The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform
Yong Soo Lee - One of the best experts on this subject based on the ideXlab platform.
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Role of reactive oxygen species in apoptosis induced by N-Ethylmaleimide in HepG2 human hepatoblastoma cells.
European journal of pharmacology, 2001Co-Authors: Jung-ae Kim, Young Shin Kang, Seung Hee Park, Hae Won Kim, Soon-yeong Cho, Yong Soo LeeAbstract:Abstract We have previously reported that N-Ethylmaleimide induces apoptosis through activation of K+, Cl−-cotransport in HepG2 human hepatoblastoma cells. In this study, we investigated the role for reactive oxygen species as a mediator of the apoptosis induced by N-Ethylmaleimide. N-Ethylmaleimide induced a significant elevation of intracellular level of reactive oxygen species. Treatment with antioxidants (N-acetyl cysteine, N,N′-diphenyl-p-phenylenediamine) which markedly suppressed generation of reactive oxygen species, significantly inhibited the N-Ethylmaleimide-induced activation of K+, Cl−-cotransport and apoptosis. Inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, d -(+)-neopterine) also significantly blunted the generation of reactive oxygen species, activation of K+, Cl−-cotransport and apoptosis induced by N-Ethylmaleimide. These results suggest that reactive oxygen species generated through activation of NADPH oxidase may play a role in the N-Ethylmaleimide-induced stimulation of K+, Cl−-cotransport and apoptosis in HepG2 cells.
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Involvement of K(+)-Cl(-)-cotransport in the apoptosis induced by N-Ethylmaleimide in HepG2 human hepatoblastoma cells.
European journal of pharmacology, 2001Co-Authors: Jung-ae Kim, Young Shin Kang, Yong Soo LeeAbstract:The role of K(+)-Cl(-)-cotransport in apoptosis in human cancer cells was investigated. N-Ethylmaleimide, a K(+)-Cl(-)-cotransport activator, induced apoptosis in a dose-dependent manner in HepG2 human hepatoblastoma cells. N-Ethylmaleimide induced Cl(-)-dependent K(+) efflux, indicating that K(+)-Cl(-)-cotransport is functionally present in HepG2 cells. Calyculin-A and genistein, inhibitors of K(+)-Cl(-)-cotransport, significantly prevented both K(+)-Cl(-)-cotransport activation and apoptosis induced by N-Ethylmaleimide. These results demonstrate, for the first time, a novel role for K(+)-Cl(-)-cotransport in apoptosis in human hepatoma cells. These results further suggest that K(+)-Cl(-)-cotransport may be a valuable target for therapeutic interventions for human hepatoma.
Mitsuo Tagaya - One of the best experts on this subject based on the ideXlab platform.
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ADP-ribosylation factor-1 is sensitive to N-Ethylmaleimide.
Journal of biochemistry, 1998Co-Authors: Tomohiro Yamaguchi, Kazuhisa Nakayama, Kiyotaka Hatsuzawa, Katsuko Tani, Masaru Himeno, Mitsuo TagayaAbstract:The treatment of normal rat kidney cells with N-Ethylmaleimide caused the release of beta-COP, a component of coatomer, from the Golgi apparatus without causing disassembly of the organelle. The release of beta-COP, which was not due to depolymerization of microtubules, was markedly blocked by the activation of GTP-binding proteins by aluminum fluoride or a nonhydrolyzable analogue of GTP. To determine which component is N-Ethylmaleimide-sensitive, we reconstituted the recruitment of coatomer from the bovine brain cytosol onto the Golgi apparatus in digitonin-permeabilized cells. In cells treated with N-Ethylmaleimide before permeabilization, beta-COP was still recruited onto the Golgi apparatus. In contrast, beta-COP was not recruited when N-Ethylmaleimide-treated bovine brain cytosol was used. These results suggest that the N-Ethylmaleimide-sensitive factor(s) are present in the cytosol. It is known that coatomer and ADP-ribosylation factor-1 (ARF1) are the only cytoplasmic proteins needed for the assembly of Golgi-derived coated vesicles. N-Ethylmaleimide treatment of a coatomer-rich fraction did not affect the binding of beta-COP to the Golgi apparatus, whereas the same treatment of an ARF-rich fraction abolished beta-COP binding. Similar results were obtained using purified recombinant ARF1. Concomitant with inactivation, 0.85 mol of N-Ethylmaleimide was incorporated into 1 mol of ARF1. ARF1 contains only one cysteine residue (Cys-159), which is located near the base moiety of the bound guanine nucleotide.
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Role of two nucleotide-binding regions in an N-Ethylmaleimide-sensitive factor involved in vesicle-mediated protein transport.
The Journal of biological chemistry, 1994Co-Authors: Mizuo Sumida, Rong-mao Hong, Mitsuo TagayaAbstract:N-Ethylmaleimide-sensitive factor (NSF) was originally characterized as the protein that restores in vitro protein transport activity of the Golgi membranes inactivated by N-Ethylmaleimide. This protein has two homologous regions, each containing the consensus sequence for nucleotide binding, and possesses ATPase activity. To investigate the role of the two nucleotide-binding regions in NSF, we have replaced two lysyl residues (Lys-274 and Lys-557) located in the consensus sequences with glutamine or methionine via site-directed mutagenesis. The mutant NSF proteins in which Lys-274 was replaced had no ability to restore protein transport between N-Ethylmaleimide-treated Golgi membranes and, in addition, inhibited the protein transport assay using normal Golgi membranes. This inhibition, which was eliminated by N-Ethylmaleimide treatment, was caused by the impairment of the function of donor Golgi membranes. Although wild-type NSF showed a protective effect against inhibition by the Lys-274 mutant NSF protein when added at the start of the protein transport assay, its protective effect diminished after the time for the formation of transport vesicles had passed. These results support the idea that NSF incorporated into transport vesicles is nonexchangeable for exogenously added NSF. On the other hand, the mutant proteins in which Lys-557 was replaced had slight but significant protein transport activity. They did not inhibit the protein transport assay using normal Golgi membranes. The mutant NSF proteins in which Lys-274 and Lys-557 were replaced had about 20 and 25% of the ATPase activities of wild-type NSF, respectively. Their ATPase activities were sensitive to N-Ethylmaleimide and dependent on their protein concentrations, as observed in wild-type NSF.
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Domain structure of an N-Ethylmaleimide-sensitive fusion protein involved in vesicular transport.
The Journal of biological chemistry, 1993Co-Authors: Mitsuo Tagaya, Duncan W. Wilson, Michael Brunner, Nancy Arango, James E RothmanAbstract:Abstract N-Ethylmaleimide-sensitive fusion protein (NSF) is an essential component for protein transport between Golgi cisternae. Sequence analysis and proteolytic dissection reveal that NSF contains two tandem "ATP domains," each containing the consensus sequence for the binding of nucleotide. When Escherichia coli-produced Chinese hamster ovary NSF is purified, it exhibits a low, but significant, ATPase activity. The ATPase activity of NSF is sensitive to N-Ethylmaleimide and influenced by monoclonal antibodies against recombinant NSF.
Richard D. Howells - One of the best experts on this subject based on the ideXlab platform.
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Studies on Inhibition of and Opioid Receptor Binding by Dithiothreitol and N-Ethylmaleimide His IS CRITICAL FOR μ OPIOID RECEPTOR BINDING AND INACTIVATION BY N-Ethylmaleimide
Journal of Biological Chemistry, 1996Co-Authors: Mandana Shahrestanifar, William W. Wang, Richard D. HowellsAbstract:Abstract The sensitivity of μ and receptor binding to dithiothreitol and N-Ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. μ receptor binding was inhibited with more rapid kinetics and at lower N-Ethylmaleimide concentrations than receptor binding. Ligand protection against N-Ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC of N-Ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-Ethylmaleimide alkylation on the μ receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-Ethylmaleimide. Serine substitution of His in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-Ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the μ opioid receptor.
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Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-Ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-Ethylmaleimide.
The Journal of biological chemistry, 1996Co-Authors: Mandana Shahrestanifar, William W. Wang, Richard D. HowellsAbstract:The sensitivity of mu and delta receptor binding to dithiothreitol and N-Ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N-Ethylmaleimide concentrations than delta receptor binding. Ligand protection against N-Ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-Ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-Ethylmaleimide alkylation on the mu receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-Ethylmaleimide. Serine substitution of His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-Ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the mu opioid receptor.
Jung-ae Kim - One of the best experts on this subject based on the ideXlab platform.
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Role of reactive oxygen species in apoptosis induced by N-Ethylmaleimide in HepG2 human hepatoblastoma cells.
European journal of pharmacology, 2001Co-Authors: Jung-ae Kim, Young Shin Kang, Seung Hee Park, Hae Won Kim, Soon-yeong Cho, Yong Soo LeeAbstract:Abstract We have previously reported that N-Ethylmaleimide induces apoptosis through activation of K+, Cl−-cotransport in HepG2 human hepatoblastoma cells. In this study, we investigated the role for reactive oxygen species as a mediator of the apoptosis induced by N-Ethylmaleimide. N-Ethylmaleimide induced a significant elevation of intracellular level of reactive oxygen species. Treatment with antioxidants (N-acetyl cysteine, N,N′-diphenyl-p-phenylenediamine) which markedly suppressed generation of reactive oxygen species, significantly inhibited the N-Ethylmaleimide-induced activation of K+, Cl−-cotransport and apoptosis. Inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, d -(+)-neopterine) also significantly blunted the generation of reactive oxygen species, activation of K+, Cl−-cotransport and apoptosis induced by N-Ethylmaleimide. These results suggest that reactive oxygen species generated through activation of NADPH oxidase may play a role in the N-Ethylmaleimide-induced stimulation of K+, Cl−-cotransport and apoptosis in HepG2 cells.
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Involvement of K(+)-Cl(-)-cotransport in the apoptosis induced by N-Ethylmaleimide in HepG2 human hepatoblastoma cells.
European journal of pharmacology, 2001Co-Authors: Jung-ae Kim, Young Shin Kang, Yong Soo LeeAbstract:The role of K(+)-Cl(-)-cotransport in apoptosis in human cancer cells was investigated. N-Ethylmaleimide, a K(+)-Cl(-)-cotransport activator, induced apoptosis in a dose-dependent manner in HepG2 human hepatoblastoma cells. N-Ethylmaleimide induced Cl(-)-dependent K(+) efflux, indicating that K(+)-Cl(-)-cotransport is functionally present in HepG2 cells. Calyculin-A and genistein, inhibitors of K(+)-Cl(-)-cotransport, significantly prevented both K(+)-Cl(-)-cotransport activation and apoptosis induced by N-Ethylmaleimide. These results demonstrate, for the first time, a novel role for K(+)-Cl(-)-cotransport in apoptosis in human hepatoma cells. These results further suggest that K(+)-Cl(-)-cotransport may be a valuable target for therapeutic interventions for human hepatoma.
Alan P. Kozikowski - One of the best experts on this subject based on the ideXlab platform.
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Cocaine and dopamine differentially protect [3H]mazindol binding sites from alkylation by N-Ethylmaleimide
European journal of pharmacology, 1992Co-Authors: Kenneth M. Johnson, John S. Bergmann, Alan P. KozikowskiAbstract:The binding of cocaine, d-amphetamine and dopamine to the site on the dopamine transporter labeled by [3H]mazindol was investigated in rat striatal membranes. N-Ethylmaleimide inhibited about 95% of the specific binding of 5 nM [3H]mazindol in a concentration-dependent manner. The effect of 10 mM N-Ethylmaleimide was completely prevented by cocaine (EC50 of 3 microM), but neither 300 microM dopamine nor d-amphetamine afforded any significant protection. On the other hand, high concentrations of cocaine, d-amphetamine and dopamine provided similar protection against inhibition by 0.1 mM N-Ethylmaleimide. Taken together these data support the hypothesis that a significant portion of the cocaine binding domain on the transporter is distinct from that of either dopamine or amphetamine. This distinction may be sufficient to allow properly designed drugs to prevent cocaine binding without inhibiting DA transport.
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cocaine and dopamine differentially protect 3h mazindol binding sites from alkylation by n ethylmaleimide
European Journal of Pharmacology, 1992Co-Authors: Kenneth M. Johnson, John S. Bergmann, Alan P. KozikowskiAbstract:The binding of cocaine, d-amphetamine and dopamine to the site on the dopamine transporter labeled by [3H]mazindol was investigated in rat striatal membranes. N-Ethylmalcimide inhibited about 95% of the specific binding of 5 nM [3H]mazindol in a concentration-dependent manner. The effect of 10 mM N-Ethylmaleimide was completely prevented by cocaine (EC50 of 3 μM). but neither 300 μM dopamine nor d-amphetamine afforded any significant protection. On the other hand, high concentrations of cocaine, d-amphetamine and dopamine provided similar protection against inhibition by 0.1 mM N-ethylmalcimide. Taken together these data support the hypothesis that a significant portion of the cocaine binding domain on the transporter is distinct from that of either dopamine or amphetamine. This distinction may be sufficient to allow properly designed drugs to prevent cocaine binding without inhibiting DA transport.
