Modulation of human lymphocyte proliferation by amino acids.

The amino acids required for phytohaemagglutinin (PHA) induced lymphocyte proliferation were determined by the 3H-thymidine incorporation in amino acid-deficient media. Results indicate that the PHA-stimulated lymphocytes require alanine and serine in addition to 13 other amino acids present in Eagle's minimal essential medium (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine). The omission of any one of the 13 amino acids would stop almost completely the proliferation of PHA-stimulated lymphocytes. The omission of serine from RPMI 1640 medium caused a mean reduction of 64% of cell proliferation, while the addition of alanine to PRMI 1640 culture medium caused a mean increment of 52%. The lymphocyte proliferation appears to be modulated by amino acids in the culture medium, and for optimal growth of lymphocytes, all these 15 amino acids are essential.     

Metabolism of the glutamate group of amino acids in rat brain as a function of age

Metabolism of the glutamate group of amino acids--glutamic acid, gamma-amino-butyric acid, glutamine, aspartic acid and alanine--was studied in the brain of rat as a function of age. The levels of glutamic acid, glutamine and aspartic acid decreased while those of gamma-aminobutyric acid, and alanine increased with age. The results on the activity of the twelve enzymes involved in the metabolism showed that five of them (glutamate dehydrogenase, glutamine synthase, gamma-aminobutyric acid transaminase, succinic semialdehyde dehydrogenase and NAD+-isocitrate dehydrogenase) decreased, while four of them (glutaminase, glutamotransferase, glutamic acid decarboxylase, and alpha-ketoglutarate dehydrogenase) increased. The other three enzymes (aspartate aminotransferase, alanine aminotransferase and NADP+-isocitrate dehydrogenase) did not show any significant change in activity. An age-related increase was seen in alpha-ketoglutarate and ammonia, the intermediates involved in the metabolism of these amino acids. The changes in the level of these amino acids are discussed in relation to the altered energy metabolism during aging.     

Expression patterns of ammonia-metabolizing enzymes in the liver,mesonephros, and gut of human embryos and their possible implications

Human and ungulate embryos can catabolize amino acids for energy production, whereas rodent embryos cannot, raising the question whether studies of rodent model systems are suitable for extrapolation to the human situation. Therefore, we investigated the expression of the amino acid- and ammonia-metabolizing enzymes glutaminase, glutamate dehydrogenase, glutamine synthase, carbamoylphosphate synthase, and arginase immunohistochemically in a graded series of human embryos and fetuses. During human development the expression of these enzymes is first seen in the liver, then in the mesonephric kidney, and finally in the small intestine. Such a simultaneous expression of nitrogen-metabolizing enzymes was not seen in any other organ. The early appearance of the enzymes involved in amino acid and ammonia metabolism in the human liver, compared to, for example, the rat liver, suggests that catabolism of amino acids may provide an important supply of metabolic energy for the human embryo. The coexpression of glutaminase, glutamate dehydrogenase, and carbamoylphosphate synthase, but not of arginase, in the mesonephros and the small intestine suggests that these organs are involved in the biosynthesis of intermediates of the ornithine cycle, e.g., arginine or citrulline. From a comparison of the developmental appearance of ornithine cycle enzymes in different mammalian species we postulate that an early appearance of these enzymes is generally associated with a relatively slow prenatal growth rate and the use of amino acids as metabolic fuel. © 1994 Wiley-Liss, Inc.     

Regulation of metabolic branch points of aromatic amino acid biosynthesis in Pichia guilliermondii

The regulatory properties of the enzymes involved in the aromatic amino acid biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory pattern found in other yeast species. 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, anthranilate synthase, chorismate mutase and prephenate dehydrogenase are key regulatory enzymes in P. guilliermondii. Two distinctly regulated isozymes of DAHP synthase, the initial pathway enzyme, which is inhibited by tyrosine or phenylalanine were separated by DEAE-cellulose chromatography and were characterized. Tryptophan is an excellent feedback inhibitor of anthranilate synthase, the first definite step in tryptophan biosynthesis. There are two controlled enzymes within the specific synthesis of phenylalanine and tyrosine, chorismate mutase and prephenate dehydrogenase. Chorismate mutase exhibits a balanced allosteric responsivity to phenylalanine and tyrosine, when these are used as inhibitor; tryptophan acts as an allosteric activator. Tyrosine is an effective inhibitor of prephenate dehydrogenase, whereas the activity of prephenate dehydratase is not affected by any of the aromatic amino acids. The synthesis of the enzymes in the yeast was not repressed by any single exogenous aromatic amino acids, nor by combinations of the same.     

Effect of aromatic acids on the influx of aromatic amino acids in rat brain slices

Summary The influx of [³H] phenylalanine, [³H]tyrosine and [³H]tryptophan into brain cells was studied using brain slices from adult rats. Each aromatic amino acid inhibited the influx of the others into the brain cells. Tryptophan inhibited non-competitively the influx of phenylalanine, and phenylalanine similarly that of tyrosine and tryptophan. On the other hand, tyrosine inhibited competitively the influx of phenylalanine, and similarly tryptophan that of tyrosine, and tyrosine that of tryptophan. Among the aromatic organic acids tested, only phenylpyruvate and homogentisate had any inhibitory effect on the influx of the aromatic amino acids. These effects were generally competitive, non-competitive inhibition being obtained only in the inhibition of phenylalanine influx by homogentisate. The existence of only one common transport system for aromatic amino acids appears to be unlikely.     

Effect of amino acids and antibodies against nerve growth factor receptors on the development of organotypic culture of lymphoid tissue

We studied the effects of 20 L-amino acids on organotypic culture of splenic lymphoid tissue from 3-month-old rats were studied in the presence of apoptosis-inducing monoclonal antibodies against low-affinity receptors for nerve growth factor NGFRp75. The influence of amino acids stimulating cell proliferation in explants (lysine, asparagine, and glutamic acid) did not depend on NGFRp75. Hydrophobic amino acids inhibiting the growth zone in isolated application and abolished the inhibition of explant development in the presence of antibodies against NGFRp75. These amino acids can mediate the proapoptotic effect on lymphoid tissue via low-affinity receptors for nerve growth factor. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 2, pp. 218–221, February, 2007     

Inhibition of polyamine biosynthesis in Crithidia fasciculata by D,L-alpha-difluoromethylornithine and D,L-alpha-difluoromethylarginine.

Using Crithidia fasciculata as a model organism for Trypanosoma cruzi, we have examined the effects of D,L-alpha-difluoromethylornithine (DFMO) and D,L-alpha-difluoromethylarginine (DFMA) on growth and polyamine synthesis. In a defined, polyamine-free medium growth was markedly inhibited by DFMO (94% at 50 mM; IC50 = 37 mM) and to a lesser extent by DFMA (65% at 50 mM). Addition of putrescine, but not agmatine, reverses inhibition of growth, suggesting that the site of inhibition is ornithine decarboxylase (ODC). Consistent with this conclusion, DFMO or DFMA results in a complete loss of putrescine and significant reductions in intracellular spermidine, glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione). In addition, significant concentrations of DFMO (0.8 mM) were present in DFMA-treated cells. However, in contrast to other organisms, conversion of DFMA to DFMO is probably not catalysed by arginase. Substantial ornithine decarboxylase activity (63.1 pmol min-1 mg-1; ODC) was observed in control cells, sufficient to account for polyamine synthesis during growth. In addition, a trace arginine decarboxylase (ADC) activity (1.19 pmol min-1 mg-1) was found. Evidence is presented showing that the apparent ADC activity is actually due to the concerted action of arginase (1.5 nmol min-1 mg-1) and ODC. Thus DFMA appears to inhibit growth of C. fasciculata via conversion to DFMO and subsequent inhibition of ODC.     

Metabolism of tryptophan in the liver: interference with decarboxylation of other aromatic amino acids

Decarboxylation of aromatic amino acid in mammalian tissues is catalyzed by aromatic amino acid decarboxylase (EC. 4.1.1.28, AAD). The enzyme differs in its affinity to individual aromatic amino acids, the best substrates being 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytroptophan. Surprisingly, AAD is abundant in the liver, where the substrates with rather low affinity to AAD as tryptophan, phenylalanine, and tyrosine are offered to decarboxylation. In the present paper, the possibility of interference of tryptophan with decarboxylation of phenylalanine, tyrosine as well as dopa in the liver was investigated. The AAD activity was measured radiometrically with 1-14C-labeled aromatic amino acid substrates using the rat liver enzyme. The influence of tryptophan on decarboxylation of tyrosine was formally competitive with Ki = 9.2 x 10(-3) M, while the inhibition of decarboxylation of phenylalanine by tryptophan was non-competitive with Ki at 2.75 x 10(-2) M. The effect of tryptophan on decarboxylation of dopa was small and it could not be expressed in terms of inhibition kinetics and inhibition constant. At physiological concentrations of aromatic amino acids in plasma, tryptophan does not seem to have remarkable effects on decarboxylation of phenylalanine, tyrosine, and dopa in the liver.     

Effects of basic fibroblast growth factor on the development of GABAergic neurons in culture.

Six-day-old neuronal cultures derived from 14-day-old embryonic rat cerebral hemispheres were highly enriched in GABAergic neurons, as was demonstrated by immunocytochemistry using an anti-glutamate decarboxylase antiserum. They contained about 64% glutamate decarboxylase-positive neurons. About 8% of these neurons proliferated, as shown by a combination of glutamate decarboxylase immunocytochemistry and [3H]thymidine incorporation into cell nuclei. The proliferative activity of GABAergic precursor cells and changes in the cellular concentrations of the non-essential amino acids, including GABA under the effect of basic fibroblast growth factor were studied. When basic fibroblast growth factor was added to the cultures 4 h after seeding, the proliferation of the GABAergic neurons was stimulated about threefold. Under this culture condition, the concentration per cell of all amino acids increased, except those of GABA and beta-alanine. When basic fibroblast growth factor was added to cultures only on day four, the proliferation of the neuronal cells was no more enhanced. Under this condition of treatment, the concentrations of all non-essential amino acids, including those of GABA and beta-alanine were enhanced. Under both basic fibroblast growth factor treatments the concentration of GABA per GABAergic cell was increased. In contrast, the specific activity of glutamate decarboxylase was not stimulated under these conditions. We hypothesize that under the effect of basic fibroblast growth factor the capabilities of the cells to store GABA are improved.     

Exchange and efflux of tryptophan from superfused cerebral cortex slices

Summary The efflux and exchange of L-tryptophan (Trp) from rat cerebral cortex slices were studied in a superfusion system. The substrate specificity of Trp exchange was assessed by measuring the stimulation of [³H]Trp exit provoked by other extracellular amino acids. Large neutral amino acids were the most potent, but also glutamic acid, lysine and glycine had some effect. The stimulation caused by extracellular Trp and phenylalanine persisted also at 0 ° C though severalfold attenuated. Only intracellular histidine provoked slight inhibition of [³H]Trp efflux and intracellular Trp, phenylalanine and lysine had a small stimulatory effect. The results suggest an involvement of carrier-mediated processes in the exchange and efflux of Trp. The substrate specificities of the exchange and efflux are not apparently identical.     

Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy

Complex regulation of T cell functions during pregnancy is required to ensure materno-fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate L-arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase-mediated L-arginine depletion induces down-regulation of CD3 zeta, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase-mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous L-arginine restored CD3 zeta chain expression and T cell proliferation. Thus, L-arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.     

The inhibition of T-lymphocyte proliferation by fatty acids is via an eicosanoid-independent mechanism.

Eicosanoids, in particular prostaglandin E2 (PGE2), are potent inhibitors of a number of immune responses, including lymphocyte proliferation. We have previously shown that fatty acids, especially polyunsaturated fatty acids (PUFA), inhibit mitogen-stimulated proliferation of lymphocytes. One mechanism by which fatty acids could exert their inhibitory effect is via modulation of eicosanoid synthesis. This possibility was investigated in the present study. PGE2 concentrations in the medium taken from lymphocytes cultured in the presence of a range of different fatty acids did not correlate with the inhibitory effects of the fatty acids upon lymphocyte proliferation. Although PGE2 at concentrations above 10 nM caused inhibition of lymphocyte proliferation, PGE2 at the concentration measured in lymphocyte culture medium (0.3-4 nM) was not inhibitory. PGE3 did not inhibit lymphocyte proliferation, except at high concentrations (greater than 250 nM). The maximal inhibition of proliferation caused by PGE2 or PGE3 was less than the inhibition caused by each of the fatty acids except myristic or palmitic acids. Inclusion of inhibitors of phospholipase A2, cyclo-oxygenase or lipoxygenase in the culture medium did not prevent the fatty acids from exerting their inhibitory effect on lymphocyte proliferation. The eicosanoids present in lymph node cell cultures originate from macrophages rather than lymphocytes. Depletion of macrophages from the cell preparation by adherence did not prevent fatty acids from inhibiting proliferation. Proliferation of thoracic duct lymphocytes, which are devoid of macrophages, is inhibited by fatty acids to a similar extent as proliferation of lymph node lymphocytes. These observations provide convincing evidence that the inhibition of lymphocyte proliferation by fatty acids is independent of the production of eicosanoids. Therefore, other mechanisms must be investigated if the effect of fatty acids upon lymphocyte proliferation is to be understood at a biochemical level.     

The influence of amino acids on mitogen-activated proliferation of human lymphocytes in vitro

Recurrent infections are common features in patients affected by various aminoacidopathies. Since these disorders are biochemically characterized by tissue accumulation of amino acids, it is possible that these compounds may act as immunosuppressants. We therefore investigated the influence of 21 amino acids on in vitro cellular growth of lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), a recognized test of cellular immunocompetence. Human peripheral lymphocytes were cultured in flat-bottomed 96-well microplates at 37°C for 96 (PHA and Con A) or 144 h (PWM) in the presence of one mitogen at different concentrations and of one amino acid added at doses of 2, 4 or 8 mM. Cell reactivity was measured by the incorporation of tritiated thymidine into cellular DNA and compared to that of identical cultures with no amino acids added (controls). We found that among the 21 amino acids tested, cysteine stimulated lymphocyte growth, whereas glutamate, tryptophan, phenylalanine and glutamine caused significant inhibition. These results may reflect an immunomodulatory role for some amino acids.     

Transport of branched-chain amino acids in brain slices of developing and adult rats

The accumulation of labelled leucine, isoleucine and valine by cerebral slices of developing and adult rats was studied. The accumulation increased with age by 15–25%. It was strongly (from 52 to 86%) inhibited by a 100-fold excess of phenylalanine, tryptophan and another branched-chain amino acid, but moderately activated by GABA and glutamate. The inhibitions evoked by leucine and isoleucine were slightly stronger in young than in adult rats. The corresponding 2-oxoacid analogs of leucine, isoleucine and valine were also inhibitory but less effective. The 30-min accumulation of ³H-labelled branched-chain amino acids was ostensibly higher than the increase in their total concentrations in incubated slices, which apparently bespeaks lively homoexchange of endogenous intracellular and labelled extracellular amino acids.     

Differential inhibition of lymphocyte function by 2-chloroadenosine

The effects of 2-chloroadenosine, a poorly metabolized adenosine analogue, on some human lymphocyte functions were studied. Mixed lymphocyte responses were strongly inhibited by very low concentrations of 2-chloroadenosine. The mitogen-induced proliferation of human lymphocytes was also generally suppressed by 2-chloroadenosine in a dose dependent manner. Blastogenesis induced by Con A and PWM was severely inhibited by low doses of 2-chloroadenosine while its inhibition of that induced by PHA was less marked. Natural killer cell activity was inhibited only about 55% by high concentrations of 2-chloroadenosine. These results suggested that many subsets of human lymphocytes are controlled by adenosine receptor.     

Role of trypanosomatid's arginase in polyamine biosynthesis and pathogenesis

L-Arginine is one of the precursor amino acids of polyamine biosynthesis in most living organisms including Leishmania parasites. L-Arginine is enzymatically hydrolyzed by arginase producing L-ornithine and urea. In Leishmania spp. and other trypanosomatids a single gene encoding arginase has been described. The product of this gene is compartmentalized in glycosomes and is the main source of L-ornithine for polyamine synthesis in these parasites. L-Ornithine is substrate of ornithine decarboxylase (ODC) - one of the key enzymes of polyamine biosynthesis and a validated target for therapeutic intervention - producing putrescine, which in turn is converted to spermidine by condensing with an aminopropyl group from decarboxylated S-adenosylmethionine. Unlike trypanosomatids, mammalian hosts have two arginases (arginase I and II), which have close structural and kinetic resemblances, but localize in different subcellular organelles, respond to different stimuli and have different immunological reactivity. Arginase I is a cytosolic enzyme, mostly expressed in the liver as a pivotal component of the urea cycle, providing in addition L-ornithine for polyamine synthesis. In contrast, arginase II localizes inside mitochondria and is metabolically involved in L-proline and L-glutamine biosynthesis. More striking is the role played by L-arginine as substrate for nitric oxide synthase (NOS2) in macrophages, the main route of clearance of many infectious agents including Leishmania and Trypanosoma cruzi. In infected macrophages L-arginine is catalysed by NOS2 or arginase, contributing to host defense or parasite killing, respectively. A balance between NOS2 and arginase activities is a crucial factor in the progression of the Leishmania infection inside macrophages. In response to T-helper type 2 (Th2) cytokines, resident macrophages induce arginase I inhibiting NO production from L-arginine, thereby promoting parasite proliferation. Conversely, the response to T-helper type 1 (Th1) cytokines is linked to NOS2 induction and parasite death. Moreover, induction of any of these enzymes is accompanied by suppression of the other. Specifically, arginase reduces NO synthesis by substrate depletion, and N(ω)-hydroxy-L-arginine, one of the intermediates of NOS2 catalysis, competitively inhibits arginase activity. In spite of abundant data concerning arginases in mammals as well their involvement in parasite killing, there are very few papers regarding the actual role of arginase in the parasite itself. This review is an update on the recent progress in research on leishmanial arginase including the role played by this enzyme in the establishment of infection in macrophages and the immune response of the host. A comparative study of arginases from other kinetoplatids is also discussed.     

Absence of GABA type A signaling in adult medial habenular neurons

Neural inhibition in the brain is mainly mediated by ionotropic GABA type A receptors. Apart from the GABA type A receptors, both K(+)-Cl(-) cotransporter isoform 2 and the GABA-synthesizing enzyme, glutamic acid decarboxylase, are essential determinants for GABA type A receptor-mediated inhibition. By using immunofluorescent staining, we observed that K(+)-Cl(-) cotransporter isoform 2, GABA type A receptor beta2/3 subunits and a presynaptically localized glutamic acid decarboxylase isoform, glutamic acid decarboxylase 65, were all absent in adult Sprague-Dawley rat medial habenular nucleus, while immunopositive staining for glutamic acid decarboxylase 67, GABA and GABA type B receptor type 2 subunit were present in the medial habenular nucleus. Consistent with the lack of GABA type A signaling as detected by immunohistochemistry, GABA (100 muM) evoked no measurable currents in the medial habenular nucleus but induced bicuculline-sensitive currents in the lateral habenular nucleus and in the CA1 area of hippocampus. We also failed to record miniature inhibitory postsynaptic currents in medial habenular nucleus neurons. These results support the idea that GABAergic transmission in medial habenular nucleus is probably not mediated by any of the most common GABA type A receptor subtypes. Our data suggest that GABA type B receptor-mediated inhibition may play a role in balancing neuronal excitation in this special region. Further exploration for factors determining medial habenular nucleus neural inhibition will lead to a more complete understanding of control of synaptic balance in the CNS.     

Transmembrane T-cell receptor peptides inhibit B- and natural killer-cell function

A synthetic hydrophobic peptide (core peptide; CP) containing two positively charged amino acids, lysine and arginine was derived from the transmembrane sequence of the T-cell receptor (TCR) alpha chain and has been shown to inhibit T-cell-mediated inflammation. In this study, we investigated the specificity of CP (10 microm) on lymphocyte function and found that it significantly inhibited interleukin-2 production in T cells and natural killer cytotoxicity by 46-58% compared to positive control. CP had no effects on B-cell proliferative responses when used at these concentrations; however, it suppressed B-cell proliferation at higher concentrations (50 microm). Inhibition by CP was not the result of membrane pore formation or cytotoxicity when examined by trypan blue, propidium iodide staining or transmission electron microscopy. CP analogues, with both lysine and arginine replaced by neutral or negatively charged amino acids, or by randomly distributing charges in the peptide sequence, had no effect on lymphocyte function. These results suggest that peptide inhibition is affected by its structure and charge interactions, and may involve common signalling molecules in T, B and natural killer cells. The potential of the immuno-inhibitory effects of CP as a novel anti-inflammatory peptide in therapy should be further explored.     

Rapid method for simultaneous detection of the arginine dihydrolase system and amino acid decarboxylases in microorganisms.

A specific procedure has been developed for the detection of the first two enzymes involved in the arginine dihydrolase system and the detection of the decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine. A loopful of growth of each organism from dihydrolase-decarboxylase induction agar medium (or broth) was washed and incubated separately with 0.2-ml samples of three test media supplemented with different amino acids. Each spent test medium was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products (citrulline, ornithine, gamma-amino-n-butyric acid, and amines) produced during incubation were estimated by comparing the fluorescent intensities of end products from the spent test media and of the corresponding parent amino acids from test medium controls after thin-layer chromatography. The method is reproducible, requiring incubation of an organism in three test media for 1 h for simultaneous detection of the first two enzymes involved in the arginine dihydrolase system and of eight amino acid decarboxylases. This method has been successfully applied to gram-positive and gram-negative microorganisms and also to Mycoplasmatales. It could simplify and improve the accuracy of the corresponding biochemical tests performed in clinical laboratories for the identification and differentiation of microorganisms, and it may prove particularly useful for the differentiation of species of Pseudomonas and Mycoplasma.