Effects of glutamine and alanine supplementation, in their free form or as dipeptide, on fatigue parameters of rats submitted to resistance training / Efeitos da suplementação com glutamina e alanina, na forma livre ou como dipeptídeo, sobre parâmetros de fadiga de ratos submetidos ao treinamento resistido

Fatigue is defined as the inability to maintain muscle power and strength, impairing performance. Nutritional interventions have been used to delay this phenomenon, such as glutamine and alanine supplementation. These amino acids might attenuate several causes of fatigue, since they are important energy substrates, transport ammonia avoiding the accumulation of this toxic metabolite and attenuate muscle damage and oxidative stress. Thus, the aim of this study was to evaluate the effects of glutamine and alanine supplementation on central and muscle fatigue parameters of rats submitted to resistance training (RT). Forty adult Wistar rats (60 days) were distributed into five groups: SED (sedentary, receiving water), CON (trained, receiving water), ALA, G+A and DIP (trained and supplemented with alanine, glutamine and alanine in their free form, and Lalanyl-L-glutamine, respectively). Trained groups underwent a ladder-climbing exercise, with progressive loads, for eight weeks. Supplements were diluted in water to a 4% concentration and offered ad libitum during the last 21 days of experiment. RT increased plasma glucose, the muscle concentrations of ammonia and glutathione (GSH) and the muscle damage parameters - plasma creatine kinase (CK) and lactate dehydrogenase (LDH), whereas decreased muscle glycogen. G+A supplementation prevented the increase of muscle ammonia by RT, while ALA and G+A administration reduced plasma CK and LDH, and DIP supplementation increased the muscle content of glycogen and LDH. Contrary to expectations, DIP administration increased central fatigue parameters, such as plasma concentration of free fatty acids (FFA), hypothalamic content of serotonin and serotonin/dopamine ratio. Despite these results, there was no difference between groups in the maximum carrying capacity (MCC) tests. In conclusion, supplementation with glutamine and alanine improves some fatigue parameters, but does not affect physical performance of rats submitted to RT

O termo fadiga é definido como a incapacidade de manutenção da força e da potência musculares, prejudicando a performance. Intervenções nutricionais têm sido utilizadas para retardar este fenômeno, como a suplementação com glutamina e alanina. Estes aminoácidos poderiam atenuar diversas causas de fadiga, pois são importantes substratos energéticos, carreiam amônia evitando o acúmulo deste metabólito tóxico e atenuam a lesão muscular e o estresse oxidativo. Logo, o objetivo deste estudo foi avaliar os efeitos da suplementação com glutamina e alanina sobre parâmetros de fadiga central e muscular em ratos submetidos ao treinamento resistido (TR). Foram utilizados 40 ratos Wistar adultos (60 dias de idade), distribuídos nos grupos: SED (não treinados, recebendo água), CON (treinados, recebendo água), ALA, G+A e DIP (treinados e suplementados com alanina, glutamina e alanina livres, e L-alanil-L-glutamina, respectivamente). Os grupos treinados realizaram um exercício de escalada em escada, com aumento progressivo de carga, durante oito semanas. A suplementação foi diluída a 4% em água e ofertada via oral, ad libitum, durante os últimos 21 dias de experimento. O TR aumentou a glicemia, as concentrações musculares de amônia e de glutationa (GSH) e os parâmetros de lesão muscular - creatina quinase (CK) e lactato desidrogenase (LDH) no plasma, enquanto reduziu o glicogênio no músculo. A suplementação com G+A preveniu o aumento de amônia muscular promovido pelo TR, enquanto a administração de ALA e G+A reduziu as concentrações de CK e LDH no plasma, e a suplementação com DIP aumentou o conteúdo muscular de glicogênio e de LDH. Ao contrário do esperado, a administração de DIP aumentou parâmetros de fadiga central, como as concentrações plasmáticas de ácidos graxos livres, o conteúdo hipotalâmico de serotonina e a razão serotonina/dopamina. Apesar disso, não houve diferença entre os grupos nos testes de carga máxima. Em conclusão, a suplementação com glutamina e alanina melhora alguns parâmetros de fadiga, mas não afeta o desempenho físico em ratos submetidos ao TR

Inhibition of d-Ala incorporation into wall teichoic acid in Staphylococcus aureus by desleucyl-oritavancin.

The mode of action for desleucyl-oritavancin was investigated by adding an antibiotic to Staphylococcus aureus during its growth in a defined medium containing l,d-[1-15N]Ala and l-[1-13C]Lys, or d-[1-15N]Ala. 13C{15N} and 15N{13C} rotational-echo double resonance NMR determined that desleucyl-oritavancin inhibited the incorporation of d-[1-15N]Ala into wall teichoic acid.

Disruption of D-alanyl esterification of Staphylococcus aureus cell wall teichoic acid by the {beta}-lactam resistance modifier (-)-epicatechin gallate.

OBJECTIVES: The naturally occurring polyphenol (-)-epicatechin gallate (ECg) increases oxacillin susceptibility in mecA-containing strains of Staphylococcus aureus. Decreased susceptibility to lysostaphin suggests alterations to the wall teichoic acid (WTA) content of ECg-grown bacteria. Changes in WTA structure in response to ECg were determined. METHODS: Nuclear magnetic resonance spectroscopy of purified monomers from S. aureus was used to elucidate WTA structures. Molecular modelling of WTA chains was employed to determine their spatial configuration. RESULTS: ECg-grown methicillin-resistant S. aureus (MRSA) strains BB568 and EMRSA-16 displayed markedly reduced resistance to oxacillin, had thickened cell walls and separated poorly. Growth in ECg-supplemented medium reduced the substitution of the WTA backbone by d-alanine (d-Ala); ratios of N-acetyl glucosamine to d-Ala were reduced from 0.6 and 0.49 (for BB568 and EMRSA-16) to 0.3 and 0.28, respectively. Molecular simulations indicated a decrease in the positive charge of the bacterial wall, confirmed by increased binding of cationized ferritin, and an increase in WTA chain flexibility to a random coil conformation. CONCLUSIONS: Structural elucidation and molecular modelling of WTA indicated that conformational changes associated with reduced d-Ala substitution may contribute to the increased susceptibility of MRSA to beta-lactam antibiotics and account for other elements of the ECg-induced phenotype.

Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections.

Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.

Topiramate prevents excitotoxic damage in the newborn rodent brain.

Brain lesions induced in newborn mice by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) and S-bromowillardiine (acting on AMPA-kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage. Topiramate (TPM), already used in children to manage newly diagnosed and refractory epilepsy, has potential neuroprotective effects that may be useful in human perinatal brain lesions. In the excitotoxic newborn mouse model, TPM provided dose-dependent and long-lasting protection of developing white matter and cortical plate against S-bromowillardiine. TPM had no significant effect on ibotenate-induced brain lesions. TPM-induced neuroprotection potentially involved increased survival of pre-oligodendrocytes, decreased neuronal apoptosis, inhibition of microglial activation and astrogliosis, and decreased seizure activity. Diazepam, phenytoin, and carbamazepine had no neuroprotective effect in this model. The present study provides experimental support for the consideration of TPM as a candidate therapy for excitotoxic perinatal brain lesions.

Does initial and delayed heart rate predict mortality in patients with acute coronary syndromes?

BACKGROUND: Lower admission heart rate (HR) is known to predict favorable outcome in ST-elevation acute myocardial infarction. However, there are limited short-term and no long-term data available regarding the prediction value of the initial HR in patients with the full spectrum of acute coronary syndromes (ACS). In addition, it is unknown whether the HR obtained later during hospitalization for ACS (i.e., Day 2 or 3) remains prognostically valuable. HYPOTHESIS: The aim of this study was to investigate the utility of the initial and delayed HR in predicting outcome in patients with ACS. METHODS: We examined mortality at 30 days and 10 months in 10,267 patients with ACS enrolled in the oral glycoprotein IIb/IIIa inhibition with Orofiban in Patients with Unstable coronary Syndromes-Thrombolysis In Myocardial Infarction (OPUS-TIMI) 16 trial. Patients were stratified by HR and day from onset of ACS into the following groups: (1) HR < 60 beats/min, (2) HR 60-80 beats/min, (3) HR 80-100 beats/min, (4) HR > 100 beats/min; and HR obtained on (1) Day 1, (2) Day 2, and (3) Day 3. RESULTS: By univariate analysis, mortality at 30 days and at 10 months increased progressively with higher HR strata (1.4 vs. 1.6 vs. 2.3 vs. 5.6%, p < 0.001, and 2.6 vs. 4.2 vs. 6.5 vs. 11.8%, p < 0.001, respectively). Elevated HR remained associated with mortality irrespective of time from onset of ACS. CONCLUSIONS: Higher initial and delayed HR is highly predictive of higher short- and long-term mortality in patients with ACS. This is a simple marker that could be easily used in risk assessment.

Troglitazone inhibits glutamine metabolism in rat mesangial cells.

Troglitazone is a peroxisome proliferator-activated receptor-gamma agonist that has been shown to halt mesangium expansion in experimental models of type 2 diabetes mellitus and to act directly on rat mesangial cells. Because glutamine serves as the precursor for cellular biosynthetic processes, we asked whether troglitazone would inhibit mesangial cell glutamine metabolism under these conditions. Confluent monolayers of rat mesangial cells were incubated in RPMI medium in the presence of troglitazone or vehicle (DMSO). Troglitazone effected a dose-dependent reduction in glutamine utilization and in alanine formation, associated with a decrease in monolayer collagen-glycosaminoglycan content. Despite the reduced glutamine uptake, ammonium formation did not decrease, consistent with increased glutamate flux through the deamination pathway. Assayable activity of the alanine aminotransferase decreased by 63%, whereas assayable glutamate dehydrogenase remained unchanged. In control monolayers, the sum of ammonium plus alanine plus glutamate nitrogen released accounted for <75% of the glutamine nitrogen uptake. In troglitazone-treated monolayers, all of the glutamine nitrogen taken up could be accounted for as ammonium nitrogen released into the medium. These results are consonant with troglitazone reducing glutamine metabolism and specifically the transamination pathway in rat mesangial cells associated with a reduction in collagen-glycosaminoglycan content.

Inhibitory effect of glycation on catalytic activity of alanine aminotransferase.

Non-enzymatic glycation is a common post-translational modification of tissue and plasma proteins which can impair their functions in living organisms. In this study, the authors have demonstrated for the first time an inhibitory effect of in vitro glycation on the catalytic activity of alanine aminotransferase (ALT, EC 2.6.1.2), a pyridoxal phosphate enzyme with several lysine residues in the molecule. The porcine heart enzyme was incubated with 50 mmol/l D-fructose, D-glucose, D,L-glyceraldehyde, or D-ribose in 0.1 mol/l phosphate buffer (pH 7.4) at 25 degrees C for up to 20 days. The strongest glycation effect was shown by D,L-glyceraldehyde, which caused complete enzyme inhibition within 6 days. After 20 days of incubation, the ALT activity in samples with D-fructose and D-ribose was less than 7% of the initial enzyme activity. A statistically significant effect of D-glucose on the enzymatic activity of ALT was not found. Incubation of ALT with D-fructose, D,L-glyceraldehyde and D-ribose minimized its catalytic activity both in the glycated and non-glycated fractions of the samples. Markedly higher activity was found in the glycated fraction with glucose. The inhibitory effect of glycation of ALT with D-fructose and D-ribose was found to be more intensive in the presence of L-alanine and weaker in the presence of 2-oxoglutarate. The findings suggest that glycation of the epsilon-amino group of Lys313 as a crucial part of the catalytic site of ALT may contribute to ALT inactivation in the presence of glycating sugars. Nevertheless, glycation of lysine residues outside the active center of ALT seems to be primary.

Induction of cyclooxygenase-2 in rat gastric mucosa by rebamipide, a mucoprotective agent.

Recent studies indicate an expression of mitogen-inducible cyclooxygenase (COX-2) in gastric mucosa. Rebamipide, a mucoprotective agent enhances prostaglandin (PG) synthesis. The present study was designed to clarify the mechanism for rebamipide-induced mucosal protection. Male Sprague-Dawley rats were administered 5, 15, or 50 mg/kg/day rebamipide for 14 days. The expression of constitutive cyclooxygenase (COX-1) and COX-2 in gastric mucosa was determined using Western blot analysis. Another series of rats was used to examine 1) the levels of PGE(2) in stomach with and without pretreatment with a COX-2 inhibitor; 2) the protective action of rebamipide against gastric damage caused by 0.6 N HCl; and 3) the effects of a COX-2 inhibitor on rebamipide-induced gastric mucosal protection. COX-2 expression was enhanced, whereas COX-1 expression did not change significantly in the gastric mucosa of rats after treatment with rebamipide. The gastric mucosal PGE(2) was higher in the rebamipide groups than in the vehicle-treated group. Rebamipide also suppressed gastric damage induced by HCl in a dose-dependent manner. A COX-2 inhibitor blocked the rebamipide-induced increase in mucosal PGE(2), and mucosal protection induced by rebamipide. The results indicate that rebamipide induces COX-2 expression, increases PGE(2) levels, and enhances gastric mucosal defense in a COX-2-dependent manner. Thus, COX-2 has an important role in the effects of rebamipide on gastric mucosal protection.

Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties.

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors.

A novel effect of rebamipide: generation of [Ca(2+)](i) oscillations through activation of CCK(1) receptors in rat pancreatic acinar cells.

The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the activation of cholecystokinin subtype 1 (CCK(1)) receptors. At concentrations higher than 5 microM, rebamipide induced [Ca(2+)](i) oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca(2+)](i) oscillations decreased with increasing concentration. The [Ca(2+)](i) oscillations evoked by rebamipide were inhibited by the CCK(1) receptor antagonist L-364,718 but not by atropine or the CCK(2) receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK(1) receptor agonist.

Amino acid fluxes in rat thin limb segments of Henle's loop during in vitro microperfusion.

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes of L-[(14)C]alanine and [(14)C]taurine in thin limbs of Henle's loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle's loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route.

Acute and neonatal capsaicin treatment inhibit jejunal amino acid absorption through a Na+-dependent mechanism.

It has recently been shown that capsaicin inhibits alanine absorption in rat jejunum via mechanisms that involve intestinal capsaicin-sensitive primary afferent (CSPA) fibers. This study provides further evidence that the effect of capsaicin is neurally mediated and demonstrates that CSPA fibers regulate Na+-dependent amino acid absorption. In vivo, basal alanine absorption in rats neonatally treated with capsaicin was reduced by 35% below control. Furthermore, intraluminal perfusion of 400 microM capsaicin reduced jejunal alanine absorption by 31% in sham rats but had no significant effect in rats neonatally treated with capsaicin. In vitro, capsaicin significantly reduced uptake of alanine and proline by jejunal strips but had no effect on uptake of lysine. Tetrodotoxin (0.2 microM) partially blocked the effects of capsaicin but did not itself affect alanine absorption. Capsaicin reduced unidirectional mucosal-to-serosal alanine (1 mM) influx by 33%, an effect that becomes significant after 5 min of preincubation with capsaicin. Neonatal capsaicin treatment reduced basal alanine influx in jejunal strips by 37%; however, preincubation of these strips with capsaicin had no significant effect. Kinetic analysis of alanine steady-state uptake and influx by jejunal strips incubated with capsaicin revealed that capsaicin reduced the Na+-dependent component of alanine influx into intestinal epithelial cells. Long-term sensory denervation by capsaicin also decreased the Na+-dependent component of alanine absorption. These data suggest that intestinal capsaicin-sensitive primary afferent fibers regulate Na+-dependent amino acid absorption.

Lipopolysaccharides stimulate Na-dependent transport in alveolar cells and protect against oxidant injury.

We have evaluated the effect of lipopolysaccharides (LPS), endotoxins from gram negative bacteria, on sodium-coupled amino acid and phosphate transport by alveolar epithelial type II cells and on their alteration induced by oxidants. Alveolar type II cells were obtained by enzymatic digestion of rat lung and grown for 24 h prior to incubation with LPS and then exposed or not exposed to H2O2 (2.5 mM; 20 min). LPS (10 micrograms/ml, 24 h) induced a significant increase in the Na-dependent component of alanine and phosphate uptake while they decreased Na,K-ATPase activity measured by ouabain-sensitive 86Rb influx. We showed that this stimulatory effect i) was independent from macrophage products since it was not mimicked either by supernatant of LPS-treated alveolar macrophages or by pretreatment with tumor necrosis factor and/or interleukin 1 and ii) was dependent on protein synthesis since it was abolished by protein synthesis inhibitors cycloheximide and actinomycin D. Moreover, LPS blunted H2O2-induced decrease of Na-dependent alanine and phosphate uptake. This protective effect of LPS against H2O2 injury i) was independent of macrophage products, ii) was abolished by cycloheximide, and iii) was not associated with either changes in extracellular H2O2 clearance or catalase and glutathione peroxidase activities. We conclude that, in alveolar type II cells, LPS stimulate sodium-coupled transport by a process involving protein synthesis and partially prevent H2O2-induced decrease of Na-coupled transport without discernible change in antioxidant activities.

In vitro toxicological investigations of isoxazolinone amino acids of Lathyrus sativus.

Two non-protein amino acids of Lathyrus sativus, beta-(isoxazoline-5-on-2-yl)-alanine (BIA) and its higher homologue alpha-amino-gamma-(isoxazoline-5-on-2-yl)-alanine (ACI) were tested for excitotoxic potential. BIA (0.5-2.0 mM) but not ACI (2.0 mM) produced a concentration-dependent neurodegeneration in mouse cortical explants. The neuronal damage was prevented by the prior and simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), indicating that it was mediated by non-N-methyl-D-aspartate type receptors. BIA (0.5-2.0 mM) activated CNQX-sensitive currents which were significantly smaller than those activated by 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the majority of neurons. In a small number of cells, BIA (2 mM) produced currents which were similar in amplitude to those activated by beta-ODAP (50 microM). These results suggest that Lathyrus sativus plants engineered to block the synthesis of beta-ODAP may accumulate a neurotoxic precursor and therefore must be tested for the presence of both BIA and beta-ODAP.

Alanine uptake by liver of mid-lactating rats.

L-Alanine transport in liver plasma membrane vesicle preparations from fed virgin and 15-day-lactating rats was studied. Lactation was found to induce a decrease of the maximal rate (Vmax) of a high-capacity-low-affinity component of the Na(+)-dependent L-alanine uptake. However, a high-affinity-low-capacity agency was significantly induced in lactating-rat livers. L-Alanine uptake was differentially inhibited by other amino acids in those preparations from lactating rats, and showed different sensitivity to Li+ as a cosubstrate instead of Na+ and to inhibition by sulfhydryl modifying reagents (N-ethylmaleimide [NEM] and p-chloromercuribenzosulfonate [PCMBS]). All of these observations taken together suggest that system A is upregulated in lactating-rat livers, thus resulting in a different contribution of both agencies A and ASC to the total Na(+)-dependent alanine transport into liver plasma membrane vesicles. This was demonstrated using the analogue alpha-methyl-aminoisobutyric acid (MeAIB), a specific system A substrate. L-Alanine uptake rates, as calculated from plasma membrane enzyme marker recoveries, were also enhanced in the physiologic range of alanine concentrations in blood. Our results prove that the physiologic adaptation to lactation involves modulation of system A activity in the liver.

Impaired alanine uptake by basolateral liver plasma membrane vesicles from rats consuming ethanol.

Chronic ethanol consumption reduces alanine transport by rat basolateral liver plasma membrane (blLPM) vesicles; however, the mechanism for this effect remains uncertain. It may be related to the ethanol-induced changes in blLPM fluidity and lipid composition; alternatively ethanol might reduce the number of transporters in the blLPM. To investigate the effect of blLPM fluidity and lipid composition on Na(+)-dependent alanine uptake these parameters were altered in vitro. Increasing the blLPM fluidity had no effect on Na(+)-dependent alanine uptake by blLPM vesicles or the activity of amino acid transport systems, A and ASC. Because ethanol is known to reduce the blLPM cholesterol content, the influence of altering blLPM cholesterol on alanine transport by these membranes was investigated next. Neither an increase nor a decrease of the cholesterol content of the blLPM altered Na(+)-dependent alanine uptake or the activity of system A or ASC. Finally, the influence of chronic ethanol consumption on the specific binding of [3H]alanine to blLPM was studied. The dissociation constant for alanine binding to blLPM from ethanol-fed rats and their pair-fed controls was similar (1.9 +/- 0.2 vs. 2.0 +/- 0.3 mM); however, the maximal binding capacity for alanine was significantly (P less than 0.05) lower in the blLPM from ethanol-fed rats (316 +/- 53 pmol/mg protein) compared with their pair-fed controls (527 +/- 79 pmol/mg protein). These studies do not support the hypothesis that ethanol-induced changes in blLPM fluidity are responsible for the impaired alanine transport; they do suggest that ethanol may reduce the

Specific inhibition of the binding of the taste stimulus, L-alanine, by sulphydryl reagents, in Ictalurus punctatus.

1. Taste receptors for L-alanine and L-arginine in the channel catfish, Ictalurus punctatus, are differentially reactive to N-ethylmaleimide (NEM) and p-chloromercuribenzenesulphonic acid (pCMBS). 2. The binding of L-[3H]alanine by a sedimentable membrane fraction (Fraction P2) isolated from taste epithelium was inhibited by both NEM and pCMBS while the binding of L[3H]arginine was unaffected. 3. Inhibition of the binding of L-[3H]alanine by pCMBS was reversible with dithiothreitol (DTT). 4. NEM (10(-3) M) inhibited multi-unit neural responses to both 10(-4) M L-alanine and 10(-4) M L-arginine, while pCMBS had little effect on neural responses. 5. Pretreatment of intact taste epithelium before the preparation of Fraction P2 with NEM caused strong inhibition of L-[3H]alanine binding, while pretreatment with pCMBS caused weak inhibition. 6. The presence of L-alanine during the reaction of pCMBS or NEM with taste plasma membranes did not substantially protect against the inhibition of L-[3H]alanine binding.

[Effect of excitant amino acid antagonists on glutamate receptors in the locust and on convulsions induced by glutamate, aspartate, kynurenine and quinolinic acid in mice]. / Vliianie antagonistov vozbuzhdaiushchikh aminokislot na glutamatnye retseptory saranchi i sudorogi, vyzvannye glutamatom, aspartatom, kinureninom i khinolinovoi kislotoi na myshakh.

All excitatory amino acid antagonists studied: diethyl esters of aspartic (DEEA) and glutamic (DEEG) acids, 2-amino-3-phosphono-propionic acid (APPA) and 2-amino-4-phosphono-butanoic acid (APBA), diminished the amplitude of excitatory postsynaptic potentials (EPP) of the locust (Locusta migratoria migratorioides) muscle fibers and arbitrary blocked glutamate (GLU) and aspartate (ASP) responses. Kynurenine (KYN) and quinolinic (QUI) acid had no effect on EPP even at a concentration of 2 X 10(-2) M. The antagonists were not strictly selective against intracerebroventricularly administered endogenous convulsants: GLU, ASP, KYN and QUI and in simulation of experimental seizures in mice. The antagonists structurally similar to ASP prevented ASP- and KYN-induced seizures in lower doses than GLU derivatives. Anti-KYN, but not anti-QUI DEEA, DEEG, APPA and APBA efficacy suggests that KYN and QUI act on different structures or binding sites.

Germination-initiation and inhibitory activities of L- and D-alanine analogues for Bacillus subtilis spores. Modification of methyl group of L- and D-alanine.

The ability of 33 compounds of L-alanine analogues over a wide range of concentrations to initiate germination of Bacillus subtilis spores was determined, and the inhibitory activity against L-alanine-initiated germination was determined for the above compounds and 22 of their D- and DL-isomers. Nineteen L-isomers were able to initiate the germination. The maximum germination rate and the apparent binding affinity of the germinant were obtained from concentration-germination response curves. Not only D-isomers but also L-isomers of many alanine analogues showed inhibitory action on L-alanine-initiated germination. The apparent binding affinity of an inhibitor was calculated by Schild's method. D-Alanine, D-serine, glycine, D-2-amino-n-butyric acid, D-cysteine, D-norvaline, and D-threonine were competitive inhibitors for the L-alanine action. Analysis of the relation between the structure of the side chain of L- and D-alanine analogues and their apparent affinity suggested that there are separate binding portions, which differ in size and electrostatic nature, for germination and for inhibition on the receptor. Certain L-alanine analogues had a dualistic property of initiating germination at low concentrations and inhibitory activity at higher concentrations, i.e., autoinhibition. The autoinhibitory phenomenon might be explained by the above postulation of the presence of separate binding portions for germination and for inhibition.