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Lothar Elling - One of the best experts on this subject based on the ideXlab platform.
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Chemoenzymatic synthesis of UDP-N-acetyl-α-d-Galactosamine
Carbohydrate Research, 1997Co-Authors: Thomas Bülter, Christian Wandrey, Lothar EllingAbstract:A novel chemoenzymatic synthesis of UDP-N-acetyl-α-d-Galactosamine starting from uridine 5′-monophosphate (UMP) and sucrose is reported. In an enzymatic repetitive batch mode UDP-glucose was generated in situ from UMP and sucrose by the combination of nucleoside monophosphate kinase (EC 2.7.7.4) and sucrose synthase (EC 2.4.1.13). The transfer of UMP from UDP-glucose by galactose-1-phosphate uridyltransferase (EC 2.7.7.12) yielded UDP-α-d-Galactosamine. The equilibrium of the synthesis was forced to the product side by the addition of phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) were used to regenerate UTP and the cofactor NAD. The yield for the enzymatic step was 42%. Finally, UDP-α-d-Galactosamine was acetylated chemically with N-acetoxysuccinimide. Product isolation was accomplished by anion-exchange chromatography and gel filtration. The overall yield was 34% and 82 mg UDP-N-acetyl-α-d-Galactosamine were isolated.
Thomas Bülter - One of the best experts on this subject based on the ideXlab platform.
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Chemoenzymatic synthesis of UDP-N-acetyl-α-d-Galactosamine
Carbohydrate Research, 1997Co-Authors: Thomas Bülter, Christian Wandrey, Lothar EllingAbstract:A novel chemoenzymatic synthesis of UDP-N-acetyl-α-d-Galactosamine starting from uridine 5′-monophosphate (UMP) and sucrose is reported. In an enzymatic repetitive batch mode UDP-glucose was generated in situ from UMP and sucrose by the combination of nucleoside monophosphate kinase (EC 2.7.7.4) and sucrose synthase (EC 2.4.1.13). The transfer of UMP from UDP-glucose by galactose-1-phosphate uridyltransferase (EC 2.7.7.12) yielded UDP-α-d-Galactosamine. The equilibrium of the synthesis was forced to the product side by the addition of phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) were used to regenerate UTP and the cofactor NAD. The yield for the enzymatic step was 42%. Finally, UDP-α-d-Galactosamine was acetylated chemically with N-acetoxysuccinimide. Product isolation was accomplished by anion-exchange chromatography and gel filtration. The overall yield was 34% and 82 mg UDP-N-acetyl-α-d-Galactosamine were isolated.
Joellyn M Mcmillan - One of the best experts on this subject based on the ideXlab platform.
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S-adenosylmethionine but not glutathione protects against Galactosamine-induced cytotoxicity in rat hepatocyte cultures.
Toxicology, 2006Co-Authors: Joellyn M Mcmillan, David C. McmillanAbstract:A gradual but extensive depletion of hepatic GSH has long been known to accompany development of Galactosamine-induced hepatotoxicity in rats, and some protection from liver injury has been observed after administration of sulfhydryl-donating compounds. Although these observations support a key role for GSH in the underlying mechanism, the impact of GSH depletion and repletion on the hepatotoxic response to Galactosamine is unclear. To investigate the role of GSH in Galactosamine-induced liver injury, we examined the effect of modulating GSH content on Galactosamine toxicity in rat primary hepatocyte cultures. Galactosamine (4 mM) cytotoxicity was assessed by release of lactate dehydrogenase into the culture medium, and hepatocellular GSH content was measured by HPLC with electrochemical detection. The data indicated that prior depletion of GSH with either diethyl maleate or buthionine sulfoximine significantly enhanced Galactosamine toxicity; however, addition of GSH-ester or alternate sulfur nucleophiles at various times during the incubation did not abrogate toxicity. In contrast, co-addition of S-adenosylmethionine (SAMe) with Galactosamine exerted a marked protective effect without significantly altering hepatocyte GSH content. These data suggest that GSH depletion is not directly involved in the sequelae for Galactosamine-induced hepatotoxicity, and raise the possibility that SAMe may have hepatoprotective effects that are not dependent on its ability to enhance GSH synthesis.
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Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: lack of involvement in cytotoxicity.
Journal of biochemical and molecular toxicology, 1999Co-Authors: Joellyn M McmillanAbstract:Galactosamine hepatotoxicity in vivo has long been associated with rapid and extensive depletion of hepatic uridine nucleotides. Depletion of uridine nucleotides is considered to be causal in the toxicity, as evidenced by the protective effect of uridine administration. However, the exact mechanism of Galactosamine-induced hepatic necrosis is still unclear. We have previously shown that the addition of Galactosamine to rat primary hepatocyte cultures dramatically decreases production of nitric oxide, as measured in the 24 hour culture medium. The present study investigates whether decreased nitric oxide production contributes to the toxicity of Galactosamine in primary hepatocyte cultures. Similar concentration-response curves were observed for the decrease in nitric oxide production and Galactosamine cytotoxicity, raising the possibility that there is a similar mechanism for these effects. Suppression of NO synthesis was a direct effect of Galactosamine, rather than an indirect effect due to loss of cells from the cultures. Both cytotoxicity and the decrease in nitric oxide production were attenuated by coaddition of 3 mM uridine. However, Galactosamine cytotoxicity was not enhanced by prior inhibition of hepatocellular NO synthesis nor was it attenuated by maintenance of culture NO levels with molsidomine or diethylamine NONOate. These data do not support a role for decreased hepatocyte nitric oxide production in Galactosamine hepatocyte toxicity.
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Galactosamine hepatotoxicity: effect of Galactosamine on glutathione resynthesis in rat primary hepatocyte cultures.
Toxicology and applied pharmacology, 1992Co-Authors: Joellyn M Mcmillan, David J. JollowAbstract:Abstract The effect of Galactosamine on the resynthesis of glutathione in rat primary hepatocyte cultures was investigated. Cultured rat hepatocytes were treated with Galactosamine (4 m m ) 1.5 hr prior to, concurrent with, or 1.5 hr after cell attachment; total cellular glutathione was then measured over time. Addition of Galactosamine at any of these times suppressed methionine-enhanced glutathione resynthesis in the cultures after a lag period of about 120 min. The lag period was not due to slow uptake of Galactosamine by the cultured cells, since cellular UTP levels fell to less than 10% of controls within 60 min, a time frame comparable to that observed in vivo . Neither was the lag period a result of interference with cellular uptake of methionine or with conversion of methionine to cysteine, since the phenomenon was observed regardless of whether methionine or cysteine was used to promote glutathione resynthesis. Addition of uridine, which protects against Galactosamine hepatotoxicity in vivo by replenishing hepatic UTP levels, did not prevent the suppression of glutathione resynthesis. The data indicate that (a) Galactosamine inhibits the time-dependent resynthesis of glutathione in primary hepatocyte cultures, (b) a lag period exists for this response, and (c) this effect is not directly related to depletion of cellular UTP stores.
J.m. Mcmillan - One of the best experts on this subject based on the ideXlab platform.
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Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: lack of involvement in cytotoxicity.
Journal of Biochemical and Molecular Toxicology, 1999Co-Authors: J.m. McmillanAbstract:Galactosamine hepatotoxicity in vivo has long been associated with rapid and extensive depletion of hepatic uridine nucleotides. Depletion of uridine nucleotides is considered to be causal in the toxicity, as evidenced by the protective effect of uridine administration. However, the exact mechanism of Galactosamine-induced hepatic necrosis is still unclear. We have previously shown that the addition of Galactosamine to rat primary hepatocyte cultures dramatically decreases production of nitric oxide, as measured in the 24 hour culture medium. The present study investigates whether decreased nitric oxide production contributes to the toxicity of Galactosamine in primary hepatocyte cultures. Similar concentration-response curves were observed for the decrease in nitric oxide production and Galactosamine cytotoxicity, raising the possibility that there is a similar mechanism for these effects. Suppression of NO synthesis was a direct effect of Galactosamine, rather than an indirect effect due to loss of cells from the cultures. Both cytotoxicity and the decrease in nitric oxide production were attenuated by coaddition of 3 mM uridine. However, Galactosamine cytotoxicity was not enhanced by prior inhibition of hepatocellular NO synthesis nor was it attenuated by maintenance of culture NO levels with molsidomine or diethylamine NONOate. These data do not support a role for decreased hepatocyte nitric oxide production in Galactosamine hepatocyte toxicity. © 1999 John Wiley & Sons, Inc. J Biochem Toxicol 13: 135–142, 1999
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Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression.
Journal of biochemical and molecular toxicology, 1999Co-Authors: J.m. McmillanAbstract:We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to Galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that Galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of Galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the Galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of Galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of Galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the Galactosamine-treated cultures. Furthermore, addition of Galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that Galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.
Christian Wandrey - One of the best experts on this subject based on the ideXlab platform.
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Chemoenzymatic synthesis of UDP-N-acetyl-α-d-Galactosamine
Carbohydrate Research, 1997Co-Authors: Thomas Bülter, Christian Wandrey, Lothar EllingAbstract:A novel chemoenzymatic synthesis of UDP-N-acetyl-α-d-Galactosamine starting from uridine 5′-monophosphate (UMP) and sucrose is reported. In an enzymatic repetitive batch mode UDP-glucose was generated in situ from UMP and sucrose by the combination of nucleoside monophosphate kinase (EC 2.7.7.4) and sucrose synthase (EC 2.4.1.13). The transfer of UMP from UDP-glucose by galactose-1-phosphate uridyltransferase (EC 2.7.7.12) yielded UDP-α-d-Galactosamine. The equilibrium of the synthesis was forced to the product side by the addition of phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) were used to regenerate UTP and the cofactor NAD. The yield for the enzymatic step was 42%. Finally, UDP-α-d-Galactosamine was acetylated chemically with N-acetoxysuccinimide. Product isolation was accomplished by anion-exchange chromatography and gel filtration. The overall yield was 34% and 82 mg UDP-N-acetyl-α-d-Galactosamine were isolated.
