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Sample records for microbial protein synthesis

  1. [Influence of dietary factors on microbial protein synthesis in the rumen].

    PubMed

    Vérité, R; Durand, M; Jouany, J P

    1986-01-01

    The effect of dietary factors (usually controlled in practice) on microbial protein synthesis is reviewed using in vivo experiments. Attention is drawn on the necessity to clearly distinguish variations in microbial growth efficiency from those of intestinal flow of microbial protein and to consider simultaneously variations in feed protein degradation. In practice, the relationship between microbial protein synthesis and energy intake depends mainly on diet composition and the nature of the forage. Microbial protein flow to the intestine, relative to energy intake, is lower with high concentrate diets (when given in restricted amounts), with silages and with antibiotic supplements. This flow is increased by some forage processing (such as dehydration and alkali treatments), by natural or induced defaunation, and occasionally by increased feeding frequency (when intake is restricted) and buffer and vitamin supplements. However, with some factors such as feeding frequency and antibiotics supplementation, these variations are partly counterbalanced by reverse effects on feed protein degradation. PMID:3517986

  2. 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.

  3. Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis

    PubMed Central

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

    2013-01-01

    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. PMID:25049798

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

    PubMed

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

    2015-07-29

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more mechanistic model were compared with those of two other models, DVE1994 and NRC-2001, that are frequently used in common international feeding practice. DVE1994 predictions for intestinally digestible rumen undegradable protein (ARUP) for starchy concentrates were higher (27 vs 18 g/kg DM, p < 0.05, SEM = 1.2) than predictions by the NRC-2001, whereas there was no difference in predictions for ARUP from protein concentrates among the three models. DVE2010 and NRC-2001 had highest estimations of intestinally digestible microbial protein for starchy (92 g/kg DM in DVE2010 vs 46 g/kg DM in NRC-2001 and 67 g/kg DM in DVE1994, p < 0.05 SEM = 4) and protein concentrates (69 g/kg DM in NRC-2001 vs 31 g/kg DM in DVE1994 and 49 g/kg DM in DVE2010, p < 0.05 SEM = 4), respectively. Potential protein supplies predicted by tested models from starchy and protein concentrates are widely different, and comparable direct measurements are needed to evaluate the actual ability of different models to predict the potential protein supply to dairy cows from different feedstuffs. PMID:26118653

  5. 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). PMID:26641055

  6. 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...

  7. Effect of monensin on in vitro fermentation of silages and microbial protein synthesis.

    PubMed

    Wischer, Gerald; Boguhn, Jeannette; Steingaß, Herbert; Schollenberger, Margit; Hartung, Karin; Rodehutscord, Markus

    2013-06-01

    The objective of the study was to investigate the effects of monensin on silage fermentation and microbial net protein synthesis. In Experiment 1, monensin (0.5, 1, 2, 4, 6, or 10 µg) was added to syringes that contained 120 mg of grass silage (GS), grass silage and concentrate (GS + C), or maize silage (MS), resulting in concentrations of 4.2, 8.3, 16.7, 33.3, 50.0 and 83.3 mg monensin/kg feed. Samples were incubated for 24 h to determine the monensin concentration that resulted in the maximum reduction in methane production without effects on the total gas production. In Experiment 2, GS and GS + C were incubated in a rumen simulation technique (Rusitec) to assess the monensin effects (133 and 266 mg/kg feed) on the production of total gas, methane and volatile fatty acids (VFA), degradation of nutrients and microbial net protein synthesis. In Experiment 1, methane production was reduced without significant effects on the total gas production; the reductions were 17% (GS), 10% (GS + C) and 13% (MS) with 16.7 (GS), 50.0 (GS + C) and 33.3 (MS) mg monensin/kg feed. Monensin reduced the total gas and methane production in GS and GS + C in Experiment 2. Propionate production was enhanced by monensin, accompanied by a decrease in acetate production. Along with a reduction in crude protein (CP) degradation, monensin reduced the ammonia nitrogen concentration in the effluent of both treatments. While the protein produced by liquid-associated microbes increased with monensin, protein production by solid-associated microbes was reduced. Total microbial net protein synthesis increased in the presence of monensin. Monensin influenced the production of total gas, methane and VFA from the silages without an effect on the degradation of organic matter (OM). Different microbial fractions were affected differently by monensin supplementation. If monensin is used as a tool to reduce methane emission, the supplementation level must be carefully chosen to avoid negative effects on

  8. Effects of forage sources on rumen fermentation characteristics, performance, and microbial protein synthesis in midlactation cows.

    PubMed

    Xu, Jun; Hou, Yujie; Yang, Hongbo; Shi, Renhuang; Wu, Caixia; Huo, Yongjiu; Zhao, Guoqi

    2014-05-01

    Eight multiparous Holstein cows (632±12 kg BW; 135±16 DIM) were used in a replicated 4×4 Latin square design to evaluate the effects of forage sources on rumen fermentation characteristics, performance, and microbial protein (MCP) synthesis. The forage portion of the diets contained alfalfa hay (AH), oat hay (OH), Leymus chinensis (LC), or rice straw (RS) as the primary source of fiber. Diets were isonitrogenous and isocaloric, and cows were fed four corn silages based total mixed rations with equivalent nonfiber carbohydrate (NFC) and forage neutral detergent fiber (NDF). Dry matter intake was not affected by the source of dietary forages, ranging from 18.83 to 19.20 kg/d, consequently, milk yield was similar among diets. Because of the numerical differences in milk fat and milk protein concentrations, 4% FCM and ECM yields were unchanged (p>0.05). Mean rumen pH, NH3-N content, and concentrations of volatile fatty acids in the rumen fluid were not affected by the treatments (p>0.05). Dietary treatments did not affect the total tract apparent digestibility of dry matter, organic matter, and crude protein (p>0.05); however, digestibility of NDF and acid detergent fiber in RS diet was higher compared with AH, OH, and LC diets (p<0.05). Total purine derivative excretion was higher in cows fed AH, OH, and LC diets compared with those fed RS diet (p<0.05), consequently, estimated MCP synthesis was 124.35 g/d higher in cows fed AH diet compared with those fed RS diet (p<0.05). The results indicated that cows fed AH, OH, LC, and RS diets with an equivalent forage NDF and NFC have no unfavourable effect on the ruminal fermentation and productive parameters. PMID:25050001

  9. Effects of Forage Sources on Rumen Fermentation Characteristics, Performance, and Microbial Protein Synthesis in Midlactation Cows

    PubMed Central

    Xu, Jun; Hou, Yujie; Yang, Hongbo; Shi, Renhuang; Wu, Caixia; Huo, Yongjiu; Zhao, Guoqi

    2014-01-01

    Eight multiparous Holstein cows (632±12 kg BW; 135±16 DIM) were used in a replicated 4×4 Latin square design to evaluate the effects of forage sources on rumen fermentation characteristics, performance, and microbial protein (MCP) synthesis. The forage portion of the diets contained alfalfa hay (AH), oat hay (OH), Leymus chinensis (LC), or rice straw (RS) as the primary source of fiber. Diets were isonitrogenous and isocaloric, and cows were fed four corn silages based total mixed rations with equivalent nonfiber carbohydrate (NFC) and forage neutral detergent fiber (NDF). Dry matter intake was not affected by the source of dietary forages, ranging from 18.83 to 19.20 kg/d, consequently, milk yield was similar among diets. Because of the numerical differences in milk fat and milk protein concentrations, 4% FCM and ECM yields were unchanged (p>0.05). Mean rumen pH, NH3-N content, and concentrations of volatile fatty acids in the rumen fluid were not affected by the treatments (p>0.05). Dietary treatments did not affect the total tract apparent digestibility of dry matter, organic matter, and crude protein (p>0.05); however, digestibility of NDF and acid detergent fiber in RS diet was higher compared with AH, OH, and LC diets (p<0.05). Total purine derivative excretion was higher in cows fed AH, OH, and LC diets compared with those fed RS diet (p<0.05), consequently, estimated MCP synthesis was 124.35 g/d higher in cows fed AH diet compared with those fed RS diet (p<0.05). The results indicated that cows fed AH, OH, LC, and RS diets with an equivalent forage NDF and NFC have no unfavourable effect on the ruminal fermentation and productive parameters. PMID:25050001

  10. Effect of Plants Containing Secondary Compounds with Palm Oil on Feed Intake, Digestibility, Microbial Protein Synthesis and Microbial Population in Dairy Cows

    PubMed Central

    Anantasook, N.; Wanapat, M.; Cherdthong, A.; Gunun, P.

    2013-01-01

    The objective of this study was to determine the effect of rain tree pod meal with palm oil supplementation on feed intake, digestibility, microbial protein synthesis and microbial populations in dairy cows. Four, multiparous early-lactation Holstein-Friesian crossbred (75%) lactating dairy cows with an initial body weight (BW) of 405±40 kg and 36±8 DIM were randomly assigned to receive dietary treatments according to a 4×4 Latin square design. The four dietary treatments were un-supplementation (control), supplementation with rain tree pod meal (RPM) at 60 g/kg, supplementation with palm oil (PO) at 20 g/kg, and supplementation with RPM at 60 g/kg and PO at 20 g/kg (RPO), of total dry matter intake. The cows were offered concentrates, at a ratio of concentrate to milk production of 1:2, and chopped 30 g/kg of urea treated rice straw was fed ad libitum. The RPM contained condensed tannins and crude saponins at 88 and 141 g/kg of DM, respectively. It was found that supplementation with RPM and/or PO to dairy cows diets did not show negative effects on feed intake and ruminal pH and BUN at any times of sampling (p>0.05). However, RPM supplementation resulted in lower crude protein digestibility, NH3-N concentration and number of proteolytic bacteria. It resulted in greater allantoin absorption and microbial crude protein (p<0.05). In addition, dairy cows showed a higher efficiency of microbial N supply (EMNS) in both RPM and RPO treatments. Moreover, NDF digestibility and cellulolytic bacteria numbers were highest in RPO supplementation (p<0.05) while, supplementation with RPM and/or PO decreased the protozoa population in dairy cows. Based on this study, supplementation with RPM and/or PO in diets could improve fiber digestibility, microbial protein synthesis in terms of quantity and efficiency and microbial populations in dairy cows. PMID:25049855

  11. Effect of plants containing secondary compounds with palm oil on feed intake, digestibility, microbial protein synthesis and microbial population in dairy cows.

    PubMed

    Anantasook, N; Wanapat, M; Cherdthong, A; Gunun, P

    2013-06-01

    The objective of this study was to determine the effect of rain tree pod meal with palm oil supplementation on feed intake, digestibility, microbial protein synthesis and microbial populations in dairy cows. Four, multiparous early-lactation Holstein-Friesian crossbred (75%) lactating dairy cows with an initial body weight (BW) of 405±40 kg and 36±8 DIM were randomly assigned to receive dietary treatments according to a 4×4 Latin square design. The four dietary treatments were un-supplementation (control), supplementation with rain tree pod meal (RPM) at 60 g/kg, supplementation with palm oil (PO) at 20 g/kg, and supplementation with RPM at 60 g/kg and PO at 20 g/kg (RPO), of total dry matter intake. The cows were offered concentrates, at a ratio of concentrate to milk production of 1:2, and chopped 30 g/kg of urea treated rice straw was fed ad libitum. The RPM contained condensed tannins and crude saponins at 88 and 141 g/kg of DM, respectively. It was found that supplementation with RPM and/or PO to dairy cows diets did not show negative effects on feed intake and ruminal pH and BUN at any times of sampling (p>0.05). However, RPM supplementation resulted in lower crude protein digestibility, NH3-N concentration and number of proteolytic bacteria. It resulted in greater allantoin absorption and microbial crude protein (p<0.05). In addition, dairy cows showed a higher efficiency of microbial N supply (EMNS) in both RPM and RPO treatments. Moreover, NDF digestibility and cellulolytic bacteria numbers were highest in RPO supplementation (p<0.05) while, supplementation with RPM and/or PO decreased the protozoa population in dairy cows. Based on this study, supplementation with RPM and/or PO in diets could improve fiber digestibility, microbial protein synthesis in terms of quantity and efficiency and microbial populations in dairy cows. PMID:25049855

  12. 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

  13. 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...

  14. Effects of BioChlor and Fermenten on microbial protein synthesis in continuous culture fermenters.

    PubMed

    Lean, I J; Webster, T K Miller; Hoover, W; Chalupa, W; Sniffen, C J; Evans, E; Block, E; Rabiee, A R

    2005-07-01

    Meta analysis models were constructed from a data-set of 15 continuous culture fermenter trials and 118 observations on studies with either BioChlor (n = 23 observations) or Fermenten (n = 95) included at 10 and 3%, respectively, of dietary dry matter (DM) to evaluate effects of the ingredients BioChlor and Fermenten (B/F) on rumen function. Digestibility of crude protein was significantly increased by 11% with B/F treatment. This was reflected in significant increases in digestibility of DM and organic matter (OM) by 3.6 and 7.9%, respectively. Increased amounts of sugar in the diet in the presence of B/F tended to reduce digestibility of non-structural carbohydrates (NSC); however, the net effect on NSC digestion was small. There was no effect of treatment on most individual volatile fatty acids (VFA) or total VFA production. Propionate production, however, was significantly reduced in treated fermenters. The main effect of B/F as well as of starch and soluble fiber when combined with the treatment was to increase propionate production; however, the interaction between B/F treatment and sugar decreased propionate production markedly, resulting in a net decrease. The acetate-to-propionate ratio increased by 6% with B/F, largely as a result of the decrease in propionate. Production of nonammonia nitrogen was 1% less in B/F-treated fermenters, and interactions between treatment and starch, sugar, or soluble fiber were significant. Treated fermenters produced 15.7% more microbial nitrogen, in association with a significant 37% increase in rumen protein digestion. Interactions between treatment and starch, soluble fiber, or sugar influenced these results. The interaction of B/F and sugar resulted in a decrease in undegradable protein N and an increase in microbial nitrogen production. Ammonia nitrogen concentrations were increased by 24.6% in treated fermenters. Efficiency of microbial nitrogen production from DM, OM, or carbohydrate was significantly increased by B

  15. 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. PMID:26885988

  16. Predicting microbial protein synthesis in beef cattle: relationship to intakes of total digestible nutrients and crude protein.

    PubMed

    Galyean, M L; Tedeschi, L O

    2014-11-01

    Prediction of microbial CP (MCP) synthesis in the rumen is an integral part of the MP system. For the NRC beef model, MCP is calculated as 0.13 multiplied by TDN intake (TDNI), with adjustment for physically effective NDF (peNDF) concentrations less than 20%. Despite its application for nearly 2 decades, MCP predictions using this approach have not been extensively evaluated. We assembled a database of 285 treatment means from 66 published papers using beef cattle and dairy or dairy × beef crossbred steers, fed diets with a wide range of TDN, CP, and ether extract (EE) concentrations, in which MCP synthesis was measured. Fat-free TDN (FFTDN) concentration was calculated by subtracting 2.25 × percent EE from the TDN concentration. Based on initial model selection procedures indicating that DMI and concentrations of TDN, FFTDN, and CP were significantly (P < 0.04) related to MCP synthesis, linear and quadratic effects of TDNI and FFTDN intake (FFTDNI) and CP intake (CPI) were considered as potential independent variables. Mixed model regression methods were used to fit 1-, 2-, and 3-independent-variable models based on either TDNI or FFTDNI (e.g., TDNI only, TDNI and CPI, and TDNI, CPI, and the quadratic effect of TDNI; or FFTDNI only, FFTDNI and CPI, and FFTDNI, CPI, and the quadratic effect of FFTDNI). True ruminal OM digested (TROMD; g/d) was highly related (r(2) = 0.84 using citation-adjusted data) to MCP synthesis. Similarly, both TDNI and FFTDNI were highly related to citation-adjusted TROMD (r(2) > 0.96) and MCP synthesis (r(2) > 0.89). Models with FFTDNI were slightly more precise with slightly smaller prediction errors than those with TDNI. Randomly dividing the citations into Development (60%) and Evaluation (40%) data sets indicated that models such as those derived from the overall database accounted for 46 to 56% of the variation in MCP synthesis, with neither mean nor linear bias (P ≥ 0.26). In contrast, calculating MCP as 0.13 × TDNI, with or

  17. 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. PMID:26254527

  18. 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. PMID:25244259

  19. Effects of Physical Form and Urea Treatment of Rice Straw on Rumen Fermentation, Microbial Protein Synthesis and Nutrient Digestibility in Dairy Steers

    PubMed Central

    Gunun, P.; Wanapat, M.; Anantasook, N.

    2013-01-01

    This study was designed to determine the effect of physical form and urea treatment of rice straw on rumen fermentation, microbial protein synthesis and nutrient digestibility. Four rumen-fistulated dairy steers were randomly assigned according to a 2 (2 factorial arrangement in a 4 (4 Latin square design to receive four dietary treatments. Factor A was roughage source: untreated rice straw (RS) and urea-treated (3%) rice straw (UTRS), and factor B was type of physical form of rice straw: long form rice straw (LFR) and chopped (4 cm) rice straw (CHR). The steers were offered the concentrate at 0.5% body weight (BW) /d and rice straw was fed ad libitum. DM intake and nutrient digestibility were increased (p<0.05) by urea treatment. Ruminal pH were decreased (p<0.05) in UTRS fed group, while ruminal ammonia nitrogen (NH3-N) and blood urea nitrogen (BUN) were increased (p<0.01) by urea treatment. Total volatile fatty acid (VFA) concentrations increased (p<0.01) when steers were fed UTRS. Furthermore, VFA concentrations were not altered by treatments (p>0.05), except propionic acid (C3) was increased (p<0.05) in UTRS fed group. Nitrogen (N) balance was affected by urea treatment (p<0.05). Microbial protein synthesis (MCP) synthesis were greater by UTRS and CHR group (p<0.05). The efficiency of microbial N synthesis was greater for UTRS than for RS (p<0.05). From these results, it can be concluded that using the long form combined with urea treatment of rice straw improved feed intake, digestibility, rumen fermentation and efficiency of microbial N synthesis in crossbred dairy steers. PMID:25049759

  20. 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

  1. Effect of carbohydrate source and cottonseed meal level in the concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes.

    PubMed

    Wanapat, M; Pilajun, R; Polyorach, S; Cherdthong, A; Khejornsart, P; Rowlinson, P

    2013-07-01

    The objective of this study was to investigate the effect of carbohydrate source and cottonseed meal level in the concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes. Four, 4-yr old rumen fistulated swamp buffaloes were randomly assigned to receive four dietary treatments according to a 2×2 factorial arrangement in a 4×4 Latin square design. Factor A was carbohydrate source; cassava chip (CC) and CC+rice bran at a ratio 3:1 (CR3:1), and factor B was level of cottonseed meal (CM); 109 g CP/kg (LCM) and 328 g CP/kg (HCM) in isonitrogenous diets (490 g CP/kg). Buffaloes received urea-treated rice straw ad libitum and supplemented with 5 g concentrate/kg BW. It was found that carbohydrate source did not affect feed intake, nutrient intake, digested nutrients, nutrient digestibility, ammonia nitrogen concentration, fungi and bacterial populations, or microbial protein synthesis (p>0.05). Ruminal pH at 6 h after feeding and the population of protozoa at 4 h after feeding were higher when buffalo were fed with CC than in the CR3:1 treatment (p<0.05). Buffalo fed with HCM had a lower roughage intake, nutrient intake, population of total viable and cellulolytic bacteria and microbial nitrogen supply than the LCM fed group (p<0.05). However, nutrient digestibility, ruminal pH, ammonia concentration, population of protozoa and fungi, and efficiency of microbial protein synthesis were not affected by cottonseed meal levels (p>0.05). Based on this experiment, concentrate with a low level of cottonseed meal could be fed with cassava chips as an energy source in swamp buffalo receiving rice straw. PMID:25049873

  2. Effect of supplementation of mustard oil cake on intake, digestibility and microbial protein synthesis of cattle in a straw-based diet in Bangladesh.

    PubMed

    Khandaker, Zahirul Haque; Uddin, Mohammad Mohi; Sultana, Nadira; Peters, Kurt J

    2012-04-01

    The objective of this study was to analyse the effects of different levels of rumen-degradable protein (RDP) on intake, digestibility and microbial protein synthesis by supplementing mustard oil cake (MOC) on rice straw-based diet of cattle (Bos indicus) in Bangladesh. A 4 × 4 Latin square design was applied. Four diets having constant energy (7.0 MJ/kg of dry matter (DM)) with varying levels of RDP (M(0) = 4.1 g/MJ (control), M(1) = 6.3 g/MJ, M(2) = 8.3 g/MJ and M(3) = 12.4 g/MJ of metabolizable energy (ME)) were received by each animal for a period of 28 days. A metabolism trial was conducted for 7 days. Results indicate that with increasing levels of RDP, crude protein (CP) and RDP intake increased significantly (P < 0.01). The significant (P < 0.01) increase in digestibility values are obtained for DM, organic matter, CP and digestible organic matter in the rumen. The digestibility of neutral detergent fibre and acid detergent fibre was also increased significantly (P < 0.05). The total nitrogen (N), ammonia-N and total volatile fatty acids increase significantly (P < 0.01) while the rumen pH increased from M(0) to M(2) and decreased thereafter. The efficiency microbial N intake increased significantly (P < 0.01) but showed a curvilinear response with higher RDP level (12.40 g/RDP/MJ ME). This study concludes that supplementation of RDP from MOC enhances the intake, digestibility and microbial protein synthesis which ultimately increases utilization of low-quality feed resources that can be used for developing cost-effective feeding systems on a straw-based diet in tropical regions. PMID:21915618

  3. 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

  4. Effect of Carbohydrate Sources and Levels of Cotton Seed Meal in Concentrate on Feed Intake, Nutrient Digestibility, Rumen Fermentation and Microbial Protein Synthesis in Young Dairy Bulls

    PubMed Central

    Wanapat, M.; Anantasook, N.; Rowlinson, P.; Pilajun, R.; Gunun, P.

    2013-01-01

    The objective of this study was to investigate the effect of levels of cottonseed meal with various carbohydrate sources in concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in dairy bulls. Four, 6 months old dairy bulls were randomly assigned to receive four dietary treatments according to a 2×2 factorial arrangement in a 4×4 Latin square design. Factor A was carbohydrate source; cassava chip (CC) and cassava chip+rice bran in the ratio of 3:1 (CR3:1), and factor B was cotton seed meal levels in the concentrate; 109 g CP/kg (LCM) and 328 g CP/kg (HCM) at similar overall CP levels (490 g CP/kg). Bulls received urea-lime treated rice straw ad libitum and were supplemented with 10 g of concentrate/kg BW. It was found that carbohydrate source and level of cotton seed meal did not have significant effects on ruminal pH, ammonia nitrogen concentration, microbial protein synthesis or feed intake. Animals which received CC showed significantly higher BUN concentration, ruminal propionic acid and butyric acid proportions, while dry matter, organic matter digestibility, populations of total viable bacteria and proteolytic bacteria were lower than those in the CR3:1 treatment. The concentration of total volatile fatty acids was higher in HCM than LCM treatments, while the concentration of butyric acid was higher in LCM than HCM treatments. The population of proteolytic bacteria with the LCM treatments was higher than the HCM treatments; however other bacteria groups were similar among the different levels of cotton seed meal. Bulls which received LCM had higher protein digestibility than those receiving HCM. Therefore, using high levels of cassava chip and cotton seed meal might positively impact on energy and nitrogen balance for the microbial population in the rumen of the young dairy bull. PMID:25049819

  5. 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

  6. 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

  7. 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

  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. 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

  10. Synthesis of Lipidated Proteins.

    PubMed

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

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

  11. 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

  12. Water Stress and Protein Synthesis

    PubMed Central

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

    1975-01-01

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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed Central

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

    1966-01-01

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

  19. Less is more, greener microbial synthesis of silver nanoparticles.

    PubMed

    Liu, Lihong; Liu, Tingting; Tade, Moses; Wang, Shaobin; Li, Xinyong; Liu, Shaomin

    2014-12-01

    Microbial nano-synthesis has been well established as a green method for the sustainable development of nanotechnology. However, the mechanism of this biotechnology has to be reconsidered with the increasing realization that microorganism culture broth plays a vital role during the synthesis, which may obviate the dependence on microbes. Here, we demonstrate that silver nanoparticles (AgNPs) could be synthesized in several types of microorganism culture broth (an aqueous solution abundant in peptone, yeast extract from Saccharomyces cerevisiae, dextrose and other reducing and stabilizing agents) without any specific living microbe involvement. Light and high pH values of broth were identified as two critical factors in ensuring pure AgNPs formation. In broths containing NaCl at high concentration (0.5wt%), silver chloride was identified as the major intermediate and could be converted to AgNPs via one-pot photoreduction. Our broth alone strategy dramatically simplifies the conventional microbial nano-synthesis process by cutting the use of microorganisms and thus provides a more eco-friendly way for nano-Ag preparation. The fundamental understanding of the microbial synthesis mechanisms and implementing of complete green methods to fabricate technologically important nanomaterials will be further promoted by this study. PMID:25442949

  20. Microbially induced synthesis of cubic and hexagonal selenium nanoparticles.

    PubMed

    Park, Bitna; Kang, Serku; Moon, Wonjin; Roh, Yul

    2013-03-01

    Nanobiotechnology represents an economic alternative for chemical and physical methods of nanoparticles formation. The objectives of this study were to synthesize selenium nanoparticles by microbial processes using anaerobic metal-reducing bacteria as well as to characterize mineralogical properties of the nanoparticles. The selenium nanoparticles were about 200 nm in size and ball shaped. Microbial processes for elemental selenium synthesis may be useful for recovery of natural selenate in the natural environments and immobilization of selenium isotope in the high level nuclear waste disposal sites. PMID:23755605

  1. 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

  2. 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

  3. 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

  4. 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

  5. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

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

    1998-01-01

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

  6. Quest for the chemical synthesis of proteins.

    PubMed

    Engelhard, Martin

    2016-05-01

    The chemical synthesis of proteins has been the wish of chemists since the early 19th century. There were decisive methodological steps necessary to accomplish this aim. Cornerstones were the introduction of the Z-protecting group of Bergmann and Zervas, the development of Solid-phase Peptide Synthesis of Merrifield, and the establishment of Native Chemical Ligation by Kent. Chemical synthesis of proteins has now become generally applicable technique for the synthesis of proteins with tailor made properties which can be applied not only in vitro but also in vivo .Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27114253

  7. Storage Protein Synthesis in Maize

    PubMed Central

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

    1976-01-01

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

  8. T-2 mycotoxin inhibits mitochondrial protein synthesis

    SciTech Connect

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

    1988-01-01

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

  9. Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli.

    PubMed

    Kim, Eun-Mi; Eom, Jin-Hee; Um, Youngsoon; Kim, Yunje; Woo, Han Min

    2015-05-13

    Myrcene, a monoterpene (C10), has gathered attention as a starting material for high-value compounds, such as geraniol/linalool and (-)-menthol. Metabolic engineering has been successfully applied to produce monoterpenes, such as pinene and limonene, at high levels in microbial hosts. However, microbial synthesis of myrcene has not yet been reported. Thus, we metabolically engineered Escherichia coli for production of myrcene by introducing a heterologous mevalonate pathway and overexpressing tailoring enzymes, such as geranyl diphosphate synthase (GPPS) and myrcene synthase (MS). Although MSs have broad ranges of functionality for producing various monoterpenes, our engineered E. coli strains harboring MS from Quercus ilex L. produced only myrcene (1.67 ± 0.029 mg/L). Subsequent engineering resulted in higher production of myrcene by optimizing the levels of GPPS in amino-acid-enriched (EZ-rich) defined medium, where glycerol as a carbon source was used. The production level of myrcene (58.19 ± 12.13 mg/L) was enhanced by 34-fold using in situ two-phase extraction to eliminate cellular toxicity and the evaporation of myrcene. PMID:25909988

  10. In vitro ruminal degradation and synthesis of protein on fractions extracted from alfalfa hay and silage.

    PubMed

    Peltekova, V D; Broderick, G A

    1996-04-01

    Net release of degraded N as NH3 and total AA plus microbial protein synthesis, quantified from incorporation of 15NH3 into microbial protein, was used to estimate the rate and extent of in vitro degradation of protein fractions isolated from alfalfa hay and silage. Seven proteins (casein, alfalfa hay, alfalfa silage, extracts from alfalfa hay and silage, and residues from alfalfa hay and silage) were studied. Results from (NH4)2SO4 and SDS-PAGE fractionations suggested that soluble proteins in alfalfa hay and silage differed in susceptibility to proteolytic attack. Although the net release of NH3 plus total AA N from alfalfa silage and alfalfa silage extract was twofold greater than that from alfalfa hay and alfalfa hay extract, net microbial protein synthesis on alfalfa hay and alfalfa hay extract was 33 and 43% greater. Despite greater NPN content in alfalfa silage, protein degradation rate and estimated escape were similar for intact alfalfa hay (0.103/h and 43%) and silage (0.067/h and 43%). This result might be explained by the less efficient microbial utilization of silage NPN, greater protozoal numbers on hay, greater soluble true protein in hay, or differences in molecular mass and stability of soluble proteins in hay versus silage. Use of a two-compartment model, based on water-soluble and insoluble CP fractions assumed to pass with the liquid and solid phases, respectively, yielded RUP estimates for alfalfa hay and silage that were similar to NRC estimates. PMID:8744226

  11. Microbial synthesis of Flower-shaped gold nanoparticles.

    PubMed

    Singh, Priyanka; Kim, Yeon Ju; Wang, Chao; Mathiyalagan, Ramya; Yang, Deok Chun

    2016-09-01

    The shape of nanoparticles has been recognized as an important attribute that determines their applicability in various fields. The flower shape (F-shape) has been considered and is being focused on, because of its enhanced properties when compared to the properties of the spherical shape. The present study proposed the microbial synthesis of F-shaped gold nanoparticles within 48 h using the Bhargavaea indica DC1 strain. The F-shaped gold nanoparticles were synthesized extracellularly by the reduction of auric acid in the culture supernatant of B. indica DC1. The shape, size, purity, and crystalline nature of F-shaped gold nanoparticles were revealed by various instrumental techniques including UV-Vis, FE-TEM, EDX, elemental mapping, XRD, and DLS. The UV-Vis absorbance showed a maximum peak at 536 nm. FE-TEM revealed the F-shaped structure of nanoparticles. The EDX peak obtained at 2.3 keV indicated the purity. The peaks obtained on XRD analysis corresponded to the crystalline nature of the gold nanoparticles. In addition, the results of elemental mapping indicated the maximum distribution of gold elements in the nanoproduct obtained. Particle size analysis revealed that the average diameter of the F-shaped gold nanoparticles was 106 nm, with a polydispersity index (PDI) of 0.178. Thus, the methodology developed for the synthesis of F-shaped gold nanoparticles is completely green and economical. PMID:25943137

  12. Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that eli...

  13. Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that el...

  14. 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.

  15. 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.

  16. Pathogenesis-related proteins protect extrafloral nectar from microbial infestation.

    PubMed

    González-Teuber, Marcia; Eilmus, Sascha; Muck, Alexander; Svatos, Ales; Heil, Martin

    2009-05-01

    Plants in more than 300 genera produce extrafloral nectar (EFN) to attract carnivores as a means of indirect defence against herbivores. As EFN is secreted at nectaries that are not physically protected from the environment, and contains carbohydrates and amino acids, EFN must be protected from infestation by micro-organisms. We investigated the proteins and anti-microbial activity in the EFN of two Central American Acacia myrmecophytes (A. cornigera and A. hindsii) and two related non-myrmecophytes (A. farnesiana and Prosopis juliflora). Acacia myrmecophytes secrete EFN constitutively at high rates to nourish the ants inhabiting these plants as symbiotic mutualists, while non-myrmecophytes secrete EFN only in response to herbivore damage to attract non-symbiotic ants. Thus, the quality and anti-microbial protection of the EFN secreted by these two types of plants were likely to differ. Indeed, myrmecophyte EFN contained significantly more proteins than the EFN of non-myrmecophytes, and was protected effectively from microbial infestation. We found activity for three classes of pathogenesis-related (PR) enzymes: chitinase, beta-1,3-glucanase and peroxidase. Chitinases and beta-1,3-glucanases were significantly more active in myrmecophyte EFN, and chitinase at the concentrations found in myrmecophyte EFN significantly inhibited yeast growth. Of the 52 proteins found in A. cornigera EFN, 28 were annotated using nanoLC-MS/MS data, indicating that chitinases and glucanases contribute more than 50% of the total protein content in the EFN of this myrmecophyte. Our study demonstrates that PR enzymes play an important role in protecting EFN from microbial infestation. PMID:19143997

  17. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

    PubMed Central

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

    2012-01-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion. PMID:22453275

  18. 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.

  19. Chlorolissoclimides: New inhibitors of eukaryotic protein synthesis

    PubMed Central

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

    2006-01-01

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

  20. EFFECT OF ANTIBIOTICS AND INHIBITORS ON M PROTEIN SYNTHESIS

    PubMed Central

    Brock, Thomas D.

    1963-01-01

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

  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. Postnatal ontogeny of skeletal muscle protein synthesis in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Postnatal ontogeny of skeletal muscle protein synthesis in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Cumulative Muscle Protein Synthesis and Protein Intake Requirements.

    PubMed

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

    2016-07-17

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

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

    PubMed Central

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

    1968-01-01

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

  6. 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.

  7. 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...

  8. 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

  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. 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

  11. 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

  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. 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

  14. 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.

  15. Metaproteomics reveals functional shifts in microbial and human proteins during a preterm infant gut colonization case.

    PubMed

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

    2015-10-01

    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. To this end, 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 increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data. More specifically, the function of the microbial community initially involved biomass growth, protein production, and lipid metabolism, and then switched to more complex metabolic functions, such as carbohydrate metabolism, once the community stabilized and matured. 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. Likewise, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. This study provides the first snapshot of coordinated human and microbial protein expression in a preterm infant's gut during early development. PMID:26077811

  16. 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

  17. Organization and Regulation of Mitochondrial Protein Synthesis.

    PubMed

    Ott, Martin; Amunts, Alexey; Brown, Alan

    2016-06-01

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

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

    PubMed

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

    2012-01-01

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

  19. 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.

  20. 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

  1. 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…

  2. 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.

  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. 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

  5. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones.

    PubMed

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

    2006-06-01

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

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

    PubMed

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

    2016-07-01

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

  7. 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

  8. 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

  9. Electro-stimulated microbial factory for value added product synthesis.

    PubMed

    Roy, Shantonu; Schievano, Andrea; Pant, Deepak

    2016-08-01

    Interplay of charge between bacteria and electrode has led to emergence of bioelectrochemical systems which leads to applications such as production of electricity, wastewater treatment, bioremediation and production of value added products. Many electroactive bacteria have been identified that have unique external electron transport systems. Coupling of electron transport with carbon metabolism has opened a new approach of carbon dioxide sequestration. The electron transport mechanism involves various cellular and sub cellular molecules. The outer membrane cytochromes, Mtr-complex and Ech-complex are few key molecules involved in electron transport in many electrogenic bacteria. Few cytochrome independent acetogenic electroactive bacteria were also discovered using Rnf complex to transport electrons. For improved productivity, an efficient bioreactor design is mandatory. It should encompass all certain critical issues such as microbial cell retention, charge dissipation, separators and simultaneous product recovery. PMID:27034155

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. 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.

  12. Benzene-free synthesis of catechol: interfacing microbial and chemical catalysis.

    PubMed

    Li, Wensheng; Xie, Dongming; Frost, J W

    2005-03-01

    The toxicity of aromatics frequently limits the yields of their microbial synthesis. For example, the 5% yield of catechol synthesized from glucose by Escherichia coli WN1/pWL1.290A under fermentor-controlled conditions reflects catechol's microbial toxicity. Use of in situ resin-based extraction to reduce catechol's concentration in culture medium and thereby its microbial toxicity during its synthesis from glucose by E. coli WN1/pWL1.290A led to a 7% yield of catechol. Interfacing microbial with chemical synthesis was then explored where glucose was microbially converted into a nontoxic intermediate followed by chemical conversion of this intermediate into catechol. Intermediates examined include 3-dehydroquinate, 3-dehydroshikimate, and protocatechuate. 3-Dehydroquinate and 3-dehydroshikimate synthesized, respectively, by E. coli QP1.1/pJY1.216A and E. coli KL3/pJY1.216A from glucose were extracted and then reacted in water heated at 290 degrees C to afford catechol in overall yields from glucose of 10% and 26%, respectively. The problematic extraction of these catechol precursors from culture medium was subsequently circumvented by high-yielding chemical dehydration of 3-dehydroquinate and 3-dehydroshikimate in culture medium followed by extraction of the resulting protocatechuate. After reaction of protocatechuate in water heated at 290 degrees C, the overall yields of catechol synthesized from glucose via chemical dehydration of 3-dehydroquinate and chemical dehydration of 3-dehydroshikimate were, respectively, 25% and 30%. Direct synthesis of protocatechuate from glucose using E. coli KL3/pWL2.46B followed by its extraction and chemical decarboxylation in water gave a 24% overall yield of catechol from glucose. In situ resin-based extraction of protocatechaute synthesized by E. coli KL3/pWL2.46B followed by chemical decarboxylation of this catechol percursor was then examined. This employment of both strategies for dealing with the microbial toxicity of

  13. 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

  14. [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

  15. 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. PMID:22010831

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

    PubMed

    Arnsburg, Kristin; Kirstein-Miles, Janine

    2014-02-01

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

  17. Microbial production of levansucrase for synthesis of fructooligosaccharides and levan.

    PubMed

    Belghith, Karima Srih; Dahech, Imen; Belghith, Hafedh; Mejdoub, Hafedh

    2012-03-01

    A newly isolated thermophilic bacterial strain from Tunisian thermal source was identified as Bacillus sp. and was selected for its ability to produce extracellular levansucrase. Following the optimization of carbon source, nitrogen source, temperature and initial pH of the growth medium in submerged liquid cultures. In fact, sucrose was found to be a good inducer of levansucrase enzymes. The optimal temperature and pH of the levansucrase were 50°C and 6.5, respectively and its activity increased four folds in the presence of 50mM Fe(2+). This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for more than 1h at pH 6.5. The half-life of the enzyme was 1h at 90°C. Crude enzyme of Bacillus sp. rich in levansucrase was established for the synthesis of fructooligosaccharides and levan. Bacillus sp. could therefore be considered as a satisfactory and promising producer of thermostable levansucrases. Contrary to other levansucrases, the one presented in the current study was able to produce high levels of levan with high molecular weight at 50°C and having an important effect as a hypoglycemic agent which was demonstrated in our previous publications (Dahech et al., 2011 [25]) and as a hypo-cholesterolemic agent which will be investigated in further research. PMID:22234294

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

    PubMed

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

    2014-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. 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

  1. Microbial synthesis of hyaluronan and chitin: New approaches.

    PubMed

    Yamada, Takashi; Kawasaki, Takeru

    2005-06-01

    Hyaluronan (HA) is an important structural element in the vitreous humor of the eye, synovial fluid, and skin of vertebrates. Moreover, HA interacts with proteins such as CD44, RHAMM, and fibrinogen, thereby influencing many natural processes such as angiogenesis, cancer, cell motility, wound healing, and cell adhesion. Reflecting such a variety of functions, HA has attracted attention from a wide range of application fields such as medicine (including surgery), cosmetics, and health foods. Traditionally HA was extracted from rooster combs, but nowadays is produced by the fermentation of streptococci. At present, quality issues such as purity and molecular weight distribution, rather than quantity, have been the focus of strain and process development in HA production. To meet ever-increasing public demand, novel systems that can yield sufficient amounts of high-quality of HA and related materials are required. PMID:16233827

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-01-01

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

  4. 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

  5. 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.

  6. Temperature-Regulated Protein Synthesis by Leptospira interrogans

    PubMed Central

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

    2001-01-01

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

  7. 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.

  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. PMID:26115345

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

    SciTech Connect

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

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  10. 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. PMID:25674807

  11. 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

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

    PubMed

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

    2007-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  14. 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.

  15. 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

  16. Monitoring protein synthesis in single live cancer cells.

    PubMed

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

    2016-05-16

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

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

    PubMed Central

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

    1979-01-01

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

  18. 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

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

    PubMed

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

    2015-07-01

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms. PMID:26054976

  20. 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

  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. The influence of an increased cobalt supply on ruminal parameters and microbial vitamin B12 synthesis in the rumen of dairy cows.

    PubMed

    Stemme, Kirsten; Lebzien, Peter; Flachowsky, Gerhard; Scholz, Henner

    2008-06-01

    The aim of the study was to examine the effects of an elevated dietary cobalt supply to dairy cows on rumen fermentation parameters and microbial vitamin B12 synthesis in the rumen. Five lactating dairy cows fitted with a ruminal and a duodenal cannula were subsequently fed either a ration containing only the native cobalt content (0.17 mg Co/ kg DM) or a ration supplemented with cobalt sulphate (0.29 mg Co/kg DM). The pH-value, the ammonia concentration as well as the concentration and the molar proportions of short chain fatty acids in the rumen were not significantly influenced by feeding the ration with the higher cobalt content. While there was no difference in microbial protein flow, the cobalamin flow at the duodenum was significantly elevated in supplemented animals (3.67 +/- 0.69 vs. 8.63 +/- 2.22 mg B12/d). The efficiency of cobalt utilisation for ruminal vitamin B12 synthesis was calculated to be 7.1 +/- 1.3% for the unsupplemented and 9.5 +/- 2.4% for the supplemented ration. Further investigation has to prove if there are any benefits for cows resulting from the elevated cobalamin synthesis measured, caused by feeding higher amounts of dietary cobalt. PMID:18610536

  3. 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

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

    SciTech Connect

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

    1986-03-30

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

  5. His6 tag-assisted chemical protein synthesis

    NASA Astrophysics Data System (ADS)

    Bang, Duhee; Kent, Stephen B. H.

    2005-04-01

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

  6. 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.

  7. 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.

  8. 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.

  9. 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

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

    PubMed

    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

    2015-07-16

    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

  11. Dietary Protein and Cellulose Effects on Chemical and Microbial Characteristics of Swine Feces and Stored Manure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to investigate the effects of dietary crude protein (14.5 or 12.0%) and cellulose (8.7 or 2.5%) levels on chemical and microbial composition of feces and stored manure after 2 and 8 weeks of storage. Twenty-two pigs were fed twice daily with urine and feces collect...

  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. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production.

    PubMed

    Mozdzierz, Nicholas J; Love, Kerry R; Lee, Kevin S; Lee, Harry L T; Shah, Kartik A; Ram, Rajeev J; Love, J Christopher

    2015-07-21

    We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing. PMID:26055071

  14. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production

    PubMed Central

    Mozdzierz, Nicholas J.; Love, Kerry R.; Lee, Kevin S.; Lee, Harry L. T.; Shah, Kartik A.; Ram, Rajeev J.

    2015-01-01

    We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing. PMID:26055071

  15. 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

  16. DNA Nanoparticles for Improved Protein Synthesis In Vitro

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    MacBeth, K J; Lee, C A

    1993-01-01

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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

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

    PubMed

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

    2008-11-01

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

  6. 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

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

    PubMed

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

    2016-01-01

    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

  8. 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.

  9. Crosstalk between cGAS DNA sensor and Beclin-1 autophagy protein shapes innate anti-microbial immune responses

    PubMed Central

    Liang, Qiming; Seo, Gil Ju; Choi, Youn Jung; Kwak, Mi-Jeong; Ge, Jianning; Rodgers, Mary A; Shi, Mude; Leslie, Benjamin J.; Hopfner, Karl-Peter; Ha, Taekjip; Oh, Byung-Ha; Jung, Jae U.

    2014-01-01

    Robust immune responses are essential for eliminating pathogens, but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase, or cGAS, produces the second messenger cGAMP to initiate the STING pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double stranded (ds)DNA stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNAs to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNAs. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced anti-microbial immune responses. PMID:24528868

  10. Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops.

    PubMed

    Raybould, Alan; Kilby, Peter; Graser, Gerson

    2013-04-01

    Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence. PMID:23065372

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    1981-01-25

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

  13. 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

  14. Microbial Synthesis of Iron Sulfide (FeS) and Iron Carbonate (FeCO3) Nanoparticles.

    PubMed

    Kim, Yumi; Lee, Yuri; Roh, Yul

    2015-08-01

    This study examined mineral transformations during anoxic bioreduction of iron hydroxide and iron oxyhydroxysulfate found in acid mine drainage (AMD) into iron sulfide (FeS) and siderite (FeCO3) nanoparticles. Glucose (10 mM) was inoculated into AMD to stimulate indigenous bacterial growth for bioreduction of Fe(III)-containing minerals. Changes in microbial, geochemical, and mineralogical characteristics were monitored via 16S rRNA, XRD, SEM-EDX, TEM-EDX, ICP-AES, and IC analyses. The AMD was found to be rich in elements, including Fe, Al, Mn, Na, and S (SO4), and had a pH of 5.2. The mineral contents mainly consisted of Fe(III)-containing minerals, such as schwertmannite [Fe8O8 (OH)8-2x(SO4)x · nH2O] and akaganeite [β-FeO(OH)]. During anoxic bioreduction of AMD, the Fe(III)-containing minerals were transformed by indigenous iron-reducing bacteria (e.g., Geobactersp.) into Fe(II)-containing minerals, such as iron sulfide (FeS) and iron carbonate, siderite (FeCO3), within 3-4 days. The microbially-formed iron sulfide (FeS) and siderite (FeCO3) were of 40-60 nm and 10 nm-3 µm in size, respectively. These results not only show that indigenous iron-reducing bacteria in AMD can aid or accelerate formation of Fe(II)-containing minerals when under anoxic environments, but can also offer a simple method for microbial synthesis of nano-sized Fe(II)-containing minerals that can be used as catalysts for environmental remediation by recycling AMD. PMID:26369153

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. 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)

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

    PubMed Central

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

    2013-01-01

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

  18. HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot

    PubMed Central

    Lima, Tania; Auchincloss, Andrea H.; Coudert, Elisabeth; Keller, Guillaume; Michoud, Karine; Rivoire, Catherine; Bulliard, Virginie; de Castro, Edouard; Lachaize, Corinne; Baratin, Delphine; Phan, Isabelle; Bougueleret, Lydie; Bairoch, Amos

    2009-01-01

    The growth in the number of completely sequenced microbial genomes (bacterial and archaeal) has generated a need for a procedure that provides UniProtKB/Swiss-Prot-quality annotation to as many protein sequences as possible. We have devised a semi-automated system, HAMAP (High-quality Automated and Manual Annotation of microbial Proteomes), that uses manually built annotation templates for protein families to propagate annotation to all members of manually defined protein families, using very strict criteria. The HAMAP system is composed of two databases, the proteome database and the family database, and of an automatic annotation pipeline. The proteome database comprises biological and sequence information for each completely sequenced microbial proteome, and it offers several tools for CDS searches, BLAST options and retrieval of specific sets of proteins. The family database currently comprises more than 1500 manually curated protein families and their annotation templates that are used to annotate proteins that belong to one of the HAMAP families. On the HAMAP website, individual sequences as well as whole genomes can be scanned against all HAMAP families. The system provides warnings for the absence of conserved amino acid residues, unusual sequence length, etc. Thanks to the implementation of HAMAP, more than 200 000 microbial proteins have been fully annotated in UniProtKB/Swiss-Prot (HAMAP website: http://www.expasy.org/sprot/hamap). PMID:18849571

  19. HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot.

    PubMed

    Lima, Tania; Auchincloss, Andrea H; Coudert, Elisabeth; Keller, Guillaume; Michoud, Karine; Rivoire, Catherine; Bulliard, Virginie; de Castro, Edouard; Lachaize, Corinne; Baratin, Delphine; Phan, Isabelle; Bougueleret, Lydie; Bairoch, Amos

    2009-01-01

    The growth in the number of completely sequenced microbial genomes (bacterial and archaeal) has generated a need for a procedure that provides UniProtKB/Swiss-Prot-quality annotation to as many protein sequences as possible. We have devised a semi-automated system, HAMAP (High-quality Automated and Manual Annotation of microbial Proteomes), that uses manually built annotation templates for protein families to propagate annotation to all members of manually defined protein families, using very strict criteria. The HAMAP system is composed of two databases, the proteome database and the family database, and of an automatic annotation pipeline. The proteome database comprises biological and sequence information for each completely sequenced microbial proteome, and it offers several tools for CDS searches, BLAST options and retrieval of specific sets of proteins. The family database currently comprises more than 1500 manually curated protein families and their annotation templates that are used to annotate proteins that belong to one of the HAMAP families. On the HAMAP website, individual sequences as well as whole genomes can be scanned against all HAMAP families. The system provides warnings for the absence of conserved amino acid residues, unusual sequence length, etc. Thanks to the implementation of HAMAP, more than 200,000 microbial proteins have been fully annotated in UniProtKB/Swiss-Prot (HAMAP website: http://www.expasy.org/sprot/hamap). PMID:18849571

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

    ERIC Educational Resources Information Center

    Hazan, Charlene Corey

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

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

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  2. 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...

  3. 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)

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

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

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

  5. 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

  6. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. 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. PMID:25930941

  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. 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

  14. 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

  15. 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

  16. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community ▿ †

    PubMed Central

    Jiao, Yongqin; D'haeseleer, Patrik; Dill, Brian D.; Shah, Manesh; VerBerkmoes, Nathan C.; Hettich, Robert 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 β-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. PMID:21685158

  17. 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.

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

    PubMed Central

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

    2012-01-01

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

  19. 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

  20. 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

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

    PubMed Central

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

    2014-01-01

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

  2. 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

  3. 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

  4. Investigations on the effect of forage source, grinding, and urea supplementation on ruminal fermentation and microbial protein flow in a semi-continuous rumen simulation system.

    PubMed

    Hildebrand, Bastian; Boguhn, Jeannette; Rodehutscord, Markus

    2011-10-01

    The objective of the present study was to compare the effect of maize silage and grass silage on microbial fermentation and protein flow in a semi-continuous rumen simulation system (Rusitec) when milling screen size (MSS) during grinding was varied. Oven-dried silages were milled through screens of 1, 4 or 9 mm pore size and incubated for 48 h in a Rusitec system. Furthermore, the effect of N supplementation to maize silage (MSS: 4 mm) was investigated and single dose vs. continuous infusion of urea-N were compared. Degradation of organic matter (OM), crude protein (CP), fibre fractions and non-structural carbohydrates (NSC) as well as short-chain fatty acid production differed significantly between forage sources. Urea-N supplementation improved the degradation of NSC, but not that of fibre fractions in maize silage. The way of urea supply had only marginal effects on fermentation characteristics. An increase in MSS, and consequently in mean feed particle size, led to an improvement in the degradation of OM, CP and NSC, but efficiency of microbial net protein synthesis (EMPS; mg microbial N flow/g degraded OM) and the microbial amino acid profile were less affected. EMPS was higher in grass silage than in maize silage and was improved by urea-N supplementation in maize silage. This study indicates that fermentation of NSC as well as EMPS during incubation of maize silage was limited by availability of NH3-N. Furthermore, an increase in MSS above 1 mm seems to improve fermentation of silages in the Rusitec system. PMID:22164961

  5. Antibiotics in development targeting protein synthesis.

    PubMed

    Sutcliffe, Joyce A

    2011-12-01

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

  6. Polyaromatic compounds alter placental protein synthesis in pregnant rats

    SciTech Connect

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

    1991-03-15

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

  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. PMID:25193103

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

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

    PubMed

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

    2011-06-01

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

  14. Synthesis of Nanogel-Protein Conjugates

    PubMed Central

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

    2014-01-01

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

  15. Synthesis of Nanogel-Protein Conjugates.

    PubMed

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

    2013-04-21

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

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

    PubMed

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

    2007-11-01

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

  17. Voluntary Exercise Regionally Augments Rates of Cerebral Protein Synthesis

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2004-01-01

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

  19. Ultrafast sonochemical synthesis of protein-inorganic nanoflowers

    PubMed Central

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

    2015-01-01

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

  20. 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. PMID:26141667

  1. 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

  2. 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.

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

    PubMed

    Marsh, E Neil G

    2014-10-21

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

  4. μ-Synthesis of dissimilation process of glycerol to 1,3-propanediol in microbial continuous culture.

    PubMed

    Zhu, Xi; Yuan, Jinlong; Wang, Xinying; Feng, Enmin; Xiu, Zhilong

    2014-02-01

    In this paper, robust control problem using μ-synthesis in microbial continuous culture is studied. The dissimilation process of glycerol to 1,3-propanediol cannot avoid the disturbances caused by uncertain factors. Based on the biodynamical model, a control system with the initial glycerol concentration as input control is proposed to simplify the controller design. μ-synthesis method is applied to find a feedback controller to assure both of robust stability and robust performance of the closed-loop system simultaneously. To solve the corresponding structured singular value optimization problem, a converged result is obtained through D-K iteration method. The μ-synthesis system is also compared with the corresponding H(∞) system. The simulation results indicate that the μ-controller might be more feasible for the continuous bioprocess controlling. PMID:24078112

  5. 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

  6. 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

  7. 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

  8. 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

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

    PubMed Central

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

    1988-01-01

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

  10. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

    PubMed Central

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

    2015-01-01

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

  11. Autophagy Protein Rubicon Mediates Phagocytic NADPH Oxidase Activation in Response to Microbial Infection or TLR Stimulation

    PubMed Central

    Yang, Chul-Su; Lee, Jong-Soo; Rodgers, Mary; Min, Chan-Ki; Lee, June-Yong; Kim, Hee Jin; Lee, Kwang-Hoon; Kim, Chul-Joong; Oh, Byungha; Zandi, Ebrahim; Yue, Zhenyu; Kramnik, Igor; Liang, Chengyu; Jung, Jae U.

    2013-01-01

    Summary Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection. PMID:22423966

  12. 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.

  13. 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.

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

    PubMed Central

    Sherman, I. W.

    1977-01-01

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

  15. 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

  16. 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. PMID:22281322

  17. Synthesis and trafficking of prion proteins in cultured cells.

    PubMed Central

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

    1992-01-01

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

  18. EFFECT OF FEEDING PROTEIN SUPPLEMENT OF DIFFERING DEGRADABILITY ON OMASAL FLOW OF MICROBIAL AND UNDEGRADED PROTEIN (CHANGE OF TITLE FROM ORIGINAL)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ten ruminally cannulated lactating Holstein cows, that were part of a larger 5 x 5 Latin square trial studying the effects of feeding different proteins on milk production, were used to quantify flows of microbial and rumen-undegraded protein (RUP) in omasal digesta. Cows were fed total mixed ration...

  19. 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

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

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. PMID:26491045

  1. 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

  2. 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

  3. 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

  4. Self-identification of protein-coding regions in microbial genomes.

    PubMed

    Audic, S; Claverie, J M

    1998-08-18

    A new method for predicting protein-coding regions in microbial genomic DNA sequences is presented. It uses an ab initio iterative Markov modeling procedure to automatically perform the partition of genomic sequences into three subsets shown to correspond to coding, coding on the opposite strand, and noncoding segments. In contrast to current methods, such as GENEMARK [Borodovsky, M. & McIninch, J. D. (1993) Comput. Chem. 17, 123-133], no training set or prior knowledge of the statistical properties of the studied genome are required. This new method tolerates error rates of 1-2% and can process unassembled sequences. It is thus ideal for the analysis of genome survey and/or fragmented sequence data from uncharacterized microorganisms. The method was validated on 10 complete bacterial genomes (from four major phylogenetic lineages). The results show that protein-coding regions can be identified with an accuracy of up to 90% with a totally automated and objective procedure. PMID:9707594

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

    PubMed Central

    Du, X; Thiem, S M

    1997-01-01

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

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

    PubMed

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

    2006-12-01

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

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

    PubMed

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

    2003-11-01

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

  8. 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.

  9. An Integrative Method for Identifying the Over-Annotated Protein-Coding Genes in Microbial Genomes

    PubMed Central

    Yu, Jia-Feng; Xiao, Ke; Jiang, Dong-Ke; Guo, Jing; Wang, Ji-Hua; Sun, Xiao

    2011-01-01

    The falsely annotated protein-coding genes have been deemed one of the major causes accounting for the annotating errors in public databases. Although many filtering approaches have been designed for the over-annotated protein-coding genes, some are questionable due to the resultant increase in false negative. Furthermore, there is no webserver or software specifically devised for the problem of over-annotation. In this study, we propose an integrative algorithm for detecting the over-annotated protein-coding genes in microorganisms. Overall, an average accuracy of 99.94% is achieved over 61 microbial genomes. The extremely high accuracy indicates that the presented algorithm is efficient to differentiate the protein-coding genes from the non-coding open reading frames. Abundant analyses show that the predicting results are reliable and the integrative algorithm is robust and convenient. Our analysis also indicates that the over-annotated protein-coding genes can cause the false positive of horizontal gene transfers detection. The webserver of the proposed algorithm can be freely accessible from www.cbi.seu.edu.cn/RPGM. PMID:21903723

  10. 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

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

    PubMed Central

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

    2015-01-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. PMID:25283437

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

    PubMed

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

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. PMID:25283437

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

    PubMed Central

    Litscher, Eveline S.; Wassarman, Paul M.

    2015-01-01

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

  14. 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

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

    PubMed Central

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

    2010-01-01

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

  16. 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

  17. 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

  18. MannDB – A microbial database of automated protein sequence analyses and evidence integration for protein characterization

    PubMed Central

    Zhou, Carol L Ecale; Lam, Marisa W; Smith, Jason R; Zemla, Adam T; Dyer, Matthew D; Kuczmarski, Thomas A; Vitalis, Elizabeth A; Slezak, Thomas R

    2006-01-01

    Background 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. Description 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. Conclusion MannDB comprises a large number of genomes and comprehensive protein sequence analyses

  19. 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

  20. 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

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

    PubMed

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

    1979-01-01

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

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

    PubMed

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

    2016-05-17

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

  3. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

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

    SciTech Connect

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

    1985-04-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infusing leucine for 1 h increases skeletal muscle protein synthesis in the neonate, but this is not sustained for 2 h unless the corresponding fall in amino acids is prevented. This study aimed to determine whether a continuous leucine infusion can stimulate protein synthesis for a prolonged period...

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

    ERIC Educational Resources Information Center

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

    2007-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 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…