EI-1941-1 and -2, novel interleukin-1beta converting enzyme inhibitors produced by Farrowia sp. E-1941. I. Biochemical characterization of EI-1941-1 and -2.
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EI-1941-1 and -2 isolated from the culture broths of Farrowia sp. selectively inhibited the human recombinant ICE activity with IC50 values of 0.086 and 0.006 microM, respectively, without inhibiting elastase and cathepsin B. EI-1941-1 and -2 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 values of 5.0 and 10.3 microM, respectively. Biochemical characterizations of EI-1941-1 and -2 are described in this article. (+info)
H-tunneling in the multiple H-transfers of the catalytic cycle of morphinone reductase and in the reductive half-reaction of the homologous pentaerythritol tetranitrate reductase.
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The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme. (+info)
Alpha,beta-unsaturated carbonyl compounds: induction of oxidative DNA damage in mammalian cells.
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Alpha,beta-unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 microM) and CHX (300 microM), HEXDI and CA (300 microM each), tGSH was depleted down to <20% of control (viability >85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals. (+info)
The role of diacylglycerol in the exocytosis of the sperm acrosome. Studies using diacylglycerol lipase and diacylglycerol kinase inhibitors and exogenous diacylglycerols.
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When ram spermatozoa were treated with Ca2+ and the ionophore A23187 to induce acrosomal exocytosis, a rise in diacylglycerol (DAG) mass was observed, concomitant with a rapid breakdown of [32P]P1-labelled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and a rise in [32P]Pi-labelled phosphatidate. Inclusion of the DAG lipase inhibitor RHC 80267 resulted in further but biphasic increases in DAG; there was an increasing accumulation of DAG with concentrations of RHC 80267 up to 10 microM, whereas higher concentrations produced lessening accumulation. Inclusion of RHC 80267 in the ionophore induction system also resulted in significant accelerations of the onset of exocytosis. In spermatozoa stimulated with Ca2+/A23187 and the DAG kinase inhibitor R59022, a similar increase in DAG levels together with stimulation of acrosomal exocytosis were observed. Preincubation of spermatozoa with sn-1-oleoyl-2-acetylglycerol, rac-1-oleoyl-2-acetylglycerol, sn-1,2-dioctanoylglycerol and sn-1,3-dioctanoylglycerol before treatment with Ca2+/A23187 resulted in a dose-dependent stimulation of exocytosis by all these isomers. Neomycin inhibited Ca2+/A23187-induced generation of DAG together with polyphosphoinositide breakdown, as well as acrosomal exocytosis. Inclusion of exogenous DAG, however, overcame the inhibitory effect of neomycin on exocytosis. Our results suggest that DAG has a key role in acrosomal exocytosis and that it acts as a messenger rather than as a substrate from which other active metabolites are generated. The lack of stereospecificity shown by the exogenous DAGs implies that DAG does not act by stimulating protein kinase C, but the metabolite's actual target in the sperm cell is as yet unclear. (+info)
The reactive site of eggplant trypsin inhibitor.
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The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds. (+info)
Carbachol stimulation of phospholipase A2 and insulin secretion in pancreatic islets.
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Arachidonic acid has been implicated as a second messenger in insulin secretion by islets of Langerhans. D-Glucose, the major physiological stimulus, increases unesterified arachidonate accumulation in islets. We now show, for the first time, that the muscarinic agonist carbachol, at concentrations which stimulate insulin secretion, causes a rapid and nearly 3-fold increase in arachidonic acid accumulation in islets. The combination of glucose and carbachol has an additive effect on unesterified arachidonate release. There is a large component of secretagogue-induced arachidonate accumulation that is independent of extracellular Ca2+. Carbachol stimulation of arachidonic acid release is mediated by activation of phospholipase A2, as demonstrated by early increases in endogenous lysophosphatidylcholine. In addition to phospholipase A2 activation, carbachol-induced arachidonic acid accumulation also appears to involve diacylglycerol hydrolysis, since the diacylglycerol lipase inhibitor RG80267 partly inhibited arachidonic acid accumulation. In contrast, glucose-induced arachidonic acid accumulation appears to reflect diacylglycerol hydrolysis entirely. Our observations indicate that phospholipase A2 has an important role in muscarinic-induced insulin secretion. (+info)
EI-1941-1 and -2, novel interleukin-1 beta converting enzyme inhibitors Produced by Farrowia sp. E-1941. II. Taxonomy of producing strain, fermentation, isolation, physico-chemical properties, and biological properties.
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EI-1941-1 and -2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Farrowia sp. E-1941. EI-1941-1 and -2 selectively inhibited the human recombinant ICE activity with IC50 values of 0.086 and 0.006 microM, respectively. Taxonomy and fermentation of the producing strain and isolation, physico-chemical properties, structure elucidation, and biological properties of EI-1941-1 and -2 are described. (+info)
In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism.
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Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases. This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha-1-antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event. Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect. Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity. Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant. Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective. Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective. Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil-mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases. (+info)