The Experts below are selected from a list of 39834 Experts worldwide ranked by ideXlab platform
Minoru Miyagoshi - One of the best experts on this subject based on the ideXlab platform.
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establishment of a triploid V79 Cell Line from tetraploid Cells obtained through polyploidization using k 252a
Cell Proliferation, 2002Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Minoru MiyagoshiAbstract:Triploid V79 Cells were established from tetraploid Cells. Diploid V79 Cells were polyploidized by K-252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the Cell population was a mixture of diploid and tetraploid Cells. Triploid Cells were obtained through the cloning of tetraploid Cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G 1 , S and G 2 /M phases was almost the same as for diploid Cells. The Cell volume of triploid V79 Cells was about two times that of the diploid Cells. An explanation for the diploid-tetraploid-triploid transition is proposed.
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Establishment of a triploid V79 Cell Line from tetraploid Cells obtained through polyploidization using K‐252a
Cell Proliferation, 2002Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Minoru MiyagoshiAbstract:Triploid V79 Cells were established from tetraploid Cells. Diploid V79 Cells were polyploidized by K-252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the Cell population was a mixture of diploid and tetraploid Cells. Triploid Cells were obtained through the cloning of tetraploid Cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G 1 , S and G 2 /M phases was almost the same as for diploid Cells. The Cell volume of triploid V79 Cells was about two times that of the diploid Cells. An explanation for the diploid-tetraploid-triploid transition is proposed.
Jacques Magdalou - One of the best experts on this subject based on the ideXlab platform.
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Expression and inducibility of UDP-glucuronosyltransferases 1-naphthol in human cultured hepatocytes and hepatocarcinoma Cell Lines
Life sciences, 1997Co-Authors: Amr Abid, Nicole Sabolovic, Jacques MagdalouAbstract:Abstract The UDP-glucuronosyltransferase (UGTs) isoforms involved in the conjugation of 1-naphthol were characterized in human cultured hepatocytes and in two human hepatocarcinoma Cell Lines, KYN-2 and Mz-Hep-1 in terms of expression, kinetics and induction by drugs. Their properties were compared to those of UGT1∗6 stably expressed in the V79 Cell Line (V79UGT1∗6), which glucuronidates 1-naphthol preferentially. The determination of kinetic constants for glucuronidation of 1-naphthol revealed a two-site model in human hepatocytes, but a one-site model in the two hepatocarcinoma Cell Lines. Southern blot analysis of RT-PCR products, showed that the UGT1∗6 mRNA was expressed in KYN-2, but not in Mz-Hep-1 Cells. However, a mRNA encoding a UGT different from UGT1∗6 was expressed in Mz-Hep-1 Cells. The two inducers, β-naphthoflavone and rifampicin exerted a differential effect, depending on the Cell Lines considered. Altogether, the results suggest that, in hepatocytes, two UGT isoforms, which glucuronidate 1-naphthol are expressed and are differentialy regulated by inducers. Both KYN-2 and Mz-Hep-1 Cells express one of the two different UGT isoforms found in hepatocytes. The UGT isoform present in KYN-2 Cells corresponds to UGT1∗6, whereas in Mz-Hep-1 Cells the UGT isoform present was different from UGT1∗6 and UGT1∗7.
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Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1∗6
Biochimica et biophysica acta, 1995Co-Authors: Eric Battaglia, Sylvie Fournel-gigleux, Jacques Magdalou, Gérard Siest, Abdelaziz Elass, Richard R. Drake, Pascal Paul, Susan Treat, Gérard Vergoten, Roger LesterAbstract:Abstract The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1∗6 stably expressed in a V79 Cell Line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, d -DPMSU, had an IC50 of 39 μM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 μM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, d -DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that d -DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1∗6 enzyme.
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Measurement of the in Vitro Glucuronidation of Peroxisome Proliferator Carboxylic Acids by Liver Microsomes and Genetically Modified V79 Cell Line
Peroxisomes, 1994Co-Authors: H. Goudonnet, Jacques Magdalou, Sylvie Fournel-gigleuxAbstract:Peroxisome proliferators (PP) belonging to the series of carboxylic acids are hypolipidemic drugs (clofibric acid, ciprofibrate, fenofibrate,...) or plastifiers (2-ethylhexanoic acid). In man, these substances are essentially excreted as acylglucuronides, which are formed by uridine-5’-diphosphate-(UDP)-glucuronosyltransferases (UGTs, EC 2.4.1.17), according to the following reaction: Open image in new window By contrast to ether glucuronides, ester (acyl) glucuronides are reactive metabolite species. At physiological pHs, they can undergo several non desirable reactions such as isomerization, hydrolysis, and transacylation, which can lead to the formation of covalently bound adducts with proteins [5]. This has been proposed as the mechanism which causes immunological reaction in patients receiving certain carboxylic acid drugs, other than fibrates, which has led to such drugs being withdrawn from the market. They are therefore toxicologically relevant.
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Glucuronidation of hyodeoxycholic acid in human liver. Evidence for a selective role of UDP-glucuronosyltransferase 2B4.
Journal of Biological Chemistry, 1993Co-Authors: T. Pillot, Brian Burchell, Mohamed Ouzzine, Sylvie Fournel-gigleux, C. Lafaurie, Gérard Siest, Anna Radominska, Jacques MagdalouAbstract:Abstract Monospecific polyclonal antibodies were raised against a variable amino-terminal domain (amino acids 14-150) of a human liver form of UDP-glucuronosyl-transferase conjugating bile acids, UGT2B4 (Jackson, M. R., McCarthy, L. R., Harding, D., Wilson, S., Coughtrie, M. W., and Burchell, B. (1987) Biochem. J. 242, 581-588), expressed as a fusion protein in Escherichia coli. The antibodies were able to recognize the protein, stably expressed in a genetically engineered eukaryotic V79 Cell Line, against which they were directed. The specificity of these antibodies allowed their use for analyzing the substrate specificity of this isoform in human liver, as well as for determining its contribution to the total hepatic and extra-hepatic glucuronidation of hyodeoxycholic acid. Western blot analysis of microsomal proteins demonstrated the presence of UGT2B4 exclusively in human liver and not in human kidney. In human liver microsomes, the antibodies were able to inhibit and precipitate up to 90% of the total hyodeoxycholic acid 6-O-glucuronidation activity, but had no effect on activities toward several other substrates, such as phenols, bilirubin, or other bile acids, especially hyocholic acid and the steroids 4-hydroxyesterone and estriol. Moreover, Western blot analysis and immunoinhibition studies of human liver microsomes from healthy patients and from patients presenting liver diseases revealed a good correlation between the glucuronidation rate of hyodeoxycholic acid and the UGT2B4 expression level. The absence of immunoinhibition of hyodeoxycholic acid conjugation with UDP sugars other than UDP-glucuronic acid suggests the involvement of different enzymatic systems in the glucosidation and xylosylation of hyodeoxycholic acid. Altogether, the results provided strong evidence for the specific and predominant involvement of UGT2B4 in the 6-O-glucuronidation of this bile acid via a UDP-glucuronic acid-dependent mechanism.
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Glucuronidation of 3'-azido-3'-deoxythymidine in human liver microsomes: enzyme inhibition by drugs and steroid hormones.
Biochimica et biophysica acta, 1992Co-Authors: Régine Herber, Jacques Magdalou, Marc Haumont, Roselyne Bidault, Helmuth Van Es, Gérard SiestAbstract:Abstract The molecular form of UDP-glucuronosyltransferase involved in the catalysis 3′-azido-3′-deoxythymidine (AZT)-5′- glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 Cell Line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steriod compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which ellicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 μM, respectively. The results suggest that these drugs were substrates of the isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 μM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.
Gérard Siest - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1∗6
Biochimica et biophysica acta, 1995Co-Authors: Eric Battaglia, Sylvie Fournel-gigleux, Jacques Magdalou, Gérard Siest, Abdelaziz Elass, Richard R. Drake, Pascal Paul, Susan Treat, Gérard Vergoten, Roger LesterAbstract:Abstract The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1∗6 stably expressed in a V79 Cell Line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, d -DPMSU, had an IC50 of 39 μM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 μM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, d -DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that d -DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1∗6 enzyme.
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Glucuronidation of hyodeoxycholic acid in human liver. Evidence for a selective role of UDP-glucuronosyltransferase 2B4.
Journal of Biological Chemistry, 1993Co-Authors: T. Pillot, Brian Burchell, Mohamed Ouzzine, Sylvie Fournel-gigleux, C. Lafaurie, Gérard Siest, Anna Radominska, Jacques MagdalouAbstract:Abstract Monospecific polyclonal antibodies were raised against a variable amino-terminal domain (amino acids 14-150) of a human liver form of UDP-glucuronosyl-transferase conjugating bile acids, UGT2B4 (Jackson, M. R., McCarthy, L. R., Harding, D., Wilson, S., Coughtrie, M. W., and Burchell, B. (1987) Biochem. J. 242, 581-588), expressed as a fusion protein in Escherichia coli. The antibodies were able to recognize the protein, stably expressed in a genetically engineered eukaryotic V79 Cell Line, against which they were directed. The specificity of these antibodies allowed their use for analyzing the substrate specificity of this isoform in human liver, as well as for determining its contribution to the total hepatic and extra-hepatic glucuronidation of hyodeoxycholic acid. Western blot analysis of microsomal proteins demonstrated the presence of UGT2B4 exclusively in human liver and not in human kidney. In human liver microsomes, the antibodies were able to inhibit and precipitate up to 90% of the total hyodeoxycholic acid 6-O-glucuronidation activity, but had no effect on activities toward several other substrates, such as phenols, bilirubin, or other bile acids, especially hyocholic acid and the steroids 4-hydroxyesterone and estriol. Moreover, Western blot analysis and immunoinhibition studies of human liver microsomes from healthy patients and from patients presenting liver diseases revealed a good correlation between the glucuronidation rate of hyodeoxycholic acid and the UGT2B4 expression level. The absence of immunoinhibition of hyodeoxycholic acid conjugation with UDP sugars other than UDP-glucuronic acid suggests the involvement of different enzymatic systems in the glucosidation and xylosylation of hyodeoxycholic acid. Altogether, the results provided strong evidence for the specific and predominant involvement of UGT2B4 in the 6-O-glucuronidation of this bile acid via a UDP-glucuronic acid-dependent mechanism.
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Establishment of a V79 transfected Cell Line highly producing recombinant human γ-glutamyltransferase
Toxicology, 1993Co-Authors: Thierry Oster, Sylvie Fournel-gigleux, C. Thioudellet, Maria Wellman, Athanase Visvikis, Gérard SiestAbstract:Abstract γ-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 Cell Line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 Cells. A Cell Line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this Cell Line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered Cell Line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
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Glucuronidation of 3'-azido-3'-deoxythymidine in human liver microsomes: enzyme inhibition by drugs and steroid hormones.
Biochimica et biophysica acta, 1992Co-Authors: Régine Herber, Jacques Magdalou, Marc Haumont, Roselyne Bidault, Helmuth Van Es, Gérard SiestAbstract:Abstract The molecular form of UDP-glucuronosyltransferase involved in the catalysis 3′-azido-3′-deoxythymidine (AZT)-5′- glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 Cell Line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steriod compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which ellicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 μM, respectively. The results suggest that these drugs were substrates of the isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 μM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.
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High-level expression of enzymatically active mature human gamma-glutamyltransferase in transgenic V79 Chinese hamster Cells.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Athanase Visvikis, Sylvie Fournel-gigleux, C. Thioudellet, Maria Wellman, Thierry Oster, Gérard SiestAbstract:gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 Cell Line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 Cells. We selected a Cell Line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this Cell Line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered Cell Line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
Sylvie Fournel-gigleux - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1∗6
Biochimica et biophysica acta, 1995Co-Authors: Eric Battaglia, Sylvie Fournel-gigleux, Jacques Magdalou, Gérard Siest, Abdelaziz Elass, Richard R. Drake, Pascal Paul, Susan Treat, Gérard Vergoten, Roger LesterAbstract:Abstract The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1∗6 stably expressed in a V79 Cell Line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, d -DPMSU, had an IC50 of 39 μM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 μM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, d -DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that d -DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1∗6 enzyme.
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Measurement of the in Vitro Glucuronidation of Peroxisome Proliferator Carboxylic Acids by Liver Microsomes and Genetically Modified V79 Cell Line
Peroxisomes, 1994Co-Authors: H. Goudonnet, Jacques Magdalou, Sylvie Fournel-gigleuxAbstract:Peroxisome proliferators (PP) belonging to the series of carboxylic acids are hypolipidemic drugs (clofibric acid, ciprofibrate, fenofibrate,...) or plastifiers (2-ethylhexanoic acid). In man, these substances are essentially excreted as acylglucuronides, which are formed by uridine-5’-diphosphate-(UDP)-glucuronosyltransferases (UGTs, EC 2.4.1.17), according to the following reaction: Open image in new window By contrast to ether glucuronides, ester (acyl) glucuronides are reactive metabolite species. At physiological pHs, they can undergo several non desirable reactions such as isomerization, hydrolysis, and transacylation, which can lead to the formation of covalently bound adducts with proteins [5]. This has been proposed as the mechanism which causes immunological reaction in patients receiving certain carboxylic acid drugs, other than fibrates, which has led to such drugs being withdrawn from the market. They are therefore toxicologically relevant.
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Glucuronidation of hyodeoxycholic acid in human liver. Evidence for a selective role of UDP-glucuronosyltransferase 2B4.
Journal of Biological Chemistry, 1993Co-Authors: T. Pillot, Brian Burchell, Mohamed Ouzzine, Sylvie Fournel-gigleux, C. Lafaurie, Gérard Siest, Anna Radominska, Jacques MagdalouAbstract:Abstract Monospecific polyclonal antibodies were raised against a variable amino-terminal domain (amino acids 14-150) of a human liver form of UDP-glucuronosyl-transferase conjugating bile acids, UGT2B4 (Jackson, M. R., McCarthy, L. R., Harding, D., Wilson, S., Coughtrie, M. W., and Burchell, B. (1987) Biochem. J. 242, 581-588), expressed as a fusion protein in Escherichia coli. The antibodies were able to recognize the protein, stably expressed in a genetically engineered eukaryotic V79 Cell Line, against which they were directed. The specificity of these antibodies allowed their use for analyzing the substrate specificity of this isoform in human liver, as well as for determining its contribution to the total hepatic and extra-hepatic glucuronidation of hyodeoxycholic acid. Western blot analysis of microsomal proteins demonstrated the presence of UGT2B4 exclusively in human liver and not in human kidney. In human liver microsomes, the antibodies were able to inhibit and precipitate up to 90% of the total hyodeoxycholic acid 6-O-glucuronidation activity, but had no effect on activities toward several other substrates, such as phenols, bilirubin, or other bile acids, especially hyocholic acid and the steroids 4-hydroxyesterone and estriol. Moreover, Western blot analysis and immunoinhibition studies of human liver microsomes from healthy patients and from patients presenting liver diseases revealed a good correlation between the glucuronidation rate of hyodeoxycholic acid and the UGT2B4 expression level. The absence of immunoinhibition of hyodeoxycholic acid conjugation with UDP sugars other than UDP-glucuronic acid suggests the involvement of different enzymatic systems in the glucosidation and xylosylation of hyodeoxycholic acid. Altogether, the results provided strong evidence for the specific and predominant involvement of UGT2B4 in the 6-O-glucuronidation of this bile acid via a UDP-glucuronic acid-dependent mechanism.
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Establishment of a V79 transfected Cell Line highly producing recombinant human γ-glutamyltransferase
Toxicology, 1993Co-Authors: Thierry Oster, Sylvie Fournel-gigleux, C. Thioudellet, Maria Wellman, Athanase Visvikis, Gérard SiestAbstract:Abstract γ-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 Cell Line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 Cells. A Cell Line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this Cell Line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered Cell Line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
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High-level expression of enzymatically active mature human gamma-glutamyltransferase in transgenic V79 Chinese hamster Cells.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Athanase Visvikis, Sylvie Fournel-gigleux, C. Thioudellet, Maria Wellman, Thierry Oster, Gérard SiestAbstract:gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 Cell Line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 Cells. We selected a Cell Line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this Cell Line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered Cell Line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
Jenifer Saffi - One of the best experts on this subject based on the ideXlab platform.
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Antioxidant and anti-mutagenic effects of ebselen in yeast and in cultured mammalian V79 Cells
Mutagenesis, 2008Co-Authors: Simone Teresinha Miorelli, Dinara Jaqueline Moura, Joao Antonio Pegas Henriques, Renato Moreira Rosa, Jaqueline Cesar Rocha, Larissa Aline Carneiro Lobo, Jenifer SaffiAbstract:Ebselen has a wide spectrum of interesting therapeutic actions including antioxidant, cytoprotective, neuroprotective and anti-inflammatory activities. Since its antioxidant effect is very well known, this paper links the effects of ebselen in redox Cellular status to its possible involvement in the maintenance of the integrity of genomic information by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defences and the mammalian V79 Cell Line. Using the alkaLine comet assay, we showed that 5–10 mM ebselen does not induce DNA damage in V79 Cells. Similarly, these same concentrations diminished the extent of the DNA damage induced by hydrogen peroxide (H2O2). The modified comet assay using DNA glycosylases (formamidopyrimidine-DNA glycosylase and endonuclease II) showed that after pre-treatment with ebselen followed by exposure to H2O2, oxidative damage as recognized by these enzymes was significantly lower. In the same way, ebselen showed strong activity against H2O2-induced oxidative damage in the anti-mutagenic assay using S.cerevisiae N123 strain and in the antioxidative assay by using S.cerevisiae strains lacking antioxidant defences. This antioxidant effect was more pronounced for thegpx3D mutant, which indicated that ebselen acts by mimicking the GPx3 catalytic activity. The results confirm that ebselen is involved in antioxidant defence and that its antioxidant ability contributes to its anti-mutagenic and anti-genotoxic action.
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Genotoxic effects of tanshinones from Hyptis martiusii in V79 Cell Line.
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 2007Co-Authors: Bruno C. Cavalcanti, Dinara Jaqueline Moura, Jenifer Saffi, Joao Antonio Pegas Henriques, Renato Moreira Rosa, Manoel Odorico De Moraes, Edigênia Cavalcante Da Cruz Araújo, Mary Anne Sousa Lima, Edilberto R. Silveira, Cláudia PessoaAbstract:Abstract The genotoxic effect of two tanshinones isolated from roots of Hyptis martiussi Benth (Labiatae) was studied using V79 (Chinese hamster lung) Cells by the alkaLine comet assay and micronucleus test. Tanshinones were incubated with the Cells at concentrations of 1, 3, 6 and 12 μg/mL for 3 h. Tanshinones were shown to be quite strongly genotoxic against V79 Cells at all tested concentrations. The data obtained provide support to the view that tanshinones has DNA damaging activity in cultured V79 Cells under the conditions of the assays.
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Antioxidant properties of β-carboLine alkaloids are related to their antimutagenic and antigenotoxic activities
Mutagenesis, 2007Co-Authors: Dinara Jaqueline Moura, Jane Marlei Boeira, Marc Francois Richter, Joao Antonio Pegas Henriques, Jenifer SaffiAbstract:The β-carboLine alkaloids found in medical plants and in a variety of foods, beverages and cigarette smoke have a range of action in various biological systems. In vitro studies have demonstrated that these alkaloids can act as scavengers of reactive oxygen species. In this paper, we report the in vivo antioxidative properties of the aromatic (harmane, harmine, harmol) and dihydro-β-carboLines (harmaLine and harmalol) studied by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. Their antimutagenic activity was also assayed in S. cerevisiae and the antigenotoxicity was tested by the comet assay in V79 Cell Line, when both eukaryotic systems were exposed to H 2 O 2 . We show that the alkaloids have a significant protective effect against H 2 O 2 and paraquat oxidative agents in yeast Cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects.
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Antioxidant properties of β-carboLine alkaloids are related to their antimutagenic and antigenotoxic activities
Mutagenesis, 2007Co-Authors: Dinara Jaqueline Moura, Jane Marlei Boeira, Marc Francois Richter, Joao Antonio Pegas Henriques, Jenifer SaffiAbstract:The beta-carboLine alkaloids found in medical plants and in a variety of foods, beverages and cigarette smoke have a range of action in various biological systems. In vitro studies have demonstrated that these alkaloids can act as scavengers of reactive oxygen species. In this paper, we report the in vivo antioxidative properties of the aromatic (harmane, harmine, harmol) and dihydro-beta-carboLines (harmaLine and harmalol) studied by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. Their antimutagenic activity was also assayed in S. cerevisiae and the antigenotoxicity was tested by the comet assay in V79 Cell Line, when both eukaryotic systems were exposed to H(2)O(2). We show that the alkaloids have a significant protective effect against H(2)O(2) and paraquat oxidative agents in yeast Cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects.