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Zoltan Gregus - One of the best experts on this subject based on the ideXlab platform.
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the glycolytic enzyme glyceraldehyde 3 phosphate dehydrogenase works as an arsenate reductase in human red blood cells and rat liver cytosol
Toxicological Sciences, 2005Co-Authors: Zoltan Gregus, Balazs NemetiAbstract:The mammalian enzymes responsible for reduction of the environmentally prevalent arsenate (AsV) to the much more toxic arsenite (AsIII) are unknown. In the previous paper (Nemeti and Gregus, 2005), we proposed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or phosphoglycerate kinase (PGK) may catalyze reduction of AsV in human red blood cells (RBC), Hemolysate, or rat liver cytosol. In testing this hypothesis, we show here that, if supplied with glutathione (GSH), NAD, and glycolytic substrate, the mixture of purified GAPDH and PGK indeed catalyzes the reduction of AsV. Further analysis revealed that GAPDH is endowed with AsV reductase activity, whereas PGK serves as an auxiliary enzyme, when 3-phosphoglycerate is the glycolytic substrate. The GAPDH-catalyzed AsV reduction required GSH, NAD, and glyceraldehyde-3-phosphate. ADP and ATP moderately, whereas NADH strongly inhibited the AsV reductase activity of the enzyme even in the presence of NAD. Koningic acid (KA), a specific and irreversible inhibitor of GAPDH, inhibited both the classical enzymatic and the AsV-reducing activities of the enzyme in a concentration-dependent fashion. To assess the contribution of GAPDH to the reduction of AsV carried out by Hemolysate, rat liver cytosol, or intact erythrocytes, we determined the concentration-dependent effect of KA on AsV reduction by these cells and extracts. Inactivation of GAPDH by KA abolished AsV reduction in intact RBC as well as in the Hemolysate and the liver cytosol, when GAPDH in the latter extracts was abundantly supplied with exogenous NAD and glycolytic substrate. However, despite complete inactivation of GAPDH by KA, the hepatic cytosol exhibited significant residual AsV-reducing activity in the absence of exogenous NAD and glycolytic substrate, suggesting that besides GAPDH, other cytosolic enzyme(s) may contribute to AsV reduction in the liver. In conclusion, the key glycolytic enzyme GAPDH can fortuitously catalyze the reduction of AsV to AsIII, if GSH, NAD, and glycolytic substrate are available. AsV reduction may take place during, or as a consequence of, the arsenolytic cleavage of the thioester bond formed between the enzyme's Cys149 and the 3-phosphoglyceroyl moiety of the substrate. Although GAPDH is exclusively responsible for reduction of AsV in human erythrocytes, its role in AsV reduction in vivo remains to be determined.
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reduction of arsenate to arsenite by human erythrocyte lysate and rat liver cytosol characterization of a glutathione and nad dependent arsenate reduction linked to glycolysis
Toxicological Sciences, 2005Co-Authors: Balazs Nemeti, Zoltan GregusAbstract:Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In Hemolysate, GSH enhanced AsV reduction in a concentrationdependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the Hemolysate, it was barely influenced by NADH, was diminished by 2phosphoglycollate, and was stimulated by NADP. Collectively, Hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identi
Balazs Nemeti - One of the best experts on this subject based on the ideXlab platform.
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the glycolytic enzyme glyceraldehyde 3 phosphate dehydrogenase works as an arsenate reductase in human red blood cells and rat liver cytosol
Toxicological Sciences, 2005Co-Authors: Zoltan Gregus, Balazs NemetiAbstract:The mammalian enzymes responsible for reduction of the environmentally prevalent arsenate (AsV) to the much more toxic arsenite (AsIII) are unknown. In the previous paper (Nemeti and Gregus, 2005), we proposed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or phosphoglycerate kinase (PGK) may catalyze reduction of AsV in human red blood cells (RBC), Hemolysate, or rat liver cytosol. In testing this hypothesis, we show here that, if supplied with glutathione (GSH), NAD, and glycolytic substrate, the mixture of purified GAPDH and PGK indeed catalyzes the reduction of AsV. Further analysis revealed that GAPDH is endowed with AsV reductase activity, whereas PGK serves as an auxiliary enzyme, when 3-phosphoglycerate is the glycolytic substrate. The GAPDH-catalyzed AsV reduction required GSH, NAD, and glyceraldehyde-3-phosphate. ADP and ATP moderately, whereas NADH strongly inhibited the AsV reductase activity of the enzyme even in the presence of NAD. Koningic acid (KA), a specific and irreversible inhibitor of GAPDH, inhibited both the classical enzymatic and the AsV-reducing activities of the enzyme in a concentration-dependent fashion. To assess the contribution of GAPDH to the reduction of AsV carried out by Hemolysate, rat liver cytosol, or intact erythrocytes, we determined the concentration-dependent effect of KA on AsV reduction by these cells and extracts. Inactivation of GAPDH by KA abolished AsV reduction in intact RBC as well as in the Hemolysate and the liver cytosol, when GAPDH in the latter extracts was abundantly supplied with exogenous NAD and glycolytic substrate. However, despite complete inactivation of GAPDH by KA, the hepatic cytosol exhibited significant residual AsV-reducing activity in the absence of exogenous NAD and glycolytic substrate, suggesting that besides GAPDH, other cytosolic enzyme(s) may contribute to AsV reduction in the liver. In conclusion, the key glycolytic enzyme GAPDH can fortuitously catalyze the reduction of AsV to AsIII, if GSH, NAD, and glycolytic substrate are available. AsV reduction may take place during, or as a consequence of, the arsenolytic cleavage of the thioester bond formed between the enzyme's Cys149 and the 3-phosphoglyceroyl moiety of the substrate. Although GAPDH is exclusively responsible for reduction of AsV in human erythrocytes, its role in AsV reduction in vivo remains to be determined.
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reduction of arsenate to arsenite by human erythrocyte lysate and rat liver cytosol characterization of a glutathione and nad dependent arsenate reduction linked to glycolysis
Toxicological Sciences, 2005Co-Authors: Balazs Nemeti, Zoltan GregusAbstract:Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In Hemolysate, GSH enhanced AsV reduction in a concentrationdependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the Hemolysate, it was barely influenced by NADH, was diminished by 2phosphoglycollate, and was stimulated by NADP. Collectively, Hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identi
Osamu Yoshida - One of the best experts on this subject based on the ideXlab platform.
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a case of a compound heterozygote for adenine phosphoribosyltransferase deficiency aprt j aprt q0 leading to 2 8 dihydroxyadenine urolithiasis review of the reported cases with 2 8 dihydroxyadenine stones in japan
The Journal of Urology, 1993Co-Authors: H Takeuchi, Yoshiyuki Kaneko, Jun Fujita, Osamu YoshidaAbstract:AbstractWe report a case of a compound heterozygote for adenine phosphoribosyltransferase deficiency (APRT*J/APRT*Q0) leading to 2,8-dihydroxyadenine urolithiasis. Polymerase chain reaction-single strand conformation polymorphism analysis demonstrated that APRT*J and APRT*Q0 alleles from the father and mother, respectively, had been transmitted to the patient. We also reviewed the literature regarding Japanese patients with 2,8-dihydroxyadenine urolithiasis. There seemed to be little difference in clinical course between type 2 homozygotes and compound heterozygotes. However, Hemolysate APRT activities of compound heterozygotes were lower than those of type 2 homozygotes.
Kefei Chen - One of the best experts on this subject based on the ideXlab platform.
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potential contribution of nuclear factor κb to cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits
Journal of Cerebral Blood Flow and Metabolism, 2007Co-Authors: Mengliang Zhou, Chunhua Hang, Huilin Cheng, Xiaoping Qi, Kefei ChenAbstract:Nuclear factor-jB (NF-jB) plays a key role in inflammation, which is involved in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). In the present study, we assessed the potential role of NF-jB in regulation of cerebral vasospasm. Nuclear factor-jB DNA-binding activity was measured in cultured vascular smooth muscle cells (VSMCs) treated with Hemolysate and pyrrolidine dithiocarbamate (PDTC, 80 lmol/L), an inhibitor of NF-jB. Forty-two rabbits were divided into three groups: control, SAH, and PDTC groups (n = 14 for each group). The caliber of the basilar artery was evaluated. Nuclear factor-jB DNA-binding activity and the gene expression levels of cytokines and adhesion molecules in the basilar artery were measured. Immunohistochemical study was performed to assess the expression and localization of tumor necrosis factor (TNF)-a, intercellular adhesion molecule (ICAM)-1, and myeloperoxidase (MPO). It was observed that NF-jB DNA-binding activity was significantly increased by treatment with Hemolysate in cultured VSCMs, but this increase was suppressed by pretreatment with PDTC. Severe vasospasm was observed in the SAH group, which was attenuated in the PDTC group. Subarachnoid hemorrhage could induce increases of NF-jB DNA-binding activity and the gene expression levels of TNF-a, interleukin (IL)-1b, ICAM-1, and vascular cell adhesion molecule (VCAM)-1, which were reduced in the PDTC group. Immunohistochemical study demonstrated that the expression levels of TNF-a, ICAM-1, and MPO were all increased in the SAH group, but these increases were attenuated in the PDTC group. Our results suggest that NF-jB is activated in the arterial wall after SAH, which potentially leads to vasospasm development through induction of inflammatory response.
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potential contribution of nuclear factor κb to cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits
Journal of Cerebral Blood Flow and Metabolism, 2007Co-Authors: Mengliang Zhou, Chunhua Hang, Huilin Cheng, Kefei Chen, Jixin Shi, Lei Mao, Hongxia YinAbstract:Nuclear factor-kappaB (NF-kappaB) plays a key role in inflammation, which is involved in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). In the present study, we assessed the potential role of NF-kappaB in regulation of cerebral vasospasm. Nuclear factor-kappaB DNA-binding activity was measured in cultured vascular smooth muscle cells (VSMCs) treated with Hemolysate and pyrrolidine dithiocarbamate (PDTC, 80 micromol/L), an inhibitor of NF-kappaB. Forty-two rabbits were divided into three groups: control, SAH, and PDTC groups (n=14 for each group). The caliber of the basilar artery was evaluated. Nuclear factor-kappaB DNA-binding activity and the gene expression levels of cytokines and adhesion molecules in the basilar artery were measured. Immunohistochemical study was performed to assess the expression and localization of tumor necrosis factor (TNF)-alpha, intercellular adhesion molecule (ICAM)-1, and myeloperoxidase (MPO). It was observed that NF-kappaB DNA-binding activity was significantly increased by treatment with Hemolysate in cultured VSCMs, but this increase was suppressed by pretreatment with PDTC. Severe vasospasm was observed in the SAH group, which was attenuated in the PDTC group. Subarachnoid hemorrhage could induce increases of NF-kappaB DNA-binding activity and the gene expression levels of TNF-alpha, interleukin (IL)-1 beta, ICAM-1, and vascular cell adhesion molecule (VCAM)-1, which were reduced in the PDTC group. Immunohistochemical study demonstrated that the expression levels of TNF-alpha, ICAM-1, and MPO were all increased in the SAH group, but these increases were attenuated in the PDTC group. Our results suggest that NF-kappaB is activated in the arterial wall after SAH, which potentially leads to vasospasm development through induction of inflammatory response.
Umberto Benatti - One of the best experts on this subject based on the ideXlab platform.
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conversion of encapsulated 5 fluoro 2 deoxyuridine 5 monophosphate to the antineoplastic drug 5 fluoro 2 deoxyuridine in human erythrocytes bioreactors deoxyribonucleotidase fluoropyrlmidine drugs encapsulation
2016Co-Authors: A De Flora, Elena Zocchi, Lucrezia Guida, Carolina Polvani, Umberto BenattiAbstract:The fluoropyrimidine deoxyribonucleotide 5- fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was en- capsulated in human erythrocytes by a procedure based on hypotonic hemolysis and isotonic resealing. Encapsulated FdUMP (up to 9 iitnol/nl of packed erythrocytes) did not affect erythrocyte metabolism or morphology. Hemolysates were found to catalyze efficient dephosphorylation of FdUMP to yield nearly stoichiometric amounts of the corresponding deoxyribonucleoside 5-fluoro-2'-deoxyuridine (FdUrd), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas. The dephosphoryla- tion reaction had an apparent Km of 7.7 + 1.2 mM FdUMP at pH 7.4 and was remarkably slower at pH 8.2. ATP, GTP, and UTP inhibited both the disappearance of FdUMP and the formation of FdUrd in Hemolysates. The enzyme responsible for the FdUMP-to-FdUrd conversion was Identified with the deoxyribonucleotide-specific isozyme of erythrocyte pyrimi- dine 5'-nucleotidase (EC 3.1.3.5). Intracellular formation and subsequent release of FdUrd were observed in intact erythro- cytes loaded with FdUMP. Inhibition of FdUrd release from these erythrocytes was obtained by raising the pH intracellu- larly and, alternatively, by coencapsulation of ATP. Autolo- gous FdUMP-loaded erythrocytes might be used as endoge- nous bioreactors designed for time-programmed and liver- targeted delivery of FdUrd.