Effects Of Amino Acid Supply Research Articles

Making Molecular Tools to Determine the Effect of Amino Acid Supply on Protein Synthesis

Making Molecular Tools to Determine the Effect of Amino Acid Supply on Protein Synthesis

Stabilization of single-chain Fv antibody production using recombinant Escherichia coli by DO-stat fed-batch culture employing yeast extract-enriched feeding medium

The stabilized production of anti-CRP single-chain Fv (scFv) antibody using recombinant E. coli by prolonged dissolved oxygen (DO)-stat fed-batch culture was demonstrated, focusing on the supply of nitrogen sources during fed-batch operation. The serious degradation of anti-CRP scFv by intracellular proteases was observed when the fermentation time was prolonged from 50 h to 100 h, which was presumably induced by the insufficient supply of free amino acids. To evaluate the effects of amino acid supply on the proteolysis of anti-CRP scFv, fed-batch experiments using yeast extract (YE)-enriched feeding medium, which had various blend ratios of glucose and YE (G/YE), were performed. By increasing the YE content in the feeding medium, the expression of intracellular proteases was significantly suppressed; consequently, the proteolysis of the scFv production was successfully prevented in 100 h fed-batch culture. The desired G/YE ratio to avoid proteolysis was 1.0 g-glucose/g-YE. The stabilization of scFv production accordingly resulted in the enhanced scFv production from 3.0 g/L in 50 h fed-batch culture to 4.0–5.2 g/L in 100 h fed-batch culture with more than 97% solubility which corresponded to 94.9% of increase in total scFv production by one fed-batch operation.

Amino Acid Availability Regulates the Effect of Hyperinsulinemia on Skin Protein Metabolism in Pigs

The effects of amino acid supply and insulin infusion on skin protein kinetics (fractional synthesis rate (FSR), fractional breakdown rate (FBR), and net balance (NB)) in pigs were investigated. Four-month-old pigs were divided into four groups as follows: control, insulin (INS), amino acid (AA), and INS + AA groups based on the nutritional and hormonal conditions. l-[ring-(13)C6]Phenylalanine was infused. FBR was estimated from the enrichment ratio of arterial phenylalanine to intracellular free phenylalanine. Plasma INS was increased (p < 0.05) in the INS and INS + AA groups. Plasma glucose was maintained by infusion of glucose in the groups receiving INS. The interventions did not change the NB of skin protein. However, the interventions affected the FSR and FBR differently. An infusion of INS significantly increased both FSR and FBR, although AA infusion did not. When an AA infusion was added to the infusion of insulin (INS + AA group), FSR and FBR were both lower when compared with the INS group. Our data demonstrate that in anesthetized pigs INS infusion did not exert an anabolic effect, but rather it increased AA cycling into and out of skin protein. Because co-infusion of AAs with INS ameliorated this effect, it is likely that the increased AA cycling during INS infusion was related to AA supply. Although protein kinetics were affected by both INS and AAs, none of the interventions affected the skin protein deposition. Thus, skin protein content is closely regulated under normal circumstances and is not subject to transient changes in AAs or hormonal concentrations.

Effect of amino acid supply on the transcription of flavour-related genes and aroma compound production during lager yeast fermentation

The objective of this study was to investigate the effect of adding selected amino acids (proline, leucine, cysteine, valine, glutamine and isoleucine) to synthetic wort on the generation of aroma compounds with respect to the transcription levels of specific genes involved in their biosynthetic pathway under brewery fermentation. The results showed that the changes in the selected amino acid levels had no eminent impact on the general course of fermentation. Addition of leucine increased the production of isoamyl alcohol and isoamyl acetate and 2-methylbutyl acetate. Adding valine and isoleucine increased the production of isobutanol and 2-methyl butanol, respectively. Overall, total higher alcohol production increased by amino acid supplementation; this effect could be associated with the upregulation of pyruvate decarboxylases and phenylpyruvate decarboxylase. Amino acid supplementation resulted in a reduction in the final total concentration of esters, especially ethyl acetate. This reduction might have been caused by the downregulation of the genes participating in ester biosynthesis. Our results demonstrate that during lager yeast fermentation, the production of aroma-active compounds can be significantly affected by changing the supply of even one amino acid.

Plasma amino acid imbalance: dangerous in chronic diseases?

In various diseases amino acid imbalances occur which have been described especially in the 1980s of the last century. It was noted that some of these imbalances may exert specific negative physiological effects. However, mainly because of economic reasons, no real attempts have been made to develop special amino acid solutions for disease processed associated with amino acid imbalances to restore normal amino acid concentrations. A recent study performed in the fruit-fly Drosophila indicated that modifying the amino acid supply may influence both lifespan and fecundity. It was shown that adding amino acids but not carbohydrates or fat to a restricted diet decreases lifespan. In contrast, administration of certain amino acids especially of methionine increased fecundity without decreasing lifespan. It is known that dietary restriction can decrease fecundity at the cost of a prolonged lifespan. Recent investigations revealed that amino acids are powerful molecules in mediating cell signalling. Therefore, it can be hypothesized that the severe amino acid imbalances as observed in uraemia or liver failure may exert a relevant impact on various physiologic processes and on organ function. The recent results described in Drosophila should stimulate a new research area on the effect of amino acid supply in various disease processes.

Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets being associated with reduced circulatory IGF-I levels. This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep. Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay. In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0xor 5.0xamino acid media (P<0.01). Although GH appeared to increase IGF-I release in this period for cells grown in 0.2xamino acid media, this effect was not statistically significant. In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P<0.05 and P<0.001 respectively). Factorial analysis of variance demonstrated a strong positive interaction (P<0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0xamino acid media (P<0.01) but had no effect on production by cells grown in 1.0xor 0.2xamino acid media. Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level. Incorporation of (35)S-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0xcompared with 5.0x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2xamino acid media (P<0.01). The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms. Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBPbeta) isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) were determined by Western blotting. When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P<0.05). These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres. The association between these effects and altered levels of C/EBPbeta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.

The effects of amino acid supply on total and individual cellular protein synthesis in ovine hepatocytes

Protein nutrition, through altered amino-acid supply, regulates the quality and quantity of animal products, and deficiencies in supply of / response to amino-acids may be a limiting factor in ruminant efficiency. The effects of amino-acid supply may be indirect, via modulation of anabolic hormonal signalling. In particular, amino-acid limitation reduces insulin-like growth factor-1 (IGF-1) - and increases IGF-1 binding protein 1 (IGFBP-1) - production by the liver, and hence modulates the sensitivity of tissues to pituitary GH as an anabolic signal (Gluckman et al, 1987). The mechanisms through which amino-acids exert these selective effects on liver protein production are not understood.The present study sought to determine whether the effects of amino-acids on total cellular protein synthesis are sufficient to account for observed reductions in IGF-1 production in cultured ovine hepatocytes. In addition the use of two dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualise changes in translation of individual proteins caused by amino-acids in cultured ovine hepatocytes is described.

The effects of amino acid supply on the sensitivity of IGF-1 secretion to growth hormone stimulation in ovine hepatocytes

Many of the anabolic effects of growth hormone (GH), including promotion of lean tissue growth, are mediated by the actions of insulin-like growth factor-1 (IGF-1) (Gluckman et al, 1987). It is known that the response of hepatic IGF-1 synthesis to GH may be modulated by alterations in levels of feeding or changes in protein supply (Pell et al, 1993). The aim of the current study was to develop an in vitro model to characterise interactions between amino acid supply and GH on hepatic IGF-1 release in ruminants.Ovine hepatocyes were prepared by a modification of a published method (Luo et al, 1992) from the median lobe of livers removed immediately after slaughter from sheep killed at a local abattoir. Free amino acid concentrations in test media were 5x, 1x and 0.2x physiological concentrations based upon in vivo ovine portal venous blood data (Lobley et al, 1995).

Organization and expression of hair follicle genes

Several families of proteins are expressed in the growth of hair and an estimated 50–100 proteins constitute the final hair fiber. The cumbersome nomenclature for naming these different proteins has led to a proposal to modify that which is currently used for epidermal keratins. Investigations of the organization of hair genes indicate that the members of each family are clustered in the genome and their expression could be under some general control. Interestingly, the protein called trichohyalin, markedly distinct from the hair proteins, is produced in the inner root sheath cells and the gene for it has been found to be located at the same human chromosome locus as the genes for profilaggrin, involucrin, and loricrin.A mainstream objective is to identify controls responsible for the production in the hair cortex of keratin intermediate filaments (IFs) and two large groups of keratin-associated proteins (KAPs) rich in the amino acids cysteine or glycine/ tyrosine. A specific family of cysteine-rich proteins is expressed in the hair cuticle. Comparisons of promoter regions of IF genes and KAP genes, including a recently characterized gene for a glycine/tyrosine-rich protein, have revealed putative hair-specific motifs in addition to known elements that regulate gene expression. In the sheep, the patterns of expression in hair differentiation are particularly interesting insofar as there are distinct segments of para-and orthocortical type cells that have significantly different pathways of expression. The testing of candidate hair-specific regulatory sequences by mouse transgenesis has produced several interesting hair phenotypes. Transgenic sheep over-expressing keratin genes but showing no hair growth change have been obtained and compared with the equivalent transgenic hairloss mice.Studies of the effects of amino acid supply on the rate of hair growth have demonstrated that with cysteine supplementation of sheep a perturbation occurs in which there is a markedly increased level of only one type of mRNA and the ration of para-to orthocortical cells is increased. A molecular explanation of this phenomenon is being sought.

Organization and expression of hair follicle genes.

Several families of proteins are expressed in the growth of hair and an estimated 50-100 proteins constitute the final hair fiber. The cumbersome nomenclature for naming these different proteins has led to a proposal to modify that which is currently used for epidermal keratins. Investigations of the organization of hair genes indicate that the members of each family are clustered in the genome and their expression could be under some general control. Interestingly, the protein called trichohyalin, markedly distinct from the hair proteins, is produced in the inner root sheath cells and the gene for it has been found to be located at the same human chromosome locus as the genes for profilaggrin, involucrin, and loricrin. A mainstream objective is to identify controls responsible for the production in the hair cortex of keratin intermediate filaments (IFs) and two large groups of keratin-associated proteins (KAPs) rich in the amino acids cysteine or glycine/tyrosine. A specific family of cysteine-rich proteins is expressed in the hair cuticle. Comparisons of promoter regions of IF genes and KAP genes, including a recently characterized gene for a glycine/tyrosine-rich protein, have revealed putative hair-specific motifs in addition to known elements that regulate gene expression. In the sheep, the patterns of expression in hair differentiation are particularly interesting insofar as there are distinct segments of para- and orthocortical type cells that have significantly different pathways of expression. The testing of candidate hair-specific regulatory sequences by mouse transgenesis has produced several interesting hair phenotypes. Transgenic sheep over-expressing keratin genes but showing no hair growth change have been obtained and compared with the equivalent transgenic hair-loss mice. Studies of the effects of amino acid supply on the rate of hair growth have demonstrated that with cysteine supplementation of sheep a perturbation occurs in which there is a markedly increased level of only one type of mRNA and the ration of para- to orthocortical cells is increased. A molecular explanation of this phenomenon is being sought.

Effect of growth conditions on the activity of ornithine decarboxylase in cultured hepatoma cells. I. Effect of amino acid supply.

AbstractOrnithine decarboxylase activity in high density, stationary phase rat hepatoma (HTC) cells in suspension culture has an extremely short half‐life of between 5 and 15 minutes, as measured after inhibiting protein synthesis. Following dilution of these cells into fresh medium there is a large increase in ornithine decarboxylase activity, reaching a peak often several hundred times the initial level at about four hours. At least part of this stimulation is due to an increase in the apparent half‐life of the enzyme, to between 30 and 90 minutes. Evidence is presented that the supply of amino acids can control the turnover of ODC under some conditions. For example supplementing high density cells with glutamine, asparagine, serine, glycine and proline, either singly or together, increases ODC activity and decreases its apparent turnover. The stimulation by amino acids is enhanced by serum.

Dietary and hormonal influences on rat liver polysome profiles; fat, glucose and insulin

1. 1. To determine which dietary components cause a restoration of disaggregated liver polysomes from fasted rats, polysome profiles from the livers of rats which had been fasted or fasted and refed diets of glucose or fat were prepared and evaluated. To investigate a possible mechanism through which dietary components act, polysome profiles on alloxan-diabetic and insulin-treated rats were prepared and analyzed. 2. 2. The gradual disaggregation of liver polysomes caused by fasting is virtually complete in 48 h. Ten h after refeeding glucose the polysome profiles of 60-h fasted rats are restored. Refeeding isocaloric amounts of fat to fasted rats is without effect. 3. 3. Production of experimental diabetes with alloxan is accompanied by a gradual disaggregation of the liver polysomes. Intraperitoneal administration of insulin restores the polysomes of an alloxan-diabetic rat within 1 h. 4. 4. Administration of insulin to fasted or fat-refed animals is without effect. 5. 5. The findings reported here appear to be unrelated to the reported effects of amino acid supply on liver polysome profiles. 6. 6. Insulin, in concert with glucose, has a rapid effect on the liver resulting in changes in the polysome profile.

Comparison of the effect of amino acid supply on hepatic polysome profiles in vivo and in vitro

Comparison of the effect of amino acid supply on hepatic polysome profiles in vivo and in vitro