in milk increased quadratically

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-11-01

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

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

    SciTech Connect

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

    1986-12-01

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

  12. Protein synthesis rates in rat brain regions and subcellular fractions during aging

    SciTech Connect

    Avola, R.; Condorelli, D.F.; Ragusa, N.; Renis, M.; Alberghina, M.; Giuffrida Stella, A.M.; Lajtha, A.

    1988-04-01

    In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

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

    PubMed

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

    2015-07-01

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

  14. Measurement of protein synthesis in human skeletal muscle: further investigation of the flooding technique.

    PubMed

    McNurlan, M A; Essen, P; Heys, S D; Buchan, V; Garlick, P J; Wernerman, J

    1991-10-01

    1. The rate of protein synthesis in quadriceps muscle of healthy subjects estimated from the incorporation of L-[1-13C]leucine given by continuous infusion was 1.1%/day. The estimate of protein synthesis from the incorporation of a flooding amount of labelled leucine was 1.8%/day (SD 0.65). The possibility that the higher rate obtained with the flooding technique arose from stimulation of protein synthesis by the large amount of leucine is unlikely. 2. The same rate of protein synthesis (1.7%/day, SD 0.3) was obtained with a flooding amount (0.05 g/kg) of a different amino acid, L-[1-13C]phenylalanine, as was obtained with leucine. 3. Incorporation of L-[1-13C]phenylalanine was not affected by simultaneous injection of leucine (1.7%/day, SD 0.7) or valine (1.6%/day, SD 0.4). 4. Protein synthesis, assessed in a completely different way from the proportion of polyribosomes isolated from the skeletal muscle, was unaltered by the injection of 0.05 g of L-leucine/kg (44.6%, SD 8.5 versus 43.8%, SD 7.7). 5. Good agreement in estimates of protein synthesis was observed in subjects in whom both legs were measured with both L-[1-13C]leucine (mean difference 0.16%/day) and L-[1-13C]phenylalanine (mean difference 0.2%/day).

  15. Synthesis, anti-microbial and molecular docking studies of quinazolin-4(3H)-one derivatives.

    PubMed

    Mabkhot, Yahia Nasser; Al-Har, Munirah S; Barakat, Assem; Aldawsari, Fahad D; Aldalbahi, Ali; Ul-Haq, Zaheer

    2014-01-01

    In this work, synthesis, antimicrobial activities and molecular docking studies of some new series of substituted quinazolinone 2a-h and 3a-d were described. Starting form 2-aminobenzamide derivatives 1, a new series of quinazolinone derivatives has been synthesized, in high yields, assisted by microwave and classical methods. Some of these substituted quinazolinones were tested for their antimicrobial activity against Gram-negative bacteria (Pseudomonas aeruginosa and Esherichia coli) and Gram-positive bacteria (Staphylococcus aureus, and Bacillus subtilis), and anti-fungal activity against (Aspergillus fumigatus, Saccharomyces cervevisiae, and Candida albicans) using agar well diffusion method. Among the prepared products, 3-benzyl-2-(4-chlorophenyl)quinazolin-4(3H)-one (3a) was found to exhibits the most potent in vitro anti-microbial activity with MICs of 25.6±0.5, 24.3±0.4, 30.1±0.6, and 25.1±0.5 µg/mL against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Esherichia coli, respectively. Compound 3a was found to exhibits the most potent in vitro anti-fungal activity with MICs of 18.3±0.6, 23.1±0.4, and 26.1±0.5 µg/mL against Aspergillus fumigatus, Saccharomyces cervevisiae, and Candidaal bicans, respectively. PMID:24968329

  16. Is carbohydrate needed to further stimulate muscle protein synthesis/hypertrophy following resistance exercise?

    PubMed Central

    2013-01-01

    It is now well established that protein supplementation after resistance exercise promotes increased muscle protein synthesis, which ultimately results in greater net muscle accretion, relative to exercise alone or exercise with supplementary carbohydrate ingestion. However, it is not known whether combining carbohydrate with protein produces a greater anabolic response than protein alone. Recent recommendations have been made that the composition of the ideal supplement post-exercise would be a combination of a protein source with a high glycemic index carbohydrate. This is based on the hypothesis that insulin promotes protein synthesis, thus maximising insulin secretion will maximally potentiate this action. However, it is still controversial as to whether raising insulin level, within the physiological range, has any effect to further stimulate muscle protein synthesis. The present commentary will review the evidence underpinning the recommendation to consume carbohydrates in addition to a protein supplementation after resistance exercise for the specific purpose of increasing muscle mass. The paucity of data will be discussed, thus our conclusions are that further studies are necessary prior to any conclusions that enable evidence-based recommendations to be made. PMID:24066806

  17. Is carbohydrate needed to further stimulate muscle protein synthesis/hypertrophy following resistance exercise?

    PubMed

    Figueiredo, Vandré Casagrande; Cameron-Smith, David

    2013-01-01

    It is now well established that protein supplementation after resistance exercise promotes increased muscle protein synthesis, which ultimately results in greater net muscle accretion, relative to exercise alone or exercise with supplementary carbohydrate ingestion. However, it is not known whether combining carbohydrate with protein produces a greater anabolic response than protein alone. Recent recommendations have been made that the composition of the ideal supplement post-exercise would be a combination of a protein source with a high glycemic index carbohydrate. This is based on the hypothesis that insulin promotes protein synthesis, thus maximising insulin secretion will maximally potentiate this action. However, it is still controversial as to whether raising insulin level, within the physiological range, has any effect to further stimulate muscle protein synthesis. The present commentary will review the evidence underpinning the recommendation to consume carbohydrates in addition to a protein supplementation after resistance exercise for the specific purpose of increasing muscle mass. The paucity of data will be discussed, thus our conclusions are that further studies are necessary prior to any conclusions that enable evidence-based recommendations to be made.

  18. Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2013-01-01

    Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame. PMID:24418875

  19. Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2013-01-01

    Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame.

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

    PubMed

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

    2014-08-25

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

  1. 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.

    PubMed

    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. PMID:25810096

  2. 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.

    PubMed

    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.

  3. Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist.

    PubMed Central

    Miller, L C; Isa, S; Vannier, E; Georgilis, K; Steere, A C; Dinarello, C A

    1992-01-01

    Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis. Images PMID:1387885

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

    PubMed

    Burkewitz, Kristopher; Choe, Keith P; Lee, Elaine Choung-Hee; Deonarine, Andrew; Strange, Kevin

    2012-01-01

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

  5. S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis.

    PubMed

    Kamiya, Noriho; Tanaka, Tsutomu; Suzuki, Tsutomu; Takazawa, Takeshi; Takeda, Shuji; Watanabe, Kimitsuna; Nagamune, Teruyuki

    2003-01-01

    We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation. PMID:12643745

  6. Circadian Rhythms in Dinoflagellates: What Is the Purpose of Synthesis and Destruction of Proteins?

    PubMed Central

    Hastings, J. Woodland

    2013-01-01

    There is a prominent circadian rhythm of bioluminescence in many species of light-emitting dinoflagellates. In Lingulodinium polyedrum a daily synthesis and destruction of proteins is used to regulate activity. Experiments indicate that the amino acids from the degradation are conserved and incorporated into the resynthesized protein in the subsequent cycle. A different species, Pyrocystis lunula, also exhibits a rhythm of bioluminescence, but the luciferase is not destroyed and resynthesized each cycle. This paper posits that synthesis and destruction constitutes a cellular mechanism to conserve nitrogen in an environment where the resource is limiting.

  7. An efficient one-pot four-segment condensation method for protein chemical synthesis.

    PubMed

    Tang, Shan; Si, Yan-Yan; Wang, Zhi-Peng; Mei, Kun-Rong; Chen, Xin; Cheng, Jing-Yuan; Zheng, Ji-Shen; Liu, Lei

    2015-05-01

    Successive peptide ligation using a one-pot method can improve the efficiency of protein chemical synthesis. Although one-pot three-segment ligation has enjoyed widespread application, a robust method for one-pot four-segment ligation had to date remained undeveloped. Herein we report a new one-pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one-pot four-segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21.

  8. Decoration of proteins with sugar chains: recent advances in glycoprotein synthesis.

    PubMed

    Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro

    2014-10-01

    Chemical or chemoenzymatic synthesis is an emerging approach to produce homogeneous glycoproteins, which are hard to obtain by conventional biotechnology methods. Recent advances in the synthetic methodologies for the decoration of protein molecules with oligosaccharides provide several remarkable syntheses of homogeneous glycoproteins. This short review highlights several of the latest syntheses of glycoproteins including therapeutically important glycoproteins, a highly glycosylated protein, and unnatural glycoproteins in order to illustrate the power of the modern glycoprotein synthesis. Structurally defined glycoproteins are a novel material for understanding the molecular basis of glycoprotein functions and for the development of the next generation of biopharmaceuticals.

  9. Rational design and asymmetric synthesis of potent and neurotrophic ligands for FK506-binding proteins (FKBPs).

    PubMed

    Pomplun, Sebastian; Wang, Yansong; Kirschner, Alexander; Kozany, Christian; Bracher, Andreas; Hausch, Felix

    2015-01-01

    To create highly efficient inhibitors for FK506-binding proteins, a new asymmetric synthesis for pro-(S)-C(5) -branched [4.3.1] aza-amide bicycles was developed. The key step of the synthesis is an HF-driven N-acyliminium cyclization. Functionalization of the C(5)  moiety resulted in novel protein contacts with the psychiatric risk factor FKBP51, which led to a more than 280-fold enhancement in affinity. The most potent ligands facilitated the differentiation of N2a neuroblastoma cells with low nanomolar potency.

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

    PubMed Central

    Shenton, Daniel; Grant, Chris M

    2003-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  12. Coingestion of protein with carbohydrate during recovery from endurance exercise stimulates skeletal muscle protein synthesis in humans.

    PubMed

    Howarth, Krista R; Moreau, Natalie A; Phillips, Stuart M; Gibala, Martin J

    2009-04-01

    Coingestion of protein with carbohydrate (CHO) during recovery from exercise can affect muscle glycogen synthesis, particularly if CHO intake is suboptimal. Another potential benefit of protein feeding is an increased synthesis rate of muscle proteins, as is well documented after resistance exercise. In contrast, the effect of nutrient manipulation on muscle protein kinetics after aerobic exercise remains largely unexplored. We tested the hypothesis that ingesting protein with CHO after a standardized 2-h bout of cycle exercise would increase mixed muscle fractional synthetic rate (FSR) and whole body net protein balance (WBNB) vs. trials matched for total CHO or total energy intake. We also examined whether postexercise glycogen synthesis could be enhanced by adding protein or additional CHO to a feeding protocol that provided 1.2 g CHO x kg(-1) x h(-1), which is the rate generally recommended to maximize this process. Six active men ingested drinks during the first 3 h of recovery that provided either 1.2 g CHO.kg(-1).h(-1) (L-CHO), 1.2 g CHO + 0.4 g protein x kg(-1) x h(-1) (PRO-CHO), or 1.6 g CHO x kg(-1) x h(-1) (H-CHO) in random order. Based on a primed constant infusion of l-[ring-(2)H(5)]phenylalanine, analysis of biopsies (vastus lateralis) obtained at 0 and 4 h of recovery showed that muscle FSR was higher (P < 0.05) in PRO-CHO (0.09 +/- 0.01%/h) vs. both L-CHO (0.07 +/- 0.01%/h) and H-CHO (0.06 +/- 0.01%/h). WBNB assessed using [1-(13)C]leucine was positive only during PRO-CHO, and this was mainly attributable to a reduced rate of protein breakdown. Glycogen synthesis rate was not different between trials. We conclude that ingesting protein with CHO during recovery from aerobic exercise increased muscle FSR and improved WBNB, compared with feeding strategies that provided CHO only and were matched for total CHO or total energy intake. However, adding protein or additional CHO to a feeding strategy that provided 1.2 g CHO x kg(-1) x h(-1) did not further

  13. [The effect of modified nano-diamonds of detonation synthesis on the protein fractions of human blood].

    PubMed

    Botvich, Iu A; Olkhovskiĭ, I A; Baron, I I; Puzyr', A P; Baron, A V; Bondar', V S

    2013-11-01

    It is established that the modified nano-diamonds of detonation synthesis are able to bind serum proteins of human blood. The relative selectivity is established concerning the effect of modified nano-diamonds of detonation synthesis on beta2- and gamma-globulin fractions of serum. The evidence of concentration dependence of effect of modified nano-diamonds of detonation synthesis from serum proteins is established. The study results make it possible to consider modified nano-diamonds of detonation synthesis as a potential sorbent in technologies of hemodialysis, plasmapheresis, isolation of blood proteins and as a foundation for development of new systems of laboratory diagnostic.

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

    PubMed

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

    2013-04-24

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

  15. Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves.

    PubMed

    Huang, Hong-Lei; Cendan, Cruz-Miguel; Roza, Carolina; Okuse, Kenji; Cramer, Rainer; Timms, John F; Wood, John N

    2008-01-01

    Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha2delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability. PMID:18700027

  16. Myocardial Reloading after Extracorporeal Membrane Oxygenation Alters Substrate Metabolism While Promoting Protein Synthesis

    SciTech Connect

    Kajimoto, Masaki; Priddy, Colleen M.; Ledee, Dolena; Xu, Chun; Isern, Nancy G.; Olson, Aaron; Des Rosiers, Christine; Portman, Michael A.

    2013-08-19

    Extracorporeal membrane oxygenation (ECMO) unloads the heart providing a bridge to recovery in children after myocardial stunning. Mortality after ECMO remains high.Cardiac substrate and amino acid requirements upon weaning are unknown and may impact recovery. We assessed the hypothesis that ventricular reloading modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Fourteen immature piglets (7.8-15.6 kg) were separated into 2 groups based on ventricular loading status: 8 hour-ECMO (UNLOAD) and post-wean from ECMO (RELOAD). We infused [2-13C]-pyruvate as an oxidative substrate and [13C6]-L-leucine, as a tracer of amino acid oxidation and protein synthesis into the coronary artery. RELOAD showed marked elevations in myocardial oxygen consumption above baseline and UNLOAD. Pyruvate uptake was markedly increased though RELOAD decreased pyruvate contribution to oxidative CAC metabolism.RELOAD also increased absolute concentrations of all CAC intermediates, while maintaining or increasing 13C-molar percent enrichment. RELOAD also significantly increased cardiac fractional protein synthesis rates by >70% over UNLOAD. Conclusions: RELOAD produced high energy metabolic requirement and rebound protein synthesis. Relative pyruvate decarboxylation decreased with RELOAD while promoting anaplerotic pyruvate carboxylation and amino acid incorporation into protein rather than to the CAC for oxidation. These perturbations may serve as therapeutic targets to improve contractile function after ECMO.

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

    PubMed

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

    1991-01-01

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

  18. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    PubMed

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.

  19. Microbial engineering of nano-heterostructures; biological synthesis of a magnetically-recoverable palladium nanocatalyst

    SciTech Connect

    Coker, V. S.; Bennett, J. A.; Telling, N.; Charnock, J. M.; van der Laan, G.; Pattrick, R. A. D.; Pearce, C. I; Cutting, R. S.; Shannon, I. J.; Wood, J.; Arenholz, E.; Vaughan, D. J.; Lloyd, J. R.

    2009-12-01

    Precious metals supported on ferrimagnetic particles form a diverse range of catalysts. Here we show a novel biotechnological route for the synthesis of a heterogeneous catalyst consisting of reactive palladium nanoparticles arrayed on a biomagnetite support. The magnetic support was synthesized at ambient temperature by the Fe(III)-reducing bacterium, Geobacter sulfurreducens, and facilitated ease of recovery of the catalyst with superior performance due to reduced agglomeration. Arrays of palladium nanoparticles were deposited on the nanomagnetite using a simple one-step method without the need to modify the biomineral surface most likely due to an organic coating priming the surface for Pd adsorption. A combination of EXAFS and XPS showed the particles to be predominantly metallic in nature. The Pd{sup 0}-biomagnetite was tested for catalytic activity in the Heck Reaction coupling iodobenzene to ethyl acrylate or styrene and near complete conversion to ethyl cinnamate or stilbene was achieved within 90 and 180 min, respectively.

  20. One-pot microbial synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase.

    PubMed

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-06-01

    A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2'-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2'-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.

  1. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells.

    PubMed

    Hill, T M; Sinden, R R; Sadler, J R

    1983-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff.

  2. Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of beta-hydroxy-beta-methylbutyrate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB). To determine the effects of HMB on protein synthesi...

  3. Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of beta-hydroxy-beta-methylbutyrate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite ß-hydroxy-ß-methylbutyrate (HMB). To determine the effects of HMB on protein synthesis and ...

  4. Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of Beta-hydroxy-Beta-methylbutyrate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite Beta-hydroxy-Beta-methylbutyrate (HMB). To determine the effects of HMB on protein synthesi...

  5. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  6. Long-Term Memory for Instrumental Responses Does Not Undergo Protein Synthesis-Dependent Reconsolidation upon Retrieval

    ERIC Educational Resources Information Center

    Hernandez, Pepe J.; Kelley, Ann E.

    2004-01-01

    Recent evidence indicates that certain forms of memory, upon recall, may return to a labile state requiring the synthesis of new proteins in order to preserve or reconsolidate the original memory trace. While the initial consolidation of "instrumental memories" has been shown to require de novo protein synthesis in the nucleus accumbens, it is not…

  7. REGULATION OF CARDIAC AND SKELETAL MUSCLE PROTEIN SYNTHESIS BY INDIVIDUAL BRANCHED-CHAIN AMINO ACIDS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle grows at a very rapid rate in the neonatal pig, due in part to an enhanced sensitivity of protein synthesis to the postprandial rise in amino acids. An increase in leucine alone stimulates protein synthesis in skeletal muscle of the neonatal pig; however, the effect of isoleucine and...

  8. Cell density modulates growth, extracellular matrix, and protein synthesis of cultured rat mesangial cells.

    PubMed

    Wolthuis, A; Boes, A; Grond, J

    1993-10-01

    Mesangial cell (MC) hyperplasia and accumulation of extracellular matrix are hallmarks of chronic glomerular disease. The present in vitro study examined the effects of cell density on growth, extracellular matrix formation, and protein synthesis of cultured rat MCs. A negative linear relationship was found between initial plating density and DNA synthesis per cell after 24 hours incubation in medium with 10% fetal calf serum (range: 1 x 10(3) to 7 x 10(5) MCs/2cm2, r = 0.996, P < 0.001). Enzyme-linked immunosorbent assay of the amount of fibronectin in the conditioned medium after 72 hours showed a negative relationship with increasing cell density. In contrast, the amount of cell-associated fibronectin increased to maximal values in confluent cultures, and no further increase was seen at supraconfluency. The relative collagen synthesis in the conditioned medium and cell layer--assessed by collagenase digestion after 5 hours [3H]proline pulse labeling--showed a similar pattern. Secreted collagen decreased with increasing cell density from 3.4% to 0.2% of total protein synthesis. In contrast, cell-associated collagen increased from 1.1% to 11.8% of newly synthesized protein until confluency followed by a decrease to 4.2% at supraconfluency. Specific immunoprecipitation of collagen types I, III, and IV revealed a significant (twofold) increase in collagen I synthesis per cell at confluency. Collagen III and IV synthesis was not affected by cell density. Specific protein expression in both the medium and cell layer were analyzed by two-dimensional polyacrylamide gel electrophoresis (150 to 20 kd, pI 5.0 to 7.0) after 20 hours steady-state metabolic labeling with [35S]methionine. Supraconfluent MCs displayed overexpression of 10, underexpression of four, new expression of five, and changed mobility of three different intracellular proteins. Of interest was the overexpression of two proteins (89 kd, pI 5.31 and 72 kd, pI 5.32) that were identified by immunoblotting as

  9. Synthesis of microbial elastomers based on soybean oily acids. Biocompatibility studies.

    PubMed

    Hazer, Derya Burcu; Hazer, Baki; Kaymaz, Figen

    2009-06-01

    Biocompatibility studies of the autoxidized and unoxidized unsaturated medium-long chain length (m-lcl) co-poly-3-hydroxyalkanoates (m-lclPHAs) derived from soya oily acids have been reported. Pseudomonas oleovorans was grown on a series of mixtures of octanoic acid (OA) and soya oily acids (Sy) with weight ratios of 20:80, 28:72 and 50:50 in order to obtain unsaturated m-lcl copolyesters coded PHO-Sy-2080, PHO-Sy-2872 and PHO-Sy-5050, respectively. The PHA films were obtained by solvent cast from CHCl(3). They were all originally sticky and waxy except PHO-Sy-5050. Autoxidation of the unsaturated copolyester films was carried out on exposure to air at room temperature in order to obtain crosslinked polymers. They became a highly flexible elastomer after being autoxidized (about 40 days of autoxidation). The in vivo tissue reactions of the autoxidized PHAs were evaluated by subcutaneous implantation in rats. The rats appeared to be healthy throughout the implantation period. No symptom such as necrosis, abscess or tumorigenesis was observed in the vicinity of the implants. Retrieved materials varied in their physical appearance after 6 weeks of implantation. In vivo biocompatibility studies of the medical applications indicated that the microbial copolyesters obtained were all biocompatible and especially the PHOSy series of copolyesters had the highest biocompatibility among them. PMID:19498224

  10. Microbially-mediated method for synthesis of non-oxide semiconductor nanoparticles

    DOEpatents

    Phelps, Tommy J.; Lauf, Robert J.; Moon, Ji Won; Rondinone, Adam J.; Love, Lonnie J.; Duty, Chad Edward; Madden, Andrew Stephen; Li, Yiliang; Ivanov, Ilia N.; Rawn, Claudia Jeanette

    2014-06-24

    The invention is directed to a method for producing non-oxide semiconductor nanoparticles, the method comprising: (a) subjecting a combination of reaction components to conditions conducive to microbially-mediated formation of non-oxide semiconductor nanoparticles, wherein said combination of reaction components comprises i) anaerobic microbes, ii) a culture medium suitable for sustaining said anaerobic microbes, iii) a metal component comprising at least one type of metal ion, iv) a non-metal component containing at least one non-metal selected from the group consisting of S, Se, Te, and As, and v) one or more electron donors that provide donatable electrons to said anaerobic microbes during consumption of the electron donor by said anaerobic microbes; and (b) isolating said non-oxide semiconductor nanoparticles, which contain at least one of said metal ions and at least one of said non-metals. The invention is also directed to non-oxide semiconductor nanoparticle compositions produced as above and having distinctive properties.

  11. Lactones 42. Stereoselective enzymatic/microbial synthesis of optically active isomers of whisky lactone.

    PubMed

    Boratyński, Filip; Smuga, Małgorzata; Wawrzeńczyk, Czesław

    2013-11-01

    Two different methods, enzyme-mediated reactions and biotrasformations with microorganisms, were applied to obtain optically pure cis- and trans-isomers of whisky lactone 4a and 4b. In the first method, eight alcohol dehydrogenases were investigated as biocatalysts to enantioselective oxidation of racemic erythro- and threo-3-methyloctane-1,4-diols (1a and 1b). Oxidation processes with three of them, alcohol dehydrogenases isolated from horse liver (HLADH) as well as recombinant from Escherichia coli and primary alcohol dehydrogenase (PADH I), were characterized by the highest degree of conversion with moderate enantioselectivity (ee=27-82%) of the reaction. In all enzymatic reactions enantiomerically enriched not naturally occurring isomers of trans-(-)-(4R,5S)-4b or cis-(+)-(4R,5R)-4a were formed preferentially. In the second strategy, based on microbial lactonization of γ-oxoacids, naturally occurring opposite isomers of whisky lactones were obtained. Trans-(+)-(4S,5R)-isomer (ee=99%) of whisky lactone 4b was stereoselectively formed as the only product of biotransformations of 3-methyl-4-oxooctanoic acid (5) catalyzed by Didimospheria igniaria KCH6651, Laetiporus sulphurens AM525, Chaetomium sp.1 KCH6670 and Saccharomyces cerevisiae AM464. Biotransformation of γ-oxoacid 5, in the culture of Beauveria bassiana AM278 and Pycnidiella resinae KCH50 afforded a mixtures of trans-(+)-(4S,5R)-4b with enantiomeric excess ee=99% and cis-(-)-(4S,5S)-4a with enantiomeric excesses ee=77% and ee=45% respectively.

  12. Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs.

    PubMed

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

    2009-05-01

    The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose-amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8-12/group) with insulin at 0, 10, 22, and 110 ng kg(-0.66) min(-1) to achieve approximately 0, 2, 6 and 30 muU ml(-1) insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of L: -[4-(3)H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids.

  13. Synthesis and renewal of proteins in duck anterior hypophysis in organ culture.

    PubMed

    Tixier-Vidal, A; Gourdji, D

    1970-07-01

    In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material. PMID:5460460

  14. Synthesis and renewal of proteins in duck anterior hypophysis in organ culture.

    PubMed

    Tixier-Vidal, A; Gourdji, D

    1970-07-01

    In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material.

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

    PubMed Central

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

    2015-01-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. PMID:26478227

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

    PubMed

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

    2015-11-01

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

  17. The Unphosphorylated EIIANtr Protein Represses the Synthesis of Alkylresorcinols in Azotobacter vinelandii

    PubMed Central

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

    2015-01-01

    Upon encystment induction, Azotobacter vinelandii produces the phenolic lipids alkylresorcinols (ARs) that are structural components of the cysts. The enzymes responsible for the ARs synthesis are encoded in the arsABCD operon, whose expression is activated by ArpR. The transcription of arpR is initiated from an RpoS dependent promoter. The nitrogen-related phosphotransferase system (PTSNtr) is a global regulatory system present in Gram negative bacteria. It comprises the EINtr, NPr and EIIANtr proteins encoded by ptsP, ptsO and ptsN genes respectively. These proteins participate in a phosphoryl-group transfer from phosphoenolpyruvate to protein EIIANtr via the phosphotransferases EINtr and NPr. In A. vinelandii, the non-phosphorylated form of EIIANtr was previously shown to repress the synthesis of poly-ß-hydroxybutyrate. In this work, we show that PTSNtr also regulates the synthesis of ARs. In a strain that carries unphosphorylated EIIANtr, the expression of arpR was reduced, while synthesis of ARs and transcription of arsA were almost abrogated. The expression of arpR from an RpoS-independent promoter in this strain restored the ARs synthesis. Taken together these results indicate that unphosphorylated EIIANtr negatively affects activation of arpR transcription by RpoS. PMID:25642700

  18. Infusion of protein synthesis inhibitors in the entorhinal cortex blocks consolidation but not reconsolidation of object recognition memory.

    PubMed

    Lima, Ramón H; Rossato, Janine I; Furini, Cristiane R; Bevilaqua, Lia R; Izquierdo, Iván; Cammarota, Martín

    2009-05-01

    Memory consolidation and reconsolidation require the induction of protein synthesis in some areas of the brain. Here, we show that infusion of the protein synthesis inhibitors anisomycin, emetine and cycloheximide in the entorhinal cortex immediately but not 180 min or 360 min after training in an object recognition learning task hinders long-term memory retention without affecting short-term memory or behavioral performance. Inhibition of protein synthesis in the entorhinal cortex after memory reactivation involving either a combination of familiar and novel objects or two familiar objects does not affect retention. Our data suggest that protein synthesis in the entorhinal cortex is necessary early after training for consolidation of object recognition memory. However, inhibition of protein synthesis in this cortical region after memory retrieval does not seem to affect the stability of the recognition trace.

  19. Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

    PubMed

    Mason, Alexander F; Thordarson, Pall

    2016-01-01

    The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers. PMID:27501061

  20. Posttranslational modification and sequence variation of redox-active proteins correlate with biofilm life cycle in natural microbial communities

    SciTech Connect

    Singer, Steven; Erickson, Brian K; Verberkmoes, Nathan C; Hwang, Mona; Shah, Manesh B; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2010-01-01

    Characterizing proteins recovered from natural microbial communities affords the opportunity to correlate protein expression and modification with environmental factors, including species composition and successional stage. Proteogenomic and biochemical studies of pellicle biofilms from subsurface acid mine drainage streams have shown abundant cytochromes from the dominant organism, Leptospirillum Group II. These cytochromes are proposed to be key proteins in aerobic Fe(II) oxidation, the dominant mode of cellular energy generation by the biofilms. In this study, we determined that posttranslational modification and expression of amino-acid sequence variants change as a function of biofilm maturation. For Cytochrome579 (Cyt579), the most abundant cytochrome in the biofilms, late developmental-stage biofilms differed from early-stage biofilms in N-terminal truncations and decreased redox potentials. Expression of sequence variants of two monoheme c-type cytochromes also depended on biofilm development. For Cyt572, an abundant membrane-bound cytochrome, the expression of multiple sequence variants was observed in both early and late developmental-stage biofilms; however, redox potentials of Cyt572 from these different sources did not vary significantly. These cytochrome analyses show a complex response of the Leptospirillum Group II electron transport chain to growth within a microbial community and illustrate the power of multiple proteomics techniques to define biochemistry in natural systems.

  1. New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation.

    PubMed

    González-Martínez, Alejandro; Calderón, Kadiya; González-López, Jesús

    2016-05-01

    High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.

  2. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins

    NASA Astrophysics Data System (ADS)

    Tang, Nicholas C.; Chilkoti, Ashutosh

    2016-04-01

    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  3. Ethionine-dependent inhibition of acute-phase plasma protein synthesis in the rat.

    PubMed Central

    Kasperczyk, H.; Koj, A.

    1983-01-01

    Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins. PMID:6882676

  4. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins.

    PubMed

    Tang, Nicholas C; Chilkoti, Ashutosh

    2016-04-01

    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

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

    PubMed

    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.

  6. Short-term muscle disuse lowers myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

    PubMed

    Wall, Benjamin T; Dirks, Marlou L; Snijders, Tim; van Dijk, Jan-Willem; Fritsch, Mario; Verdijk, Lex B; van Loon, Luc J C

    2016-01-15

    Disuse leads to rapid loss of skeletal muscle mass and function. It has been hypothesized that short successive periods of muscle disuse throughout the lifespan play an important role in the development of sarcopenia. The physiological mechanisms underlying short-term muscle disuse atrophy remain to be elucidated. We assessed the impact of 5 days of muscle disuse on postabsorptive and postprandial myofibrillar protein synthesis rates in humans. Twelve healthy young (22 ± 1 yr) men underwent a 5-day period of one-legged knee immobilization (full leg cast). Quadriceps cross-sectional area (CSA) of both legs was assessed before and after immobilization. Continuous infusions of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine were combined with the ingestion of a 25-g bolus of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled dietary protein to assess myofibrillar muscle protein fractional synthetic rates in the immobilized and nonimmobilized control leg. Immobilization led to a 3.9 ± 0.6% decrease in quadriceps muscle CSA of the immobilized leg. Based on the l-[ring-(2)H5]phenylalanine tracer, immobilization reduced postabsorptive myofibrillar protein synthesis rates by 41 ± 13% (0.015 ± 0.002 vs. 0.032 ± 0.005%/h, P < 0.01) and postprandial myofibrillar protein synthesis rates by 53 ± 4% (0.020 ± 0.002 vs. 0.044 ± 0.003%/h, P < 0.01). Comparable results were found using the l-[1-(13)C]leucine tracer. Following protein ingestion, myofibrillar protein bound l-[1-(13)C]phenylalanine enrichments were 53 ± 18% lower in the immobilized compared with the control leg (0.007 ± 0.002 and 0.015 ± 0.002 mole% excess, respectively, P < 0.05). We conclude that 5 days of muscle disuse substantially lowers postabsorptive myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

  7. Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose.

    PubMed

    Chen, Zhen; Geng, Feng; Zeng, An-Ping

    2015-02-01

    Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products.

  8. Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis.

    PubMed

    Balke, Kathleen; Kadow, Maria; Mallin, Hendrik; Sass, Stefan; Bornscheuer, Uwe T

    2012-08-21

    Baeyer-Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio- and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in protein sequence databases using distinct motifs to discover new Baeyer-Villiger monooxygenases as well as high-throughput assays to facilitate protein engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.

  9. Enteral β-hydroxy-β-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs.

    PubMed

    Kao, Michelle; Columbus, Daniel A; Suryawan, Agus; Steinhoff-Wagner, Julia; Hernandez-Garcia, Adriana; Nguyen, Hanh V; Fiorotto, Marta L; Davis, Teresa A

    2016-06-01

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were studied immediately (F) or fed one of five diets for 24 h: low-protein (LP), high-protein (HP), or LP diet supplemented with 4 (HMB4), 40 (HMB40), or 80 (HMB80) μmol HMB·kg body wt(-1)·day(-1) Cell replication was assessed from nuclear incorporation of BrdU in the longissimus dorsi (LD) muscle and jejunum crypt cells. Protein synthesis rates in LD, gastrocnemius, rhomboideus, and diaphragm muscles, lung, and brain were greater in HMB80 and HP and in brain were greater in HMB40 compared with LP and F groups. Formation of the eIF4E·eIF4G complex and S6K1 and 4E-BP1 phosphorylation in LD, gastrocnemius, and rhomboideus muscles were greater in HMB80 and HP than in LP and F groups. Phosphorylation of eIF2α and eEF2 and expression of SNAT2, LAT1, MuRF1, atrogin-1, and LC3-II were unchanged. Numbers of BrdU-positive myonuclei in the LD were greater in HMB80 and HP than in the LP and F groups; there were no differences in jejunum. The results suggest that enteral supplementation with HMB increases skeletal muscle protein anabolism in neonates by stimulation of protein synthesis and satellite cell proliferation.

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

    SciTech Connect

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

    1983-10-01

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

  11. Understanding of Protein Synthesis in a Living Cell

    ERIC Educational Resources Information Center

    Mustapha, Y.; Muhammad, S.

    2006-01-01

    The assembly of proteins takes place in the cytoplasm of a cell. There are three main steps. In initiation, far left, all the necessary parts of the process are brought together by a small molecule called a ribosome. During elongation, amino acids, the building blocks of proteins, are joined to one another in a long chain. The sequence in which…

  12. Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification.

    PubMed

    Meier, Jordan L; Mercer, Andrew C; Rivera, Heriberto; Burkart, Michael D

    2006-09-20

    In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.

  13. Effects of chilling on protein synthesis in tomato suspension cultures

    SciTech Connect

    Matadial, B.; Pauls, K.P. )

    1989-04-01

    The effect of chilling on cell growth, cell viability, protein content and protein composition in suspension cultures of L. esculentum and L. hirsutum was investigated. Cell growth for both species was arrested at 2{degrees}C but when cultures were transferred to 25{degree}C cell growth resumed. There was no difference in viability between control and chilled cultures of L. esculentum, however, L. hirsutum control cultures exhibited larger amounts of Fluorescein Diacetate induced fluorescence than chilled cultures. {sup 35}S-methionine incorporation into proteins was 2.5-2 times higher in L. hirsutum than in L. esculentum. Quantitative and qualitative differences, in {sup 35}S-methionine labelled proteins, between chilled and control cultures were observed by SDS-PAGE and fluorography. Protein content in chilled cultures decreased over time but then increased when cultures were transferred to 25{degrees}C.

  14. Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives.

    PubMed

    Vijeetha, Tadla; Balakrishna, Marrapu; Karuna, Mallampalli Sri Lakshmi; Surya Koppeswara Rao, Bhamidipati Venkata; Prasad, Rachapudi Badari Narayana; Kumar, Koochana Pranay; Surya Narayana Murthy, Upadyaula

    2015-01-01

    The study involved synthesis of five novel amino acid derivatives of phosphatidylethanolamine isolated from egg yolk lecithin employing a three step procedure i) N-protection of L-amino acids with BOC anhydride in alkaline medium ii) condensation of - CO2H group of N-protected amino acid with free -NH2 of PE by a peptide linkage and iii) deprotection of N-protected group of amino acids to obtain phosphatidylethanolamine-N-amino acid derivatives in 60-75% yield. The five L-amino acids used were L glycine, L-valine, L-leucine, L-isoleucine and L-phenylalanine. The amino acid derivatives were screened for anti-baterial activity against B. subtilis, S. aureus, P. aeroginosa and E. coli taking Streptomycin as reference compound and anti-fungal activity against C. albicans, S. cervisiae, A. niger taking AmphotericinB as reference compound. All the amino acid derivatives exhibited extraordinary anti-bacterial activities about 3 folds or comparable to Streptomycin and moderate or no anti-fungal activity against Amphotericin-B.

  15. Origins of tmRNA: the missing link in the birth of protein synthesis?

    PubMed Central

    Macé, Kevin; Gillet, Reynald

    2016-01-01

    The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis. PMID:27484476

  16. Some Uses of Tissue Explants in the Teaching of Protein Synthesis

    ERIC Educational Resources Information Center

    King, B.

    1977-01-01

    Experiments are described in which inhibitors are used to investigate the timing of transcription and translation of the messenger RNA for the enzyme invertase. It is suggested that plant tissue slices provide adaptable material with which to study enzyme induction, protein synthesis, and cell differentiation at sixth-form level. (Author/MA)

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

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

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

  18. Energetics of Polymerization: A Contribution to an Understanding of Protein Synthesis.

    ERIC Educational Resources Information Center

    Friedmann, Herbert C.

    1986-01-01

    Discusses the various ways that textbooks treat the energetics of protein synthesis. Offers an approach to explaining the phenomenon by emphasizing the ordering aspects of the process. Describes the participation of compounds such as ATP and GTP in the ordering process. (TW)

  19. Origins of tmRNA: the missing link in the birth of protein synthesis?

    PubMed

    Macé, Kevin; Gillet, Reynald

    2016-09-30

    The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis.

  20. Protein synthesis in artificial cells: using compartmentalisation for spatial organisation in vesicle bioreactors.

    PubMed

    Elani, Yuval; Law, Robert V; Ces, Oscar

    2015-06-28

    Whereas spatial organisation of function is ubiquitous in biology, it has been lacking in artificial cells. We rectify this by using multi-compartment vesicles as chassis for artificial cells, allowing distinct biological processes to be isolated in space. This is demonstrated by in vitro synthesis of two proteins in predefined vesicle regions.

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

    PubMed Central

    Ramiro-Cortés, Yazmín; Israely, Inbal

    2013-01-01

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

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

    NASA Technical Reports Server (NTRS)

    1982-01-01

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

  3. Reconsolidation of a Context Long-Term Memory in the Terrestrial Snail Requires Protein Synthesis

    ERIC Educational Resources Information Center

    Gainutdinova, Tatiana H.; Tagirova, Rosa R.; Ismailova, Asja I.; Muranova, Lyudmila N.; Samarova, Elena I.; Gainutdinov, Khalil L.; Balaban, Pavel M.

    2005-01-01

    We investigated the influence of the protein synthesis blocker anisomycin on contextual memory in the terrestrial snail "Helix." Prior to the training session, the behavioral responses in two contexts were similar. Two days after a session of electric shocks (5 d) in one context only, the context conditioning was observed as the significant…

  4. Using Simple Manipulatives to Improve Student Comprehension of a Complex Biological Process: Protein Synthesis

    ERIC Educational Resources Information Center

    Guzman, Karen; Bartlett, John

    2012-01-01

    Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article…

  5. Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain.

    PubMed

    Bonova, Petra; Burda, Jozef; Danielisova, Viera; Nemethova, Miroslava; Gottlieb, Miroslav

    2013-05-01

    In the clinic delayed post-conditioning would represent an attractive strategy for the survival of vulnerable neurons after an ischemic event. In this paper we studied the impact of ischemia and delayed post-conditioning on blood and brain tissue concentrations of glutamate and protein synthesis. We designed two groups of animals for analysis of brain tissues and blood after global ischemia and post-conditioning, and one for analysis of blood glutamate after transient focal ischemia. Our results showed elevated blood glutamate in two models of transient brain ischemia and decreases in blood glutamate to control in the first 20min of post-conditioning recirculation followed by a consecutive drop of about 20.5% on the first day. Similarly, we recorded reduced protein synthesis in hippocampus and cortex 2 and 3days after ischemia. However, increased glutamate was registered only in the hippocampus. Post-conditioning improves protein synthesis in CA1 and dentate gyrus and, surprisingly, leads to 50% reduction in glutamate in whole hippocampus and cortex. In conclusion, ischemia leads to meaningful elevation of blood and tissue glutamate. Post-conditioning activates mechanisms resulting in rapid elimination of glutamate from brain tissue and/or in the circulatory system that could otherwise impede brain-to-blood glutamate efflux mechanisms. Moreover, post-conditioning induces protein synthesis renewing in ischemia affected tissues that could also contribute to elimination of excitotoxicity. In addition, the potential of glutamate for monitoring the progress of ischemia and efficacy of therapy was shown.

  6. Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography

    SciTech Connect

    Pentelute, Brad L.; Mandal, Kalyaneswar; Gates, Zachary P.; Sawaya, Michael R.; Yeates, Todd O.; Kent, Stephen B.H.

    2010-11-05

    Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space groupP that diffracted to atomic-resolution (0.95 {angstrom}), enabling the X-ray structure of kaliotoxin to be determined by direct methods.

  7. Leptin stimulates protein synthesis-activating translation machinery in human trophoblastic cells.

    PubMed

    Pérez-Pérez, Antonio; Maymó, Julieta; Gambino, Yésica; Dueñas, José L; Goberna, Raimundo; Varone, Cecilia; Sánchez-Margalet, Víctor

    2009-11-01

    Leptin was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it may work as an autocrine hormone, mediating angiogenesis, growth, and immunomodulation. Leptin receptor (LEPR, also known as Ob-R) shows sequence homology to members of the class I cytokine receptor (gp130) superfamily. In fact, leptin may function as a proinflammatory cytokine. We have previously found that leptin is a trophic and mitogenic factor for trophoblastic cells. In order to further investigate the mechanism by which leptin stimulates cell growth in JEG-3 cells and trophoblastic cells, we studied the phosphorylation state of different proteins of the initiation stage of translation and the total protein synthesis by [(3)H]leucine incorporation in JEG-3 cells. We have found that leptin dose-dependently stimulates the phosphorylation and activation of the translation initiation factor EIF4E as well as the phosphorylation of the EIF4E binding protein EIF4EBP1 (PHAS-I), which releases EIF4E to form active complexes. Moreover, leptin dose-dependently stimulates protein synthesis, and this effect can be partially prevented by blocking mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PIK3) pathways. In conclusion, leptin stimulates protein synthesis, at least in part activating the translation machinery, via the activation of MAPK and PIK3 pathways.

  8. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    PubMed

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins.

  9. Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia

    PubMed Central

    Rieu, Isabelle; Balage, Michèle; Sornet, Claire; Giraudet, Christophe; Pujos, Estelle; Grizard, Jean; Mosoni, Laurent; Dardevet, Dominique

    2006-01-01

    The present study was designed to assess the effects of dietary leucine supplementation on muscle protein synthesis and whole body protein kinetics in elderly individuals. Twenty healthy male subjects (70 ± 1 years) were studied before and after continuous ingestion of a complete balanced diet supplemented or not with leucine. A primed (3.6 μmol kg−1) constant infusion (0.06 μmol kg−1 min−1) of l-[1-13C]phenylalanine was used to determine whole body phenylalanine kinetics as well as fractional synthesis rate (FSR) in the myofibrillar fraction of muscle proteins from vastus lateralis biopsies. Whole body protein kinetics were not affected by leucine supplementation. In contrast, muscle FSR, measured over the 5-h period of feeding, was significantly greater in the volunteers given the leucine-supplemented meals compared with the control group (0.083 ± 0.008 versus 0.053 ± 0.009% h−1, respectively, P < 0.05). This effect was due only to increased leucine availability because only plasma free leucine concentration significantly differed between the control and leucine-supplemented groups. We conclude that leucine supplementation during feeding improves muscle protein synthesis in the elderly independently of an overall increase of other amino acids. Whether increasing leucine intake in old people may limit muscle protein loss during ageing remains to be determined. PMID:16777941

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

    SciTech Connect

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

    1985-06-01

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

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

    PubMed

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

    2015-02-01

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

  12. Protein Stable Isotope Fingerprinting (P-SIF): A New Tool to Understand Natural Isotopic Heterogeneity of Mixed Microbial Ecosystems

    NASA Astrophysics Data System (ADS)

    Pearson, A.; Mohr, W.; Tang, T.; Sattin, S.; Bovee, R.

    2014-12-01

    Protein stable isotope fingerprinting (P-SIF) is a method to measure the carbon isotope ratios of whole proteins separated from complex mixtures, including cultures and environmental samples. The goal of P-SIF is to expose the links between identity and function in microbial ecosystems by (i) determining the ratios of 13C/12C (values of δ13C) for different taxonomic divisions, and (ii) using those values as clues to the metabolic pathways employed by the respective organisms, while (iii) not perturbing the system, i.e., not adding exogenous substrates or isotope labels. To accomplish this, we employ two-dimensional HPLC to resolve a sample containing ca. 5-10 mg of mixed proteins into 960-1440 fractions. Each fraction then is split in two aliquots: The first is digested with trypsin for peptide sequencing, while the second is measured in triplicate using an isotope-ratio mass spectrometer interfaced with a spooling wire microcombustion device. Data from pure cultures show that bacteria have a narrow distribution of protein δ13C values within individual taxa (±0.7-1.2‰, 1σ). This is moderately larger than the mean precision of the triplicate isotope measurements (±0.5‰, 1σ) and may reflect heterogeneous distribution of 13C among the amino acids. When cells from different species are mixed together prior to protein extraction and separation, the results can predict accurately (to within ±1σ) the δ13C values of the original taxa. The number of data points required for this endmember prediction is ≥20/taxon, yielding a theoretical resolution of ca. 10 taxonomic units/sample. Initial tests on environmental samples suggest the approach will be useful to determine the overall trophic breadth of mixed microbial ecosystems.

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

    PubMed

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

    2013-09-28

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

  14. Comparison of the Effects of Ornithine and Arginine on the Brain Protein Synthesis Rate in Young Rats.

    PubMed

    Suzumura, Shoko; Tujioka, Kazuyo; Yamada, Takashi; Yokogoshi, Hidehiko; Akiduki, Saori; Hishida, Yukihiro; Tsutsui, Kazumi; Hayase, Kazutoshi

    2015-01-01

    Brain protein synthesis and the plasma concentration of growth hormone (GH) are sensitive to dietary ornithine. The purpose of this study was to determine whether dietary arginine, the metabolite of ornithine, affects the brain protein synthesis, and to that end, the effects of arginine on brain protein synthesis were compared with that of ornithine treatment in young rats. Two experiments were done on five or three groups of young rats (5-wk-old) given 0%, 0.25%, 0.5%, 0.7% arginine or 0.7% ornithine-HCl added to a 20% casein diet for 1 d (only one 3 h period) (Experiment 1), or given a diet containing 0% or 0.7% ornithine-HCl or 0.7% arginine added to a 20% casein diet (Experiment 2). The concentrations of plasma growth hormone (GH) and fractional rates of protein synthesis in the brains increased significantly with the 20% casein+0.7% arginine diet and still more with the 20% casein+0.7% ornithine diet compared with the 20% casein diet alone. In the cerebral cortex and cerebellum, the RNA activity [g protein synthesized/(g RNA•d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was also related to the fractional rate of protein synthesis in these organs. The results suggest that the treatment with arginine is likely to increase the concentrations of GH and the rate of brain protein synthesis in rats, and that the effects of arginine on brain protein synthesis and GH concentration were lower than that of ornithine. The RNA activity is at least partly related to the fractional rate of brain protein synthesis.

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

    SciTech Connect

    Scalise, F.W.

    1988-01-01

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

  16. Testosterone and Progesterone, But Not Estradiol, Stimulate Muscle Protein Synthesis in Postmenopausal Women

    PubMed Central

    Smith, Gordon I.; Yoshino, Jun; Reeds, Dominic N.; Bradley, David; Burrows, Rachel E.; Heisey, Henry D.; Moseley, Anna C.

    2014-01-01

    Context: The effect of the female sex steroids, estradiol and progesterone, on muscle protein turnover is unclear. Therefore, it is unknown whether the changes in the hormonal milieu throughout the life span in women contribute to the changes in muscle protein turnover and muscle mass (eg, age associated muscle loss). Objective: The objective of this study was to provide a comprehensive evaluation of the effect of sex hormones on muscle protein synthesis and gene expression of growth-regulatory factors [ie, myogenic differentiation 1 (MYOD1), myostatin (MSTN), follistatin (FST), and forkhead box O3 (FOXO3)]. Subjects and Design: We measured the basal rate of muscle protein synthesis and the expression of muscle growth-regulatory genes in 12 premenopausal women and four groups of postmenopausal women (n = 24 total) who were studied before and after treatment with T, estradiol, or progesterone or no intervention (control group). All women were healthy, and pre- and postmenopausal women were carefully matched on body mass, body composition, and insulin sensitivity. Results: The muscle protein fractional synthesis rate was approximately 20% faster, and MYOD1, FST, and FOXO3 mRNA expressions were approximately 40%–90% greater (all P < .05) in postmenopausal than premenopausal women. In postmenopausal women, both T and progesterone treatment increased the muscle protein fractional synthesis rate by approximately 50% (both P < .01), whereas it was not affected by estradiol treatment and was unchanged in the control group. Progesterone treatment increased MYOD1 mRNA expression (P < .05) but had no effect on MSTN, FST, and FOXO3 mRNA expression. T and estradiol treatment had no effect on skeletal muscle MYOD1, MSTN, FST, and FOXO3 mRNA expression. Conclusion: Muscle protein turnover is faster in older, postmenopausal women compared with younger, premenopausal women, but these age-related differences do not appear to be explained by the age- and menopause-related changes

  17. Continued protein synthesis at low [ATP] and [GTP] enables cell adaptation during energy limitation.

    PubMed

    Jewett, Michael C; Miller, Mark L; Chen, Yvonne; Swartz, James R

    2009-02-01

    One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K(m)(ATP), 27 +/- 4 microM; K(m)(GTP), 14 +/- 2 microM). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K(m)(ATP) is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

  18. Myocardial oxidative metabolism and protein synthesis during mechanical circulatory support by extracorporeal membrane oxygenation.

    PubMed

    Priddy, Colleen M O'Kelly; Kajimoto, Masaki; Ledee, Dolena R; Bouchard, Bertrand; Isern, Nancy; Olson, Aaron K; Des Rosiers, Christine; Portman, Michael A

    2013-02-01

    Extracorporeal membrane oxygenation (ECMO) provides essential mechanical circulatory support necessary for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur, which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative metabolism and protein synthesis. We focused on the amino acid leucine and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart 1) the fractional contribution of leucine (FcLeucine) and pyruvate to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and 2) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 h of normal circulation or ECMO) and intracoronary infusion [(13)C(6),(15)N]-L-leucine (3.7 mM) alone or with [2-(13)C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (∼40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining 1) metabolic flexibility indicated by ability to respond to pyruvate and 2) a normal or increased capacity for global protein synthesis.

  19. A Model for the Origin of Protein Synthesis as Coreplicational Scanning of Nascent RNA

    NASA Astrophysics Data System (ADS)

    Yakhnin, Alexander V.

    2007-12-01

    The origin of protein synthesis is one of the major riddles of molecular biology. It was proposed a decade ago that the ribosomal RNA evolved from an earlier RNA-replisome (a ribozyme fulfilling RNA replication) while transfer RNA (tRNA) evolved from a genomic replication origin. Applying these hypotheses, I suggest that protein synthesis arose for the purpose of segregating copy and template RNA during replication through the conventional formation of a complementary strand. Nascent RNA was scanned in 5' to 3' direction following the progress of replication. The base pairing of several tRNA-like molecules with nascent RNA released the replication intermediates trapped in duplex. Synthesis of random peptides evolved to fuel the turnover of tRNAs. Then the combination of replication-coupled peptide formation and the independent development of amino acid-specific tRNA aminoacylation resulted in template-based protein synthesis. Therefore, the positioning of tRNAs adjacent to each other developed for the purpose of replication rather than peptide synthesis. This hypothesis does not include either selection for useful peptides or specific recognition of amino acids at the initial evolution of translation. It does, however, explain a number of features of modern translation apparatus, such as the relative flexibility of genetic code, the number of proteins shared by the transcription and translation machines, the universal participation of an RNA subunit in co-translational protein secretion, ‘unscheduled translation’, and factor-independent translocation. Assistance of original ribosomes in keeping apart the nascent transcript from its template is still widely explored by modern bacteria and perhaps by other domains of life.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27721985