Effects Of Other Amino Acids Research Articles

Neither glutamate nor alanine but arginine sensitizes BY-2 cells to arsenate.

Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

L-Glutamine in sickle cell disease.

L-Glutamine is a conditionally essential amino acid required for synthesis of the pyridines for nucleotides, including nicotinamide adenine dinucleotide (NAD) and glutathione, as well as glutamate, and becomes essential during oxidative stress exposure. The NADH:[NAD⁺ + NADH] (redox) ratio in sickle red blood cells (RBCs) is lower than in normal RBCs, consistent with oxidative stress, therefore glutamine availability is important in sickle cell disease (SCD). RBC glutamine levels vary between SCD studies but the ratio glutamine:glutamate was inversely related to tricuspid regurgitant jet velocity in one. Oral L-glutamine was associated with an increase in NADH and reduction in RBC endothelium adhesion in small studies of SCD patients. In a sickle mouse model, glutamine levels were directly related to cerebral blood flow. Phase II and III randomized, double-blind, controlled trials of L-glutamine 0.6 g/kg/day compared with placebo in children and adults with SCD and = 2 episodes of pain in the previous year provide evidence that L-glutamine is safe and associated with a reduction in painful episodes and in hospitalizations. However, L-glutamine was only tolerated in two-thirds of patients, anemia and hemolysis did not improve and there are few data on mortality and organ complications. Future studies should investigate the effect of other amino acids and total protein intake.

STRUCTURAL CHARACTERISTICS AND ANTIHYPERTENSIVE EFFECTS OF ANGIOTENSIN-I-CONVERTING ENZYME INHIBITORY PEPTIDES IN THE RENIN-ANGIOTENSIN AND KALLIKREIN KININ SYSTEMS.

Background:The commercially available synthetic angiotensin-I-converting enzyme (ACE) inhibitors are known to exert negative side effects which have driven many research groups globally to discover the novel ACE inhibitors.Method:Literature search was performed within the PubMed, ScienceDirect.com and Google Scholar.Results:The presence of proline at the C-terminal tripeptide of ACE inhibitor can competitively inhibit the ACE activity. The effects of other amino acids are less studied leading to difficulties in predicting potent peptide sequences. The broad specificity of the enzyme may be due to the dual active sites observed on the somatic ACE. The inhibitors may not necessarily competitively inhibit the enzyme which explains why some reported inhibitors do not have the common ACE inhibitor characteristics. Finally, the in vivo assay has to be carried out before the peptides as the antihypertensive agents can be claimed. The peptides must be absorbed into circulation without being degraded, which will affect their bioavailability and potency. Thus, peptides with strong in vitro IC50 values do not necessarily have the same effect in vivo and vice versa.Conclusion:The relationship between peptide amino acid sequence and inhibitory activity, in vivo studies of the active peptides and bioavailability must be studied before the peptides as antihypertensive agents can be claimed.

A novel potentiometric biosensor for determination of L-lysine in commercial pharmaceutical L-lysine tablet and capsule

The construction of an L-lysine biosensor on ammonium-selective poly(vinylchloride) (PVC) membrane electrode is described in this study. The construction procedure occurs in two stages: (I) the preparation of ammonium-selective poly(vinylchloride) (PVC) membrane electrode and (II) the chemical immobilization of lysine oxidase on this ammonium-selective electrode by using glutaraldehyde. The ammonium ions produced after enzymatic reaction were determined potentiometrically. The sensitivity of the lysine biosensor against ammonium ions and lysine were studied. The response time, linear working range, reproducibility and life time of the biosensor were also determined. The interfering effect of other amino acids on the biosensor performance was also studied and potentiometric selectivity coefficients were calculated. Although the biosensor responded mainly against tyrosine, a lot of amino acids and ascorbic acid that can be present in some real samples did not show any important interference. Additionally, lysine assay in commercial pharmaceutical lysine tablets and capsules was also successfully carried out. The results were in good agreement with previously reported values.

Quantitative Determination of Glycine in Aqueous Solution Using Glutamate Dehydrogenase-Immobilized Glyoxal Agarose Beads

In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD(+) in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1-10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).

Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells

Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24h and then treated with MTX (100ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P<0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P<0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P<0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated invivo.

Anti‐obesity effects of amino acid in high‐fat diet induced obese mice

Anti‐obesity effects of dietary supplemented amino acid have been reported. Dietary supplementation of glutamine, alanine, or leucine decreases high‐fat diet induced obesity in C57BL/6J mice. It was reported that histidine supplementation suppressed food intake and fat accumulation in Wistar rats. And it was shown that dietary arginine supplementation reduced fat mass in Zucker diabetic fatty rats. However there is no information about the anti‐obesity effects of other amino acids than above mentioned. Therefore, we conducted this series of experiments to compare the anti‐obesity effects of 20 kinds of proteinogenic amino acids. Male C57BL/6J mice were fed a high‐fat diet supplemented with either one of 20 proteinogenic amino acids (1‐6 %) for 4 to 8 weeks. Body weight, food intake, epididymal fat weight, and plasma triglyceride concentration were measured. Results showed that supplementation with any one of cystine (3%), histidine (5%), leucine (3%), lysine HCl (3%), threonine (3%), and tryptophan (3%) suppressed body weight gain and visceral fat deposition. Reduction of food intake was observed in cystine and histidine group but not in leucine, lysine, threonine, and tryptophan group. Plasma triglyceride concentration reduced only in lysine group. In conclusion, dietary supplementation of any one of leucine, lysine, threonine, and tryptophan prevents high‐fat diet induced obesity without reduction in food intake, which suggests increased energy expenditure. Among these amino acids, lysine is the most potent amino acid to reduce adiposity, since it decreases diet induced obesity as well as hyperlipidemia.

Effect of 40% restriction of dietary amino acids (except methionine) on mitochondrial oxidative stress and biogenesis, AIF and SIRT1 in rat liver

Previous studies have shown that the decrease in mitochondrial reactive oxygen species (mitROS) generation and oxidative damage to mitochondrial DNA (mtDNA) that occurs during life extending dietary restriction also occurs during protein or methionine restriction, whereas it does not take place during carbohydrate or lipid restriction. In order to study the possible effects of other amino acids, in this investigation all the dietary amino acids, except methionine, were restricted by 40% in male Wistar rats (RESTAAS group). After 6-7 weeks, experimental parameters were measured in the liver. Amino acid restriction did not change the levels of the methionine metabolites S-adenosylmethionine and S-adenosylhomocysteine, mitochondrial oxygen consumption and ROS generation, oxidative damage to mtDNA, amounts of the respiratory complexes I-IV, and the mitochondrial biogenesis factors PGC-1alpha and NRF-2. On the other hand, adenylate energy charge, mitochondrial protein oxidation, lipooxidation and glycooxidation, the degree of mitochondrial fatty acid unsaturation, and the amount of the apoptosis inducing factor (AIF) were decreased in the RESTAAS group. Amino acid restriction also increased SIRT1 protein. These results, together with previous ones, strongly suggest that the decrease in mitROS generation and oxidative damage to mtDNA that occurs during dietary restriction is due to restriction of a single aminoacid: methionine. They also show for the first time that restriction of dietary amino acids different from methionine decreases mitochondrial protein oxidative modification and AIF, and increases SIRT1, in rat liver.

Short Communication: Amino Acids Antagonistic to the Amino Acids Inhibitory for Growth Rate of Mixed Ruminal Bacteria

Antagonism of some amino acids (AA) to the inhibitory effects of other AA (Ile, Phe, and Thr) on the growth rate of mixed ruminal bacteria was investigated. In vitro growth rate of the mixed ruminal bacteria was inhibited when the 3 inhibitory AA (1mM each) were each added to individual control treatments in which an ammonium salt was included as a sole N source. The inhibitory effect caused by Ile was relieved by addition of Leu or Val (equimolar to Ile), and no significant inhibition was shown when both Leu and Val were added together with Ile. The growth inhibition caused by Phe was also alleviated by supplementing with Trp, and was completely negated by adding Tyr. The inhibitory effect of Thr, on the other hand, was not affected by addition of Lys or Met (which are synthesized using a common pathway with Thr), but was mitigated by supplementation with Glu, Ser, Val, Ala, or Gln. Among the antagonistic AA, Leu, Val, Trp, Tyr, and Glu were indispensable for the maximum growth rate of the ruminal bacteria under the experimental condition of supplementation of amino-N, the removal of which from a mixture of 20 protein AA caused the growth rate to decline. Removals of Ile along with Leu or Val or both, of Phe along with Trp or Tyr, and of Thr along with Glu recovered the promotion of bacterial growth rate. It was concluded that inhibitions of the bacterial growth rate caused by Ile, Phe, or Thr could be antagonized by some other AA (Leu, Val, Tyr, Trp, or Glu), and the role of these latter AA as relievers of the inhibitory effects could explain why they are indispensable for maximum growth rate of ruminal bacteria.

Leader Peptide-mediated Transcriptional Attenuation of Lysine Biosynthetic Gene Cluster in Thermus thermophilus

The molecular mechanism for regulation of the genes involved in the biosynthesis of amino acids is poorly identified in Thermus thermophilus. In this study, we analyzed the transcriptional control of the major lysine biosynthetic gene cluster in T. thermophilus. S1 nuclease mapping revealed that the transcription, which is repressed by lysine, starts at 111 bp, upstream of the translational start codon, ATG, for the homocitrate synthase (hcs) gene. The 5'-leader region of 111 bp carries a sequence that can encode a short peptide of 14 amino acids with tandem-arranged lysine residues in its sequence. The nucleotide sequence of the region suggests that the transcript can form complicated secondary structures. Deletion of most of the 5'-leader region or mutation of the tandem lysine codons suppressed the transcriptional repression by lysine. Mutation of the tandem codons from lysine to glutamine resulted in glutamine-dependent repression of the gene connected downstream, indicating that the leader peptide mediated the transcriptional attenuation of the gene expression. This is the first report demonstrating the transcriptional regulation of amino acid biosynthesis in T. thermophilus.

Effects of glycine and proline on the calcium activation properties of skinned muscle fibre segments from crayfish and rat.

The effects of the polar amino acid glycine (20 mmol l(-1)) and the non-polar amino acid proline (20 mmol l(-1)) on Ca(2+)-activated contraction have been examined in four types of striated muscle fibres. Single fibres dissected from the claw muscle of a crustacean (long- and short-sarcomere) and the hindlimb muscles of the rat (slow-twitch from soleus and fast-twitch from extensor digitorum longus) were activated in matched solutions that either contained the amino acid ('test') or not ('control'). The steady-state force produced in these solutions was used to determine the relation between force production and pCa (-log10[Ca2+]). The results show that in the concentrations used, glycine and proline had only small effects on the maximum Ca(2+)-activated force, pCa corresponding to 10, 50 and 90% maximum force (pCa10, pCa50, pCa90, respectively) or on the slope of the force-pCa curves in the four different fibre types. The relative lack of effects of glycine and proline on contractile activation would confer a distinct physiological advantage to force production of muscle of Cherax, where the concentrations of glycine and proline vary considerably. Finally, the results show that glycine and proline may be useful to balance control solutions when the effects of other amino acids or zwitterions on contractile activation are examined.

Construction of a l-lysine biosensor by immobilizing lysine oxidase on a gold-poly( o-phenylenediamine) electrode

The construction of a l-lysine biosensor on a Si–gold strip electrode (SGSE) is described in this study. The construction comprises (a) the formation of poly( o-phenylenediamine, o-PD) membrane on the electrode surface via electropolymerization and (b) the immobilization of lysine oxidase on the gold/poly( o-PD) electrode with glutaraldehyde. The behavior of the gold/poly( o-PD) electrode against H 2O 2 and lysine, as well as the repeatability of the electropolymerization and the time stability of the polymer were studied. The study showed that the electropolymerization procedure is repeatable, and that the polymer is quite stable for at least 40 days. The biosensor showed a linear calibration curve in the range 0.01–1×10 −5 M (0.1–10 μM) lysine. The interfering effect of other amino acids on the biosensor performance was also studied and amperometric selectivity coefficients were calculated. The biosensor responded mainly against tyrosine and cysteine, while the response to phenylalanine, arginine, histidine and ornithine was very low. By changing the electropolymerization conditions, the effect of interferents was further reduced.

Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley.

To investigate the regulation of HvNRT2, genes that encode high-affinity NO(3)(-) transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mM NO(3)(-) in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH(4)(+), and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity (13)NO(3)(-) influx and HvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO(3)(-) influx by 50%, indicating that NO(3)(-) itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH(4)(+) may down-regulate HvNRT2 gene expression and high-affinity NO(3)(-) influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO(3)(-) influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2 expression, although it does not preclude a contributory effect of other amino acids.

Mutual amino acid catalysis in salt-induced peptide formation supports this mechanism's role in prebiotic peptide evolution.

The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 degrees C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive organisms.

Endogenous mutagens derived from amino acids

l-Cysteine, glutathione and the therapeutically used l-cysteine precursor, N- acetyl- l-cysteine , induced strong mutagenic effects in Salmonella typhimurium (reversion of the his − strains TA97, TA92 and TA104), when tested in the presence of subcellular kidney preparations. The tyrosine metabolites, levodopa (an ortho-hydroquinone) and homogentisic acid (a para-hydroquinone) reverted various his − strains as well. This mutagenicity did not require the presence of mammalian enzymes, and was relatively weak. The induction of gene mutations was also studied in mammalian cells (V79 Chinese hamster cells), using acquisition of resistance toward 6-thioguanine as the marker. l-Cysteine and N- acetyl- l-cysteine were found to be inactive, levodopa was weakly mutagenic, and homogentisic acid was strongly mutagenic (enhancing the mutation frequency 135-fold above background at an exposure concentration of 50 μM). This finding is striking as the urinary concentration of homogentisic acid is about 1000 times higher in patients with a genetic defect in homogentisic acid 1,2-dioxygenase (alkaptonuria). Genotoxic and carcinogenic effects of other amino acids and metabolites, reported in the literature, are discussed as well.

Spectrophotometric determination of thiols using N,N-dimethyl-1,4-phenylenediammonium dichloride and chloramine-T

A simple, rapid, and sensitive Spectrophotometric method for the determination of thiols, based on the formation of colored species with N,N-dimethyl-1,4-phenylenediammonium dichloride and chloramine-T is described. Some important features such as the effects of other amino acids and the best conditions for forming colored complexes are studied. The method is applicable to the determination of cysteine, cystine, and glutathione in pure samples and protein hydrolysates.

Regulation of protein turnover by glucose, insulin, and amino acids in adipose tissue.

Protein synthesis and degradation were measured simultaneously in epididymal fat pads of rats by use of the incorporation of [14C]phenylalanine into protein and the sum of net protein breakdown and protein synthesis, respectively. Neither glucose nor insulin altered protein synthesis, but together they promoted this process; pyruvate could be substituted for glucose. Separately, glucose or insulin diminished proteolysis, and these effects were additive. In the presence of glucose and insulin, leucine, alanine, glutamine, glutamate, and aspartate lowered protein degradation to varying degrees but did not alter protein synthesis. Glutamate, but not leucine or alanine, was inhibitory without glucose and insulin present. When aminooxyacetic acid was provided to decrease the rate of transamination of amino acids, the inhibitory effects of leucine, alanine, and aspartate, but not of glutamate, appeared to be diminished. alpha-Ketoglutarate, but neither alpha-ketoisocaproate nor pyruvate, could diminish proteolysis. Inhibition of proteolysis was associated with a higher tissue content of glutamate and a greater production of glutamate and glutamine. These results suggest that glutamate itself may inhibit proteolysis in adipose tissue and mediate, at least in part, the effects of other amino acids.

Spectrophotometric determination of amino acids using metol and sodium hypochlorite or chloramine-T

Simple and sensitive spectrophotometric methods for the determination of proline, hydroxyproline, aspartic acid, arginine and histidine, based on the formation of a coloured species with metol and sodium hypochlorite or chloramine-T are described. The effect of other amino acids and the best conditions for forming coloured complexes have been studied. The methods are applicable to the determination of the aforementioned amino acids in pure samples and in protein hydrolysates.

Gluconeogenesis from alanine and serine by trout hepatocytes: Effects of other amino acids

Gluconeogenesis from alanine and serine by trout hepatocytes: Effects of other amino acids

Characteristics of synaptosomal tyrosine uptake in various brain regions: Effect of other amino acids

Tyrosine uptake by rat synaptosomes was maximal after 5–10 min of incubation and at 30°C; uptake was inhibited by dinitrophenol (10 −4 M) or ATP (10 −3 M) and increased by reducing sodium concentrations or increasing calcium or potassium. The best model for uptake is a two-carrier system, in which one carrier shows high-affinity uptake and the other may be diffusional. Both uptake mechanisms are more concentrated in catecholamine-rich brain areas, and are inhibited in vitro by other large, neutral amino acids. At physiologic amino acid brain concentrations, each system probably carries about half of the tyrosine into the nerve terminal.