Role of gamma-aminobutyricacidB(GABA(B)) receptors in the regulation of kainic acid-induced cell death in mouse hippocampus.
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Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of gamma-aminobutyric acid B (GABA(B)) receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABA(B) receptors antagonist, 20 mug) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca(2+)/calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABA(B) receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice. (+info)
Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland.
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Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation. (+info)
Heterologous expression, purification, and characterization of an l-ornithine N(5)-hydroxylase involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa.
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Pseudomonas aeruginosa is an opportunistic pathogen that produces the siderophore pyoverdine, which enables it to acquire the essential nutrient iron from its host. Formation of the iron-chelating hydroxamate functional group in pyoverdine requires the enzyme PvdA, a flavin-dependent monooxygenase that catalyzes the N(5) hydroxylation of l-ornithine. pvdA from P. aeruginosa was successfully overexpressed in Escherichia coli, and the enzyme was purified for the first time. The enzyme possessed its maximum activity at pH 8.0. In the absence of l-ornithine, PvdA has an NADPH oxidase activity of 0.24 +/- 0.02 micromol min(-1) mg(-1). The substrate l-ornithine stimulated this activity by a factor of 5, and the reaction was tightly coupled to the formation of hydroxylamine. The enzyme is specific for NADPH and flavin adenine dinucleotide (FAD(+)) as cofactors, as it cannot utilize NADH and flavin mononucleotide. By fluorescence titration, the dissociation constants for NADPH and FAD(+) were determined to be 105.6 +/- 6.0 microM and 9.9 +/- 0.3 microM, respectively. Steady-state kinetic analysis showed that the l-ornithine-dependent NADPH oxidation obeyed Michaelis-Menten kinetics with apparent K(m) and V(max) values of 0.58 mM and 1.34 micromol min(-1) mg(-1). l-Lysine was a nonsubstrate effector that stimulated NADPH oxidation, but uncoupling occurred and hydrogen peroxide instead of hydroxylated l-lysine was produced. l-2,4-Diaminobutyrate, l-homoserine, and 5-aminopentanoic acid were not substrates or effectors, but they were competitive inhibitors of the l-ornithine-dependent NADPH oxidation reaction, with K(ic)s of 3 to 8 mM. The results indicate that the chemical nature of effectors is important for simulation of the NADPH oxidation rate in PvdA. (+info)
Quantification of protein synthesis in the human brain using L-[1-11C]-leucine PET: incorporation of factors for large neutral amino acids in plasma and for amino acids recycled from tissue.
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The rate of incorporation of exogenous amino acids into brain proteins is indicative of the protein synthesis rate (PSR). The objective of this study was to assess the effect of plasma concentrations of leucine and large neutral amino acids (LNAAs) on the unidirectional uptake rate constant (Kcplx) of l-[1-(11)C]-leucine in the brain and to estimate the amino acid pool recycled from tissue. METHODS: Twenty-seven healthy adult volunteers (11 men and 16 women; age range, 20-50 y) underwent dynamic l-[1-(11)C]-leucine PET with arterial blood sampling. Data were analyzed with a standard 2-tissue-compartment model yielding the unidirectional uptake rate of plasma leucine into tissue (Kcplx = K(1)k(3)/(k(2) + k(3))) and the fraction of leucine originating from exogenous sources (lambda = k(2)/(k(2) + k(3))). PSR in brain was calculated as PSR = [Kcplx/lambda] x leucine. RESULTS: The mean plasma concentration of the sum of all LNAAs was 13% higher in men (981 +/- 86 micromol/L) than in women (850 +/- 76 micromol/L, P = 0.012), whereas the plasma leucine concentration was found to be similar in both sexes (men, 64 +/- 20 micromol/L; women, 58 +/- 21 micromol/L, P = 0.57). The whole-brain value for lambda was determined to be 0.64 +/- 0.03 and did not show a sex difference (P = 0.66). Whole-brain Kcplx values were significantly higher in women (0.0162 +/- 0.0024) than in men (0.0121 +/- 0.0031, P = 0.011); however, after normalization of the Kcplx to a standard plasma concentration of the sum of all LNAAs (Kcplx'), the Kcplx' was similar between the sexes (P = 0.21), as was the PSR' (1.24 +/- 0.49 micromol/L/min in men; 1.29 +/- 0.62 micromol/L/min in women, P = 0.87). No relationship between plasma leucine and Kcplx (r = -0.13, P = 0.63) was observed. Finally, there was a significant correlation between the PSR and the Kcplx derived using Patlak graphical analysis (rho = 0.65, P < 0.001). CONCLUSION: We conclude that both the Kcplx macroparameter and the PSR are stable indices of brain protein synthesis and are appropriate measures for testing altered protein synthesis in neurologic disorders. (+info)
Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells.
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System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-L-cysteine (l-CSNO). In this study, we examined the characteristics of L-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The K(m) for transport was 57 +/- 8 microM for 4F2HC-LAT1 and 520 +/- 52 microM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of L-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of L-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-alpha-mediated IkappaB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and L-cysteine but not with d-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin. (+info)
Prevalence of positive selection among nearly neutral amino acid replacements in Drosophila.
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We have estimated the selective effects of amino acid replacements in natural populations by comparing levels of polymorphism in 91 genes in African populations of Drosophila melanogaster with their divergence from Drosophila simulans. The genes include about equal numbers whose level of expression in adults is greater in males, greater in females, or approximately equal in the sexes. Markov chain Monte Carlo methods were used to sample key parameters in the stationary distribution of polymorphism and divergence in a model in which the selective effect of each nonsynonymous mutation is regarded as a random sample from some underlying normal distribution whose mean may differ from one gene to the next. Our analysis suggests that approximately 95% of all nonsynonymous mutations that could contribute to polymorphism or divergence are deleterious, and that the average proportion of deleterious amino acid polymorphisms in samples is approximately 70%. On the other hand, approximately 95% of fixed differences between species are positively selected, although the scaled selection coefficient (N(e)s) is very small. We estimate that approximately 46% of amino acid replacements have N(e)s < 2, approximately 84% have N(e)s < 4, and approximately 99% have N(e)s < 7. Although positive selection among amino acid differences between species seems pervasive, most of the selective effects could be regarded as nearly neutral. There are significant differences in selection between sex-biased and unbiased genes, which relate primarily to the mean of the distributions of mutational effects and the fraction of slightly deleterious and weakly beneficial mutations that are fixed. (+info)
Distinct sensor pathways in the hierarchical control of SNAT2, a putative amino acid transceptor, by amino acid availability.
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Mammalian nutrient sensors are novel targets for therapeutic intervention in disease states such as insulin resistance and muscle wasting; however, the proteins responsible for this important task are largely uncharacterized. To address this issue we have dissected an amino acid (AA) sensor/effector regulon that controls the expression of the System A amino acid transporter SNAT2 in mammalian cells, a paradigm nutrient-responsive process, and found evidence for the convergence of at least two sensor/effector pathways. During AA withdrawal, JNK is activated and induces the expression of SNAT2 in L6 myotubes by stimulating an intronic nutrient-sensitive domain. A sensor for large neutral AA (e.g. Tyr, Gln) inhibits JNK activation and SNAT2 up-regulation. Additionally, shRNA and transporter chimeras demonstrate that SNAT2 provides a repressive signal for gene transcription during AA sufficiency, thus echoing AA sensing by transceptor (transporter-receptor) orthologues in yeast (Gap1/Ssy1) and Drosophila (PATH). Furthermore, the SNAT2 protein is stabilized during AA withdrawal. (+info)
Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS.
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The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131. (+info)