Progression of hepatic stellate cell activation is associated with the level of oxidative stress rather than cytokines during CCl4-induced fibrogenesis.
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In order to identify a fibrogenic factor associated with the potential of hepatic stellate cells (HSC) activation that arises during the CCl4-induced fibrogenic process, the relationship between the activation of HSC and levels of several fibrogenic factors were investigated. After isolation of HSC from the liver at different stages of CCl4 intoxication, the activation of HSC was assessed by the expression of alpha-smooth muscle actin. Levels of cytokines and oxidative stress in liver homogenates and plasma were measured by enzyme linked immunosorbent assay and the colorimetric method. In primary culture, HSC isolated from a rat liver were gradually activated in a time-dependent manner according to CCl4 administration. The progression of HSC activation was closely correlated with parameters related to oxidative stress in liver homogenates rather than the tissue levels of several cytokines. Also, the levels of antioxidants and arginase activity were inversely correlated with HSC activation. In plasma, the levels of oxidative stress and cytokines in CCl4-treated rat livers were not associated with the activation of HSC found during the CCl4-induced fibrogenic process. The relationship between HSC activation and oxidative stress was also confirmed through several factor-treated HSC cultures. In conclusion, the activation of HSC was accelerated according to CCl4 administration, and the progression of HSC activation is absolutely related to the oxidative stress. These results show that enhanced oxidative stress is an important signal for activation of HSC in experimental liver fibrogenesis. (+info)
The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production.
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In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages. (+info)
Phylogeny of related functions: the case of polyamine biosynthetic enzymes.
(27/943)
Genome annotation requires explicit identification of gene function. This task frequently uses protein sequence alignments with examples having a known function. Genetic drift, co-evolution of subunits in protein complexes and a variety of other constraints interfere with the relevance of alignments. Using a specific class of proteins, it is shown that a simple data analysis approach can help solve some of the problems posed. The origin of ureohydrolases has been explored by comparing sequence similarity trees, maximizing amino acid alignment conservation. The trees separate agmatinases from arginases but suggest the presence of unknown biases responsible for unexpected positions of some enzymes. Using factorial correspondence analysis, a distance tree between sequences was established, comparing regions with gaps in the alignments. The gap tree gives a consistent picture of functional kinship, perhaps reflecting some aspects of phylogeny, with a clear domain of enzymes encoding two types of ureohydrolases (agmatinases and arginases) and activities related to, but different from ureohydrolases. Several annotated genes appeared to correspond to a wrong assignment if the trees were significant. They were cloned and their products expressed and identified biochemically. This substantiated the validity of the gap tree. Its organization suggests a very ancient origin of ureohydrolases. Some enzymes of eukaryotic origin are spread throughout the arginase part of the trees: they might have been derived from the genes found in the early symbiotic bacteria that became the organelles. They were transferred to the nucleus when symbiotic genes had to escape Muller's ratchet. This work also shows that arginases and agmatinases share the same two manganese-ion-binding sites and exhibit only subtle differences that can be accounted for knowing the three-dimensional structure of arginases. In the absence of explicit biochemical data, extreme caution is needed when annotating genes having similarities to ureohydrolases. (+info)
Modulation of cholinergic airway reactivity and nitric oxide production by endogenous arginase activity.
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Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L-arginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea-pig tracheal tube preparation, we investigated the modulation of methacholine-induced airway constriction by the recently developed, potent and specific arginase inhibitor N(Omega)-hydroxy-nor-L-arginine (nor-NOHA). Intraluminal (IL) administration of nor-NOHA caused a concentration-dependent inhibition of the maximal effect (E(max)) in response to IL methacholine, which was maximal in the presence of 5 microM nor-NOHA (E(max)=31.2+/-1.6% of extraluminal (EL) 40 mM KCl-induced constriction versus 51.6+/-2.1% in controls, P<0.001). In addition, the pEC(50) (-log(10) EC(50)) was slightly but significantly reduced in the presence of 5 microM nor-NOHA. The inhibition of E(max) by 5 microM nor-NOHA was concentration-dependently reversed by the NOS inhibitor N(Omega)-nitro-L-arginine methyl ester (L-NAME), reaching an E(max) of 89.4+/-7.7% in the presence of 0.5 mM L-NAME (P<0.01). A similar E(max) in the presence of 0.5 mM L-NAME was obtained in control preparations (85.2+/-9.7%, n.s.). In the presence of excess of exogenously applied L-arginine (5 mM), 5 microM nor-NOHA was ineffective (E(max)=33.1+/-5.8 versus 31.1+/-7.5% in controls, n.s.). The results indicate that endogenous arginase activity potentiates methacholine-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L-arginine. This finding may represent an important novel regulation mechanism of airway reactivity. (+info)
Klebsiella pneumoniae and Staphylococcus aureus infection in mice: difference in uremia and ammoniagenesis.
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Lethal infections by Staphylococcus aureus and Klebsiella pneumoniae were compared for kidney-related effects in mice. K. pneumoniae caused uremia and an increase in blood ammonia that could reach 2.5 times normal. These events did not occur in mice inoculated with S. aureus. Use of germfree animals indicated that most of the increase in ammonia arose from the gut, presumably due to greater availability of urea and ureolysis. Injected ornithine restored blood ammonia to nearly normal levels and extended survival. (+info)
Interaction between arginase and L-ornithine carbamoyltransferase in Saccharomyces cerevisiae. The regulatory sites of arginase.
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The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control or arginine and ornithine, involves a regulatory site for arginine on the arginase distinct from its catalytic site. This regulatory site is responsible for the reinforcement effect of arginine on the inhibition of ornithine carbamoyltransferase by arginase. The binding site of ornithine carbamoyltransferase on arginase is also shown by our analysis. (+info)
Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines.
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Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription-PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis. (+info)
Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells.
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Endothelial cells (EC) metabolize L-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II. To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC). Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC. For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM L-arginine plus [1-(14)C]arginine. Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC. Arginine uptake was 31-33% greater in AI-EC and AII-EC than in lacZ-EC. Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC. Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC. Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC. Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC. Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function. (+info)