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Sample records for protein synthesis rate

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

    Science.gov (United States)

    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.

  2. Determination of human muscle protein fractional synthesis rate

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    Science.gov (United States)

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

  4. Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

    Science.gov (United States)

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

    2016-09-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P protein synthesis rates increased after ingesting MCas-35 (P protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an equal amount of wheat protein. Ingesting a larger amount of wheat protein (i.e., 60 g) substantially increases myofibrillar protein synthesis rates in healthy older men. This trial was registered at clinicaltrials.gov as NCT01952639. © 2016 American Society for Nutrition.

  5. Contraction intensity and feeding affect collagen and myofibrillar protein synthesis rates differently in human skeletal muscle

    DEFF Research Database (Denmark)

    Holm, Lars; Hall, Gerrit van; Rose, Adam John

    2010-01-01

    Exercise stimulates muscle protein fractional synthesis rate (FSR) but the importance of contractile intensity and whether it interplays with feeding is not understood. This was investigated following two distinct resistance exercise (RE) contraction intensities using an intra-subject design...... to feeding. Further, although functionally linked, the contractile and the supportive matrix structures upregulate their protein synthesis rate quite differently in response to feeding and contractile-activity and -intensity....

  6. Measuring Protein Synthesis Rate In Living Object Using Flooding Dose And Constant Infusion Methods

    OpenAIRE

    Ulyarti, Ulyarti

    2018-01-01

    Constant infusion is a method used for measuring protein synthesis rate in living object which uses low concentration of amino acid tracers. Flooding dose method is another technique used to measure the rate of protein synthesis which uses labelled amino acid together with large amount of unlabelled amino acid.  The latter method was firstly developed to solve the problem in determination of precursor pool arise from constant infusion method.  The objective of this writing is to com...

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

    Science.gov (United States)

    Hursel, Rick; Martens, Eveline A P; Gonnissen, Hanne K J; Hamer, Henrike M; Senden, Joan M G; van Loon, Luc J C; Westerterp-Plantenga, Margriet S

    2015-01-01

    Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;Pprotein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;Psynthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;Pprotein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620). In the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Science.gov (United States)

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Kouw, Imre Wk; Wall, Benjamin T; Burd, Nicholas A; de Groot, Lisette Cpgm; van Loon, Luc Jc

    2017-02-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis rates and the ability to increase muscle protein synthesis after protein ingestion. To our knowledge, no previous studies have evaluated the impact of habituation to either low protein intake (LOW PRO) or high protein intake (HIGH PRO) on the postprandial muscle protein synthetic response. We assessed the impact of LOW PRO compared with HIGH PRO on basal and postprandial muscle protein synthesis rates after the ingestion of 25 g whey protein. Twenty-four healthy, older men [age: 62 ± 1 y; body mass index (in kg/m 2 ): 25.9 ± 0.4 (mean ± SEM)] participated in a parallel-group randomized trial in which they adapted to either a LOW PRO diet (0.7 g · kg -1 · d -1 ; n = 12) or a HIGH PRO diet (1.5 g · kg -1 · d -1 ; n = 12) for 14 d. On day 15, participants received primed continuous l-[ring- 2 H 5 ]-phenylalanine and l-[1- 13 C]-leucine infusions and ingested 25 g intrinsically l-[1- 13 C]-phenylalanine- and l-[1- 13 C]-leucine-labeled whey protein. Muscle biopsies and blood samples were collected to assess muscle protein synthesis rates as well as dietary protein digestion and absorption kinetics. Plasma leucine concentrations and exogenous phenylalanine appearance rates increased after protein ingestion (P 0.05). Plasma exogenous phenylalanine availability over the 5-h postprandial period was greater after LOW PRO than after HIGH PRO (61% ± 1% compared with 56% ± 2%, respectively; P protein synthesis rates increased from 0.031% ± 0.004% compared with 0.039% ± 0.007%/h in the fasted state to 0.062% ± 0.005% compared with 0.057% ± 0.005%/h in the postprandial state after LOW PRO compared with HIGH PRO, respectively (P protein-derived amino acids in the circulation and does not lower basal muscle protein synthesis rates or increase postprandial muscle protein synthesis rates after ingestion of 25 g protein in older men. This trial was registered at clinicaltrials.gov as NCT

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

    Directory of Open Access Journals (Sweden)

    Rick Hursel

    Full Text Available Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d or low protein (0.4 g protein/kg/d energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001. Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03, synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01 and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001 were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042

  11. Brain tumors : L-[1-C-11]tyrosine PET for visualization and quantification of protein synthesis rate

    NARCIS (Netherlands)

    Pruim, J; Willemsen, A T; Molenaar, W M; Waarde, A van; Paans, A M; Heesters, M A; Go, K G; Visser, Gerben; Franssen, E J; Vaalburg, W

    1995-01-01

    PURPOSE: Positron emission tomography (PET) with the amino acid tracer L-[1-C-11]-tyrosine was evaluated in 27 patients with primary and recurrent brain tumors. MATERIALS AND METHODS: Patients underwent either static (n = 14) or dynamic PET (n = 13), with quantification of protein synthesis rate

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

    International Nuclear Information System (INIS)

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

    1981-01-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  14. Estimation of the protein synthesis rates of the whole body of growing broilers

    International Nuclear Information System (INIS)

    Koehler, R.; Pahle, T.; Gruhn, K.; Zander, R.; Jeroch, H.; Gebhardt, G.

    1988-01-01

    The purpose of the investigations was to prove a method, developed for monogastric mammalians, based on a 3-compartment model and assuming a proportional growth of the pools of total N, whether it is applicable to growing poultry. The tracer, 15 N-L-lysine, was given quasi-continuously for four days. In this time and in the following period of five days without tracer intake, the 15 N excretion in the urine was measured. The average of the live weight of the broiler cockerels was 1724 g. The animals were colostomized for sampling the urine separately. Using the fluxes of lysine, the calculation of the whole-body protein synthesis rate was 64.1 g/d. The protein degradation rate was 54.4 g/d. The adequate values of the fractional rates of protein synthesis and degradation for the whole body (without feathers) were 23.3% and 19.8%, resp. Thus it is clearly shown, that the method applied gives real data of the parameters of the N metabolism for growing broilers, being in the range of values for muscle proteins and proteins of the whole body of growing poultry, published by other authors. (author)

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

    Science.gov (United States)

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

    1978-01-01

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

  16. Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis.

    Science.gov (United States)

    Ferguson, Angelica; Wang, Leyi; Altman, Roger B; Terry, Daniel S; Juette, Manuel F; Burnett, Benjamin J; Alejo, Jose L; Dass, Randall A; Parks, Matthew M; Vincent, C Theresa; Blanchard, Scott C

    2015-11-05

    The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    Science.gov (United States)

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Directory of Open Access Journals (Sweden)

    Sophia Rudorf

    2014-10-01

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

  19. Carbon-11 tyrosine PET for visualization and protein synthesis rate assessment of laryngeal and hypopharyngeal carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Boer, Jurjan R. de; Laan, Bernard F.A.M. van der; Albers, Frans W.J. [Department of Otorhinolaryngology and Head and Neck Surgery, Groningen University Hospital, Hanzeplein 1, 9700 RB Groningen (Netherlands); Pruim, Jan; Que, Tjin H.; Willemsen, Antoon T.M.; Vaalburg, Willem [PET Center of Groningen University Hospital, Groningen (Netherlands); Burlage, Fred [Department of Radiotherapy, Groningen University Hospital, Groningen (Netherlands); Krikke, Allard [Department of Radiology, Groningen University Hospital, Groningen (Netherlands); Tiebosch, Anton T.M.G. [Department of Pathology, Groningen University Hospital, Groningen (Netherlands)

    2002-09-01

    Accurate assessment of tumour extent and lymph node involvement in squamous cell carcinomas of the head and neck region is essential for therapy planning. Unfortunately, conventional diagnostic examination and imaging techniques, which monitor tumours on the basis of anatomical parameters, have drawbacks in clinical practice. The aim of this study was to investigate the feasibility of L-[1-{sup 11}C]-tyrosine (TYR) positron emission tomography (PET) for visualisation of squamous cell carcinoma of the larynx and hypopharynx and quantification of tumour activity by assessment of protein synthesis rate (PSR). Dynamic TYR PET was performed on 31 patients with T1-T4 laryngeal or hypopharyngeal carcinoma before therapy. Plasma activity of TYR, {sup 11}CO{sub 2} and {sup 11}C-protein levels were measured, and PSRs were calculated for primary malignancies. All 31 laryngeal and hypopharyngeal tumours were visualised as a hotspot (sensitivity 100%). The median PSR of the tumours (2.06 nmol ml{sup -1} min{sup -1}; range 0.72-6.96) was significantly higher (P<0.001) than that of non-tumour (background) tissue (0.51 nmol ml{sup -1} min{sup -1}; range 0.22-0.89). L-[1-{sup 11}C]-Tyrosine PET appears to be a potential method for visualisation of primary laryngeal and hypopharyngeal tumours. In vivo quantification of tumour activity by assessment of PSR is possible and may have a future role in the therapy planning and therapy evaluation of laryngeal and hypopharyngeal tumours. (orig.)

  20. Relationship Between Hepatic Albumin and Sulphate Synthesis and its Use in Measurement of the Absolute Rate of Synthesis of Liver-Produced Plasma Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Awwad, H. K.; Sheraki, A. S. [Department of Radiology and Radiological Sciences, Cancer Institute, University of Cairo, Cairo (Egypt); Radioisotope Unit, Medical Research Institute, Alexandria (Egypt)

    1971-02-15

    A model is proposed whereby serum albumin synthesis is expressed in terms of production of inorganic sulphate in the liver and the entire organism, following the administration of {sup 35}S-L-cystine. The basis assumption involved is that the precursor amino acid pool for albumin synthesis in the liver is either identical with that of inorganic sulphate synthesis or that the two pools concerned are in rapid equilibrium with each other so that they can be treated as a single pool. The feasibility of the proposed model was tested by comparing the synthesis rate of rat serum albumin with the catabolic rate of the radioiodinated protein measured in the same animal. A good agreement between the two rates was noted in a group of adult rats, whereas an excess of anabolism was noted in young growing animals. In rats fed low-protein diet, the synthesis rate exceeded the catabolic rate; both being subnormal. The equilibrium between hepatic and plasma radiosulphate concentration was complete within four hours following the injection of {sup 35}S-cystine. The total radiosulphate production could then be evaluated after such an interval from the urinary excretion and serum concentration multiplied by the volume of the sulphate space. Lack of significant re-utilization was demonstrated following the injection of radiosulphate. This is a decided advantage of the proposed method. However, extensive re-utilization of selenate selenium in the synthesis of the seleno-analogues of sulphur-amino acids was shown. This could explain the poor yield of radioselenate following the injection of {sup 75}Se-selenocystine and precludes the use of the latter agent as a tracer for measurement of synthesis of plasma proteins. (author)

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

    Science.gov (United States)

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

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

    Directory of Open Access Journals (Sweden)

    Brita Singers Sørensen

    Full Text Available The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect, which weakens the spatial linkage between hypoxia and acidosis.Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15 were treated with hypoxia, acidosis (pH 6.3, or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe, genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2, and Ribosomal protein L37 (RPL37. Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa and protein synthesis (both cell lines was observed when hypoxia and low pHe were combined.We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de

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

    Science.gov (United States)

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

    2015-01-01

    The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo

  4. Both basal and post-prandial muscle protein synthesis rates, following the ingestion of a leucine-enriched whey protein supplement, are not impaired in sarcopenic older males.

    Science.gov (United States)

    Kramer, Irene Fleur; Verdijk, Lex B; Hamer, Henrike M; Verlaan, Sjors; Luiking, Yvette C; Kouw, Imre W K; Senden, Joan M; van Kranenburg, Janneau; Gijsen, Annemarie P; Bierau, Jörgen; Poeze, Martijn; van Loon, Luc J C

    2017-10-01

    Studying the muscle protein synthetic response to food intake in elderly is important, as it aids the development of interventions to combat sarcopenia. Although sarcopenic elderly are the target group for many of these nutritional interventions, no studies have assessed basal or post-prandial muscle protein synthesis rates in this population. To assess the basal and post-prandial muscle protein synthesis rates between healthy and sarcopenic older men. A total of 15 healthy (69 ± 1 y) and 15 sarcopenic (81 ± 1 y) older men ingested a leucine-enriched whey protein nutritional supplement containing 21 g of protein, 9 g of carbohydrate, and 3 g of fat. Stable isotope methodology combined with frequent collection of blood and muscle samples was applied to assess basal and post-prandial muscle protein fractional synthetic rates. Handgrip strength, muscle mass, and gait speed were assessed to identify sarcopenia, according to international criteria. Basal mixed muscle protein fractional synthetic rates (FSR) averaged 0.040 ± 0.005 and 0.032 ± 0.003%/h (mean ± SEM) in the sarcopenic and healthy group, respectively (P = 0.14). Following protein ingestion, FSR increased significantly to 0.055 ± 0.004 and 0.053 ± 0.004%/h in the post-prandial period in the sarcopenic (P = 0.003) and healthy groups (P protein synthesis rates during the early (0.058 ± 0.007 vs 0.060 ± 0.008%/h, sarcopenic vs healthy, respectively) and late (0.052 ± 0.004 vs 0.048 ± 0.003%/h) stages of the post-prandial period (P = 0.93 and P = 0.34, respectively). Basal muscle protein synthesis rates are not lower in sarcopenic older men compared to healthy older men. The ingestion of 21 g of a leucine-enriched whey protein effectively increases muscle protein synthesis rates in both sarcopenic and healthy older men. Public trial registry number: NTR3047. Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights

  5. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    Science.gov (United States)

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  6. Skeletal Muscle Myofibrillar and Sarcoplasmic Protein Synthesis Rates Are Affected Differently by Altitude-Induced Hypoxia in Native Lowlanders

    DEFF Research Database (Denmark)

    Holm, Lars; Lyhne Haslund, Mads; Robach, Paul

    2010-01-01

    As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O(2). With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-(13)C...... and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%⋅hr(-1) (p0.05). Trends...... to increments in whole body protein kinetics were seen: Degradation rate elevated from 2.51±0.21 at sea level to 2.73±0.13 µmol⋅kg(-1)⋅min(-1) (p = 0.05) at high altitude and synthesis rate similar; 2.24±0.20 at sea level and 2.43±0.13 µmol⋅kg(-1)⋅min(-1) (p>0.05) at altitude. We conclude that whole body amino...

  7. Ethylene and protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Osborne, D J

    1973-01-01

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

  8. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Woloschak, G.E.

    1994-01-01

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  9. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Woloschak, G.E.; Felcher, P.; Chang-Liu, Chin-Mei

    1992-01-01

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  10. A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

    Science.gov (United States)

    Fierro-Monti, Ivo; Racle, Julien; Hernandez, Celine; Waridel, Patrice; Hatzimanikatis, Vassily; Quadroni, Manfredo

    2013-01-01

    Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538. PMID:24312217

  11. A novel pulse-chase SILAC strategy measures changes in protein decay and synthesis rates induced by perturbation of proteostasis with an Hsp90 inhibitor.

    Directory of Open Access Journals (Sweden)

    Ivo Fierro-Monti

    Full Text Available Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc variant of SILAC (stable isotope labeling by amino acids in cell culture. pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

  12. Alcohol ingestion impairs maximal post-exercise rates of myofibrillar protein synthesis following a single bout of concurrent training.

    Directory of Open Access Journals (Sweden)

    Evelyn B Parr

    Full Text Available INTRODUCTION: The culture in many team sports involves consumption of large amounts of alcohol after training/competition. The effect of such a practice on recovery processes underlying protein turnover in human skeletal muscle are unknown. We determined the effect of alcohol intake on rates of myofibrillar protein synthesis (MPS following strenuous exercise with carbohydrate (CHO or protein ingestion. METHODS: In a randomized cross-over design, 8 physically active males completed three experimental trials comprising resistance exercise (8×5 reps leg extension, 80% 1 repetition maximum followed by continuous (30 min, 63% peak power output (PPO and high intensity interval (10×30 s, 110% PPO cycling. Immediately, and 4 h post-exercise, subjects consumed either 500 mL of whey protein (25 g; PRO, alcohol (1.5 g·kg body mass⁻¹, 12±2 standard drinks co-ingested with protein (ALC-PRO, or an energy-matched quantity of carbohydrate also with alcohol (25 g maltodextrin; ALC-CHO. Subjects also consumed a CHO meal (1.5 g CHO·kg body mass⁻¹ 2 h post-exercise. Muscle biopsies were taken at rest, 2 and 8 h post-exercise. RESULTS: Blood alcohol concentration was elevated above baseline with ALC-CHO and ALC-PRO throughout recovery (P<0.05. Phosphorylation of mTOR(Ser2448 2 h after exercise was higher with PRO compared to ALC-PRO and ALC-CHO (P<0.05, while p70S6K phosphorylation was higher 2 h post-exercise with ALC-PRO and PRO compared to ALC-CHO (P<0.05. Rates of MPS increased above rest for all conditions (∼29-109%, P<0.05. However, compared to PRO, there was a hierarchical reduction in MPS with ALC-PRO (24%, P<0.05 and with ALC-CHO (37%, P<0.05. CONCLUSION: We provide novel data demonstrating that alcohol consumption reduces rates of MPS following a bout of concurrent exercise, even when co-ingested with protein. We conclude that alcohol ingestion suppresses the anabolic response in skeletal muscle and may therefore impair recovery and adaptation

  13. Contraction mode and whey protein intake affect the synthesis rate of intramuscular connective tissue

    DEFF Research Database (Denmark)

    Holm, Lars; Klejs Rahbek, Stine; Farup, Jean

    2016-01-01

    either WPH or maltodextrin [carbohydrate (CHO)] immediately after completion of unilateral shortening and lengthening knee extensions. Ring-13C6-phenylalanine was infused, and muscle biopsies were obtained. IMCT protein FSR was measured at 1–5, as well as 1–3 and 3–5 hours after contractions and nutrient...

  14. Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

    Science.gov (United States)

    Newsom, Sean A; Miller, Benjamin F; Hamilton, Karyn L; Ehrlicher, Sarah E; Stierwalt, Harrison D; Robinson, Matthew M

    2017-11-01

    Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H 2 O 2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance. Copyright © 2017 the

  15. Noncovalent synthesis of protein dendrimers

    NARCIS (Netherlands)

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

    2009-01-01

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

  16. Synthesis of Lipidated Proteins.

    Science.gov (United States)

    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.

  17. Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

    Science.gov (United States)

    Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William J

    2016-03-15

    Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM. Copyright © 2016 the American Physiological Society.

  18. Protein synthesis in the growing rat lung

    International Nuclear Information System (INIS)

    Kelley, J.; Chrin, L.

    1986-01-01

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

  19. Tendon protein synthesis rate in classic Ehlers-Danlos patients can be stimulated with insulin-like growth factor-I

    DEFF Research Database (Denmark)

    Nielsen, Rie Harboe; Holm, Lars; Jensen, Jacob Kildevang

    2014-01-01

    tissue protein turnover is unknown. We investigated whether cEDS affected the protein synthesis rate in skin and tendon, and whether this could be stimulated in tendon tissue with insulin-like growth factor-I (IGF-I). Five patients with cEDS and 10 healthy, matched controls (CTRL) were included. One...... patellar tendon of each participant was injected with 0.1 ml IGF-I (Increlex, Ipsen, 10 mg/ml) and the contralateral tendon with 0.1 ml isotonic saline as control. The injections were performed at both 24 and 6 h prior to tissue sampling. The fractional synthesis rate (FSR) of proteins in skin and tendon.......002 (cEDS) and 0.007 ± 0.002 (CTRL); tendon: 0.008 ± 0.001 (cEDS) and 0.009 ± 0.002 (CTRL) %/h, mean ± SE]. IGF-I injections significantly increased FSR values in cEDS patients but not in controls (delta values: cEDS 0.007 ± 0.002, CTRL 0.001 ± 0.001%/h). In conclusion, baseline protein synthesis rates...

  20. Tyrosine positron emission tomography and protein synthesis rate in pituitary adenoma: Different effects of surgery and radiation therapy

    International Nuclear Information System (INIS)

    Bergh, Alfons C.M. van den; Pruim, Jan; Links, Thera P.; Vliet, Anton M. van der; Sluiter, Wim; Wolffenbuttel, Bruce H.R.; Langendijk, Johannes A.; Hoving, Eelco W.; Dullaart, Robin P.F.

    2011-01-01

    Introduction: Positron emission tomography (PET) using amino acid tracers is able to establish biochemical tumour characterization in vivo. The use of PET in the follow-up of non-functioning pituitary adenomas (NFA) and growth hormone producing pituitary adenomas (GHA) after surgery and radiation treatment is not yet clear. Methods: To determine the value of PET before and after transsphenoidal neurosurgery in NFA and GHA, we investigated 12 patients with pituitary adenoma (9 NFA and 3 GHA) before and 4 months after surgery with magnetic resonance imaging (MRI) and tyrosine PET (TYR-PET). Three years after radiation therapy TYR-PET was used to document residual activity in 6 of these patients (4 NFA- and 2 GHA). Tumour size was quantified by computerized MRI measurements. In TYR-PET, tumour activity was assessed by computerized measurements of the hot spot and by determination of protein synthesis rate (PSR). Results: In response to surgery, MRI showed a median tumour volume reduction of 58% (P 0.30). After radiation therapy the MRI-volumes of the residual pituitary adenomas did not change but the volume of the hot spot on TYR-PET-imaging was reduced by 58% (P = 0.02), and PSR decreased in 5 of 6 patients (P = 0.12). Conclusion: Amino acid PET tumour activity is reduced parallel with MRI volume changes after surgery. The decrease in TYR-PET activity after radiation therapy, despite unaltered MRI tumour volume, supports the concept that it is possible to follow biological tumour activity with this technique. The diagnostic merit of this tracer technique, predicting pituitary adenoma re-growth, needs to be validated in a large prospective study.

  1. Leucine stimulation of skeletal muscle protein synthesis

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-09-01

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

  3. Albumin synthesis in protein energy malnutrition

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  4. Determination of muscle protein synthesis rates in fish using (2)H2O and (2)H NMR analysis of alanine.

    Science.gov (United States)

    Marques, Cátia; Viegas, Filipa; Rito, João; Jones, John; Viegas, Ivan

    2016-09-15

    Following administration of deuterated water ((2)H2O), the fractional synthetic rate (FSR) of a given endogenous protein can be estimated by (2)H-enrichment quantification of its alanine residues. Currently, this is measured by mass spectrometry following a derivatization procedure. Muscle FSR was measured by (1)H/(2)H NMR analysis of alanine from seabass kept for 6 days in 5% (2)H-enriched saltwater, following acid hydrolysis and amino acid isolation by cation-exchange chromatography of muscle tissue. The analysis is simple and robust, and provides precise measurements of excess alanine (2)H-enrichment in the 0.1-0.4% range from 50 mmol of alanine recovered from muscle protein. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Albumin synthesis in protein energy malnutrition

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-31

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

  6. Effects of feeding grains naturally contaminated with Fusarium mycotoxins on hepatic fractional protein synthesis rates of laying hens and the efficacy of a polymeric glucomannan mycotoxin adsorbent.

    Science.gov (United States)

    Chowdhury, S R; Smith, T K

    2005-11-01

    Experiments were conducted to evaluate the effects of feeding grains naturally contaminated with a combination of Fusarium mycotoxins on hepatic fractional protein synthesis rates (FSR) of laying hens. Thirty-six 32-wk-old laying hens were fed diets formulated with 1) uncontaminated grains, 2) contaminated grains, or 3) contaminated grains + 0.2% polymeric glucomannan mycotoxin adsorbent for a period of 4 wk. Hepatic FSR were measured in vivo by the flooding-dose method. The feeding of contaminated grains decreased hepatic FSR in laying hens compared with controls after 4 wk. The hepatic FSR of birds fed contaminated grains and contaminated grains + glucomannan mycotoxin adsorbent were not different. It was concluded that the in vivo hepatic FSR of laying hens was inhibited by the feeding of grains naturally contaminated with Fusarium mycotoxins and that this may explain some of the adverse effects seen when contaminated grains were fed to laying hens.

  7. Protein synthesis controls phosphate homeostasis.

    Science.gov (United States)

    Pontes, Mauricio H; Groisman, Eduardo A

    2018-01-01

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

  8. Control of the Protein Turnover Rates in Lemna minor

    Science.gov (United States)

    Trewavas, A.

    1972-01-01

    The control of protein turnover in Lemna minor has been examined using a method described in the previous paper for determining the rate constants of synthesis and degradation of protein. If Lemna is placed on water, there is a reduction in the rate constants of synthesis of protein and an increase (3- to 6-fold) in the rate constant of degradation. The net effect is a loss of protein from the tissue. Omission of nitrate, phosphate, sulfate, magnesium, or calcium results in increases in the rate constant of degradation of protein. An unusual dual effect of benzyladenine on the turnover constants has been observed. Treatment of Lemna grown on sucrose-mineral salts with benzyladenine results in alterations only in the rate constant of synthesis. Treatment of Lemna grown on water with benzyladenine alters only the rate constant of degradation. Abscisic acid on the other hand alters both rate constants of synthesis and degradation of protein together. Inclusion of growth-inhibiting amino acids in the medium results in a reduction in the rate constants of synthesis and increases in the rate constant of degradation of protein. It is concluded that the rate of turnover of protein in Lemna is very dependent on the composition of the growth medium. Conditions which reduce growth rates also reduce the rates of synthesis of protein and increase those of degradation. PMID:16657895

  9. Studies on the distribution of radioactivity in the organism during constant intravenous infusion of tracer amino acids and on the calculation of the rate of tissue protein synthesis in rats

    International Nuclear Information System (INIS)

    Simon, O.; Bergner, H.; Wolf, E.

    1978-01-01

    Male wistar rats (100 p body weight) were infused into the tail vein with 14 C-leucine and 14 C-lysine simultaneously for 0.5; 1.0; 2.0; 3.0; 4.5; 6.0 and 7.0 hours. At the end of the infusion the specific radioactivity was determined of the free leucine and lysine in the blood plasma, liver, M. gastrocnemius, small intestine, and colon as well as of the protein-bound leucine and lysine. In all the tissues tested the specific radioactivity of the free amino acids attained a plateau during the 6-hour and 7-hour infusions. The rate constants for the increase were calculated for each organ tested. The two amino acids used are suitable for calculating the fractional rate of protein synthesis in tissues. The values of the fractional rate of protein synthesis calculated on the basis of the 6-hour and 7-hour infusions were: 54+-7.7%/day for the liver, 9.4+-1.2%/day for the muscles, 89+-12.2%/day for the small intestine, and 42+-5.9%/day for the colon. The simultaneous application of two tracer amino acids is recommendable for estimating the precursor pool of the protein synthesis and the more accurate calculation of the rate of protein synthesis. (author)

  10. [The mechanism of phenoptosis: I. Age-dependent decrease of the overall rate of protein synthesis is caused by the programmed attenuation of bio-energetics].

    Science.gov (United States)

    Trubitsyn, A G

    2009-01-01

    The age-dependent degradation of all vital processes of an organism can be result of influences of destructive factors (the stochastic mechanism of aging), or effect of realizations of the genetic program (phenoptosis). The stochastic free-radical theory of aging dominating now contradicts the set of empirical data, and the semicentenial attempts to create the means to slow down aging did not give any practical results. It makes obvious that the stochastic mechanism of aging is incorrect. At the same time, the alternative mechanism of the programmed aging is not developed yet but preconditions for it development have already been created. It is shown that the genes controlling process of aging exist (contrary to the customary opinion) and the increase in the level of damaged macromolecules (basic postulate of the free-radical theory) can be explained by programmed attenuation of bio-energetics. As the bio-energetics is a driving force of all vital processes, decrease of its level is capable to cause degradation of all functions of an organism. However to transform this postulate into a basis of the theory of phenoptosis it is necessary to show, that attenuation of bio-energetics predetermines such fundamental processes accompanying aging as decrease of the overall rate of protein biosynthesis, restriction of cellular proliferations (Hayflick limit), loss of telomeres etc. This article is the first step in this direction: the natural mechanism of interaction of overall rate of protein synthesis with a level of cellular bio-energetics is shown. This is built-in into the translation machine and based on dependence of recirculation rate of eukaryotic initiation factor 2 (elF2) from ATP/ADP value that is created by mitochondrial bio-energetic machine.

  11. Predictors of muscle protein synthesis after severe pediatric burns.

    Science.gov (United States)

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

    2015-04-01

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

  12. Inhibition of chloroplast protein synthesis following light chilling of tomato

    International Nuclear Information System (INIS)

    Kent, J.; Ort, D.

    1989-01-01

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

  13. Synthesis of acid-soluble spore proteins by Bacillus subtilis.

    OpenAIRE

    Leventhal, J M; Chambliss, G H

    1982-01-01

    The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phos...

  14. Cell-specific monitoring of protein synthesis in vivo.

    Directory of Open Access Journals (Sweden)

    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  15. Rate in template-directed polymer synthesis.

    Science.gov (United States)

    Saito, Takuya

    2014-06-01

    We discuss the temporal efficiency of template-directed polymer synthesis, such as DNA replication and transcription, under a given template string. To weigh the synthesis speed and accuracy on the same scale, we propose a template-directed synthesis (TDS) rate, which contains an expression analogous to that for the Shannon entropy. Increasing the synthesis speed accelerates the TDS rate, but the TDS rate is lowered if the produced sequences are diversified. We apply the TDS rate to some production system models and investigate how the balance between the speed and the accuracy is affected by changes in the system conditions.

  16. Chronological protein synthesis in regenerating rat liver.

    Science.gov (United States)

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

    2015-07-01

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

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

    Science.gov (United States)

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

    2016-06-03

    Whey protein (WP) is characterized as a "fast" protein and caseinate (CA) as a "slow" protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP.

  18. Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

    Science.gov (United States)

    Ishihara, Hirofumi; Moraes, Thiago Alexandre; Pyl, Eva-Theresa; Schulze, Waltraud X; Obata, Toshihiro; Scheffel, André; Fernie, Alisdair R; Sulpice, Ronan; Stitt, Mark

    2017-08-01

    Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO 2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day -1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    2016-06-01

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

  1. Modulation of protein synthesis by polyamines.

    Science.gov (United States)

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

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

  2. Predictors of muscle protein synthesis after severe pediatric burns

    Science.gov (United States)

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

  3. Chemical Synthesis of Circular Proteins*

    Science.gov (United States)

    Tam, James P.; Wong, Clarence T. T.

    2012-01-01

    Circular proteins, once thought to be rare, are now commonly found in plants. Their chemical synthesis, once thought to be difficult, is now readily achievable. The enabling methodology is largely due to the advances in entropic chemical ligation to overcome the entropy barrier in coupling the N- and C-terminal ends of large peptide segments for either intermolecular ligation or intramolecular ligation in end-to-end cyclization. Key elements of an entropic chemical ligation consist of a chemoselective capture step merging the N and C termini as a covalently linked O/S-ester intermediate to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide. Many ligation methods exploit the supernucleophilicity of a thiol side chain at the N terminus for the capture reaction, which makes cysteine-rich peptides ideal candidates for the entropy-driven macrocyclization. Advances in desulfurization and modification of the thiol-containing amino acids at the ligation sites to other amino acids add extra dimensions to the entropy-driven ligation methods. This minireview describes recent advances of entropy-driven ligation to prepare circular proteins with or without a cysteinyl side chain. PMID:22700959

  4. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  6. Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

    OpenAIRE

    sprotocols

    2015-01-01

    Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as...

  7. RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. Pt. 2

    International Nuclear Information System (INIS)

    Skog, S.; Tribukait, B.; Nygard, O.; Wenner-Gren-Center foer Vetenskaplig Forskning, Stockholm

    1985-01-01

    Poly(A)-containing RNA (m-RNA) was studied in in vivo growing Ehrlich ascites tumour cells following a roentgen irradiation dose of 5 Gy. m-RNA increased significantly during the first 12 hours after irradiation. Thus, the observed decrease in protein synthesis rate during this time seems not to be due to radiation induced changes at the transcriptional level. The protein synthesis rate of in vivo irradiated cells incubated in vitro in culture medium was unchanged. On the other hand, the protein synthesis rate of non-irradiated cells incubated in vitro in ascites fluid from irradiated animals was decreased. We concluded that factor(s) inhibiting protein synthesis or the lack of factor(s) promoting protein synthesis in the ascites fluid is(are) of significance for the reduced protein synthesis of tumour cells found in irradiated in vivo growing cells. (orig.)

  8. Retinal protein synthesis in relationship to environmental lighting

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  9. Albumin synthesis in protein energy malnutrition

    International Nuclear Information System (INIS)

    Duggan, C.; Hardy, S.; Kleinman, R.E.; Harvard Medical School, Children's Hospital, Boston, MA; Lembcke, J.; Young, V.R.

    1996-01-01

    Assessment of protein nutritional status during re-feeding children with protein energy malnutrition (PEM) can be difficult. We hypothesized that the fractional synthesis rate (FSR) of albumin, as measured by stable isotope technology, would serve as an objective measure of changes in protein status, and that increased amounts of dietary protein (15% of calories vs 10%) would lead to higher FSR. Eight (5 M, 3 F) Peruvian children (mean age 15.5 months) with PEM (mean wt/ht Z score = -2.47) were studied twice during the first week of admission by the flooding dose technique. An intravenous dose of 13 C-leucine (57 mg/kg, 99 atom%) was given and serial blood samples were drawn in intervals up to 90 minutes in order to measure isotopic enrichment of serum albumin. Mean FSR for the day one infusion was 6.11% (range 3.07 - 15.37%) (n = 8). Mean FSR for the follow-up infusion was 7.67% (range 3.63 - 12.37%) (n = 5), and FSR was no different between the two dietary groups. FSR on day one was inversely related to age (r = -0.62), and one patient with Shigella dysentery had the highest FSR (15.9%). We conclude that FSR of albumin can be measured successfully in children with PEM using the flooding dose technique, and that assessment of albumin FSR holds promise to help determine protein requirements and status during recovery from PEM. (author). 14 refs, 6 figs, 3 tabs

  10. Albumin synthesis in protein energy malnutrition

    Energy Technology Data Exchange (ETDEWEB)

    Duggan, C; Hardy, S; Kleinman, R E [Massachusetts General Hospital, Boston, MA (United States); [Harvard Medical School, Children` s Hospital, Boston, MA (United States). Combined Program in Pediatric GI and Nutrition; Lembcke, J [Av. La Universidad S/N - La Molina, Lima (Peru). Inst. de Investigacion Nutricional; Young, V R [Massachussetts Inst. of Technology, Cambridge, MA (United States). Lab. of Human Nutrition

    1997-12-31

    Assessment of protein nutritional status during re-feeding children with protein energy malnutrition (PEM) can be difficult. We hypothesized that the fractional synthesis rate (FSR) of albumin, as measured by stable isotope technology, would serve as an objective measure of changes in protein status, and that increased amounts of dietary protein (15% of calories vs 10%) would lead to higher FSR. Eight (5 M, 3 F) Peruvian children (mean age 15.5 months) with PEM (mean wt/ht Z score = -2.47) were studied twice during the first week of admission by the flooding dose technique. An intravenous dose of {sup 13}C-leucine (57 mg/kg, 99 atom%) was given and serial blood samples were drawn in intervals up to 90 minutes in order to measure isotopic enrichment of serum albumin. Mean FSR for the day one infusion was 6.11% (range 3.07 - 15.37%) (n = 8). Mean FSR for the follow-up infusion was 7.67% (range 3.63 - 12.37%) (n = 5), and FSR was no different between the two dietary groups. FSR on day one was inversely related to age (r = -0.62), and one patient with Shigella dysentery had the highest FSR (15.9%). We conclude that FSR of albumin can be measured successfully in children with PEM using the flooding dose technique, and that assessment of albumin FSR holds promise to help determine protein requirements and status during recovery from PEM. (author). 14 refs, 6 figs, 3 tabs.

  11. Protein synthesis in geostimulated root caps

    Science.gov (United States)

    Feldman, L. J.

    1982-01-01

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

  12. Regulation of protein synthesis during sea urchin early development

    International Nuclear Information System (INIS)

    Kelso, L.C.

    1989-01-01

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

  13. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

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

    2012-07-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  16. Synthesis of acid-soluble spore proteins by Bacillus subtilis.

    Science.gov (United States)

    Leventhal, J M; Chambliss, G H

    1982-12-01

    The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

  17. Origins of the protein synthesis cycle

    Science.gov (United States)

    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.

  18. Synthesis and thermotolerance of heat shock proteins in Campylobacter jejuni

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  19. Measuring protein breakdown rate in individual proteins in vivo

    DEFF Research Database (Denmark)

    Holm, Lars; Kjaer, Michael

    2010-01-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  2. Measurements of Protein Crystal Face Growth Rates

    Science.gov (United States)

    Gorti, S.

    2014-01-01

    Protein crystal growth rates will be determined for several hyperthermophile proteins.; The growth rates will be assessed using available theoretical models, including kinetic roughening.; If/when kinetic roughening supersaturations are established, determinations of protein crystal quality over a range of supersaturations will also be assessed.; The results of our ground based effort may well address the existence of a correlation between fundamental growth mechanisms and protein crystal quality.

  3. Protein synthesis rate measured with l-[1-11C]tyrosine positron emission tomography correlates with mitotic activity and MIB-1 antibody-detected proliferation in human soft tissue sarcomas

    International Nuclear Information System (INIS)

    Plaat, B.; Mastik, M.; Molenaar, W.; Kole, A.; Vaalburg, W.; Hoekstra, H.

    1999-01-01

    Protein synthesis rate (PSR) can be assessed in vivo using positron emission tomography with l-[1- 11 C]tyrosine (TYR-PET). Biological activity of soft tissue sarcomas (STS) can be measured in vitro by the mitotic rate and number of proliferating cells. In STS the grade of malignancy, in which the mitotic index plays a major role, is considered to be the major standard in predicting biological tumour behaviour. This study was designed to test the validity of TYR-PET in relation to different histopathological features. In 21 patients with untreated STS, the PSR was measured using TYR-PET. The number of mitoses was counted and tumours were graded according to the grading system of Coindre et al. (Cancer 1986; 58:306-309). Proliferative activity was assessed by immunohistological detection of the Ki-67 nuclear antigen using MIB-1 monoclonal antibody. To test the association between the PSR and these tumour parameters, a correlation analysis was performed. A significant (P<0.05) correlation was found between PSR and the Ki-67 proliferation index (R = 0.54), and between PSR and mitotic rate (R = 0.64). There was no correlation between PSR and tumour grade. The present study in malignant soft tissue tumours relates in vivo tumour metabolism as established with TYR-PET to tumour activity measured in vitro and indicates that the non-invasive method of TYR-PET can estimate the mitotic and proliferative activity in STS. (orig.)

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

    Science.gov (United States)

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

    2015-01-01

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

  5. Glucocorticoid effects on hippocampal protein synthesis

    International Nuclear Information System (INIS)

    Schlatter, L.K.

    1988-01-01

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

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

    Science.gov (United States)

    Mizutani, H.; Ponnamperuma, C.

    1977-01-01

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

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

    International Nuclear Information System (INIS)

    Horst, M.N.

    1990-01-01

    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

  8. Mitochondrial Protein Synthesis, Import, and Assembly

    Science.gov (United States)

    Fox, Thomas D.

    2012-01-01

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

  9. The limits of adaptation of functional protein synthesis to severe undernutrition

    International Nuclear Information System (INIS)

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

    1996-01-01

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

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  15. Protein synthesis rate measured with l-[1-{sup 11}C]tyrosine positron emission tomography correlates with mitotic activity and MIB-1 antibody-detected proliferation in human soft tissue sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Plaat, B.; Mastik, M.; Molenaar, W. [Department of Pathology, University Hospital Groningen (Netherlands); Kole, A.; Vaalburg, W. [PET Centre, University Hospital Groningen (Netherlands); Hoekstra, H. [Department of Surgical Oncology, University Hospital Groningen (Netherlands)

    1999-04-29

    Protein synthesis rate (PSR) can be assessed in vivo using positron emission tomography with l-[1-{sup 11}C]tyrosine (TYR-PET). Biological activity of soft tissue sarcomas (STS) can be measured in vitro by the mitotic rate and number of proliferating cells. In STS the grade of malignancy, in which the mitotic index plays a major role, is considered to be the major standard in predicting biological tumour behaviour. This study was designed to test the validity of TYR-PET in relation to different histopathological features. In 21 patients with untreated STS, the PSR was measured using TYR-PET. The number of mitoses was counted and tumours were graded according to the grading system of Coindre et al. (Cancer 1986; 58:306-309). Proliferative activity was assessed by immunohistological detection of the Ki-67 nuclear antigen using MIB-1 monoclonal antibody. To test the association between the PSR and these tumour parameters, a correlation analysis was performed. A significant (P<0.05) correlation was found between PSR and the Ki-67 proliferation index (R = 0.54), and between PSR and mitotic rate (R = 0.64). There was no correlation between PSR and tumour grade. The present study in malignant soft tissue tumours relates in vivo tumour metabolism as established with TYR-PET to tumour activity measured in vitro and indicates that the non-invasive method of TYR-PET can estimate the mitotic and proliferative activity in STS. (orig.) With 2 figs., 2 tabs., 30 refs.

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

    Science.gov (United States)

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

    2015-10-21

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p 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.

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

    DEFF Research Database (Denmark)

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

    2018-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    International Nuclear Information System (INIS)

    Inwood, W.; Bernardin, J.

    1990-01-01

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

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

    Science.gov (United States)

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

    2015-05-01

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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. Understanding Protein Synthesis: An Interactive Card Game Discussion

    Science.gov (United States)

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  5. Effect of thyroid hormone on the protein turnover rate of mouse pancreas

    International Nuclear Information System (INIS)

    He Xinjun; Zhou Hui; Wang Shizhen; Zhou Zhonming; Li Liangxue; Wei Huaiwei; Sun Xiaomiao; Wang Yanli

    1986-01-01

    The effects of thyroid hormone on the protein turnover of pancreas in mice were studied using labelled amino acid incorporation, double isotopic and labelled protein decay methods. After injection of L-thyroxine (100 μ g/mouse) for 5 days, the amino acid incorporation into pancreatic proteins of mice was profoundly decreased, the ratio of 3 H/ 14 C in labelled proteins and the fractional turnover rate of pancreatic proteins were also decreased, the protein half-lives being consequently prolonged. These findings suggest that large doses of thyroid hormone may reduce the trunover rate of pancreatic proteins, by inhibiting not only the synthesis but also the degradation

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

    Science.gov (United States)

    Pain, Debkumar; Dancis, Andrew

    2016-06-01

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

  7. Measurement of the rate of bacterial protein synthesis in rumen. Part of a coordinated programme on tracer techniques in studies on the use of non-protein nitrogen in ruminants

    International Nuclear Information System (INIS)

    Leng, R.A.

    1975-10-01

    A technique has been developed to differentiate between rumen microbial and dietary protein at the duodenum. Mature whethers fitted with rumen, duodenal and ileal cannulas were fed protein poor diets with added urea, casein and formaldehyde treated casein. These contained 0.30, 1.54, 3.26 and 3.26 % N respectively. The isotopes 35 S or 15 N were infused into the rumen to label microbial protein so that microbial protein could be differentiated from dietary protein. By using the 35 S or 15 N system to mark microbes, it was found that 40% of the formaldehyde treated protein is fermented in the rumen and about 10% is lost in the feces. This indicates that approximately 50% of the protected protein was utilized by the sheep

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

    International Nuclear Information System (INIS)

    Kefalides, N.A.

    1986-01-01

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

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

    Science.gov (United States)

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

    2017-09-06

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

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

    Science.gov (United States)

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

    2007-11-01

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

  11. Nitrogen control of photosynthetic protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, G.W.

    1986-09-01

    Plant growth is severely affected by impaired photosynthesis resulting from nitrogen deficiency. The molecular aspects of this effect are being studied in the green alga Chlamydomonas grown in continuous culture systems. Photosynthetic membranes of nitrogen-limited cells are dramatically depleted in chlorophylls, xanthophylls and proteins of the light-harvesting complexes. In contrast, enzymes of the reductive pentose phosphate cycle and electron transport chain complexes are reduced only 40 to 65% on a per cell basis comparison with nitrogen-sufficient cultures. From analyses of mRNA levels by in vitro translation and hybridization analyses with cloned DNA sequences for photosynthetic proteins, we have found there are rather minor effects of nitrogen deficiency on nuclear or chloroplast gene transcription. Maturation of a transcript of the nuclear-encoded small subunit of ribulose 1,5-bisphosphate carboxylase is inhibited in nitrogen-deficient cells and causes accumulation of large amounts of mRNA precursors. Most of the effects of nitrogen deficiency on photosynthetic proteins appear to result from posttranscriptional regulatory processes: light-harvesting protein synthesis may be sustained but their import into chloroplasts or translocation to photosynthetic membranes is impaired. Nitrogen-deficient cells lack violaxanthin, a pigment that is essential for the structure, function and biogenesis of the major antenna complexes. The absence of this pigment may be a causative factor for the deficiency of light harvesting complexes. Finally, the accumulation of massive amounts of starch and triglycerides in nitrogen-limited cells indicate there are some genes whose maximal expression is dependent upon nitrogen-limiting conditions. 10 refs.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  15. Protein degradation and protein synthesis in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2014-06-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

  17. Dendritic protein synthesis in the normal and diseased brain

    Science.gov (United States)

    Swanger, Sharon A.; Bassell, Gary J.

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-12-09

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  20. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

    2017-12-06

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

  1. Stimulation of protein synthesis by internalized insulin

    International Nuclear Information System (INIS)

    Miller, D.S.; Sykes, D.B.

    1991-01-01

    Previous studies showed that microinjected insulin stimulates transcription and translation in Stage 4 Xenopus oocytes by acting at nuclear and cytoplasmic sites. The present report is concerned with the question of whether hormone, internalized from an external medium, can act on those sites to alter cell function. Both intracellular accumulation of undegraded 125I-insulin and insulin-stimulated 35S-methionine incorporation into oocyte protein were measured. Anti-insulin antiserum and purified anti-insulin antibody were microinjected into the cytoplasm of insulin-exposed cells to determine if insulin derived from the medium acted through internal sites. In cells exposed for 2 h to 7 or 70 nM external insulin, methionine incorporation was stimulated, but intracellular hormone accumulation was minimal and microinjected antibody was without effect. In cells exposed for 24 h, methionine incorporation again increased, but now accumulation of undegraded, intracellular hormone was substantial (2.6 and 25.3 fmol with 7 and 70 nM, respectively), and microinjected anti-insulin antibody significantly reduced the insulin-stimulated component of incorporation; basal incorporation was not affected. For cells exposed to 70 nM insulin for 24 h, inhibition of the insulin-stimulated component was maximal at 39%. Thus under those conditions, about 40% of insulin's effects were mediated by the internal sites. Together, the data show that inhibition of insulin-stimulated protein synthesis by microinjected antibody was associated with the intracellular accumulation of insulin. They indicate that when oocytes are exposed to external insulin, hormone eventually gains access to intracellular sites of action and through these stimulates translation. Control of translation appears to be shared between the internal sites and the surface receptor

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  3. Protein synthesis in the presence of carbamoyl-amino acids

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  4. High-deposition-rate ceramics synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Allendorf, M.D.; Osterheld, T.H.; Outka, D.A. [Sandia National Laboratories, Livermore, CA (United States)] [and others

    1995-05-01

    Parallel experimental and computational investigations are conducted in this project to develop validated numerical models of ceramic synthesis processes. Experiments are conducted in the High-Temperature Materials Synthesis Laboratory in Sandia`s Combustion Research Facility. A high-temperature flow reactor that can accommodate small preforms (1-3 cm diameter) generates conditions under which deposition can be observed, with flexibility to vary both deposition temperature (up to 1500 K) and pressure (as low as 10 torr). Both mass spectrometric and laser diagnostic probes are available to provide measurements of gas-phase compositions. Experiments using surface analytical techniques are also applied to characterize important processes occuring on the deposit surface. Computational tools developed through extensive research in the combustion field are employed to simulate the chemically reacting flows present in typical industrial reactors. These include the CHEMKIN and Surface-CHEMKIN suites of codes, which permit facile development of complex reaction mechanisms and vastly simplify the implementation of multi-component transport and thermodynamics. Quantum chemistry codes are also used to estimate thermodynamic and kinetic data for species and reactions for which this information is unavailable.

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

    OpenAIRE

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

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential a...

  6. Viral protein synthesis in cowpea mosaic virus infected protoplasts

    International Nuclear Information System (INIS)

    Rottier, P.

    1980-01-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

    He, Mingyue

    2008-01-01

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

  11. The origin of polynucleotide-directed protein synthesis

    Science.gov (United States)

    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.

  12. Protein synthesis in the rat brain: a comparative in vivo and in vitro study in immature and adult animals

    International Nuclear Information System (INIS)

    Shahbazian, F.M.

    1985-01-01

    Rates of protein synthesis of CNS and other organs were compared in immature and adult rats by in vivo and slice techniques with administration of flooding doses of labeled precursor. The relationship between synthesis and brain region, cell type, subcellular fraction, or MW was examined. Incorporation of [ 14 C]valine into protein of CNS regions in vivo was about 1.2% per hour for immature rats and 0.6% for adults. For slices, the rates decreased significantly more in adults. In adult organs, the highest synthesis rate in vivo was found in liver (2.2% per hour) followed by kidney, spleen, lung, heart, brain, and muscle (0.5% per hour). In immature animals synthesis was highest in liver and spleen (2.5% per hour) and lowest in muscle (0.9% per hour). Slices all showed lower rates than in vivo, especially in adults. In vivo, protein synthesis rates of immature neurons and astrocytes and adult neurons exceeded those of whole brain, while that in adult astrocytes was the same. These results demonstrate a developmental difference of protein synthesis (about double in immature animals) in all brain cells, cell fractions and most brain protein. Similarly the decreased synthesis in brain slices - especially in adults, affects most proteins and structural elements

  13. Protein synthesis in x-irradiated rabbit lens

    International Nuclear Information System (INIS)

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

    1984-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  15. Determination of possible effects of mineral concentration on protein synthesis by rumen microbes in vitro

    International Nuclear Information System (INIS)

    Nikolic, J.A.; Jovanovic, M.; Andric, R.

    1976-01-01

    The aim of the present investigation was to determine the effect of different concentrations of sulphide, magnesium and zinc on protein synthesis by rumen micro-organisms in vitro. Rumen content was taken from a young bull fed a diet based on maize and dried sugar beet pulp (2/1) supplemented with urea. The rate of incorporation of 35 S from Na 2 35 SO 4 in relation to the mean specific radioactivity of the sulphide pool was used to estimate the overall rate of microbial protein synthesis. It was found that the rate of protein synthesis and the net rate of utilization of ammonia-N were not affected by differences in mean sulphide concentration from 3.6-8.0 mg/litre. The rate of reduction of sulphate appeared not to be affected by the addition of sodium sulphide to the medium. The rate and efficiency of protein synthesis by rumen micro-organisms were not significantly affected by increasing the concentration of total magnesium from 8.4-15.3 mg/100 ml. The values for soluble magnesium varied widely (1.2-7.8 mg/100 ml), and appeared to be partly dependent on the pH of the medium. Zinc concentrations varying from 5.2-12.4 mg/litre did not influence the overall rate of protein synthesis, although the efficiency tended to be higher when the concentration of zinc was greater. Concentrations of soluble zinc were low (0.3-1.15 mg/litre), and not influenced by changes in the concentration of total zinc. It was concluded that increasing the concentrations of the examined elements above the basic values did not lead consistently to an improved production of microbial protein but, on the other hand, had no obvious detrimental effect on microbial metabolic activity within the limits studied. (author)

  16. In vitro estimation of rumen microbial protein synthesis of water buffaloes using 30S as tracer

    International Nuclear Information System (INIS)

    Hendratno, C.; Abidin, Z.; Bahaudin, R.; Sastrapradja, D.

    1977-01-01

    An experiment to study the effect of diet and individual differences of animals on the in vitro estimation of rumen microbial protein synthesis in young female water buffaloes using the technique of inorganic 35 S incorporation, is described. The dietary treatments were four combinations of roughage supplemented with cassava meal. From the value of rate constant for dilution of radioactivity in the sulphide pool and percentage of inorganic 35 S incorporated into microbial protein, it can be concluded that individual differences of animals have no influence on the efficiency of microbial protein synthesis. Feed composition, on the other hand, tends to have some influence on the efficiency of protein synthesis(P3O.15). (author)

  17. Measurement of rates of cholesterol synthesis using tritiated water

    International Nuclear Information System (INIS)

    Dietschy, J.M.; Spady, D.K.

    1984-01-01

    Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14 C-labeled substrates or [ 3 H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14 C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [ 3 H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14 C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3 H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3 H are incorporated into cholesterol from [ 3 H]water in different tissues of several animal species, so that the 3 H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [ 3 H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [ 3 H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo

  18. Protein synthesis and sublethal damage repair in synchronized CHO cells

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  19. Nuclear transport factor directs localization of protein synthesis during mitosis

    NARCIS (Netherlands)

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

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

  20. In situ synthesis of protein arrays.

    Science.gov (United States)

    He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

    2008-02-01

    In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

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

    Science.gov (United States)

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

    2017-05-01

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

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

    International Nuclear Information System (INIS)

    Mortensen, R.M.

    1983-01-01

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

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

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

  5. Rewiring protein synthesis: From natural to synthetic amino acids.

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2016-02-18

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

  9. Induction of protein synthesis in Escherichia coli following UV- or γ-irradiation, mitomycin C treatment or tif expression

    International Nuclear Information System (INIS)

    West, S.C.; Emmerson, P.T.

    1977-01-01

    The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or γ-rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in the protein X. Synthesis of large quantities of protein X is induced by UV, γ-rays, MMC treatment or tif expression in rec + but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA + cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation. Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage lambda or septation. (orig./MG) [de

  10. Serotonin synthesis rate and the tryptophan hydroxylase-2

    DEFF Research Database (Denmark)

    Furmark, Tomas; Marteinsdottir, Ina; Frick, Andreas

    2016-01-01

    It is disputed whether anxiety disorders, like social anxiety disorder, are characterized by serotonin over- or underactivity. Here, we evaluated whether our recent finding of elevated neural serotonin synthesis rate in patients with social anxiety disorder could be reproduced in a separate cohor...

  11. No effect of menstrual cycle on myofibrillar and connective tissue protein synthesis in contracting skeletal muscle

    DEFF Research Database (Denmark)

    Miller, B.F.; Hansen, M.; Olesen, J.L.

    2006-01-01

    We tested the hypothesis that acute exercise would stimulate synthesis of myofibrillar protein and intramuscular collagen in women and that the phase of the menstrual cycle at which the exercise took place would influence the extent of the change. Fifteen young, healthy female subjects were studied...... in the follicular (FP, n=8) or the luteal phase (LP, n=7, n=1 out of phase) 24 h after an acute bout of one-legged exercise (60 min of kicking at 67% W(max)), samples being taken from the vastus lateralis in both the exercised and resting legs. Rates of synthesis of myofibrillar and muscle collagen proteins were...... measured by incorporation of [(13)C]leucine. Myofibrillar protein synthesis (means+/-SD; rest FP: 0.053+/-0.009%/h, LP: 0.055+/-0.013%/h) was increased at 24-h postexercise (FP: 0.131+/-0.018%/h, Psynthesis...

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

  14. Selective memory generalization by spatial patterning of protein synthesis.

    Science.gov (United States)

    O'Donnell, Cian; Sejnowski, Terrence J

    2014-04-16

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

  15. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  17. Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation

    International Nuclear Information System (INIS)

    Smith, C.B.; Deibler, G.E.; Eng, N.; Schmidt, K.; Sokoloff, L.

    1988-01-01

    A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1- 14 C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steady-state degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account

  18. Effects of starvation on protein synthesis and nucleic acid metabolism in the muscle of the barred sand bass Paralabrax nebulifer

    Energy Technology Data Exchange (ETDEWEB)

    Lowery, M.S.

    1987-01-01

    Starvation induced different protein synthesis responses in red and white muscle of the barred sand bass Paralabrax nebulifer. Red muscle had /sup 14/C-leucine incorporation rates into total protein which were several times higher than white muscle in both the fed and starved states. Muscle was separated into a myofibrillar fraction consisting of the structural proteins and a sarcoplasmic fraction consisting of soluble proteins. Synthesis of the myofibrillar fraction of white muscle decreased by 90%, while red muscle myofibrillar synthesis remained essentially unchanged. Changes in the labeling of several enzymes purified from the sarcoplasmic fraction were different even though the overall loss of enzyme activity was similar, suggesting that changes in synthesis rates were important in maintaining appropriate relative enzyme concentrations.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  1. Effects of putrescine, kinetin and IAA on protein synthesis in 'Phaseolus vulgaris' coleoptiles

    Energy Technology Data Exchange (ETDEWEB)

    Crocomo, O J; Lee, T S.G. [Centro de Energia Nuclear na Agricultura, Piracicaba (Brazil)

    1975-01-01

    Incubation of etiolated 'Phaseolus vulgaris' coleoptiles shows a converse flux between soluble protein and reducing sugar. The rate of incorporation of radioactive arginine into protein was higher than that of radioactive leucine. Radioactive arginine incorporation into protein was linear up to 120 min and then started to decline. The rate of incorporation of radioactive leucine was increased by preincubation of the tissue in the incubation medium. Roots were found to contain more soluble protein and much less reducing sugar than the coleoptile. The optimum pH value for protein synthesis in coleoptile sections was found to be 6 for control tissues and 4 for those treated with 10-/sup 3/M IAA. This high concentration of IAA was also found to inhibit soluble protein synthesis, the incorporation rate of radioactive arginine and leucine into protein fraction, the secretion of hydrogen ion into the incubation medium and elongation of the bean segment. Kinetin at 2x10/sup -4/M and putrescine at 5mM both decreased the rate of /sup 14/C-arginine incorporation into soluble protein, but for /sup 14/C-leucine, this rate of incorporation was found to be increased after 90 min incubation with a preincubation of 30 min. In general, the change pattern of the soluble protein content, the reducing sugar level and the incorporation rate of radioactive arginine and leucine into protein in the kinetin and putrescine treated tissues were about the same although tissues that incubated with kinetin always contain more soluble protein and less reducing sugar than that of incubated with putrescine.

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

    Directory of Open Access Journals (Sweden)

    Richa Tyagi

    2015-02-01

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

  3. Assessing the reliability of calculated catalytic ammonia synthesis rates

    DEFF Research Database (Denmark)

    Medford, Andrew James; Wellendorff, Jess; Vojvodic, Aleksandra

    2014-01-01

    We introduce a general method for estimating the uncertainty in calculated materials properties based on density functional theory calculations. We illustrate the approach for a calculation of the catalytic rate of ammonia synthesis over a range of transition-metal catalysts. The correlation...... between errors in density functional theory calculations is shown to play an important role in reducing the predicted error on calculated rates. Uncertainties depend strongly on reaction conditions and catalyst material, and the relative rates between different catalysts are considerably better described...

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

    Science.gov (United States)

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

    2013-01-01

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

  5. The limits of adaptation of functional protein synthesis to sever undernutrition

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  6. The limits of adaptation of functional protein synthesis to sever undernutrition

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-31

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

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

    Science.gov (United States)

    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).

  8. Sulfur in human nutrition - effects beyond protein synthesis

    NARCIS (Netherlands)

    Gertjan Schaafsma

    2008-01-01

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

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

    Science.gov (United States)

    Szeberenyi, Jozsef

    2009-01-01

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

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

    Science.gov (United States)

    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…

  11. Intestinal DNA concentration and protein synthesis in response to ...

    African Journals Online (AJOL)

    Performance, protein synthesis and mucosal DNA in small intestine of Leghorn hens may be affected by low quality feedstuff. An experiment was conducted in completely randomized design (CRD) in 2 × 2 factorial arrangement. Main factors included diets containing 20 and 40 % barley and black and blue strains of leghorn ...

  12. Digestion and microbial protein synthesis in sheep as affected by ...

    African Journals Online (AJOL)

    Useni , Alain

    enzyme (EFE) on the in vitro gas production (GP) and ANKOM digestion systems on the mixture of milled ... determine the EFE effect on the DM, CP and NDF digestion of a mixture of lucerne hay and wheat straw .... and the microbial protein synthesis (MPS) measured as purine derivates (RNA equivalent in µg/DM g) on.

  13. 46 CFR 108.463 - Foam rate: Protein.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Foam rate: Protein. 108.463 Section 108.463 Shipping... EQUIPMENT Fire Extinguishing Systems Foam Extinguishing Systems § 108.463 Foam rate: Protein. (a) If the outlets of a protein foam extinguishing system are in a space, the foam rate at each outlet must be at...

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  15. Control of protein synthesis in the female pupa of Bombyx mori

    International Nuclear Information System (INIS)

    Yamao, Masami; Koga, Katsumi

    1975-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Matthew B Hudson

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

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

    Science.gov (United States)

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

    2015-01-01

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

  18. Effect of administration of oral contraceptives on the synthesis and breakdown of myofibrillar proteins in young women

    DEFF Research Database (Denmark)

    Hansen, M; Langberg, Henning; Holm, L

    2011-01-01

    Oral contraceptive (OC) treatment has an inhibiting effect on protein synthesis in tendon and muscle connective tissue. We aimed to investigate whether OC influence myofibrillar protein turnover in young women. OC-users (24±2 years; Lindynette® n=7, Cilest® n=4) and non-OC-users (controls, 24......±4 years n=12) performed one-legged kicking exercise. The next day, the myofibrillar protein fractional synthesis rate (FSR) was measured using stable isotopic tracers ((13)C-proline) while the subjects were fed standardized nutrient drinks. Simultaneously, a marker for myofibrillar protein breakdown, 3...

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

    Science.gov (United States)

    Fernie, Alisdair R.; Stitt, Mark

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    1961-01-01

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

  1. Protein synthesis and intestinal flora in piglets

    International Nuclear Information System (INIS)

    Namioka, Shigeo

    1980-01-01

    Utilization of non-protein nitrogen (NPN) by the flora in piglet colon was studied by administration of 15 N-urea and 15 N-ammonium salt to aseptic piglets and to SPF piglets which had been acclimatized to a clean environment after settling of intestinal flora. Administration of 15 N-urea did not result in 15 N uptake by any tissue-constituting protein at any site of the aseptic piglets, almost all 15 N being excreted into the urine. In contrast, the tissue and skeletal muscle of the SPF piglets showed incorporated 15 N from urea. Urea was converted, by urease of the intestinal flora, into NH 3 , which was absorbed from the mucosa of the intestinal tract to reach the liver where it was synthesized into glutamic acid, followed by conversion into various amino acids. 15 N-ammonium administration produced a significant amount of 15 N even in the tissue protein of the aseptic piglets. After NPN administration, the liver protein-constituting amino acid fraction showed 15 N-labeling of almost all essential, as well as non-essential amino acids. Culture of colonic flora with 15 N-urea revealed 15 N-labeling of all amino acids that constituted bacterial cells, indicating the presence of urea recycling mediated by bacterial urease in single rumen animals.(Chiba, N.)

  2. Ribosomal history reveals origins of modern protein synthesis.

    Directory of Open Access Journals (Sweden)

    Ajith Harish

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

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

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

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

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

    Science.gov (United States)

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

    2007-08-01

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

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

    Science.gov (United States)

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

    2015-09-15

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

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

    Directory of Open Access Journals (Sweden)

    Takehiro Nishikawa

    2012-01-01

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

  7. Enteral β-hydroxy-β-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs

    Science.gov (United States)

    Kao, Michelle; Columbus, Daniel A.; Suryawan, Agus; Steinhoff-Wagner, Julia; Hernandez-Garcia, Adriana; Nguyen, Hanh V.; Fiorotto, Marta L.

    2016-01-01

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were studied immediately (F) or fed one of five diets for 24 h: low-protein (LP), high-protein (HP), or LP diet supplemented with 4 (HMB4), 40 (HMB40), or 80 (HMB80) μmol HMB·kg body wt−1·day−1. Cell replication was assessed from nuclear incorporation of BrdU in the longissimus dorsi (LD) muscle and jejunum crypt cells. Protein synthesis rates in LD, gastrocnemius, rhomboideus, and diaphragm muscles, lung, and brain were greater in HMB80 and HP and in brain were greater in HMB40 compared with LP and F groups. Formation of the eIF4E·eIF4G complex and S6K1 and 4E-BP1 phosphorylation in LD, gastrocnemius, and rhomboideus muscles were greater in HMB80 and HP than in LP and F groups. Phosphorylation of eIF2α and eEF2 and expression of SNAT2, LAT1, MuRF1, atrogin-1, and LC3-II were unchanged. Numbers of BrdU-positive myonuclei in the LD were greater in HMB80 and HP than in the LP and F groups; there were no differences in jejunum. The results suggest that enteral supplementation with HMB increases skeletal muscle protein anabolism in neonates by stimulation of protein synthesis and satellite cell proliferation. PMID:27143558

  8. MATHEMATICAL AND COMPUTATIONAL MODELLING OF RIBOSOMAL MOVEMENT AND PROTEIN SYNTHESIS: AN OVERVIEW

    Directory of Open Access Journals (Sweden)

    Tobias von der Haar

    2012-04-01

    Full Text Available Translation or protein synthesis consists of a complex system of chemical reactions, which ultimately result in decoding of the mRNA and the production of a protein. The complexity of this reaction system makes it difficult to quantitatively connect its input parameters (such as translation factor or ribosome concentrations, codon composition of the mRNA, or energy availability to output parameters (such as protein synthesis rates or ribosome densities on mRNAs. Mathematical and computational models of translation have now been used for nearly five decades to investigate translation, and to shed light on the relationship between the different reactions in the system. This review gives an overview over the principal approaches used in the modelling efforts, and summarises some of the major findings that were made.

  9. Cell growth and protein synthesis of unicellular green alga Chlamydomonas in heavy water

    International Nuclear Information System (INIS)

    Ishida, M.R.

    1983-01-01

    The effects of heavy water on the cell growth and protein synthesis of the photoautotrophically growing Chlamydomonas cells were studied. The growth rate of the cells is inversely proportional to the concentrations of heavy water. The cells can barely live in 90% heavy water, but they die in 99.85% heavy water within a few days. Incorporation of 14 Cleucine into cells is markedly stimulated by heavy water of various concentrations between 30 and 99.85% in the case of the short time incubation. Contrary to this, in the long time incubation as several days, heavy water inhibits the protein synthesis. Such two modes of the protein synthetic activities are dependent upon the incubation time of the cells grown photoautotrophically in the heavy water media. (author)

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  11. Synthesis and functioning of the colicin E1 lysis protein: Comparison with the colicin A lysis protein

    International Nuclear Information System (INIS)

    Cavard, D.

    1991-01-01

    The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [ 35 S]cysteine or [ 3 H]lysine. This 3-kDa protein was acylated, as shown by [2- 3 H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants

  12. Towards single-molecule observation of protein synthesis

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  13. Effects of estrogen replacement and lower androgen status on skeletal muscle collagen and myofibrillar protein synthesis in postmenopausal women

    DEFF Research Database (Denmark)

    Hansen, Mette; Skovgaard, Dorthe; Reitelseder, Søren

    2012-01-01

    Our aim was to determine synthesis rate of myofibrillar and collagen proteins in 20 postmenopausal women, who were either nonusers (Controls) or users of estrogen replacement therapy (ERT) after hysterectomy/oophorectomy. Myofibrillar and muscle collagen protein fractional synthesis rate (FSR) were...... determined in a nonexercised leg and 24 hours after exercise in the contralateral leg. A significant interaction between treatment and mechanical loading was observed in myofibrillar protein FSR. At rest, myofibrillar protein FSR was found to be lower in ERT users than in Controls. Exercise enhanced...... myofibrillar protein FSR only in ERT users. Similarly, muscle collagen FSR tended to be lower in ERT users compared with Controls. In ERT participants, the androgen profile was reduced, whereas estradiol and sex hormone–binding globulin were higher. In conclusion, at rest, myofibrillar protein FSR was lower...

  14. Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

    Science.gov (United States)

    Taupin, Philippe

    2011-09-01

    The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

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

    Science.gov (United States)

    Kent, Stephen

    2017-09-15

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

  16. Differential effects of methylmercury on the synthesis of protein species in dorsal root ganglia of the rat

    International Nuclear Information System (INIS)

    Kasama, Hidetaka; Itoh, Kazuo; Omata, Saburo; Sugano, Hiroshi

    1989-01-01

    Dorsal root ganglia from control and methylmercury(MeHg)-treated rats were incubated in vitro with 35 S-methionine and the proteins synthesized were analyzed by two-dimensional electrophoresis. The double labelling method, in which proteins of control dorsal root ganglia labelled in vitro with 3 H-leucine were added to each of the two samples as an internal standard, was used to minimize unavoidable errors arising from the resolving procedure itself. The results obtained showed that the effect of MeHg on the synthesis of proteins in dorsal root ganglia was not uniform for individual protein species in the latent period of MeHg intoxication. Among 200 protein species investigated, 157 showed inhibition of synthesis close to that of the total proteins in the tissue (68% of the control). Among the remaining protein species, 20 showed real stimulation of synthesis, whereas 7 were moderately inhibited and 16 were inhibited more strongly than the total proteins in the tissue. These results suggest that the effect of MeHg on the synthetic rates for protein species in dorsal root ganglia differs with the species, and that unusual elevation or reduction of the synthesis of some protein species caused by MeHg may lead to impairment of normal nerve functions. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Proud Christopher G

    2002-05-01

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

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

    Science.gov (United States)

    Kent, Stephen Bh

    2018-04-04

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1978-10-01

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

  20. Twister Protein: a ludic tool involving protein synthesis

    Directory of Open Access Journals (Sweden)

    Aline Weyh

    2015-07-01

    Full Text Available Several studies show that students of various grade levels report the Genetics as an abstract theme and difficult to assimilate by the students, with multiple problems in the teaching-learning process and becoming necessary the development of auxiliary practices. Among the teaching tools, the game is the most currently opted playful activity by stimulating multiple intelligences, allowing greater student-teacher interaction. This work seeks the production of an innovative and dynamic educational game, Twister Protein, as a pedagogical resource for Genetics discipline. The development of the game was based on the use of easily accessible and low cost materials by teachers, allowing the knowledge of transcription, translation and protein folding. The activity was proposed and applied in the classroom with pilot undergraduate students. The fun associated with the knowledge of science not only allowed a better memorization of the content addressed, as aroused the curiosity, theme reflection, character building and collaborative spirits, as well as competitiveness through the interaction between class. This practice proved to be an effective tool in the escape from routine and fault repair of the theoretical process.

  1. Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

    Science.gov (United States)

    Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens Høgh; Alfonta, Lital

    2017-06-16

    Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

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

    OpenAIRE

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

    1989-01-01

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

  3. Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

    Science.gov (United States)

    Munoz, L; Sadaie, Y; Doi, R H

    1978-10-10

    The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

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

    Directory of Open Access Journals (Sweden)

    Boyce Mark

    2012-08-01

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

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

    Science.gov (United States)

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

    2012-08-29

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

  6. Protein synthesis in vitro by Micrococcus luteus.

    Science.gov (United States)

    Farwell, M A; Rabinowitz, J C

    1991-01-01

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

  7. Myofibrillar protein synthesis following ingestion of soy protein isolate at rest and after resistance exercise in elderly men

    Directory of Open Access Journals (Sweden)

    Yang Yifan

    2012-06-01

    Full Text Available Abstract Background Increased amino acid availability stimulates muscle protein synthesis, however, aged muscle appears less responsive to the anabolic effects of amino acids when compared to the young. We aimed to compare changes in myofibrillar protein synthesis (MPS in elderly men at rest and after resistance exercise following ingestion of different doses of soy protein and compare the responses to those we previously observed with ingestion of whey protein isolate. Methods Thirty elderly men (age 71 ± 5 y completed a bout of unilateral knee-extensor resistance exercise prior to ingesting no protein (0 g, or either 20 g or 40 g of soy protein isolate (0, S20, and S40 respectively. We compared these responses to previous responses from similar aged men who had ingested 20 g and 40 g of whey protein isolate (W20 and W40. A primed constant infusion of L-[1-13 C]leucine and L-[ring-13 C6]phenylalanine and skeletal muscle biopsies were used to measure whole-body leucine oxidation and MPS over 4 h post-protein consumption in both exercised and non-exercised legs. Results Whole-body leucine oxidation increased with protein ingestion and was significantly greater for S20 vs. W20 (P = 0.003. Rates of MPS for S20 were less than W20 (P = 0.02 and not different from 0 g (P = 0.41 in both exercised and non-exercised leg muscles. For S40, MPS was also reduced compared with W40 under both rested and post-exercise conditions (both P P = 0.04. Conclusions The relationship between protein intake and MPS is both dose and protein source-dependent, with isolated soy showing a reduced ability, as compared to isolated whey protein, to stimulate MPS under both rested and post-exercise conditions. These differences may relate to the lower postprandial leucinemia and greater rates of amino acid oxidation following ingestion of soy versus whey protein.

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

    Science.gov (United States)

    Heude, M; Chanet, R; Moustacchi, E

    1975-04-01

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

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

    Science.gov (United States)

    Liu, Han-Hsuan; Cline, Hollis T

    2016-07-06

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

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

    Science.gov (United States)

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

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

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

    International Nuclear Information System (INIS)

    Hurewitz, J.; Jagendorf, A.T.

    1986-01-01

    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 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

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

    Science.gov (United States)

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

    2008-10-01

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

  13. Neuronal response of the hippocampal formation to injury: blood flow, glucose metabolism, and protein synthesis

    International Nuclear Information System (INIS)

    Kameyama, M.; Wasterlain, C.G.; Ackermann, R.F.; Finch, D.; Lear, J.; Kuhl, D.E.

    1983-01-01

    The reaction of the hippocampal formation to entorhinal lesions was studied from the viewpoints of cerebral blood flow ([ 123 I]isopropyl-iodoamphetamine[IMP])-glucose utilization ([ 14 C]2-deoxyglucose), and protein synthesis ([ 14 C]leucine), using single- and double-label autoradiography. Researchers' studies showed decreased glucose utilization in the inner part, and increased glucose utilization in the outer part of the molecular layer of the dentate gyrus, starting 3 days after the lesion; increased uptake of [ 123 I]IMP around the lesion from 1 to 3 days postlesion; and starting 3 days after the lesion, marked decrease in [ 14 C]leucine incorporation into proteins and cell loss in the dorsal CA1 and dorsal subiculum in about one-half of the rats. These changes were present only in animals with lesions which invaded the ventral hippocampal formation in which axons of CA1 cells travel. By contrast, transsection of the 3rd and 4th cranial nerves resulted, 3 to 9 days after injury, in a striking increase in protein synthesis in the oculomotor and trochlear nuclei. These results raise the possibility that in some neurons the failure of central regeneration may result from the cell's inability to increase its rate of protein synthesis in response to axonal injury

  14. Differential effect of NMDA and AMPA receptor blockade on protein synthesis in the rat infarct borderzone

    DEFF Research Database (Denmark)

    Christensen, Thomas; Bruhn, T; Frank, L

    1996-01-01

    treated with either saline, MK-801 (5 mg/kg i.p.) or NBQX (30 mg/kg i.p. x 3) were subjected to permanent MCAO. Regional CPSR and volumes of gray matter structures displaying normal CPSR were measured in coronal cryosections of the brain by quantitative autoradiography following an i.v. bolus injection....... Treatment with MK-801 significantly increased the volume of tissue with normal CPSR in the ischemic hemisphere compared to controls, whereas this was not seen with NBQX treatment. The results suggest that MK-801 and NBQX have different effects on peri-infarct protein synthesis after MCAO. Since both......We investigated whether the known neuroprotective effects of two selective glutamate receptor antagonists, the NMDA antagonist MK-801 and the AMPA antagonist NBQX, are reflected in the regional cerebral protein synthesis rates (CPSR) in rats with middle cerebral artery occlusion (MCAO). Rats...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  16. Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals

    International Nuclear Information System (INIS)

    Woodson, W.R.; Handa, A.K.

    1986-01-01

    Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of 3 H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide

  17. Role for tryptophan in regulation of protein synthesis in porcine muscle

    International Nuclear Information System (INIS)

    Lin, F.D.; Smith, T.K.; Bayley, H.S.

    1988-01-01

    Experiments were conducted to determine the effect of varying concentrations of dietary tryptophan on growth rate and protein synthesis in edible muscle tissues of growing swine. A total of 45 immature swine (initial weight approximately 24 kg) were fed corn-gelatin diets containing 0.5 (n = 8), 0.8 (n = 10), 1.3 (n = 10), 1.5 (n = 7) or 2.0 (n = 10) g tryptophan/kg diet for 35 d. Animals fed 0.5 and 0.8 g tryptophan/kg grew more slowly, consumed less feed and had a lower efficiency of feed utilization than animals fed higher concentrations of tryptophan. Thirty similar animals were used in a second experiment. Diets containing 0.5, 0.8, 1.0, 1.5 or 2.0 g tryptophan/kg diet (n = 6) were fed for 14 d, after which all animals were killed and samples were taken of longissimus dorsi, triceps brachii and biceps femoris. Protein synthetic activity was determined by monitoring the incorporation of [ 14 C]phenylalanine into protein in vitro. There was no significant difference in synthetic activity between different muscle types. There was no effect of diet on the activity of the muscle soluble protein fraction. The activity of the muscle ribosomal fraction, however, was positively correlated with increasing concentrations of dietary tryptophan. It was concluded that tryptophan has the potential to regulate muscle protein synthesis in a manner beyond serving simply as a component of protein

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

    Science.gov (United States)

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

    2006-07-28

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    1984-08-01

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

  1. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    Science.gov (United States)

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-04-01

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

  3. Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues

    OpenAIRE

    Yao, Lihang; Jenkins, Katie; Horn, Paul S.; Lichtenberg, M. Hayden; Woollett, Laura A.

    2007-01-01

    The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only margina...

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

    Energy Technology Data Exchange (ETDEWEB)

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

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  6. Protein composition and synthesis in the adult mouse spinal cord

    International Nuclear Information System (INIS)

    Stodieck, L.S.; Luttges, M.W.

    1983-01-01

    Properties of spinal cord proteins were studied in adult mice subjected to unilateral crush or electrical stimulation of sciatic nerve. The protein composition of spinal tissue was determined using SDS-polyacrylamide gel electrophoresis coupled with subcellular fractionation. Comparisons of mouse spinal cord and brain revealed similarities in the types but differences in the concentrations of myelin associated proteins, nuclear histones and other proteins. Comparisons with sciatic nerve proteins demonstrated differences in types of proteins but similarities in the concentration of myelin proteins and nuclear histones. The short term (less than 2 hrs.) incorporation of radioactive amino acids into spinal cord proteins revealed heterogeneous rates of incorporation. Neither nerve crush six days prior to testing nor sciatic nerve stimulation had a significant effect on the protein composition or amino acid incorporation rates of spinal cord tissue. These observations suggest that known differences in spinal cord function following alterations in nerve input may be dependent upon different mechanisms than have been found in the brain

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

    Science.gov (United States)

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.

  8. Effect of exercise and recovery on muscle protein synthesis in human subjects

    International Nuclear Information System (INIS)

    Carraro, F.; Stuart, C.A.; Hartl, W.H.; Rosenblatt, J.; Wolfe, R.R.

    1990-01-01

    Previous studies using indirect means to assess the response of protein metabolism to exercise have led to conflicting conclusions. Therefore, in this study we have measured the rate of muscle protein synthesis in normal volunteers at rest, at the end of 4 h of aerobic exercise (40% maximal O2 consumption), and after 4 h of recovery by determining directly the rate of incorporation of 1,2-[13C]leucine into muscle. The rate of muscle protein breakdown was assessed by 3-methylhistidine (3-MH) excretion, and total urinary nitrogen excretion was also measured. There was an insignificant increase in 3-MH excretion in exercise of 37% and a significant increase (P less than 0.05) of 85% during 4 h of recovery from exercise (0.079 +/- 0.008 vs. 0.147 +/- 0.0338 mumol.kg-1.min-1 for rest and recovery from exercise, respectively). Nonetheless, there was no effect of exercise on total nitrogen excretion. Muscle fractional synthetic rate was not different in the exercise vs. the control group at the end of exercise (0.0417 +/- 0.004 vs. 0.0477 +/- 0.010%/h for exercise vs. control), but there was a significant increase in fractional synthetic rate in the exercise group during the recovery period (0.0821 +/- 0.006 vs. 0.0654 +/- 0.012%/h for exercise vs. control, P less than 0.05). Thus we conclude that although aerobic exercise may stimulate muscle protein breakdown, this does not result in a significant depletion of muscle mass because muscle protein synthesis is stimulated in recovery

  9. Upregulation of circulating myomiR following short-term energy restriction is inversely associated with whole body protein synthesis

    Science.gov (United States)

    The objective of the present investigation was to determine whether energy restriction (ER) influences expression of skeletal muscle-specific microRNA (miRNA) in circulation (c-myomiR) and whether changes in c-myomiR are associated with rates of whole body protein synthesis. Sixteen older (64 +/- 2 ...

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

    International Nuclear Information System (INIS)

    Gailit, James

    1990-01-01

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

  11. Effect of Insulin Infusion on Liver Protein Synthesis during Hemodialysis

    DEFF Research Database (Denmark)

    Reinhard, Mark; Frystyk, Jan; Jespersen, Bente

    2011-01-01

    Background Hemodialysis (HD) is a catabolic procedure that may contribute to the high frequency of protein-energy wasting among patients receiving maintenance HD. The present study investigated the additional effect of glucose and glucose-insulin infusion on liver protein synthesis during HD...... compared with a meal alone. Methods In a randomized cross-over study with three arms, 11 non-diabetic HD patients were assigned to receive a conventional HD session with either: • no treatment (NT) • IV infusion of glucose (G) • IV infusion of glucose-insulin (GI) During infusions blood glucose levels were...... maintained at 8.0-10.0 mmol/L by additional glucose infusion. Glucose and glucose-insulin infusions were commenced 2 h prior to HD and continued throughout the HD session. Fasting blood samples were collected at baseline before infusion and followed by the only meal allowed during the study. Results Blood...

  12. Effect of inhibition of protein synthesis on the development of thermotolerance

    International Nuclear Information System (INIS)

    Chang, P.Y.; Blakely, E.A.; Gonzalez-Flores, I.

    1986-01-01

    The authors have chosen to use a temperature-sensitive mutant line, CHO-TSH1, which shuts down protein synthesis at nonpermissive temperatures of 40 0 C and above by the inactivation of its cytoplasmic nonmitochondrial leucyl-transfer RNA (t-RNA) synthetase enzyme. The parent cell line, CHO-SC1, was used as the control for these experiments. Exponentially growing, asynchronous CHO-TSH1 and CHO-SC1 cell populations were treated for times up to 8 hours at 41.5 0 C, 42 0 C, and 42.5 0 C. The wild-type cells showed the development of tolerance to heat killing at 41.5 0 C, 42 0 C, and possibly at 42.5 0 C, although the survival level at which tolerance developed at 42.5 0 C was too low to be statistically significant. The CHO-TSH1 mutant cell showed no tolerance at any of those temperatures. The rate of total protein synthesis was measured in both cell lines in pulse-labeling experiments with 3 H-leucine under the conditions of the experiment. Results indicated that the rate of synthesis dropped precipitously within the initial hour of exposure to 42 0 C and remained low during the 3 hours of 42 0 C treatment. When each cell line was returned to 35 0 C after the 3-hour treatment at 42 0 C, protein synthesis immediately resumed and eventually returned to control levels after 7 hours at 35 0 C

  13. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

    Science.gov (United States)

    Manjarín, Rodrigo; Columbus, Daniel A; Suryawan, Agus; Nguyen, Hanh V; Hernandez-García, Adriana D; Hoang, Nguyet-Minh; Fiorotto, Marta L; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.

  14. Whey protein supplementation preserves postprandial myofibrillar protein synthesis during short-term energy restriction in overweight and obese adults.

    Science.gov (United States)

    Hector, Amy J; Marcotte, George R; Churchward-Venne, Tyler A; Murphy, Caoileann H; Breen, Leigh; von Allmen, Mark; Baker, Steven K; Phillips, Stuart M

    2015-02-01

    Higher dietary energy as protein during weight loss results in a greater loss of fat mass and retention of muscle mass; however, the impact of protein quality on the rates of myofibrillar protein synthesis (MPS) and lipolysis, processes that are important in the maintenance of muscle and loss of fat, respectively, are unknown. We aimed to determine how the consumption of different sources of proteins (soy or whey) during a controlled short-term (14-d) hypoenergetic diet affected MPS and lipolysis. Men (n = 19) and women (n = 21) (age 35-65 y; body mass index 28-50 kg/m(2)) completed a 14-d controlled hypoenergetic diet (-750 kcal/d). Participants were randomly assigned, double blind, to receive twice-daily supplements of isolated whey (27 g/supplement) or soy (26 g/supplement), providing a total protein intake of 1.3 ± 0.1 g/(kg · d), or isoenergetic carbohydrate (25 g maltodextrin/supplement) resulting in a total protein intake of 0.7 ± 0.1 g/(kg · d). Before and after the dietary intervention, primed continuous infusions of L-[ring-(13)C6] phenylalanine and [(2)H5]-glycerol were used to measure postabsorptive and postprandial rates of MPS and lipolysis. Preintervention, MPS was stimulated more (P whey than with soy or carbohydrate. Postintervention, postabsorptive MPS decreased similarly in all groups (all P whey group, which was less (P whey. We conclude that whey protein supplementation attenuated the decline in postprandial rates of MPS after weight loss, which may be of importance in the preservation of lean mass during longer-term weight loss interventions. This trial was registered at clinicaltrials.gov as NCT01530646. © 2015 American Society for Nutrition.

  15. Age-related differences in lean mass, protein synthesis and skeletal muscle markers of proteolysis after bed rest and exercise rehabilitation

    DEFF Research Database (Denmark)

    Tanner, Ruth E; Brunker, Lucille B; Agergaard, Jakob

    2015-01-01

    during a constant stable isotope infusion in the postabsorptive state and after essential amino acid (EAA) ingestion on three occasions: before (PRE), after bed rest and after rehabilitation. Samples were assessed for protein synthesis, mTORC1 signalling, REDD1/2 expression and molecular markers related...... to muscle proteolysis (MURF1, MAFBX, AMPKα, LC3II/I, Beclin1). We found that leg lean mass and strength decreased in older but not younger adults after bedrest (P protein synthesis increased before bed rest in both age groups...... (P protein synthesis rates and increased MAFBX mRNA, p-AMPKα and the LC3II/I ratio (P

  16. Stimulation of the synthesis of bacteriophage T4 gene 32 protein by ultraviolet light irradiation

    International Nuclear Information System (INIS)

    Krisch, H.M.; Van Houwe, G.

    1976-01-01

    The synthesis of bacteriophage T4 gene 32 product, P32, has been followed by gel electrophoresis of lysates of infected cells which have been irradiated with ultraviolet light. In wild-type infections irradiation after the commencement of late gene expression results in a rapid stimulation of the rate of P32 synthesis. Within four minutes after irradiation P32 is synthesized at 11 times the rate of the unirradiated control infection. P32 seems to be the only T4 protein which exhibits such u.v. inducibility. This inducibility is dependent on the function of genes 46 and 47 and to a lesser extent on several other T4 genes thought to be involved in repair (P43, w and y). An infection defective in both P43 and P46 shows essentially no stimulation of the rate of P32 synthesis after irradiation. In the absence of DNA replication the parental DNA is degraded after irradiation in a dose-dependent manner. The extent of P32 induction in such an infection is also proportional to the dose. It is suggested that the production of gaps during repair of u.v.-irradiated DNA is responsible for the stimulation of P32 synthesis. A model is proposed in which such regions of single-stranded DNA compete for P32 by binding it nonspecifically, thus reducing the amount of P32 free to block the expression of gene 32. Because the expression of gene 32 is self-regulatory this would result in increased P32 synthesis. The possible role of P32 in the repair of u.v.-damaged DNA is discussed. (author)

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

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  18. Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of β-hydroxy-β-methylbutyrate

    Science.gov (United States)

    Wheatley, Scott M.; El-Kadi, Samer W.; Suryawan, Agus; Boutry, Claire; Orellana, Renán A.; Nguyen, Hanh V.; Davis, Steven R.

    2013-01-01

    Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB). To determine the effects of HMB on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were infused with HMB at 0, 20, 100, or 400 μmol·kg body wt−1·h−1 for 1 h (HMB 0, HMB 20, HMB 100, or HMB 400). Plasma HMB concentrations increased with infusion and were 10, 98, 316, and 1,400 nmol/ml in the HMB 0, HMB 20, HMB 100, and HMB 400 pigs. Protein synthesis rates in the longissimus dorsi (LD), gastrocnemius, soleus, and diaphragm muscles, lung, and spleen were greater in HMB 20 than in HMB 0, and in the LD were greater in HMB 100 than in HMB 0. HMB 400 had no effect on protein synthesis. Eukaryotic initiation factor (eIF)4E·eIF4G complex formation and ribosomal protein S6 kinase-1 and 4E-binding protein-1 phosphorylation increased in LD, gastrocnemius, and soleus muscles with HMB 20 and HMB 100 and in diaphragm with HMB 20. Phosphorylation of eIF2α and elongation factor 2 and expression of system A transporter (SNAT2), system L transporter (LAT1), muscle RING finger 1 protein (MuRF1), muscle atrophy F-box (atrogin-1), and microtubule-associated protein light chain 3 (LC3-II) were unchanged. Results suggest that supplemental HMB enhances protein synthesis in skeletal muscle of neonates by stimulating translation initiation. PMID:24192287

  19. No additional effect of different types of physical activity on 10-hour muscle protein synthesis in elderly men on a controlled energy- and protein-sufficient diet

    DEFF Research Database (Denmark)

    Bulow, Jacob; Agergaard, Jakob; Kjær, Michael

    2016-01-01

    protein synthesis (MPS) are less investigated. The aim of this study was to determine the effects of daily physical activities upon MPS in elderly individuals. Methods: A total of 24 elderly men (70 +/- 1 year) were recruited and randomly assigned: inactivity in form of bed-rest (IA), daily physical......-hour myofibrillar protein fractional synthesis rates (FSR), and typical prerequisites for calculating FSR were fulfilled. Physical activities were monitored, and venous blood and muscle biopsies collected. Results: Physical activity was highest in the DA compared to both the IA and RE groups. Nutrient...... activities (DA), or heavy resistance exercise (RE). All groups undertook a normal eating routine containing carbohydrates (52 E%), fat (32 E%), and protein (16 E%). Ingestion of labeled milk protein ([1-C-13] leucine-labeled whey and caseinate) served to maintain tracer enrichment for determination of 10...

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

    Science.gov (United States)

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

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  1. Cholesterol metabolism: use of D2O for determination of synthesis rate in cell culture

    International Nuclear Information System (INIS)

    Esterman, A.L.; Cohen, B.I.; Javitt, N.B.

    1985-01-01

    Cholesterol synthesis in cell culture in the presence of D 2 O yields a spectrum of enriched molecules having a relative abundance that indicates random substitution of deuterium for hydrogen. Quantitation of the absolute rate of cholesterol synthesis is obtained by isotope ratio mass spectrometry. Mevinolin and 26-hydroxycholesterol both decrease cholesterol synthesis rate but have a discordant effect on HMG-CoA reductase activity

  2. Heterotopic cardiac transplantation decreases the capacity for rat myocardial protein synthesis

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  3. Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men.

    Science.gov (United States)

    Murphy, Caoileann H; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Kassis, Amira; Karagounis, Leonidas G; Burke, Louise M; Hawley, John A; Phillips, Stuart M

    2015-05-01

    Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during energy balance (EB), energy restriction (ER), and energy restriction plus resistance training (ER + RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 yr, 31 ± 5 kg/m(2)) who were randomized to either a balanced (BAL: 25% daily protein/meal × 4) or skewed (SKEW: 7:17:72:4% daily protein/meal; n = 10/group) pattern. Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of l-[ring-(13)C6]phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2 wk ER, and after 2 wk ER + RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (P < 0.001), but was greater in BAL than SKEW (P = 0.014). In ER + RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (P = 0.903). In SKEW myofibrillar FSR remained lower than EB (P = 0.002) and lower than BAL (P = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER + RT compared with EB (P < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution "rescued" the lower rates of myofibrillar protein synthesis during moderate ER. Copyright © 2015 the American Physiological Society.

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

    Science.gov (United States)

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

    2017-09-15

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

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

    Science.gov (United States)

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

    2018-06-01

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

  6. Betaine is as effective as folate at re-synthesizing methionine for protein synthesis during moderate methionine deficiency in piglets.

    Science.gov (United States)

    McBreairty, Laura E; Robinson, Jason L; Harding, Scott V; Randell, Edward W; Brunton, Janet A; Bertolo, Robert F

    2016-12-01

    Both folate and betaine (synthesized from choline) are nutrients used to methylate homocysteine to reform the amino acid methionine following donation of its methyl group; however, it is unclear whether both remethylation pathways are of equal importance during the neonatal period when remethylation rates are high. Methionine is an indispensable amino acid that is in high demand in neonates not only for protein synthesis, but is also particularly important for transmethylation reactions, such as creatine and phosphatidylcholine synthesis. The objective of this study was to determine whether supplementation with folate, betaine, or a combination of both can equally re-synthesize methionine for protein synthesis when dietary methionine is limiting. Piglets were fed a low methionine diet devoid of folate, choline, and betaine, and on day 6, piglets were supplemented with either folate, betaine, or folate + betaine (n = 6 per treatment) until day 10. [1- 13 C]-phenylalanine oxidation was measured as an indicator of methionine availability for protein synthesis both before and after 2 days of supplementation. Prior to supplementation, piglets had lower concentrations of plasma folate, betaine, and choline compared to baseline with no change in homocysteine. Post-supplementation, phenylalanine oxidation levels were 20-46 % lower with any methyl donor supplementation (P = 0.006) with no difference among different supplementation groups. Furthermore, both methyl donors led to similarly lower concentrations of homocysteine following supplementation (P folate to remethylate methionine for protein synthesis, as indicated by lower phenylalanine oxidation.

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

    Directory of Open Access Journals (Sweden)

    Robert W Tilghman

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

  8. Efficiency and fidelity of cell-free protein synthesis by transfer RNA from aged mice

    Energy Technology Data Exchange (ETDEWEB)

    Foote, R.S.; Stulberg, M.P.

    1980-01-01

    Transfer RNAs (tRNAs) from heart, kidney, liver, and spleen of mature (10 to 12 months old) and aged (29 months old) C57BL/6 mice were tested for their ability to translate encephalomyocarditis viral RNA in a tRNA-dependent cell-free system derived from mouse ascites tumor cells. The rates of in vitro protein synthesis were compared as a function of tRNA concentration, and the fidelity of translation was examined by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing of the viral polypeptides synthesized in vitro. No significant age-related differences in either the efficiency or fidelity of synthesis were discovered, indicating that alternations in tRNAs are probably not involved in the cellular aging of these tissues.

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

    Science.gov (United States)

    Wolf, Gerald; Engelmann, Mario; Richter, Karin

    2005-01-01

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

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

    Science.gov (United States)

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

    2010-07-01

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

  11. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients.

    Directory of Open Access Journals (Sweden)

    Jakob G Jespersen

    Full Text Available BACKGROUND: Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR, glycogen synthase kinase 3β (GSK3β and forkhead box O (FoxO pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU patients compared with healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, and muscle ring finger protein 1 (MuRF1; and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1, FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6, tumor necrosis factor α (TNF-α and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2=0.36, p<0.05 between insulin infusion dose and phosphorylated Akt was demonstrated. CONCLUSIONS/SIGNIFICANCE: We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

  12. Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin.

    Science.gov (United States)

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

    2008-07-01

    Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.

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

    International Nuclear Information System (INIS)

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

    1978-01-01

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

  14. Design, synthesis, and evaluation of an alpha-helix mimetic library targeting protein-protein interactions.

    Science.gov (United States)

    Shaginian, Alex; Whitby, Landon R; Hong, Sukwon; Hwang, Inkyu; Farooqi, Bilal; Searcey, Mark; Chen, Jiandong; Vogt, Peter K; Boger, Dale L

    2009-04-22

    The design and solution-phase synthesis of an alpha-helix mimetic library as an integral component of a small-molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate alpha-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 x 20 x 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i + 4, i + 7 amino acid side chains of an alpha-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long-term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead alpha-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an alpha-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

  15. Protein synthesis in the embryo of Pinus thunbergii seed, 2

    International Nuclear Information System (INIS)

    Yamamoto, Naoaki; Sasaki, Satohiko.

    1977-01-01

    14 C-Amino acid incorporating activity in the absence of exogenous mRNA was found in a cell-free system from embryos of light-germinated Pinus thunbergii seeds, but not in that from dark-imbibed seed embryos. Template activity in the cell-free system from the light-germinated seed embryos was observed in the ribosome fraction, especially the polyribosome fraction, but not in the 100,000 x g supernatant fraction (s100). These facts suggest that the nature of the block in protein synthesis during the imbibition of seeds in the dark is due to the lack or inactivity of mRNA. The s100 from light-germinated seed embryos was found to be less active in amino acid incorporation than that from dark-imbibed seed embryos. (auth.)

  16. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  18. Changes in the synthesis of DNA, RNA and protein during somatic embryogenesis in wheat (triticum aestivum L.)

    International Nuclear Information System (INIS)

    Cui Kairong; Wang Xiaozhe; Chen Xiong; Wang Yafu

    1997-01-01

    Embryogenic and non-embryogenic callus formed from immature embryo of wheat (Triticum aestivum L.) in N 6 B 5 MS medium I supplemented with 2,4-D 2 mg/L, KT 0.5 mg/L, LH300 mg/L, sucrose 3% were sub-cultured and transferred respectively to N 6 B 5 MS medium II (2,4-D was decreased to 0.5 mg/L and 4 mol/L proline was added). Somatic embryos obtained from embryogenic callus, and plantlet formed from non-embryogenic callus through organogenesis respectively. By incorporation of 3 H-thymidine, 3 H-uridine and 3 H-leucine into DNA, RNA and protein respectively, the rate of synthesis of DNA, RNA and protein during somatic embryogenesis were measured. A large amount of RNA and protein synthesized during the early somatic embryogenesis. The activities of RNA and protein synthesis reached the peak on the 4th and the 8th day respectively, then decreased a little, but kept a high level. The synthesis of DNA increased apparently during the early stage. No apparent change occurred when the embryogenic cell masses formed. The synthesis rate of RNA and protein in non-embryogenic callus were much less than that in embryogenic callus. Actinomycin and cycloheximide inhibited not only the synthesis of nucleic acid and protein, but also the growth of embryogenic callus and somatic embryogenesis. The earlier the inhibitors were added, the greater the influence was caused. The results indicate that the active expression of corresponding genes of wheat is the molecular base of somatic embryogenesis

  19. Analyzing the effects of mechanical and osmotic loading on glycosaminoglycan synthesis rate in cartilaginous tissues.

    Science.gov (United States)

    Gao, Xin; Zhu, Qiaoqiao; Gu, Weiyong

    2015-02-26

    The glycosaminoglycan (GAG) plays an important role in cartilaginous tissues to support and transmit mechanical loads. Many extracellular biophysical stimuli could affect GAG synthesis by cells. It has been hypothesized that the change of cell volume is a primary mechanism for cells to perceive the stimuli. Experimental studies have shown that the maximum synthesis rate of GAG is achieved at an optimal cell volume, larger or smaller than this level the GAG synthesis rate decreases. Based on the hypothesis and experimental findings in the literature, we proposed a mathematical model to quantitatively describe the cell volume dependent GAG synthesis rate in the cartilaginous tissues. Using this model, we investigated the effects of osmotic loading and mechanical loading on GAG synthesis rate. It is found our proposed mathematical model is able to well describe the change of GAG synthesis rate in isolated cells or in cartilage with variations of the osmotic loading or mechanical loading. This model is important for evaluating the GAG synthesis activity within cartilaginous tissues as well as understanding the role of mechanical loading in tissue growth or degeneration. It is also important for designing a bioreactor system with proper extracellular environment or mechanical loading for growing tissue at the maximum synthesis rate of the extracellular matrix. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2008-10-01

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

  1. Glucagon infusion increases rate of purine synthesis de novo in rat liver

    International Nuclear Information System (INIS)

    Itakura, Mitsuo; Maeda, Noriaki; Tsuchiya, Masami; Yamashita, Kamejiro

    1987-01-01

    Based on the parallel increases of glucagon, the second peak of hepatic cAMP, and the rate of purine synthesis de novo in the prereplicative period in regenerating rate liver after a 70% hepatectomy, it was hypothesized that glucagon is responsible for the increased rate of purine synthesis de novo. To test this hypothesis, the effect of glucagon or dibutyryl cAMP infusion on the rate of purine synthesis de novo in rat liver was studied. Glucagon infusion but not insulin or glucose infusion increased the rate of purine synthesis de novo, which was assayed by [ 14 C]glycine or [ 14 C]formate incorporation, by 2.7- to 4.3-fold. Glucagon infusion increased cAMP concentrations by 4.9-fold and 5-phosphoribosyl-1-pyrophosphate concentrations by 1.5-fold in liver but did not change the specific activity of amidophosphoribosyltransferase or purine ribonucleotide concentrations. Dibutyryl cAMP infusion also increased the rate of purine synthesis de novo by 2.2- to 4.0-fold. Because glucagon infusion increased the rate of purine synthesis de novo in the presence of unchanged purine ribonucleotide concentrations, it is concluded that glucagon after infusion or in animals after a 70% hepatectomy is playing an anabolic role to increase the rate of purine synthesis de novo by increasing cAMP and 5-phosphoribosyl-1-pyrophosphate concentrations

  2. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    Science.gov (United States)

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

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

    International Nuclear Information System (INIS)

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

    1977-01-01

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

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

    International Nuclear Information System (INIS)

    Ortiz, W.

    1990-01-01

    Growing cultures of photoheterotrophic Euglena gracilis experience an increase in chlorophyll accumulation during the initial phase of the temperature-induced bleaching response suggesting an increase in the synthesis of plastid components at the bleaching temperature of 33 degree C. A primary goal of this work was to establish whether an increase in the synthesis of plastid proteins accompanies the observed increase in chlorophyll accumulation. In vivo pulse-labeling experiments with [ 35 S]sodium sulfate were carried out with cells grown at room temperature or at 33 degree C. The synthesis of a number of plastid polypeptides of nucleocytoplasmic origin, including some presumably novel polypeptides, increased in cultures treated for 15 hours at 33 degree C. In contrast, while synthesis of thylakoid proteins by the plastid protein synthesis machinery decreased modestly, synthesis of the large subunit of the enzyme ribulosebisphosphate carboxylase was strongly affected at the elevated temperature. Synthesis of novel plastid-encoded polypeptides was not induced at the bleaching temperature. It is concluded that protein synthesis in plastids declines during the initial phase of the temperature response in Euglena despite an overall increase in cellular protein synthesis and an increase in chlorophyll accumulation per cell

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

    International Nuclear Information System (INIS)

    Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

    1984-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

    ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

  7. Age-related changes in the synthesis and phosphorylation of proteins

    International Nuclear Information System (INIS)

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

    1986-01-01

    It is well documented that the protein synthetic activity of liver tissue decreases significantly with age. However, very little information is available on the effect of age on the synthesis or phosphorylation of individual proteins. Hepatocytes were isolated from 5- to 30-month-old male Fischer F344 rats, and proteins were labeled with either [ 3 H]-valine or [ 32 P]-phosphate. Two-dimensional polyacrylamide gel electrophoresis was used to monitor the synthesis and phosphorylation of a wide variety of proteins. A dramatic increase or decrease in the synthesis of approximately 2 to 3% of the proteins was observed. Most of the proteins whose synthesis increased with age were found to be plasma proteins, e.g., acute phase proteins, synthesized by the liver. In general, the synthesis of most proteins decreased 20 to 40% with age. The phosphorylation of most proteins (over 200) did not appear to change with age. However the phosphorylation of two acidic proteins (molecular weights of 148 Kd and 130 Kd and pIs of 5.4 and 5.36, respectively) decreased with age while the phosphorylation of a basic protein (molecular weight of 57 Kd and pI of 8.09) increased with age

  8. Origins of the Mechanochemical Coupling of Peptide Bond Formation to Protein Synthesis.

    Science.gov (United States)

    Fritch, Benjamin; Kosolapov, Andrey; Hudson, Phillip; Nissley, Daniel A; Woodcock, H Lee; Deutsch, Carol; O'Brien, Edward P

    2018-04-18

    Mechanical forces acting on the ribosome can alter the speed of protein synthesis, indicating that mechanochemistry can contribute to translation control of gene expression. The naturally occurring sources of these mechanical forces, the mechanism by which they are transmitted 10 nm to the ribosome's catalytic core, and how they influence peptide bond formation rates are largely unknown. Here, we identify a new source of mechanical force acting on the ribosome by using in situ experimental measurements of changes in nascent-chain extension in the exit tunnel in conjunction with all-atom and coarse-grained computer simulations. We demonstrate that when the number of residues composing a nascent chain increases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that are fully transmitted to the ribosome's P-site. The route of force transmission is shown to be through the nascent polypetide's backbone, not through the wall of the ribosome's exit tunnel. Utilizing quantum mechanical calculations we find that a consequence of such a pulling force is to decrease the transition state free energy barrier to peptide bond formation, indicating that the elongation of a nascent chain can accelerate translation. Since nascent protein segments can start out as largely unfolded structural ensembles, these results suggest a pulling force is present during protein synthesis that can modulate translation speed. The mechanism of force transmission we have identified and its consequences for peptide bond formation should be relevant regardless of the source of the pulling force.

  9. Altered synthesis of some secretory proteins in pancreatic lobules isolated from streptozotocin-induced diabetic rats

    International Nuclear Information System (INIS)

    Duan, R.D.; Erlanson-Albertsson, C.

    1990-01-01

    The in vitro incorporation of [35S]cysteine into lipase, colipase, amylase, procarboxypeptidase A and B, and the serine proteases and total proteins was studied in pancreatic lobules isolated from normal and diabetic rats with or without insulin treatment. The incorporation of [35S]cysteine into total proteins was 65% greater in pancreatic lobules from diabetic animals than from normal rats. The increased incorporation was partly reversed by insulin treatment (2 U/100 g/day for 5 days) of diabetic rats. The relative rates of biosynthesis for amylase and the procarboxypeptidases in diabetic pancreatic lobules were decreased by 75 and 25%, respectively, after 1 h of incubation, while those for lipase, colipase, and the serine proteases were increased by 90, 85, and 35%, respectively. The absolute rates of synthesis for these enzymes changed in the same direction as the relative rates in diabetic lobules, except that for the procarboxypeptidases, which did not change. The changed rates of biosynthesis for the pancreatic enzymes were reversed by insulin treatment of the diabetic rats. Kinetic studies showed that the incorporation of [35S]cysteine into amylase, lipase, and colipase was linear until up to 2 h of incubation in normal pancreatic lobules, while in the diabetic lobules the incorporation into lipase and colipase was accelerated, reaching a plateau level already after 1 h of incubation. It is concluded that the biosynthesis of pancreatic secretory proteins in diabetic rats is greatly changed both in terms of quantity and kinetics

  10. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors invol...... involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls....

  11. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors invol...... involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls....

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

    Science.gov (United States)

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

    2009-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Cem Gerçek

    2018-02-01

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

  14. Protein synthesis in TE 671/RD (human rabdomiosarcoma) cells treated with thapsigargin and hyperthermia: impairment of HSP 70 induction.

    Science.gov (United States)

    Delpino, A; Piselli, P; Mangano, G

    1995-01-01

    In this study we considered the quantitative and qualitative changes of protein synthetic activity occurring in TE 671/RD cells treated with thapsigargin (TG), with hyperthermia (HT) or with a combination of both these agents. In cells treated with TG (100 nM, continuous exposure), the overall protein synthetic activity was initially inhibited but subsequently recovered to about 60% of the initial level. Chronic TG exposure was also able to induce the expression of GRP 78. The rate of synthesis of GRP 78, after a lag period of about 2 h, increased gradually to reach a maximum (9-fold induction) after 6 h of TG-treatment and was then maintained at that level up to 18 h. A weak induction of GRP 94 was observed following 6-8 h of continuous exposure to TG. In cells treated with HT (43 degrees C for 30 min), a typical heat shock response was observed: in particular, the relative rate of synthesis of HSP 70 (the major heat-inducible mammalian heat shock protein) was increased 10-fold over the constitutive level. The heat-promoted induction of HSP 70 was significantly reduced by concomitant or previous exposure to TG. When TG and HT were administred simultaneously, the increase in HSP 70 synthesis was only 4.7-fold over the control level, while in cells pre-treated for 1 h with TG before the hyperthermic challenge the rate of HSP 70 synthesis was only stimulated 2-fold. In both these conditions, by contrast, it was apparent that HT did not affect the TG-promoted induction of GRP 78. The correlations between the TG-induced mobilization of cytosolic Ca2+ and the effects on protein synthesis are discussed.

  15. Leucine content of dietary proteins is a determinant of postprandial skeletal muscle protein synthesis in adult rats

    Directory of Open Access Journals (Sweden)

    Norton Layne E

    2012-07-01

    Full Text Available Abstract Background Leucine (Leu regulates muscle protein synthesis (MPS producing dose-dependent plasma Leu and MPS responses from free amino acid solutions. This study examined the role of Leu content from dietary proteins in regulation of MPS after complete meals. Methods Experiment 1 examined 4 protein sources (wheat, soy, egg, and whey with different Leu concentrations (6.8, 8.0, 8.8, and 10.9% (w/w, respectively on the potential to increase plasma Leu, activate translation factors, and stimulate MPS. Male rats (~250 g were trained for 14 day to eat 3 meals/day consisting of 16/54/30% of energy from protein, carbohydrates and fats. Rats were killed on d14 either before or 90 min after consuming a 4 g breakfast meal. Experiment 2 compared feeding wheat, whey, and wheat + Leu to determine if supplementing the Leu content of the wheat meal would yield similar anabolic responses as whey. Results In Experiment 1, only whey and egg groups increased post-prandial plasma Leu and stimulated MPS above food-deprived controls. Likewise, greater phosphorylation of p70 S6 kinase 1 (S6K1 and 4E binding protein-1 (4E-BP1 occurred in whey and egg groups versus wheat and soy groups. Experiment 2 demonstrated that supplementing wheat with Leu to equalize the Leu content of the meal also equalized the rates of MPS. Conclusion These findings demonstrate that Leu content is a critical factor for evaluating the quantity and quality of proteins necessary at a meal for stimulation of MPS.

  16. Rate and extent of ruminal degradation of crude protein from ...

    African Journals Online (AJOL)

    Predicted crude protein degradation was calculated at rate constants for outflow of 0.04 and 0.06/h respect- ively. ... as buffers, an ionophore and an antibiotic according to general .... the non-bird resistant ('sweet') varieties. Ruminal .... have been affected by both the particle type and the math- ematical model we used.

  17. Leucine Supplementation in a Chronically Protein-Restricted Diet Enhances Muscle Weight and Postprandial Protein Synthesis of Skeletal Muscle by Promoting the mTOR Pathway in Adult Rats

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2017-10-01

    Full Text Available Low protein intake causes a decrease in protein deposition in most animal tissues. The purpose of this study was to investigate whether leucine supplementation would increase the synthesis rate of protein and muscle weight in adult rats, which chronically consume only 58.8% of their protein requirements. Thirty-six male Sprague-Dawley rats were assigned to one of three dietary treatments including a 20% casein diet (CON, a 10% casein + 0.44% alanine diet (R, and a 10% casein + 0.87% leucine diet (RL. After a 10 d dietary treatment, plasma amino acid levels were measured after feeding, the gastrocnemius muscles and soleus muscles were harvested and weighed, and the fractional synthesis rate (FSR and mammalian target of rapamycin (mTOR signaling proteins in skeletal muscle were measured. Regarding the plasma amino acid level, the RL group had the highest concentration of leucine (P < 0.05 and the lowest concentration of isoleucine (P < 0.05 among the three groups, and the CON group had a lower concentration of valine (P < 0.05 than the R and RL groups. Compared with the R and RL groups, the CON group diet significantly increased (P < 0.05 feed intake, protein synthesis rate, and the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1, and decreased the weight of abdominal adipose. Compared with the R group, the RL group significantly increased in gastrocnemius muscle weight, protein synthesis rate, and phosphorylation of both ribosomal protein S6 kinase 1 (S6K1 and 4E-BP1. In conclusion, when protein is chronically restricted in adult rat diets, leucine supplementation moderately improves body weight gain and increases muscle protein synthesis through mTOR activation.

  18. The effect of chloramphenicol on synthesis of ΦX 174-specific proteins and detection of the cistron A protein

    NARCIS (Netherlands)

    Mei, D. Van Der; Zandberg, J.; Jansz, H.S.

    1972-01-01

    Synthesis of ΦX 174-specific proteins in Escherichia coli H 502 was examined on sodium dodecyl sulphate-acrylamide gels by coelectrophoresis of proteins from [3H]leucine-labelled infected cells and [14C]leucine-labelled reference cells, which had been infected with ultraviolet-light irradiated

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1975-01-01

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

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

    International Nuclear Information System (INIS)

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

    1975-01-01

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-01-01

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

  3. Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

    International Nuclear Information System (INIS)

    Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

    1987-01-01

    The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 μg/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  5. Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

    Science.gov (United States)

    Everman, Sarah; Meyer, Christian; Tran, Lee; Hoffman, Nyssa; Carroll, Chad C; Dedmon, William L; Katsanos, Christos S

    2016-10-01

    Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. Copyright © 2016 the American Physiological Society.

  6. L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446 in a Nitric Oxide-Dependent Manner in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Ruxia Wang

    2018-01-01

    Full Text Available Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS, the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446 and p70S6K (Thr 389. L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside, an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446/p70S6K signaling pathway in an NO-dependent manner.

  7. High rate flame synthesis of highly crystalline iron oxide nanorods

    International Nuclear Information System (INIS)

    Merchan-Merchan, W; Taylor, A M; Saveliev, A V

    2008-01-01

    Single-step flame synthesis of iron oxide nanorods is performed using iron probes inserted into an opposed-flow methane oxy-flame. The high temperature reacting environment of the flame tends to convert elemental iron into a high density layer of iron oxide nanorods. The diameters of the iron oxide nanorods vary from 10 to 100 nm with a typical length of a few microns. The structural characterization performed shows that nanorods possess a highly ordered crystalline structure with parameters corresponding to cubic magnetite (Fe 3 O 4 ) with the [100] direction oriented along the nanorod axis. Structural variations of straight nanorods such as bends, and T-branched and Y-branched shapes are frequently observed within the nanomaterials formed, opening pathways for synthesis of multidimensional, interconnected networks

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

    International Nuclear Information System (INIS)

    Chen Shali; Liu Shuzheng

    1997-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

    Relief learning refers to the association of a stimulus with the relief from an aversive event. The thus-learned relief stimulus then can induce, e.g., an attenuation of the startle response or approach behavior, indicating positive valence. Previous studies revealed that the nucleus accumbens is essential for the acquisition and retrieval of relief memory. Here, we ask whether the nucleus accumbens is also the brain site for consolidation of relief memory into a long-term form. In rats, we blocked local protein synthesis within the nucleus accumbens by local infusions of anisomycin at different time points during a relief conditioning experiment. Accumbal anisomycin injections immediately after the relief conditioning session, but not 4 h later, prevented the consolidation into long-term relief memory. The retention of already consolidated relief memory was not affected by anisomycin injections. This identifies a time window and site for relief memory consolidation. These findings should complement our understanding of the full range of effects of adverse experiences, including cases of their distortion in humans such as post-traumatic stress disorder and/or phobias. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. BDNF-induced local protein synthesis and synaptic plasticity.

    Science.gov (United States)

    Leal, Graciano; Comprido, Diogo; Duarte, Carlos B

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is an important regulator of synaptic transmission and long-term potentiation (LTP) in the hippocampus and in other brain regions, playing a role in the formation of certain forms of memory. The effects of BDNF in LTP are mediated by TrkB (tropomyosin-related kinase B) receptors, which are known to be coupled to the activation of the Ras/ERK, phosphatidylinositol 3-kinase/Akt and phospholipase C-γ (PLC-γ) pathways. The role of BDNF in LTP is best studied in the hippocampus, where the neurotrophin acts at pre- and post-synaptic levels. Recent studies have shown that BDNF regulates the transport of mRNAs along dendrites and their translation at the synapse, by modulating the initiation and elongation phases of protein synthesis, and by acting on specific miRNAs. Furthermore, the effect of BDNF on transcription regulation may further contribute to long-term changes in the synaptic proteome. In this review we discuss the recent progress in understanding the mechanisms contributing to the short- and long-term regulation of the synaptic proteome by BDNF, and the role in synaptic plasticity, which is likely to influence learning and memory formation. This article is part of the Special Issue entitled 'BDNF Regulation of Synaptic Structure, Function, and Plasticity'. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2010-01-01

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

  12. An unusual correlation between ppGpp pool size and rate of ribosome synthesis during partial pyrimidine starvation of Escherichia coli

    DEFF Research Database (Denmark)

    Vogel, Ulla; Pedersen, Steen; Jensen, Kaj Frank

    1991-01-01

    Escherichia coli was exposed to partial pyrimidine starvation by feeding a pyrBI strain orotate as the only pyrimidine source. Subsequently, differential rates of synthesis of rRNA and of a few ribosome-associated proteins as well as the pool sizes of nucleoside triphosphates and ppGpp were measu...

  13. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis...

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  15. Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis.

    Science.gov (United States)

    Xue, Qiao; Cui, Ying-Lu; Zheng, Qing-Chuan; Zhang, Hong-Xing

    2016-05-01

    BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Araceli eMartínez-Moreno

    2011-09-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    OpenAIRE

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    Maisonhaute, Claude

    1976-01-01

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

  2. Effect of plaster cast immobilization on the turnover rates of soluble proteins and lactate dehydrogenase isoenzymes of rabbit M. soleus

    Energy Technology Data Exchange (ETDEWEB)

    Edes, I.; Dosa, E.; Sohar, I.; Guba, F. (Orvostudomanyi Egyetem, Szeged (Hungary). Biokemiai Tanszek)

    1982-01-01

    In atrophized muscle the decreases of the activity of LDH isoenzymes can be explained partly by a 15 per cent decrease of the enzyme synthesis and partly by a 25 per cent increase in catabolism. The quantities of the soluble proteins and LDH were measured after intravenously administered /sup 3/H-leucin incorporation, from the musculus soleus. LDH was isolated by means of affinity chromatography. Radioactivity was determined in a Packard Tri-Carb scintillation counter. The synthesis rate of soluble proteins barely changed during immobilization. In the atrophized muscle the decrease of the amount of soluble proteins could be almost exclusively interpreted in terms of a 25 per cent enchancement of degradative process. The accelerated catabolism is most probably due to the proteolytic enzymes activated by immobilization.

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

    DEFF Research Database (Denmark)

    Holm, Lars; Reitelseder, Søren; Dideriksen, Kasper

    2014-01-01

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

  4. Noise analysis of genome-scale protein synthesis using a discrete computational model of translation

    Energy Technology Data Exchange (ETDEWEB)

    Racle, Julien; Hatzimanikatis, Vassily, E-mail: vassily.hatzimanikatis@epfl.ch [Laboratory of Computational Systems Biotechnology, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland); Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne (Switzerland); Stefaniuk, Adam Jan [Laboratory of Computational Systems Biotechnology, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland)

    2015-07-28

    Noise in genetic networks has been the subject of extensive experimental and computational studies. However, very few of these studies have considered noise properties using mechanistic models that account for the discrete movement of ribosomes and RNA polymerases along their corresponding templates (messenger RNA (mRNA) and DNA). The large size of these systems, which scales with the number of genes, mRNA copies, codons per mRNA, and ribosomes, is responsible for some of the challenges. Additionally, one should be able to describe the dynamics of ribosome exchange between the free ribosome pool and those bound to mRNAs, as well as how mRNA species compete for ribosomes. We developed an efficient algorithm for stochastic simulations that addresses these issues and used it to study the contribution and trade-offs of noise to translation properties (rates, time delays, and rate-limiting steps). The algorithm scales linearly with the number of mRNA copies, which allowed us to study the importance of genome-scale competition between mRNAs for the same ribosomes. We determined that noise is minimized under conditions maximizing the specific synthesis rate. Moreover, sensitivity analysis of the stochastic system revealed the importance of the elongation rate in the resultant noise, whereas the translation initiation rate constant was more closely related to the average protein synthesis rate. We observed significant differences between our results and the noise properties of the most commonly used translation models. Overall, our studies demonstrate that the use of full mechanistic models is essential for the study of noise in translation and transcription.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

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

    Science.gov (United States)

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

    2016-11-01

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Science.gov (United States)

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

    2017-09-15

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

  9. Ethylene-induced senescence-related gene expression requires protein synthesis

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  11. Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin-independent pathway.

    Science.gov (United States)

    Coëffier, Moïse; Claeyssens, Sophie; Bôle-Feysot, Christine; Guérin, Charlène; Maurer, Brigitte; Lecleire, Stéphane; Lavoinne, Alain; Donnadieu, Nathalie; Cailleux, Anne-Françoise; Déchelotte, Pierre

    2013-02-01

    Glutamine modulates duodenal protein metabolism in fasted healthy humans, but its effects in a fed state remain unknown. We aimed to assess the effects of either glutamine or an isonitrogenous protein mixture on duodenal protein metabolism in humans in the fed state. Twenty-four healthy volunteers were randomly included in 2 groups. Each volunteer was studied on 2 occasions in a random order and received, during 5 h, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹; both groups) that mimicked a carbohydrate fed state or maltodextrins with glutamine (group 1) or an isonitrogenous (22.4 mg N · kg⁻¹ · h⁻¹) protein powder (group 2). Simultaneously, a continuous intravenous infusion of ¹³C-leucine and ²H₅-phenylalanine (both 9 μmol · kg⁻¹ · h⁻¹) was performed. Endoscopic duodenal biopsies were taken. Leucine and phenylalanine enrichments were assessed by using gas chromatography-mass spectrometry in duodenal proteins and the intracellular free amino acids pool to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates and macroarrays, respectively. The FSR and proteasome activity were not different after the glutamine supply compared with after maltodextrins alone. In contrast, the FSR increased (1.7-fold increase; P protein-powder delivery without modification of total proteasome activity. The protein powder increased insulinemia, PI3 kinase, and erk phosphorylation but did not affect the mammalian target of rapamycin (mTOR) pathway and mitogen-activated protein kinase signal-integrating kinase 1 phosphorylation. A trend for an increase of eukaryotic translation initiation factor 4E phosphorylation was observed (P = 0.07). In the carbohydrate fed state, enteral proteins but not glutamine increased duodenal protein synthesis through an mTOR independent pathway in humans.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Science.gov (United States)

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

    2016-04-26

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

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

    Directory of Open Access Journals (Sweden)

    Je H

    2011-01-01

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

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

    Science.gov (United States)

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

    1984-01-01

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

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

    Science.gov (United States)

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

    1978-09-01

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

  17. Studies on protein synthesis by protoplasts of saccharomyces carlsbergensis III. Studies on the specificity and the mechanism of the action of ribonuclease on protein synthesis

    NARCIS (Netherlands)

    Kloet, S.R. de; Dam, G.J.W. van; Koningsberger, V.V.

    1962-01-01

    In this paper, the experimental results are presented of a continued study on the specificity and the mechanism of the inhibition by ribonuclease of protein synthesis in protoplasts of Saccharomyces carlsbergensis. By comparing the effects of native pancreatic ribonuclease with those of

  18. An emergency brake for protein synthesis The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

    Czech Academy of Sciences Publication Activity Database

    Hronová, Vladislava; Valášek, Leoš

    2017-01-01

    Roč. 6, APR 25 (2017), s. 1-3, č. článku e27085. ISSN 2050-084X Institutional support: RVO:61388971 Keywords : synthesis of proteins * eukaryotic cells * eIF2 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 7.725, year: 2016

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

    NARCIS (Netherlands)

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

    1961-01-01

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

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

    Directory of Open Access Journals (Sweden)

    J. K. Seo

    2013-03-01

    Full Text Available 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.

  1. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis

    Science.gov (United States)

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

    1996-01-01

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

  2. Protein thermodynamics can be predicted directly from biological growth rates.

    Directory of Open Access Journals (Sweden)

    Ross Corkrey

    Full Text Available Life on Earth is capable of growing from temperatures well below freezing to above the boiling point of water, with some organisms preferring cooler and others hotter conditions. The growth rate of each organism ultimately depends on its intracellular chemical reactions. Here we show that a thermodynamic model based on a single, rate-limiting, enzyme-catalysed reaction accurately describes population growth rates in 230 diverse strains of unicellular and multicellular organisms. Collectively these represent all three domains of life, ranging from psychrophilic to hyperthermophilic, and including the highest temperature so far observed for growth (122 °C. The results provide credible estimates of thermodynamic properties of proteins and obtain, purely from organism intrinsic growth rate data, relationships between parameters previously identified experimentally, thus bridging a gap between biochemistry and whole organism biology. We find that growth rates of both unicellular and multicellular life forms can be described by the same temperature dependence model. The model results provide strong support for a single highly-conserved reaction present in the last universal common ancestor (LUCA. This is remarkable in that it means that the growth rate dependence on temperature of unicellular and multicellular life forms that evolved over geological time spans can be explained by the same model.

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

    International Nuclear Information System (INIS)

    Sardinia, L.M.

    1985-01-01

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

  4. Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

    Science.gov (United States)

    Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

    2012-12-14

    Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  6. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

  7. Enteral B-hydroxy-B-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs

    Science.gov (United States)

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite B-hydr...

  8. Leucine pulses enhance skeletal muscle protein synthesis during continuous feeding in neonatal pigs

    Science.gov (United States)

    Infants unable to maintain oral feeding can be nourished by orogastric tube. We have shown that orogastric continuous feeding restricts muscle protein synthesis compared with intermittent bolus feeding in neonatal pigs. To determine whether leucine leu infusion can be used to enhance protein synthes...

  9. Sepsis and development impede muscle protein synthesis in neonatal pigs by different ribosomal mechanisms

    Science.gov (United States)

    In muscle, sepsis reduces protein synthesis (MPS) by restraining translation in neonates and adults. Even though protein accretion decreases with development as neonatal MPS rapidly declines by maturation, the changes imposed by development on the sepsis-associated decrease in MPS have not been desc...

  10. Ingestion of Casein in a Milk Matrix Modulates Dietary Protein Digestion and Absorption Kinetics but Does Not Modulate Postprandial Muscle Protein Synthesis in Older Men.

    Science.gov (United States)

    Churchward-Venne, Tyler A; Snijders, Tim; Linkens, Armand M A; Hamer, Henrike M; van Kranenburg, Janneau; van Loon, Luc J C

    2015-07-01

    The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix. The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men. In a parallel-group design, 32 healthy older men (aged 71 ± 1 y) received a primed continuous infusion of L-[ring-(2)H5]-phenylalanine, L-[ring-3,5-(2)H2]-tyrosine, and L-[1-(13)C]-leucine, and ingested 25 g intrinsically L-[1-(13)C]-phenylalanine and L-[1-(13)C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism. Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-(13)C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein-derived phenylalanine availability did not differ between Cas+Serum (47 ± 2%, mean ± SEM) and Cas (46 ± 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min (0.038 ± 0.005 vs. 0.031 ± 0.007%/h) or from 120 to 300 min (0.052 ± 0.004 vs. 0.067 ± 0.005%/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed at 120 min (0.003 ± 0.001 vs. 0.002 ± 0.001) or 300 min (0.015 ± 0.002 vs. 0.016 ± 0.002 mole

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

    Science.gov (United States)

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  13. Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Ana Ferreras

    2002-04-01

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

  14. Measurement of Hepatic Protein Fractional Synthetic Rate with Stable Isotope Labeling Technique in Thapsigargin Stressed HepG2 Cells

    Science.gov (United States)

    Song, Juquan; Zhang, Xiao-jun; Boehning, Darren; Brooks, Natasha C.; Herndon, David N.; Jeschke, Marc G.

    2012-01-01

    Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-13C2-glycine and L-[ring-13C6]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2-13C2-glycine and L-[ring-13C6]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (pisotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis. PMID:22298954

  15. Is there any relationship between decreased AgNOR protein synthesis and human hair loss?

    Science.gov (United States)

    Eroz, R; Tasdemir, S; Dogan, H

    2012-11-01

    Argyrophilic nucleolar organizing region associated proteins (AgNORs) play roles in cell proliferation and a variety of diseases. We attempted to determine whether decreased NOR protein synthesis causes human hair loss. We studied 21 healthy males who suffered hair loss on the frontal/vertex portion of the head. Hair root cells from normal and hair loss sites were stained for AgNOR. One hundred nuclei per site were evaluated and the AgNOR number and NORa/TNa proportions of individual cells were determined using a computer program. The cells from normal sites had significantly higher AgNOR counts than those from hair loss sites. Also, the cells from the normal sites had significantly higher NORa/TNa than cells from the hair loss sites. In the normal sites, the cells demonstrated more NOR protein synthesis than cells in hair loss sites. Therefore, decreased NOR protein synthesis appears to be related to hair loss in humans.

  16. A global synthesis of plant extinction rates in urban areas.

    Science.gov (United States)

    Hahs, Amy K; McDonnell, Mark J; McCarthy, Michael A; Vesk, Peter A; Corlett, Richard T; Norton, Briony A; Clemants, Steven E; Duncan, Richard P; Thompson, Ken; Schwartz, Mark W; Williams, Nicholas S G

    2009-11-01

    Plant extinctions from urban areas are a growing threat to biodiversity worldwide. To minimize this threat, it is critical to understand what factors are influencing plant extinction rates. We compiled plant extinction rate data for 22 cities around the world. Two-thirds of the variation in plant extinction rates was explained by a combination of the city's historical development and the current proportion of native vegetation, with the former explaining the greatest variability. As a single variable, the amount of native vegetation remaining also influenced extinction rates, particularly in cities > 200 years old. Our study demonstrates that the legacies of landscape transformations by agrarian and urban development last for hundreds of years, and modern cities potentially carry a large extinction debt. This finding highlights the importance of preserving native vegetation in urban areas and the need for mitigation to minimize potential plant extinctions in the future.

  17. Comparison of two methods forecasting binding rate of plasma protein.

    Science.gov (United States)

    Hongjiu, Liu; Yanrong, Hu

    2014-01-01

    By introducing the descriptors calculated from the molecular structure, the binding rates of plasma protein (BRPP) with seventy diverse drugs are modeled by a quantitative structure-activity relationship (QSAR) technique. Two algorithms, heuristic algorithm (HA) and support vector machine (SVM), are used to establish linear and nonlinear models to forecast BRPP. Empirical analysis shows that there are good performances for HA and SVM with cross-validation correlation coefficients Rcv(2) of 0.80 and 0.83. Comparing HA with SVM, it was found that SVM has more stability and more robustness to forecast BRPP.

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

    International Nuclear Information System (INIS)

    Scalise, F.W.

    1988-01-01

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

  19. Anaerobiosis and ethanol effects on germination, growth, and protein synthesis of five Echinochloa species

    International Nuclear Information System (INIS)

    Dybiec, L.D.; Rumpho, M.E.; Kennedy, R.A.

    1989-01-01

    Five Echinochloa species, encompassing a spectrum from flood tolerant to flood intolerant, were studied to determine the mechanisms of anaerobic germination and growth. Seeds were germinated in air or N 2 , plus 0, 1 or 3% ethanol, and germination rates and growth measurements recorded for 7 days. In air or N 2 increasing ethanol levels did not affect total germination per se, although the rate of germination was delayed in N 2 . Shoot/root lengths in air were highest for tolerant species and increased with increasing ethanol, whereas, in intolerant species, shoot/root lengths decreased with increasing ethanol. Aerobic vs. anaerobic polypeptide profiles of each of the species were compared by SDS/PAGE. For all species, the number of polypeptides decreased under anaerobiosis and several quantitative differences were apparent relative to the aerobic profile. In addition, amino acid incorporation into protein was analyzed by [ 35 S]-Met labeling of 3 day old seedlings grown in air or N 2 . Significant protein synthesis was measured in tolerant seedlings under N 2 and several polypeptides were specifically induced. These results are being compared with labeling patterns of the other semi-tolerant and intolerant Echinochloa species to determine their importance in flooding tolerance

  20. Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

    Science.gov (United States)

    Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

    2017-07-21

    Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

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

    Science.gov (United States)

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

    2017-11-01

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

  2. The Chemical Basis for the Origin of the Genetic Code and the Process of Protein Synthesis

    Science.gov (United States)

    Lacey, James C., Jr.

    1990-01-01

    A model for the origin of protein synthesis. The essential features of the model are that 5'-AMP and perhaps other monoribonucleotides can serve as catalysts for the selective synthesis of L-based peptides. A unique set of characteristics of 5'-AMP is responsible for the selective catalysts and these characteristics are described in detail. The model involves the formation of diesters as intermediates and selectivity for use of the L-isomer occurs principally at the step of forming the diester. However, in the formation of acetyl phenylalanine-AMP monoester there is a selectivity for esterification by the D-isomer. Data showing this selectivity is presented. This selectivity for D-isomer disappears after the first step. The identity was confirmed of all four of possible diesters of acetyl-D- and -L phenylaline with 5'-AMP by nuclear magnetic resonance (NMR). The data using flourescence and NMR show the Trp ring can associate with the adenine ring more strongly when the D-isomer is in the 2' position than it can when in the 3' position. These same data also suggest a molecular mechanisim for the faster esterificaton of 5'-AMP by acetyl-D-phenylaline. Some new data is also presented on the possible structure of the 2' isomer of acetyl-D-tryptophan-AMP monoester. The HPLC elution times of all four possible acetyl diphenylalanine esters of 5'-AMP were studied, these peptidyl esters will be products in the studies of peptide formation on the ribose of 5'-AMP. Other studies were on the rate of synthesis and the identity of the product when producing 3'Ac-Phe-2'tBOC-Phe-AMP diester. HPLC purification and identification of this product were accomplished.

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

    Science.gov (United States)

    Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

    2017-07-01

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

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

    Science.gov (United States)

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

    2005-02-23

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

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Use of protein cages as a template for confined synthesis of inorganic and organic nanoparticles.

    Science.gov (United States)

    Uchida, Masaki; Qazi, Shefah; Edwards, Ethan; Douglas, Trevor

    2015-01-01

    Protein cages are hollow spherical proteins assembled from a defined number of subunits. Because they are extremely homogeneous in size and structure, their interior cavities can serve as ideal templates to encapsulate and synthesize well-defined nanoparticles. Here, we describe the exemplary synthesis of a hard and a soft material in two representative protein cages, i.e., magnetite nanoparticles in ferritin and a poly(2-aminoethyl)methacrylate inside a viral capsid derived from the bacteriophage P22.

  8. Anabolic sensitivity of postprandial muscle protein synthesis to the ingestion of a protein-dense food is reduced in overweight and obese young adults.

    Science.gov (United States)

    Beals, Joseph W; Sukiennik, Richard A; Nallabelli, Julian; Emmons, Russell S; van Vliet, Stephan; Young, Justin R; Ulanov, Alexander V; Li, Zhong; Paluska, Scott A; De Lisio, Michael; Burd, Nicholas A

    2016-10-01

    Excess body fat diminishes muscle protein synthesis rates in response to hyperinsulinemic-hyperaminoacidemic clamps. However, muscle protein synthetic responses after the ingestion of a protein-dense food source across a range of body mass indexes (BMIs) have not been compared. We compared the myofibrillar protein synthetic response and underlying nutrient-sensing mechanisms after the ingestion of lean pork between obese, overweight, and healthy-weight adults. Ten healthy-weight [HW; BMI (in kg/m 2 ): 22.7 ± 0.4], 10 overweight (OW; BMI: 27.1 ± 0.5), and 10 obese (OB; BMI: 35.9 ± 1.3) adults received primed continuous l-[ring- 13 C 6 ]phenylalanine infusions. Blood and muscle biopsy samples were collected before and after the ingestion of 170 g pork (36 g protein and 3 g fat) to assess skeletal muscle anabolic signaling, amino acid transporters [large neutral and small neutral amino acid transporters (LAT1, SNAT2) and CD98], and myofibrillar protein synthesis. At baseline, OW and OB groups showed greater relative amounts of mammalian target of rapamycin complex 1 (mTORC1) protein than the HW group. Pork ingestion increased mTORC1 phosphorylation only in the HW group (P = 0.001). LAT1 and SNAT2 protein content increased during the postprandial period in all groups (time effect, P anabolic signals, that reduces muscle sensitivity to food ingestion. This trial was registered at clinicaltrials.gov as NCT02613767. © 2016 American Society for Nutrition.

  9. Hypophosphatemia promotes lower rates of muscle ATP synthesis

    DEFF Research Database (Denmark)

    Pesta, Dominik H; Tsirigotis, Dimitrios N; Befroy, Douglas E

    2016-01-01

    . Rates of VATP normalized in both hypophosphatemic groups after restoring plasma Pi concentrations. Furthermore, VATP was directly related to cellular and mitochondrial Pi uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic...

  10. Measurement of glomerular filtration rate by impulse synthesis: Clinical validation and optimization

    International Nuclear Information System (INIS)

    Palagi, B.; Verga, P.; Broggi, A.; Picozzi, R.; Villa, F.; Guzzini, F.; Cozzi, C.; Tomasi, A.

    1988-01-01

    Impulse synthesis is a technique which relies upon the logic of continuous infusion but extracts the clearance value from single-injection data by shifting and adding them until an asymptotic value is attained. This study has been aimed at validating and optimizing clinically the measurement of glomerular filtration rate by impulse synthesis. A single intravenous injection of 51 Cr-EDTA has been made in 32 patients and plasma activity monitored over the next 6 h. Glomerular filtration rate computed by a single-exponential fit method (GFR-SEF) has been shown to be significantly (p [de

  11. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis -acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times. IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli , synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  13. Post-Exercise Muscle Protein Synthesis in Rats after Ingestion of Acidified Bovine Milk Compared with Skim Milk.

    Science.gov (United States)

    Nakayama, Kyosuke; Kanda, Atsushi; Tagawa, Ryoichi; Sanbongi, Chiaki; Ikegami, Shuji; Itoh, Hiroyuki

    2017-09-27

    Bovine milk proteins have a low absorption rate due to gastric acid-induced coagulation. Acidified milk remains liquid under acidic conditions; therefore, the absorption rate of its protein may differ from that of untreated milk. To investigate how this would affect muscle protein synthesis (MPS), we compared MPS after ingestion of acidified versus skim milk in rats. Male Sprague-Dawley rats swam for 2 h and were immediately administered acidified or skim milk, then euthanized at 30, 60, 90, and 120 min afterwards. Triceps muscle samples were excised for assessing fractional synthetic rate (FSR), plasma components, intramuscular free amino acids and mTOR signaling. The FSR in the acidified milk group was significantly higher than in the skim milk group throughout the post-ingestive period. Plasma essential amino acids, leucine, and insulin levels were significantly increased in the acidified milk group at 30 min after administration compared to the skim milk group. In addition, acidified milk ingestion was associated with greater phosphorylation of protein kinase B (Akt) and ribosomal protein S6 kinase (S6K1), and sustained phosphorylation of 4E-binding protein 1 (4E-BP1). These results indicate that compared with untreated milk, acidified milk ingestion is associated with greater stimulation of post-exercise MPS.

  14. Post-Exercise Muscle Protein Synthesis in Rats after Ingestion of Acidified Bovine Milk Compared with Skim Milk

    Directory of Open Access Journals (Sweden)

    Kyosuke Nakayama

    2017-09-01

    Full Text Available Bovine milk proteins have a low absorption rate due to gastric acid-induced coagulation. Acidified milk remains liquid under acidic conditions; therefore, the absorption rate of its protein may differ from that of untreated milk. To investigate how this would affect muscle protein synthesis (MPS, we compared MPS after ingestion of acidified versus skim milk in rats. Male Sprague-Dawley rats swam for 2 h and were immediately administered acidified or skim milk, then euthanized at 30, 60, 90, and 120 min afterwards. Triceps muscle samples were excised for assessing fractional synthetic rate (FSR, plasma components, intramuscular free amino acids and mTOR signaling. The FSR in the acidified milk group was significantly higher than in the skim milk group throughout the post-ingestive period. Plasma essential amino acids, leucine, and insulin levels were significantly increased in the acidified milk group at 30 min after administration compared to the skim milk group. In addition, acidified milk ingestion was associated with greater phosphorylation of protein kinase B (Akt and ribosomal protein S6 kinase (S6K1, and sustained phosphorylation of 4E-binding protein 1 (4E-BP1. These results indicate that compared with untreated milk, acidified milk ingestion is associated with greater stimulation of post-exercise MPS.

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

    International Nuclear Information System (INIS)

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

    1978-01-01

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

  16. Pyridine induction of cytochrome P450IIE1: Evidence for enhanced protein synthesis

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  17. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    Directory of Open Access Journals (Sweden)

    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  18. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    Science.gov (United States)

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  20. Synthesis and characterization of recombinant abductin-based proteins.

    Science.gov (United States)

    Su, Renay S-C; Renner, Julie N; Liu, Julie C

    2013-12-09

    Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

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

    Science.gov (United States)

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

    2017-12-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  3. The course of protein synthesis during grain filling in normal and high lysine barley

    International Nuclear Information System (INIS)

    Giese, H.; Andersen, B.

    1984-01-01

    A study of the course of protein synthesis during grain filling in Bomi and the high lysine barleys Hily 82/3 and Risoe 56 showed that the four salt-soluble proteins, protein Z, β-amylase and the chymotrypsin inhibitors CI-1 and CI-2, are synthesized in greater amounts earlier in the high lysine lines than in Bomi. On the other hand, the hordeins are synthesized in greater amounts earlier during grain filling in Bomi than in Hily 82/3 and Risoe 56. There is no indication of a significant reduction of total protein synthesis in the high lysine lines compared with the standard lines Bomi and Pirrka. Hily 82/3 and Risoe 56 are very similar in protein composition in that they have a lower hordein content and higher levels, particularly of β-amylase and the chymotrypsin inhibitors, than Bomi. (author)

  4. Data-driven modelling of protein synthesis : A sequence perspective

    NARCIS (Netherlands)

    Gritsenko, A.

    2017-01-01

    Recent advances in DNA sequencing, synthesis and genetic engineering have enabled the introduction of choice DNA sequences into living cells. This is an exciting prospect for the field of industrial biotechnology, which aims at using microorganisms to produce foods, beverages, pharmaceuticals and

  5. Synthesis of the major storage protein, hordein, in barley

    DEFF Research Database (Denmark)

    Giese, Nanna Henriette; Andersen, B.; Doll, Hans

    1983-01-01

    A liquid culture system for culturing detached spikes of barley (Hordeum vulgare L.) at different nutritional levels was established. The synthesis of hordein polypeptides was studied by pulse-labeling with [14C]sucrose at different stages of development and nitrogen (N) nutrition. All polypeptides...

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

    Science.gov (United States)

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

    2015-09-11

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

  7. Dependency on de novo protein synthesis and proteomic changes during metamorphosis of the marine bryozoan Bugula neritina

    KAUST Repository

    Wong, Yue Him; Arellano, Shawn M; Zhang, Huoming; Ravasi, Timothy; Qian, Pei-Yuan

    2010-01-01

    synthesis of proteins and, instead, involves post-translational modifications of existing proteins, providing a simple mechanism to quickly initiate metamorphosis. To test our hypothesis, we challenged B. neritina larvae with transcription and translation

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

    OpenAIRE

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

    2011-01-01

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

  9. Inhibition of acetaminophen oxidation by cimetidine and the effects on glutathione and activated sulphate synthesis rates

    DEFF Research Database (Denmark)

    Dalhoff, K; Poulsen, H E

    1993-01-01

    inhibition of cytochrome P-450 drug oxidation by cimetidine in isolated rat hepatocytes. The synthesis rates of glutathione and PAPS were determined simultaneously by an established method based on trapping of radioactivity (35S) in the prelabelled glutathione and PAPS pools. Preincubation of the hepatocytes...

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

    Science.gov (United States)

    Heude, M; Chanet, R

    1975-04-01

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

  11. Protein synthesis directed by cowpea mosaic virus RNAs

    International Nuclear Information System (INIS)

    Stuik, E.

    1979-01-01

    The thesis concerns the proteins synthesized under direction of Cowpea mosaic virus RNAs. Sufficient radioactive labelling of proteins was achieved when 35 S as sulphate was administered to intact Vigna plants, cultivated in Hoagland solution. The large polypeptides synthesized under direction of B- and M-RNA are probably precursor molecules from which the coat proteins are generated by a mechanism of posttranslational cleavage. (Auth.)

  12. Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  13. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.

    Science.gov (United States)

    Hector, Amy J; McGlory, Chris; Damas, Felipe; Mazara, Nicole; Baker, Steven K; Phillips, Stuart M

    2018-01-01

    Preservation of lean body mass (LBM) may be important during dietary energy restriction (ER) and requires equal rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Currently, the relative contribution of MPS and MPB to the loss of LBM during ER in humans is unknown. We aimed to determine the impact of dietary protein intake and resistance exercise on MPS and MPB during a controlled short-term energy deficit. Adult men (body mass index, 28.6 ± 0.6 kg/m 2 ; age 22 ± 1 yr) underwent 10 d of 40%-reduced energy intake while performing unilateral resistance exercise and consuming lower protein (1.2 g/kg/d, n = 12) or higher protein (2.4 g/kg/d, n = 12). Pre- and postintervention testing included dual-energy X-ray absorptiometry, primed constant infusion of ring -[ 13 C 6 ]phenylalanine, and 15 [N]phenylalanine to measure acute postabsorptive MPS and MPB; D 2 O to measure integrated MPS; and gene and protein expression. There was a decrease in acute MPS after ER (higher protein, 0.059 ± 0.006 to 0.051 ± 0.009%/h; lower protein, 0.061 ± 0.005 to 0.045 ± 0.006%/h; P resistance exercise (higher protein, 0.067 ± 0.01%/h; lower protein, 0.061 ± 0.006%/h), and integrated MPS followed a similar pattern. There was no change in MPB (energy balance, 0.080 ± 0.01%/hr; ER rested legs, 0.078 ± 0.008%/hr; ER exercised legs, 0.079 ± 0.006%/hr). We conclude that a reduction in MPS is the main mechanism that underpins LBM loss early in ER in adult men.-Hector, A. J., McGlory, C., Damas, F., Mazara, N., Baker, S. K., Phillips, S. M. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise. © FASEB.

  14. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    Science.gov (United States)

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  16. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells

    International Nuclear Information System (INIS)

    Hill, T.M.; Sinden, R.R.; Sadler, J.R.

    1983-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff

  17. Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of Beta-hydroxy-Beta-methylbutyrate

    Science.gov (United States)

    Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite Beta-hydroxy-Beta-methylbutyrate (HMB). To determine the effects of HMB on protein synthesi...

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

    International Nuclear Information System (INIS)

    Faust, H.

    1984-01-01

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

  19. Understanding of Protein Synthesis in a Living Cell

    Science.gov (United States)

    Mustapha, Y.; Muhammad, S.

    2006-01-01

    The assembly of proteins takes place in the cytoplasm of a cell. There are three main steps. In initiation, far left, all the necessary parts of the process are brought together by a small molecule called a ribosome. During elongation, amino acids, the building blocks of proteins, are joined to one another in a long chain. The sequence in which…

  20. Effects of chilling on protein synthesis in tomato suspension cultures

    International Nuclear Information System (INIS)

    Matadial, B.; Pauls, K.P.

    1989-01-01

    The effect of chilling on cell growth, cell viability, protein content and protein composition in suspension cultures of L. esculentum and L. hirsutum was investigated. Cell growth for both species was arrested at 2 degrees C but when cultures were transferred to 25 degree C cell growth resumed. There was no difference in viability between control and chilled cultures of L. esculentum, however, L. hirsutum control cultures exhibited larger amounts of Fluorescein Diacetate induced fluorescence than chilled cultures. 35 S-methionine incorporation into proteins was 2.5-2 times higher in L. hirsutum than in L. esculentum. Quantitative and qualitative differences, in 35 S-methionine labelled proteins, between chilled and control cultures were observed by SDS-PAGE and fluorography. Protein content in chilled cultures decreased over time but then increased when cultures were transferred to 25 degrees C

  1. Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry

    Science.gov (United States)

    Yarasheski, Kevin E.; Smith, Kenneth; Rennie, Michael J.; Bier, Dennis M.

    2014-01-01

    The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuous-flow technique using capillary GC/combustion IRMS. Quadriceps muscles were removed from four Sprague–Dawley rats after each was infused at a different rate with (1-13C)leucine for 6–8 h. Muscle leucine enrichment (at.% excess) measured by both methods differed by less than 4%, except at low (13C)leucine enrichments (IRMS was used to assess muscle (13C)leucine enrichment and fractional muscle protein synthesis rate in ten normal young men and women infused with (1,2-13C2)leucine for 12–14 h. This approach reduced the variability of the isotope abundance measure and gave estimates of muscle protein synthesis rate (0.050 ± 0.011% h−1 (mean ± SEM); range = 0.023–0.147% h−1) that agree with published values determined using the standard analytical approach. The measurement of (13C)leucine enrichment from skeletal muscle protein by capillary GC/combustion IRMS provides a simple, acceptable and practical alternative to preparative GC/ninhydrin IRMS. PMID:1420371

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

    International Nuclear Information System (INIS)

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

    1975-01-01

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

  3. Higher skeletal muscle protein synthesis and lower breakdown after chemotherapy in cachectic mice.

    Science.gov (United States)

    Samuels, S E; Knowles, A L; Tilignac, T; Debiton, E; Madelmont, J C; Attaix, D

    2001-07-01

    The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P synthesis (-38%; P synthesis (~-54 to -69%; P synthesis (+46 to +73%; P synthesis and degradation.

  4. Relationship between the rate of hepatic sterol synthesis and the incorporation of [3H]water

    International Nuclear Information System (INIS)

    Pullinger, C.R.; Gibbons, G.F.

    1983-01-01

    The true rate of sterol synthesis in liver cells was determined by measurement of the weight of desmosterol produced over a given time period during incubations in the presence of triparanol. The simultaneous presence of tritiated water ( 3 H 2 O) during the incubations permitted a direct observation of the weight of tritium incorporated into a given mass of newly synthesized sterol. The incorporation of tritium per atom of sterol carbon (H/C ratio) was lower than some previously reported values and suggests that a sizeable proportion of the reducing equivalents (NADPH) required for sterol synthesis arises via the pentose phosphate pathway. The H/C ratio changed significantly with length of the incubation period. The value of the ratio was also dependent upon whether the acetyl-CoA units utilized for sterol synthesis were derived predominantly from a carbohydrate or a fatty acid source

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

    Science.gov (United States)

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

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

  6. Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth-restricted fetal sheep.

    Science.gov (United States)

    Rozance, Paul J; Zastoupil, Laura; Wesolowski, Stephanie R; Goldstrohm, David A; Strahan, Brittany; Cree-Green, Melanie; Sheffield-Moore, Melinda; Meschia, Giacomo; Hay, William W; Wilkening, Randall B; Brown, Laura D

    2018-01-01

    Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass, which may contribute to insulin resistance and the development of diabetes. We demonstrate slower hindlimb linear growth and muscle protein synthesis rates that match the reduced hindlimb blood flow and oxygen consumption rates in IUGR fetal sheep. These adaptations resulted in hindlimb blood flow rates in IUGR that were similar to control fetuses on a weight-specific basis. Net hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was significantly lower in IUGR fetal sheep. Among all fetuses, blood O 2 saturation and plasma glucose, insulin and insulin-like growth factor-1 were positively associated and norepinephrine was negatively associated with hindlimb weight. These results further our understanding of the metabolic and hormonal adaptations to reduced oxygen and nutrient supply with placental insufficiency that develop to slow hindlimb growth and muscle protein accretion. Reduced skeletal muscle mass in the fetus with intrauterine growth restriction (IUGR) persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake and protein accretion rates in skeletal muscle, late gestation control (CON) (n = 8) and IUGR (n = 13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate and fractional synthetic rate were lower in IUGR compared to CON (P fetal norepinephrine and reduced IGF-1 and insulin. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

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

    Directory of Open Access Journals (Sweden)

    Ying Wang

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

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

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

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

  9. Detection of carriers and genetic counseling in duchenne muscular dystrophy by ribosomal protein synthesis.

    Science.gov (United States)

    Ionasescu, V; Zellweger, H; Burmeister, L

    1976-11-01

    The in vitro protein synthesis by polyribosomes extracted from biopsied muscle (vastus lateralis) was studied in 47 known carriers, 87 possible carriers and in 60 normal females. A significant increase in specific activity of monomeric ribosomes, total polyribosomes and collagen synthesis was found in 46 (97.8 per cent) known carriers and 47 (54 per cent) possible carriers of Duchenne muscular dytrophy. The latter showed an increase in ribosomal protein synthesis in 10 (52.6 per cent) of 19 mothers of isolated cases, 31 (53.3 per cent) of 58 sisters, and 6 (60 per cent) of other female relatives. Serum creatine phosphokinase was increased in 30 (63.8 per cent) of 47 known carriers.

  10. Protein synthesis in muscle cultures from patients with duchenne muscular dystrophy

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  11. Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

    Science.gov (United States)

    Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

    2011-06-01

    Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

  12. Ammonia treatment of wheat straw. 2. Efficiency of microbial protein synthesis, rumen microbial protein pool size and turnover, and small intestinal protein digestion in sheep.

    NARCIS (Netherlands)

    Oosting, S.J.; Viets, T.C.; Lammers-Wienhoven, S.C.W.; Bruchem, van J.

    1993-01-01

    Ammonia-treated wheat straw (AWS) was compared with untreated wheat straw (UWS) and untreated wheat straw supplemented with urea (SWS) in an experiment with 6 wether sheep. Microbial protein synthesis increased after ammonia treatment due to the higher intake of rumen degradable organic matter (OM).

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

    Science.gov (United States)

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

    2018-02-27

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

  14. Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development

    International Nuclear Information System (INIS)

    Cenedella, R.J.; Fleschner, C.R.

    1989-01-01

    The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3 H 2 O and [ 14 C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14 C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3 H of 3 H 2 O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered

  15. Protein synthesis and antioxidant capacity in aging mice : Effects of long-term voluntary exercise

    NARCIS (Netherlands)

    Vaanholt, Lobke M.; Speakman, John R.; Garland, Theodore; Lobley, Gerald E.; Visser, G. Henk

    2008-01-01

    Exercise increases metabolic rate and the production of reactive oxygen species (ROS) but also elevates protein turnover. ROS cause damage to macromolecules (e. g., proteins) and thereby contribute to aging. Protein turnover removes and replaces damaged proteins. The balance between these two

  16. Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis

    International Nuclear Information System (INIS)

    Hatch, T.P.; Miceli, M.; Silverman, J.A.

    1985-01-01

    Synthesis of protein by the obligate intracellular parasitic bacteria Chlamydia psittaci (6BC) and Chlamydia trachomatis (serovar L2) isolated from host cells (host-free chlamydiae) was demonstrated for the first time. Incorporation of [ 35 S]methionine and [ 35 S]cysteine into trichloroacetic acid-precipitable material by reticulate bodies of chlamydiae persisted for 2 h and was dependent upon a exogenous source of ATP, an ATP-regenerating system, and potassium or sodium ions. Magnesium ions and amino acids stimulated synthesis; chloramphenicol, rifampin, oligomycin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a proton ionophore) inhibited incorporation. Ribonucleoside triphosphates (other than ATP) had little stimulatory effect. The optimum pH for host-free synthesis was between 7.0 and 7.5. The molecular weights of proteins synthesized by host-free reticulate bodies closely resembled the molecular weights of proteins synthesized by reticulate bodies in an intracellular environment, and included outer membrane proteins. Elementary bodies of chlamydiae were unable to synthesize protein even when incubated in the presence of 10 mM dithiothreitol, a reducing agent which converted the highly disulfide bond cross-linked major outer membrane protein to monomeric form

  17. In Profile: Models of Ribosome Biogenesis Defects and Regulation of Protein Synthesis

    NARCIS (Netherlands)

    Essers, P.B.M.

    2013-01-01

    Ribosomes are the mediators of protein synthesis in the cell and therefore crucial to proper cell function. In addition, ribosomes are highly abundant, with ribosomal RNA making up 80% of the RNA in the cell. A large amount of resources go into maintaining this pool of ribosomes, so ribosome

  18. Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2008-01-01

    The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

  19. Solid-phase synthesis of an apoptosis-inducing tetrapeptide mimicking the Smac protein

    DEFF Research Database (Denmark)

    Le Quement, Sebastian Thordal; Ishøy, Mette; Petersen, Mette Terp

    2011-01-01

    An approach for the solid-phase synthesis of apoptosis-inducing Smac peptidomimetics is presented. Using a Rink linker strategy, tetrapeptides mimicking the N-4-terminal residue of the Smac protein [(N-Me)AVPF sequence] were synthesized on PEGA resin in excellent purities and yields. Following two...

  20. Using Simple Manipulatives to Improve Student Comprehension of a Complex Biological Process: Protein Synthesis

    Science.gov (United States)

    Guzman, Karen; Bartlett, John

    2012-01-01

    Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article…

  1. Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

    DEFF Research Database (Denmark)

    Brøndsted Nielsen, Mogens; Andersen, Lars Henrik; Rinza, Tomás Rocha

    2011-01-01

    The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase ...

  2. Degradation and de novo synthesis of D1 protein and psbA ...

    Indian Academy of Sciences (India)

    This shows that synthesis of D1 protein is not the only component involved in the recovery process. Our events, which ... transcript levels in the green alga Chlamydomonas reinhardtii in ..... and Gaba V 1996 Accelerated degradation of the D2 ...

  3. Myelin Basic Protein synthesis is regulated by small non-coding RNA 715

    NARCIS (Netherlands)

    Bauer, N.M.; Moos, C.; van Horssen, J.; Witte, M.E.; van der Valk, P.; Altenhein, B.; Luhmann, H.J.; White, R.

    2012-01-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP

  4. Reconsolidation of a Context Long-Term Memory in the Terrestrial Snail Requires Protein Synthesis

    Science.gov (United States)

    Gainutdinova, Tatiana H.; Tagirova, Rosa R.; Ismailova, Asja I.; Muranova, Lyudmila N.; Samarova, Elena I.; Gainutdinov, Khalil L.; Balaban, Pavel M.

    2005-01-01

    We investigated the influence of the protein synthesis blocker anisomycin on contextual memory in the terrestrial snail "Helix." Prior to the training session, the behavioral responses in two contexts were similar. Two days after a session of electric shocks (5 d) in one context only, the context conditioning was observed as the significant…

  5. Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography.

    Science.gov (United States)

    Pentelute, Brad L; Mandal, Kalyaneswar; Gates, Zachary P; Sawaya, Michael R; Yeates, Todd O; Kent, Stephen B H

    2010-11-21

    Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space group P1 that diffracted to atomic-resolution (0.95 Å), enabling the X-ray structure of kaliotoxin to be determined by direct methods.

  6. Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

    Science.gov (United States)

    Kleihues, P.; Magee, P. N.

    1973-01-01

    1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2–3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[14C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA. PMID:4774397

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

    Directory of Open Access Journals (Sweden)

    Yazmín Ramiro-Cortés

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

  8. Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

    Science.gov (United States)

    Dos Santos, Maísa Pavani; Batistela, Emanuele; Pereira, Mayara Peron; Paula-Gomes, Silvia; Zanon, Neusa Maria; Kettelhut, Isis do Carmo; Karatzaferi, Christina; Andrade, Claudia Marlise Balbinotti; de França, Suélem Aparecida; Baviera, Amanda Martins; Kawashita, Nair Honda

    2016-08-01

    Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. DETERMINATION OF PROTEIN CATABOLIC RATE IN PATIENTS ON CHRONIC INTERMITTENT HEMODIALYSIS - UREA OUTPUT MEASUREMENTS COMPARED WITH DIETARY-PROTEIN INTAKE AND WITH CALCULATION OF UREA GENERATION RATE

    NARCIS (Netherlands)

    STEGEMAN, CA; HUISMAN, RM; DEROUW, B; JOOSTEMA, A; DEJONG, PE

    We assessed the agreement between different methods of determining protein catabolic rate (PCR) in hemodialysis patients and the possible influence of postdialysis urea rebound and the length of the interdialytic interval on the PCR determination. Protein catabolic rate derived from measured total

  10. Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

    International Nuclear Information System (INIS)

    Singh, Jaspal; Singh, Ajit; Ganguli, N.C.

    1976-01-01

    The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate- 14 C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

  11. Dasatinib synergises with irinotecan to suppress hepatocellular carcinoma via inhibiting the protein synthesis of PLK1.

    Science.gov (United States)

    Xu, Li; Zhu, Yuanrun; Shao, Jinjin; Chen, Min; Yan, Hao; Li, Guanqun; Zhu, Yi; Xu, Zhifei; Yang, Bo; Luo, Peihua; He, Qiaojun

    2017-04-11

    Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumour and has poor prognosis. Currently, systematic chemotherapy is the only approach to prolong survival. Thus the development of new treatment regimens is urgently needed to improve the therapeutic efficacy. Our study intended to assess the combination of dasatinib and irinotecan against HCC and made an effort to develop a potential medical choice for advanced HCC patients. We used SRB colorimetric assay and clonogenic assay to assess antitumour effect in vitro and HCC xenograft model to assess antitumour effect in vivo. We applied flow cytometry and western blotting to explore the mechanism of the combined therapy. Knockdown and overexpression of PLK1 are also applied for validation. We confirmed that dasatinib has synergistic effect with irinotecan (or SN38) on HCC both in vitro and in vivo. The effect is due to arisen apoptosis rate of HCC cells that is accompanied by mitochondria dysfunction. The enhanced antitumour efficacy of SN38 could be explained by additional inhibition of PLK1, which is triggered by dasatinib. Unlike existed PLK1 inhibitors, dasatinib does not inhibit PLK1 activity in a direct way. Instead, we found that dasatinib reduces PLK1 level by interfering with its protein synthesis progress. We validated that this kind of downregulation of PLK1 level has a key role in the synergistic effect of the two agents. Dasatinib is able to reinforce the anti-HCC efficacy of irinotecan/SN38 by downregulation of PLK1 synthesis. The combination of the two agents might be a potential medical choice for HCC therapy.

  12. Supplementing Breakfast with a Vitamin D and Leucine-Enriched Whey Protein Medical Nutrition Drink Enhances Postprandial Muscle Protein Synthesis and Muscle Mass in Healthy Older Men.

    Science.gov (United States)

    Chanet, Audrey; Verlaan, Sjors; Salles, Jérôme; Giraudet, Christophe; Patrac, Véronique; Pidou, Véronique; Pouyet, Corinne; Hafnaoui, Nordine; Blot, Adeline; Cano, Noël; Farigon, Nicolas; Bongers, Anke; Jourdan, Marion; Luiking, Yvette; Walrand, Stéphane; Boirie, Yves

    2017-12-01

    Background: A promising strategy to help older adults preserve or build muscle mass is to optimize muscle anabolism through providing an adequate amount of high-quality protein at each meal. Objective: This "proof of principle" study investigated the acute effect of supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink on postprandial muscle protein synthesis and longer-term effect on muscle mass in healthy older adults. Methods: A randomized, placebo-controlled, double-blind study was conducted in 24 healthy older men [mean ± SD: age 71 ± 4 y; body mass index (in kg/m 2 ) 24.7 ± 2.8] between September 2012 and October 2013 at the Unit of Human Nutrition, University of Auvergne, Clermont-Ferrand, France. Participants received a medical nutrition drink [test group; 21 g leucine-enriched whey protein, 9 g carbohydrates, 3 g fat, 800 IU cholecalciferol (vitamin D 3 ), and 628 kJ] or a noncaloric placebo (control group) before breakfast for 6 wk. Mixed muscle protein fractional synthesis rate (FSR) was measured at week 0 in the basal and postprandial state, after study product intake with a standardized breakfast with the use of l-[ 2 H 5 ]-phenylalanine tracer methodology. The longer-term effect of the medical nutrition drink was evaluated by measurement of appendicular lean mass, representing skeletal muscle mass at weeks 0 and 6, by dual-energy X-ray absorptiometry. Results: Postprandial FSR (0-240 min) was higher in the test group than in the control group [estimate of difference (ED): 0.022%/h; 95% CI: 0.010%/h, 0.035%/h; ANCOVA, P = 0.001]. The test group gained more appendicular lean mass than the control group after 6 wk (ED: 0.37 kg; 95% CI: 0.03, 0.72 kg; ANCOVA, P = 0.035), predominantly as leg lean mass (ED: 0.30 kg; 95% CI: 0.03, 0.57 kg; ANCOVA, P = 0.034). Conclusions: Supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink stimulated postprandial muscle protein

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

    Science.gov (United States)

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

    2016-05-01

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

  14. The induction of the oxidative burst in Elodea densa by sulfhydryl reagent does not depend on de novo protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Amicucci, Enrica [Milan, Univ. (Italy). Dipt. di Fisiologia e Biochimica delle Piante

    1997-12-31

    In Elodea densa Planchon leaves, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a marked and temporary increase of respiration that is insensitive to cyanide, hydroxamate and propylgallate and completely inhibited by diphenylene iodonium (DPI) and by quinacrine. In this paper the author investigates whether the mechanism that causes the oxidative burst depends on the activation of preexisting oxidative systems or on the activation of de novo protein synthesis. The inhibitors used were cycloheximide (CHI) which inhibits protein synthesis in plant cells by depressing the incorporation of aminoacids into proteins and cordycepin, an effective inhibitor of mRNA synthesis. The data support the idea that the mechanism investigated depends on the activation of a long lived protein(s) and not on de novo protein synthesis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    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.

  16. Deficiency in plasma protein synthesis caused by x-ray-induced lethal albino alleles in mouse

    International Nuclear Information System (INIS)

    Garland, R.C.; Satrustegui, J.; Gluecksohn-Waelsch, S.; Cori, C.F.

    1976-01-01

    Plasma protein synthesis was studied in mice bearing x-ray induced lethal mutations at the albino locus. Newborn albino mutants showed a decrease in each of the three principal plasma proteins, albumin, α-fetoprotein, and transferrin, when compared with colored littermate controls. Incorporation of [ 14 C] leucine into plasma proteins of the newborn albinos 30 min after injection was only 1 / 5 that of the controls, but incorporation into total liver protein was only slightly diminished. Incorporation of [ 14 C] leucine into an albumin fraction obtained by immunoprecipitation from livers incubated in vitro in an amino acid mixture was also strongly diminished. Thus, the liver of 18-day-old albino fetuses incorporated into this fraction 1 / 3 and that of newborn albinos 1 / 8 as much as the controls, but in both cases the incorporation into total liver protein was only 25 percent less than in the respective controls. These results indicate that the rather severe structural abnormalities observed in the mutants in the endoplasmic reticulum and the Golgi apparatus are not associated with a general deficiency of hepatic protein synthesis. Instead the data from this and previous work show that the progressive deficiency from fetal life to birth involves certain specific proteins represented by several perinatally developing enzymes and by plasma proteins. It is suggested that the mutational effects observed in these mice are due to deletions involving regulatory rather than structural genes at or near the albino locus

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    KAUST Repository

    Ruchti, E.

    2016-10-08

    The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

  19. Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

    International Nuclear Information System (INIS)

    Kopecky, J.; Baudysova, M.; Zanotti, F.; Janikova, D.; Pavelka, S.; Houstek, J.

    1990-01-01

    In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria

  20. Effect of transcutaneous electrical muscle stimulation on postoperative muscle mass and protein synthesis

    DEFF Research Database (Denmark)

    Vinge, O; Edvardsen, L; Jensen, F

    1996-01-01

    In an experimental study, 13 patients undergoing major elective abdominal surgery were given postoperative transcutaneous electrical muscle stimulation (TEMS) to the quadriceps femoris muscle on one leg; the opposite leg served as control. Changes in cross-sectional area (CSA) and muscle protein ...... protein synthesis and muscle mass after abdominal surgery and should be evaluated in other catabolic states with muscle wasting.......In an experimental study, 13 patients undergoing major elective abdominal surgery were given postoperative transcutaneous electrical muscle stimulation (TEMS) to the quadriceps femoris muscle on one leg; the opposite leg served as control. Changes in cross-sectional area (CSA) and muscle protein...... synthesis were assessed by computed tomography and ribosome analysis of percutaneous muscle biopsies before surgery and on the sixth postoperative day. The percentage of polyribosomes in the ribosome suspension decreased significantly (P

  1. Direct vs. indirect pathway of hepatic glycogen synthesis as a function of glucose infusion rate

    International Nuclear Information System (INIS)

    Bagby, G.J.; Lang, C.H.; Johnson, J.L.; Blakesly, H.L.; Spitzer, J.J.

    1986-01-01

    This study was initiated to determine the influence of the rate of exogenous glucose administration on liver glycogen synthesis by the direct (glucose uptake and incorporation into glycogen) vs the indirect pathway (glucose degradation to 3-carbon intermediates, e.g., lactate, prior to incorporation into glycogen). Catheterized rats were fasted 2 days prior to receiving a 3 hr infusion of glucose at rates of 0 to 230 μmol/min/kg containing tracer [6- 3 H]- and [U- 14 C]-glucose. Plasma glucose (r = 0.80), insulin (r = 0.90) and lactate (r = 0.84) were correlated with glucose infusion rate. The rate of liver glycogen deposition (0.46 +/- 0.03 μmol/min/g) did not differ between a glucose infusion rate of 20 and 230 μmol/min/kg. At the lowest and highest glucose infusion rates hepatic glycogenesis accounted for 87 +/- 6 and 9 +/- 1% of the total glucose load, respectively. The percent contribution of the direct pathways to glycogen deposition ([ 3 H] specific activity in hepatic glycogen/[ 3 H] specific activity in plasma glucose) increased from 16 +/- 3 to 83 +/- 5% from lowest to highest glucose infusion rates (prevailing plasma glucose concentrations: 9 +/- 1 and 21 +/- 2 mM, respectively). The results indicate that the relative contribution of the direct and indirect pathways of glucogen synthesis are dependent upon the glucose load or plasma glucose concentration

  2. Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

    Science.gov (United States)

    Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

    1997-05-02

    Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

  3. Enteral leucine and protein synthesis in skeletal and cardiac muscle

    Science.gov (United States)

    There are three members of the Branch Chain Amino Acids: leucine, isoleucine, and valine. As essential amino acids, these amino acids have important functions which include a primary role in protein structure and metabolism. It is intriguing that the requirement for BCAA in humans comprise about 40–...

  4. Intestinal DNA concentration and protein synthesis in response to ...

    African Journals Online (AJOL)

    Jane

    2011-10-05

    Oct 5, 2011 ... Full Length Research Paper. Intestinal ... transporters are membrane-bound proteins and operate ... sporters that are similar to those found on other plasma ... on fastDNA® kit (application manual revision 6540-400-4H01) and.

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  6. Whole body synthesis rates of DHA from α-linolenic acid are greater than brain DHA accretion and uptake rates in adult rats[S

    OpenAIRE

    Domenichiello, Anthony F.; Chen, Chuck T.; Trepanier, Marc-Olivier; Stavro, P. Mark; Bazinet, Richard P.

    2014-01-01

    Docosahexaenoic acid (DHA) is important for brain function, however, the exact amount required for the brain is not agreed upon. While it is believed that the synthesis rate of DHA from α-linolenic acid (ALA) is low, how this synthesis rate compares with the amount of DHA required to maintain brain DHA levels is unknown. The objective of this work was to assess whether DHA synthesis from ALA is sufficient for the brain. To test this, rats consumed a diet low in n-3 PUFAs, or a diet containing...

  7. Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    International Nuclear Information System (INIS)

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

    1977-01-01

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

  11. Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

    Directory of Open Access Journals (Sweden)

    Ysabel Montoya

    2000-08-01

    Full Text Available Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V. peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V. braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

  12. Age-related differences in lean mass, protein synthesis and skeletal muscle markers of proteolysis after bed rest and exercise rehabilitation.

    Science.gov (United States)

    Tanner, Ruth E; Brunker, Lucille B; Agergaard, Jakob; Barrows, Katherine M; Briggs, Robert A; Kwon, Oh Sung; Young, Laura M; Hopkins, Paul N; Volpi, Elena; Marcus, Robin L; LaStayo, Paul C; Drummond, Micah J

    2015-09-15

    Bed rest-induced muscle loss and impaired muscle recovery may contribute to age-related sarcopenia. It is unknown if there are age-related differences in muscle mass and muscle anabolic and catabolic responses to bed rest. A secondary objective was to determine if rehabilitation could reverse bed rest responses. Nine older and fourteen young adults participated in a 5-day bed rest challenge (BED REST). This was followed by 8 weeks of high intensity resistance exercise (REHAB). Leg lean mass (via dual-energy X-ray absorptiometry; DXA) and strength were determined. Muscle biopsies were collected during a constant stable isotope infusion in the postabsorptive state and after essential amino acid (EAA) ingestion on three occasions: before (PRE), after bed rest and after rehabilitation. Samples were assessed for protein synthesis, mTORC1 signalling, REDD1/2 expression and molecular markers related to muscle proteolysis (MURF1, MAFBX, AMPKα, LC3II/I, Beclin1). We found that leg lean mass and strength decreased in older but not younger adults after bedrest (P protein synthesis increased before bed rest in both age groups (P protein synthesis rates and increased MAFBX mRNA, p-AMPKα and the LC3II/I ratio (P protein synthesis and a marginal increase in proteolytic markers. Finally, rehabilitation restored bed rest-induced deficits in lean mass and strength in older adults. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  13. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    Science.gov (United States)

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

  14. Relationship between synthesis and cleavage of poliovirus-specific proteins.

    OpenAIRE

    Thomas, A A; Voorma, H O; Boeye, A

    1983-01-01

    Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

  15. Effects of moisture stress on germination and protein synthesis in ...

    African Journals Online (AJOL)

    ... 3, 5 triphenyl tetrazolium chloride (TTC), and their abilities to synthesize protein after stress by incorporating L- 4,5-3H leucine into their root tips. Les graines de dolique non pigmentées, TVX 3236 (crème et brune) et IT81S-818 (blanche), étaient exposées aux conditions d'humidité constantes plus stressantes (-0.1 et ...

  16. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part C: Protein Synthesis and Post-Translational Processing in Eukaryotic Cells

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available The translation of mRNA constitutes the first step in the synthesis of a functional protein. The polypeptide chain is subsequently folded into the appropriate three-dimensional configuration and undergoes a variety of processing steps before being converted into its active form. These processing steps are intimately related to the cellular events that occur in the endoplasmic reticulum and Golgi compartments, and determine the sorting and transport of different proteins to their appropriate destinations within the cell. While the regulation of gene expression occurs primarily at the level of transcription, the expression of many genes can also be controlled at the level of translation. Most proteins can be regulated in response to extracellular signals. In addition, intracellular protein levels can be controlled by differential rates of protein degradation. Thus, the regulation of both the amounts and activities of intracellular proteins ultimately determines all aspects of cell behaviour.

  17. Synthesis of protein in intestinal cells exposed to cholera toxin

    International Nuclear Information System (INIS)

    Peterson, J.W.; Berg, W.D. Jr.; Coppenhaver, D.H.

    1987-01-01

    The mechanism by which cyclic adenosine monophosphate (AMP), formed by intestinal epithelial cells in response to cholera toxin, ultimately results in alterations in water and electrolyte transport is poorly understood. Several studies have indicated that inhibitors of transcription or translation block much of the transport of ions and water in the intestine and edema formation in tissue elicited by cholera toxin. Data presented in this study confirmed the inhibitory effects of cycloheximide on cholera toxin-induced fluid accumulation in the rabbit intestinal loop model. Neither cycloheximide nor actinomycin D altered the amount of cyclic AMP that accumulated in intestinal cells and Chinese hamster ovary cells exposed to cholera toxin. An increase in [ 3 H] leucine incorporation was readily demonstrable in intestinal epithelial cells from rabbits challenged with Vibrio cholerae. Similarly, intestinal epithelial cells incubated with cholera toxin for 4 hr synthesized substantially more protein than controls as determined by relative incorporation of [ 35 S] methionine. Most of the new protein synthesized in response to cholera toxin was membrane associated and of high molecular weight. The possible significance of the toxin-induced protein relative to cholera pathogenesis was discussed

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  19. Change detection in the dynamics of an intracellular protein synthesis model using nonlinear Kalman filtering.

    Science.gov (United States)

    Rigatos, Gerasimos G; Rigatou, Efthymia G; Djida, Jean Daniel

    2015-10-01

    A method for early diagnosis of parametric changes in intracellular protein synthesis models (e.g. the p53 protein - mdm2 inhibitor model) is developed with the use of a nonlinear Kalman Filtering approach (Derivative-free nonlinear Kalman Filter) and of statistical change detection methods. The intracellular protein synthesis dynamic model is described by a set of coupled nonlinear differential equations. It is shown that such a dynamical system satisfies differential flatness properties and this allows to transform it, through a change of variables (diffeomorphism), to the so-called linear canonical form. For the linearized equivalent of the dynamical system, state estimation can be performed using the Kalman Filter recursion. Moreover, by applying an inverse transformation based on the previous diffeomorphism it becomes also possible to obtain estimates of the state variables of the initial nonlinear model. By comparing the output of the Kalman Filter (which is assumed to correspond to the undistorted dynamical model) with measurements obtained from the monitored protein synthesis system, a sequence of differences (residuals) is obtained. The statistical processing of the residuals with the use of x2 change detection tests, can provide indication within specific confidence intervals about parametric changes in the considered biological system and consequently indications about the appearance of specific diseases (e.g. malignancies).

  20. Effect of Nitrogen, Phosphorus and Potassium on the Dynamics of Synthesis of Nucleic Acids and Proteins in the Different Phases of Wheat Development

    Energy Technology Data Exchange (ETDEWEB)

    Martinovic, B.; Grujic-Injac, B.; Jelenic, Dj. [Institute for the Application of Nuclear Energy in Agriculture, Veterinary Medicine and Forestry, Belgrade (Yugoslavia)

    1968-07-01

    The influence of different nitrogen, phosphorus and potassium levels on the dynamics of RNA synthesis and raw proteins, as well as the uptake of phosphorus in the different phases of wheat development, were studied by applying radioactive phosphorus {sup 32}P . The rate of the uptake of phosphorus is proportional to its concentration in the nutrient supply in all phases of wheat development, in spite of the fact that the uptake of phosphorus during vegetation decreases with the plant's maturity. The influence of nitrogen on the uptake of phosphorus is inversely proportional to the concentration of nitrogen in the nutrient, while the influence of potassium depends on the relation of the amounts of nitrogen and phosphorus. The increased levels of nitrogen and phosphorus are directly proportional to the increase of RNA synthesis, and conversely the decrease of these levels decreases the RNA synthesis. The RNA synthesis decreases with the wheat's maturity during vegetation and is considerably greater in the first phase than in the later phases of the plant's development. Nitrogen and phosphorus have the greatest influence on protein synthesis; potassium has far less influence. These investigations show that the synthesis of nucleic acids and the building up of phosphorus in the RNA-fraction is directly proportional to the concentration of nitrogen in the nutrient supply, even though the uptake of phosphorus by wheat is not proportional to the increased nitrogen levels. Investigations now being carried out provide further explanations concerning the mutual relationship of some forms of RNA of the cells and the amino acids in the synthesis of certain protein fractions of wheat under the influence of different levels of nitrogen, phosphorus and potassium. (author)

  1. Time course of protein synthesis-dependent phase of olfactory memory in the cricket Gryllus bimaculatus.

    Science.gov (United States)

    Matsumoto, Yukihisa; Noji, Sumihare; Mizunami, Makoto

    2003-04-01

    The cricket Gryllus bimaculatus forms a stable olfactory memory that lasts for practically a lifetime. As a first step to elucidate the cellular mechanisms of olfactory learning and memory retention in crickets, we studied the dependency of memory retention on the de novo brain protein synthesis by injecting the protein synthesis inhibitor cycloheximide (CHX) into the head capsule. Injection of CHX inhibited (3)H-leucine incorporation into brain proteins by > 90% for 3 hr. Crickets were trained to associate peppermint odor with water (reward) and vanilla odor with saline solution (non-reward) and were injected with CHX before or at different times after training. Their odor preferences were tested at 2 hr, 1 day and 4 days after training. Memory retention at 2 hr after training was unaffected by CHX injection. However, the level of retention at 1 day and 4 days after training was lowered when CHX was injected 1 hour before training or at 1 hr or 6 hr after training. To study the time course of the development of CHX-sensitive memory phase, crickets that had been injected with CHX at 1 hr after training were tested at different times from 2 to 12 hr after training. The level of retention was unaffected up to 4 hr after training but significantly lowered at 5 hr after training, and the CHX-sensitive memory phase developed gradually during the next several hours. CHX dissociates two phases of olfactory memory in crickets: earlier protein synthesis-independent phase ( 5 hr) protein synthesis-dependent phase.

  2. Bringing the science of proteins into the realm of organic chemistry: total chemical synthesis of SEP (synthetic erythropoiesis protein).

    Science.gov (United States)

    Kent, Stephen B H

    2013-11-11

    Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as "arguably the most successful drug spawned by the revolution in recombinant DNA technology". Recently, the EPO glycoprotein molecule has re-emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this "synthetic erythropoiesis protein" was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Whole body synthesis rates of DHA from α-linolenic acid are greater than brain DHA accretion and uptake rates in adult rats.

    Science.gov (United States)

    Domenichiello, Anthony F; Chen, Chuck T; Trepanier, Marc-Olivier; Stavro, P Mark; Bazinet, Richard P

    2014-01-01

    Docosahexaenoic acid (DHA) is important for brain function, however, the exact amount required for the brain is not agreed upon. While it is believed that the synthesis rate of DHA from α-linolenic acid (ALA) is low, how this synthesis rate compares with the amount of DHA required to maintain brain DHA levels is unknown. The objective of this work was to assess whether DHA synthesis from ALA is sufficient for the brain. To test this, rats consumed a diet low in n-3 PUFAs, or a diet containing ALA or DHA for 15 weeks. Over the 15 weeks, whole body and brain DHA accretion was measured, while at the end of the study, whole body DHA synthesis rates, brain gene expression, and DHA uptake rates were measured. Despite large differences in body DHA accretion, there was no difference in brain DHA accretion between rats fed ALA and DHA. In rats fed ALA, DHA synthesis and accretion was 100-fold higher than brain DHA accretion of rats fed DHA. Also, ALA-fed rats synthesized approximately 3-fold more DHA than the DHA uptake rate into the brain. This work indicates that DHA synthesis from ALA may be sufficient to supply the brain.

  4. Whole body synthesis rates of DHA from α-linolenic acid are greater than brain DHA accretion and uptake rates in adult rats[S

    Science.gov (United States)

    Domenichiello, Anthony F.; Chen, Chuck T.; Trepanier, Marc-Olivier; Stavro, P. Mark; Bazinet, Richard P.

    2014-01-01

    Docosahexaenoic acid (DHA) is important for brain function, however, the exact amount required for the brain is not agreed upon. While it is believed that the synthesis rate of DHA from α-linolenic acid (ALA) is low, how this synthesis rate compares with the amount of DHA required to maintain brain DHA levels is unknown. The objective of this work was to assess whether DHA synthesis from ALA is sufficient for the brain. To test this, rats consumed a diet low in n-3 PUFAs, or a diet containing ALA or DHA for 15 weeks. Over the 15 weeks, whole body and brain DHA accretion was measured, while at the end of the study, whole body DHA synthesis rates, brain gene expression, and DHA uptake rates were measured. Despite large differences in body DHA accretion, there was no difference in brain DHA accretion between rats fed ALA and DHA. In rats fed ALA, DHA synthesis and accretion was 100-fold higher than brain DHA accretion of rats fed DHA. Also, ALA-fed rats synthesized approximately 3-fold more DHA than the DHA uptake rate into the brain. This work indicates that DHA synthesis from ALA may be sufficient to supply the brain. PMID:24212299

  5. Myocardial Oxidative Metabolism and Protein Synthesis during Mechanical Circulatory Support by Extracorporeal Membrane Oxygenation

    Energy Technology Data Exchange (ETDEWEB)

    Priddy, MD, Colleen M.; Kajimoto, Masaki; Ledee, Dolena; Bouchard, Bertrand; Isern, Nancy G.; Olson, Aaron; Des Rosiers, Christine; Portman, Michael A.

    2013-02-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support essential for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative. We focused on the amino acid leucine, and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart (i) the fractional contribution of leucine (FcLeucine) and pyruvate (FCpyruvate) to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and (ii) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 hours of normal circulation or ECMO) and intracoronary infusion [13C6,15N]-L-leucine (3.7 mM) alone or with [2-13C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (~ 40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. Conclusion: The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining (i) metabolic flexibility indicated by ability to respond to pyruvate, and (ii) a normal or increased capacity for global protein synthesis, suggesting an improved protein balance.

  6. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    International Nuclear Information System (INIS)

    Tzeng, W.-P.; Frey, Teryl K.

    2005-01-01

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  8. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  9. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  10. Gamma-ray-induced changes in the synthesis of tomato pericarp protein

    International Nuclear Information System (INIS)

    Ferullo, J.M.; Nespoulous, L.; Triantaphylides, C.

    1994-01-01

    The application of massive doses of gamma rays (1–8 kGy) to mature green cherry-tomato fruits led to a transient fall in pericarp tissue protein metabolism within 6h. A separate 3 kGy treatment resulted in the appearance of certain transcripts and proteins, and a reduction in the abundance of others. At the same dose, protein synthesis regained the control level within 24 h, and in addition a new set of proteins was induced. Gamma-induced proteins (referred to as GIPs) were divided into three groups, depending on the time-course of their induction. Group 1 GIPs were synthesized only during the first few hours following treatment, whereas group 2 GIPs were synthesized for at least 48 h. Group 3 GIPs were progressively induced when the control level of synthesis was restored. These results demonstrated that, despite its deleterious effects on DNA and cell structures, irradiation induced an active response in plant tissue. Comparative experiments suggest that the majority of group 1 GIPs might belong to the heat shock protein family. GIPs might play a role in the protection and repair of cellular structures, or may be implicated in physiological disorders triggered by irradiation. (author)

  11. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  12. Comparison of the Rate and Extent of Deoxyribonucleic Acid Repair and Semi-Conservative Synthesis in Bacteria Exposed to Ultra-Violet Light

    Energy Technology Data Exchange (ETDEWEB)

    Billen, D. [Radiation Biology Laboratory and Departments of Microbiology and Radiology, College of Medicine, University of Florida, Gainesville, FL (United States)

    1968-08-15

    Many bacterial strains possess the ability to repair genetic damage resulting from ultra-violet light (u.v. ) exposure. Of major importance is the occurrence of a 'repair' type of deoxyribonucleic acid (DNA) replication during 'dark repair', which presumably results in the replacement of the damaged portion of the genome. With deuterium, {sup 15}N and {sup 13}C as a density label, and buoyant density centrifugation in CsCl as a means of separating pre and post-irradiation synthesized DNA strands, the rate and extent of DNA repair synthesis in exponential - phase Escherichia coli strain B/r were determined. After u.v. exposure, {sup 3}H-thymine incorporation into the 'heavy' parental DNA strands was used to measure repair synthesis, while {sup 3}H-thymine incorporation into 'light' and newly synthesized DNA strands measured semi-conservative replication. The rate of bases incorporated by repair synthesis in the initial 15 minures of post-irradiation incubation at 37 Degree-Sign C appears to be saturated at a dose of approximately 100 ergs/mm{sup 2}. At higher doses (up to 600 ergs/mm{sup 2}) the increase observed was not proportional to dose. During this initial 15 minutes, less than 1% of the chromosomal DNA was replaced. The amount of DNA synthesized by semi-conservative replication during the initial 15 minutes was reduced with increasing u.v. dose. After exposure to 600 ergs/mm{sup 2}, repair and semiconservative DNA synthesis were nearly equivalent in the irradiated cells after 15 minutes of incubation. Repair synthesis was observed to be terminated by 45 minutes in bacteria exposed to 160 or 500 ergs/mm{sup 2} (64% and 10% survivors, respectively). The amount of genome replaced by repair synthesis at several doses was determined. Starvation for a required amino acid (resulting in an inhibition of protein and ribonucleic acid synthesis) did not prevent the repair synthesis nor grossly alter its extent. The restoration of the semi-conservative mo d e of DNA

  13. Influence of heat and shear induced protein aggregation on the in vitro digestion rate of whey proteins.

    Science.gov (United States)

    Singh, Tanoj K; Øiseth, Sofia K; Lundin, Leif; Day, Li

    2014-11-01

    Protein intake is essential for growth and repair of body cells, the normal functioning of muscles, and health related immune functions. Most food proteins are consumed after undergoing various degrees of processing. Changes in protein structure and assembly as a result of processing impact the digestibility of proteins. Research in understanding to what extent the protein structure impacts the rate of proteolysis under human physiological conditions has gained considerable interest. In this work, four whey protein gels were prepared using heat processing at two different pH values, 6.8 and 4.6, with and without applied shear. The gels showed different protein network microstructures due to heat induced unfolding (at pH 6.8) or lack of unfolding, thus resulting in fine stranded protein networks. When shear was applied during heating, particulate protein networks were formed. The differences in the gel microstructures resulted in considerable differences in their rheological properties. An in vitro gastric and intestinal model was used to investigate the resulting effects of these different gel structures on whey protein digestion. In addition, the rate of digestion was monitored by taking samples at various time points throughout the in vitro digestion process. The peptides in the digesta were profiled using SDS-polyacrylamide gel electrophoresis, reversed-phase-HPLC and LC-MS. Under simulated gastric conditions, whey proteins in structured gels were hydrolysed faster than native proteins in solution. The rate of peptides released during in vitro digestion differed depending on the structure of the gels and extent of protein aggregation. The outcomes of this work highlighted that changes in the network structure of the protein can influence the rate and pattern of its proteolysis under gastrointestinal conditions. Such knowledge could assist the food industry in designing novel food formulations to control the digestion kinetics and the release of biologically

  14. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    International Nuclear Information System (INIS)

    Klinefelter, G.R.; Hamilton, D.W.

    1985-01-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-[35S]methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-[35S]methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB[3H]4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  16. Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line

    International Nuclear Information System (INIS)

    O'Reilly, M.A.; Gazdar, A.F.; Clark, J.C.; Pilot-Matias, T.J.; Wert, S.E.; Hull, W.M.; Whitsett, J.A.

    1989-01-01

    Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of [35S]methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression

  17. Synthesis of nanoparticles with frog foam nest proteins

    International Nuclear Information System (INIS)

    Choi, Hyo-Jick; Ebersbacher, Charles F.; Myung, Nosang V.; Montemagno, Carlo D.

    2012-01-01

    Microemulsions provide an efficient means of synthesizing monodispersed nanoparticles. Recent studies have demonstrated potential problems of surfactant due to the interaction with nanoparticles/precursors. To solve the problems, various types of chemical surfactants have been tested, but natural biosurfactants have not received a great deal of attention in engineering application. Here, we report the formation of microemulsions using frog foam nest protein, ranaspumin-2 (RSN-2), based on the hypothesis that RSN-2 assembles at the water–oil interface as a result of conformational change into an extended form. Fluorescence spectroscopic studies showed that RSN-2 undergoes a reversible transition between extended and globular conformation in foams/microemulsions and aqueous solution, respectively. Microemulsions were formulated with RSN-2 to synthesize 8–10 nm superparamagnetic iron oxide nanoparticles by mixing precursor-containing microemulsions with base-containing microemulsions. RSN-2 proteins were recovered from microemulsions and found to be recycled to make foams and microemulsions. Fluorescence spectroscopic analyses showed that RSN-2 maintained its mechanical agitation-induced amphiphilicity throughout multiple foaming/defoaming processes. These results suggest that conformational flexibility and structural stability of RSN-2 in aggressive environments enable the recycled use of RSN-2, elucidating the cost-effective advantage.

  18. Synthesis of nanoparticles with frog foam nest proteins

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hyo-Jick, E-mail: choihc@ucmail.uc.edu; Ebersbacher, Charles F. [University of Cincinnati, School of Energy, Environmental, Biological and Medical Engineering (United States); Myung, Nosang V. [University of California, Riverside, Department of Chemical and Environmental Engineering (United States); Montemagno, Carlo D., E-mail: montemcd@ucmail.uc.edu [University of Cincinnati, School of Energy, Environmental, Biological and Medical Engineering (United States)

    2012-09-15

    Microemulsions provide an efficient means of synthesizing monodispersed nanoparticles. Recent studies have demonstrated potential problems of surfactant due to the interaction with nanoparticles/precursors. To solve the problems, various types of chemical surfactants have been tested, but natural biosurfactants have not received a great deal of attention in engineering application. Here, we report the formation of microemulsions using frog foam nest protein, ranaspumin-2 (RSN-2), based on the hypothesis that RSN-2 assembles at the water-oil interface as a result of conformational change into an extended form. Fluorescence spectroscopic studies showed that RSN-2 undergoes a reversible transition between extended and globular conformation in foams/microemulsions and aqueous solution, respectively. Microemulsions were formulated with RSN-2 to synthesize 8-10 nm superparamagnetic iron oxide nanoparticles by mixing precursor-containing microemulsions with base-containing microemulsions. RSN-2 proteins were recovered from microemulsions and found to be recycled to make foams and microemulsions. Fluorescence spectroscopic analyses showed that RSN-2 maintained its mechanical agitation-induced amphiphilicity throughout multiple foaming/defoaming processes. These results suggest that conformational flexibility and structural stability of RSN-2 in aggressive environments enable the recycled use of RSN-2, elucidating the cost-effective advantage.

  19. Synthesis of nanoparticles with frog foam nest proteins

    Science.gov (United States)

    Choi, Hyo-Jick; Ebersbacher, Charles F.; Myung, Nosang V.; Montemagno, Carlo D.

    2012-09-01

    Microemulsions provide an efficient means of synthesizing monodispersed nanoparticles. Recent studies have demonstrated potential problems of surfactant due to the interaction with nanoparticles/precursors. To solve the problems, various types of chemical surfactants have been tested, but natural biosurfactants have not received a great deal of attention in engineering application. Here, we report the formation of microemulsions using frog foam nest protein, ranaspumin-2 (RSN-2), based on the hypothesis that RSN-2 assembles at the water-oil interface as a result of conformational change into an extended form. Fluorescence spectroscopic studies showed that RSN-2 undergoes a reversible transition between extended and globular conformation in foams/microemulsions and aqueous solution, respectively. Microemulsions were formulated with RSN-2 to synthesize 8-10 nm superparamagnetic iron oxide nanoparticles by mixing precursor-containing microemulsions with base-containing microemulsions. RSN-2 proteins were recovered from microemulsions and found to be recycled to make foams and microemulsions. Fluorescence spectroscopic analyses showed that RSN-2 maintained its mechanical agitation-induced amphiphilicity throughout multiple foaming/defoaming processes. These results suggest that conformational flexibility and structural stability of RSN-2 in aggressive environments enable the recycled use of RSN-2, elucidating the cost-effective advantage.

  20. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    Science.gov (United States)

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.