ABSTRACT A method for the determination of taurine and hypotaurine in biological samples involvin... more ABSTRACT A method for the determination of taurine and hypotaurine in biological samples involving the preparation of their 3,5-dinitrobenzoyl derivatives followed by HPLC was established. Taurine and hypotaurine in aqueous media were reacted with 3,5-dinitrobenzoyl chloride in the presence of triethylamine to prepare 3,5-dinitrobenzoyl derivatives. These derivatives were separated on a C18 reversed-phase column and detected by recording the absorbance at 254 nm. Derivatives of taurine and hypotaurine were obtained in yields of 91.4 +/- 3.3 and 85.6 +/- 2.6%, respectively. The calibration graphs for taurine and hypotaurine were linear between 2.5 and 500 microM with correlation coefficients of 0.999. The method was applied to the determination of taurine and hypotaurine in human and rat urine and blood and in rat liver and heart.
Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cello... more Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cellobiose or maltose. The key steps in the synthesis were the Wittig reaction and trichloroacetimidate glycosylation. Tests for tyrosinase inhibitory activity showed that all were significantly active, indicating that they are unique hydrophilic tyrosinase inhibitors. Bibenzyl xyloside 2 is a particularly potent inhibitor (IC(50) = 0.43 μM, 17 times higher than that of kojic acid). These results suggest that the hydrophilic cavity of tyrosinase might accommodate the bulky carbohydrate on the bibenzyl scaffold.
Japanese burdock roots were extracted with aqueous methanol solution. A compound having the inhib... more Japanese burdock roots were extracted with aqueous methanol solution. A compound having the inhibitory activity of angiotensin I-converting enzyme (ACE)[EC 3.4.15.1] was purified and identified as nicotianamine, (2S, 3’S, 3”S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid. The compound content in burdock roots and related food products were determined. Data revealed that burdock root is a good source of nicotianamine (NA).
Problem statement: Though alloxan-induced mouse hyperglycemia was ameliorated by feeding of 5 % A... more Problem statement: Though alloxan-induced mouse hyperglycemia was ameliorated by feeding of 5 % Asperagillus awamori (A. awamori)-fermented burdock root diet (fermented burdock diet), it is unclear whether the anti-hyperglycemia activity is due to A. awamori or antioxidant activity induced by the fermentation.Methods: A 0.05 % A. awamori diet was prepared. Acatalasemic mice, having a quite low catalase activity in blood, were divided three groups, and each group fed control, A. awamori and the fermented burdock diets for 14 weeks, separately. Then, alloxan monohydrate (200 mg/ kg of body weight) was intraperitoneally administrated to each mouse. Glucose, insulin, C-peptide contents in blood and glucose tolerance tests (GTTs) were examined. Results: Incidence of alloxan-induced hyperglycemia in acatalasemic mice maintained with the A. awamori diet or the fermented burdock diet was low (20 or 25%) compared to that (75%) maintained with the control diet. Feeding the A. awamori diet ame...
Synthesis of ester-linked glycoside prodrug of taxol, i.e., 7-glycolyltaxol 2″- O-α-D-glucopyrano... more Synthesis of ester-linked glycoside prodrug of taxol, i.e., 7-glycolyltaxol 2″- O-α-D-glucopyranoside, was investigated by chemical synthetic procedure. The encapsulation efficiency and loading efficiency of taxol for liposomes was much improved by modification with glucosyl ester group. The immunoliposomes containing the glycoside prodrug of taxol, i.e., 7-glycolyltaxol 2″- O-α-D-glucopyranoside, exhibited effective anti-tumor activities.
beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous... more beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous plants and is used as a medicine, a food additive, and a preservative, and in cosmetics as hair tonic. The cultured plant cells of Nicotiana tabacum glycosylated beta-thujaplicin to two glucosides, 4-isopropyltropolone 2-O-beta-D-glucoside (6%) and 6-isopropyltropolone 2-O-beta-D-glucoside (12%), and two gentiobiosides, 4-isopropyltropolone 2-O-beta-D-gentiobioside (2%) and 6-isopropyltropolone 2-O-beta-D-gentiobioside (5%) after 48 h incubation. The use of immobilized cells of N. tabacum in sodium alginate gel much improved the yield of the products; the glycosylation of beta-thujaplicin with immobilized N. tabacum gave the glycoside products, 4-isopropyltropolone 2-O-beta-D-glucoside (11%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (6%), 6-isopropyltropolone 2-O-beta-D-glucoside (20%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (10%). On the other hand, 4-isopropyltropolone 2-O-beta-D-glucoside (14%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (7%), 6-isopropyltropolone 2-O-beta-D-glucoside (33%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (13%) were obtained through the biotransformation with immobilized cells in the medium without iron ions. In comparison with the case of bioconversion in the normal medium containing iron ions, removal of iron ions improved the yields of products.
International journal of food sciences and nutrition, 2016
Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclea... more Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclear about the effect of caffeic acid and alkyl caffeates on superoxide anion (O2(-)) generation catalyzed by xanthine oxidase. Effects of caffeic acid and alkyl caffeates on the uric acid formation and O2(-) generation catalyzed by xanthine oxidase were analyzed. The scavenging activities of 1,1-diphenyl-2-picryhydrazyl (DPPH) radical and O2(-) generated with phenazine methosulfate (PMS) and NADH were examined. Caffeic acid derivatives equally suppressed O2(-) generation, and the suppression is stronger than inhibition of xanthine oxidase. Scavenging activity of O2(-) is low compared to the suppression of O2(-) generation. Suppression of O2(-) generation catalyzed by xanthine oxidase with caffeic acid derivatives was not due to enzyme inhibition or O2(-) scavenging but due to the reduction of xanthine oxidase molecules. Alkyl caffeates are effective inhibitors of uric acid and O2(-) cataly...
Anacardic acid C15:3 and cardol C15:3 sigmoidally suppressed superoxide anion (O2-) generation us... more Anacardic acid C15:3 and cardol C15:3 sigmoidally suppressed superoxide anion (O2-) generation using xanthine oxidase. To study this suppression activity, anacardic acids, cardanols and cardols having different numbers of double bonds in alk(en)yl chains were prepared. The O2- scavenging activity and H2O2 formation from O2- using PMS-NADH were examined. Anacardic acids and cardols indicated sigmoidal O2- scavenging activity but cardanols did not. The O2- scavenging activity of anacardic acid C15:3 was weaker than the suppression activity using xanthine oxidase, but the scavenging activity of cardol C15:3 was quite similar to the suppression using xanthine oxidase. The H2O2 formation from O2- decreased by the addition of anacardic acids, cardanols and cardols but increased by the addition of gallic and caffeic acids. From these results, we deduced that the O2- suppression activity of xanthine oxidase reaction with cardols is the O2- scavenging activity and that anacardic acids and ca...
ABSTRACT More and more chemicals are synthesized for industrial and consumer use, it is impossibl... more ABSTRACT More and more chemicals are synthesized for industrial and consumer use, it is impossible to finish long-term rodent bioassay for identification of environmental hazards in all chemicals because it involves large numbers of animals and is extremely expensive. Therefore, simple and efficient pre-screening alternatives to animal experimentation are desirable. This study attempted to develop a risk assessment method to accurately identify environmental oxidative chemicals and assess their potential toxic effect at low doses on biological systems. Primary cultured hepatocytes from catalase-mutant mice (Csb) and wild type (Csa) were isolated using collagenase perfusion techniques. The catalase level of Csb was 73% of Csa. We preliminarily examined hydroquinone, a widely used chemical in clinical situations and cosmetics industry because of its inhibitive effect of melanin formation. We found that even low dose of hydroquinone exposure reduced the cell viability significantly and increased cytochrome P450 CYP2E1 mRNA expression with negative correlation to catalase levels. The mRNA level of CYP2E1 was increased about 1.5-fold after 24hr exposure to 0.4mM hydroquinone in Csb relative to Csa. Pretreatment of resveratrol markedly suppressed hydroquinone toxicity, particularly in Csb. Oxidized glutathione (GSSG) levels in Csb was significantly increased by hydroquinone in comparison with Csa. Hydroquinone-induced cell apoptosis was also observed in both Csa and Csb. The present results suggest a possibility of this test system for environmental hazard assessment of oxidative chemicals.
Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargy... more Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.
Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among t... more Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among these metabolites, cystathionine ketimine significantly enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in parallel with tyrosyl phosphorylation of 45 kDa protein in human neutrophils. We investigated the effect of various sulfur amino acids on fMLP-, phorbol-12-myristate-13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in human neutrophils. In addition, the effects of these sulfur amino acids on the membrane translocation of cytosolic compounds p47(phox) and p67(phox) and on the scavenging of superoxide anions were investigated. When the cells were preincubated with various sulfur amino acids, fMLP-induced superoxide generation was enhanced by D,L-homocysteine and D,L-homocysteine-thiolactone but was inhibited by other sulfur amino acids in a concentration-dependent manner. The AA-induced superoxide was enhanced by L-cysteine, N-acetyl-L-cysteine and D,L-homocysteine. The strength of enhancing effect was: L-cysteine>N-acetyl-L-cysteine>D,L-homocysteine. On the other hand, the superoxide generation was weakly inhibited by L-cystathionine. The superoxide generation induced by PMA was weakly inhibited by L-cysteine, N-acetyl-L-cysteine and L-cystathionine. Homocysteine and D,L-homocysteine-thiolactone had no effect. In addition, D,L-homocysteine also enhanced translocation to the cell membrane of cytosolic compounds p47(phox) and p67(phox). Conversely, L-cystathionine and N-acetyl-L-cysteine inhibited the translocation to membrane of p47(phox) and p67(phox) in a concentration-dependent manner. N-acetyl-L-cysteine and L-cysteine revealed scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The sulfur amino acids tested also indicated radical scavenging activity on superoxide anion generated by phenazine methoxysulfate (PMS)-NADH system. D,L-homocysteine and D,L-homocysteine-thiolactone enhanced fMLP-induced superoxide generation by the increment of translocation to membrane of p47(phox) and p67(phox). L-cystathionine and N-acetyl-L-cysteine suppressed fMLP- and PMA-induced superoxide generation by the inhibition of translocation to membrane of p47(phox) and p67(phox). N-acetyl-L-cysteine also had scavenging activity against DPPH radicals and superoxide anion.
A new method for the determination of glutathione peroxidase activity in erythrocytes was develop... more A new method for the determination of glutathione peroxidase activity in erythrocytes was developed. The present method was applied to the measurement of hydrogen peroxide removal rates by glutathione peroxidase in erythrocytes at 70 microM hydrogen peroxide under simulated in vivo conditions. The removal rates by glutathione peroxidase in mouse erythrocytes were twenty-times faster than those in human ones and were 5.2 mumol/sec/g of Hb. The removal rates in acatalasemic mouse erythrocytes indicate that glutathione peroxidase is the main means of hydrogen peroxide removal in acatalasemic mouse erythrocytes. Based on these results, we concluded that glutathione peroxidase in mouse erythrocytes had sufficient ability to remove hydrogen peroxide at even relatively high concentrations. This may be one of the reasons why acatalasemic mice suffer no health problems while Japanese acatalasemic patients suffer from Takahara disease when infected with hydrogen peroxide-generating bacteria.
ABSTRACT A method for the determination of taurine and hypotaurine in biological samples involvin... more ABSTRACT A method for the determination of taurine and hypotaurine in biological samples involving the preparation of their 3,5-dinitrobenzoyl derivatives followed by HPLC was established. Taurine and hypotaurine in aqueous media were reacted with 3,5-dinitrobenzoyl chloride in the presence of triethylamine to prepare 3,5-dinitrobenzoyl derivatives. These derivatives were separated on a C18 reversed-phase column and detected by recording the absorbance at 254 nm. Derivatives of taurine and hypotaurine were obtained in yields of 91.4 +/- 3.3 and 85.6 +/- 2.6%, respectively. The calibration graphs for taurine and hypotaurine were linear between 2.5 and 500 microM with correlation coefficients of 0.999. The method was applied to the determination of taurine and hypotaurine in human and rat urine and blood and in rat liver and heart.
Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cello... more Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cellobiose or maltose. The key steps in the synthesis were the Wittig reaction and trichloroacetimidate glycosylation. Tests for tyrosinase inhibitory activity showed that all were significantly active, indicating that they are unique hydrophilic tyrosinase inhibitors. Bibenzyl xyloside 2 is a particularly potent inhibitor (IC(50) = 0.43 μM, 17 times higher than that of kojic acid). These results suggest that the hydrophilic cavity of tyrosinase might accommodate the bulky carbohydrate on the bibenzyl scaffold.
Japanese burdock roots were extracted with aqueous methanol solution. A compound having the inhib... more Japanese burdock roots were extracted with aqueous methanol solution. A compound having the inhibitory activity of angiotensin I-converting enzyme (ACE)[EC 3.4.15.1] was purified and identified as nicotianamine, (2S, 3’S, 3”S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid. The compound content in burdock roots and related food products were determined. Data revealed that burdock root is a good source of nicotianamine (NA).
Problem statement: Though alloxan-induced mouse hyperglycemia was ameliorated by feeding of 5 % A... more Problem statement: Though alloxan-induced mouse hyperglycemia was ameliorated by feeding of 5 % Asperagillus awamori (A. awamori)-fermented burdock root diet (fermented burdock diet), it is unclear whether the anti-hyperglycemia activity is due to A. awamori or antioxidant activity induced by the fermentation.Methods: A 0.05 % A. awamori diet was prepared. Acatalasemic mice, having a quite low catalase activity in blood, were divided three groups, and each group fed control, A. awamori and the fermented burdock diets for 14 weeks, separately. Then, alloxan monohydrate (200 mg/ kg of body weight) was intraperitoneally administrated to each mouse. Glucose, insulin, C-peptide contents in blood and glucose tolerance tests (GTTs) were examined. Results: Incidence of alloxan-induced hyperglycemia in acatalasemic mice maintained with the A. awamori diet or the fermented burdock diet was low (20 or 25%) compared to that (75%) maintained with the control diet. Feeding the A. awamori diet ame...
Synthesis of ester-linked glycoside prodrug of taxol, i.e., 7-glycolyltaxol 2″- O-α-D-glucopyrano... more Synthesis of ester-linked glycoside prodrug of taxol, i.e., 7-glycolyltaxol 2″- O-α-D-glucopyranoside, was investigated by chemical synthetic procedure. The encapsulation efficiency and loading efficiency of taxol for liposomes was much improved by modification with glucosyl ester group. The immunoliposomes containing the glycoside prodrug of taxol, i.e., 7-glycolyltaxol 2″- O-α-D-glucopyranoside, exhibited effective anti-tumor activities.
beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous... more beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous plants and is used as a medicine, a food additive, and a preservative, and in cosmetics as hair tonic. The cultured plant cells of Nicotiana tabacum glycosylated beta-thujaplicin to two glucosides, 4-isopropyltropolone 2-O-beta-D-glucoside (6%) and 6-isopropyltropolone 2-O-beta-D-glucoside (12%), and two gentiobiosides, 4-isopropyltropolone 2-O-beta-D-gentiobioside (2%) and 6-isopropyltropolone 2-O-beta-D-gentiobioside (5%) after 48 h incubation. The use of immobilized cells of N. tabacum in sodium alginate gel much improved the yield of the products; the glycosylation of beta-thujaplicin with immobilized N. tabacum gave the glycoside products, 4-isopropyltropolone 2-O-beta-D-glucoside (11%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (6%), 6-isopropyltropolone 2-O-beta-D-glucoside (20%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (10%). On the other hand, 4-isopropyltropolone 2-O-beta-D-glucoside (14%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (7%), 6-isopropyltropolone 2-O-beta-D-glucoside (33%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (13%) were obtained through the biotransformation with immobilized cells in the medium without iron ions. In comparison with the case of bioconversion in the normal medium containing iron ions, removal of iron ions improved the yields of products.
International journal of food sciences and nutrition, 2016
Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclea... more Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclear about the effect of caffeic acid and alkyl caffeates on superoxide anion (O2(-)) generation catalyzed by xanthine oxidase. Effects of caffeic acid and alkyl caffeates on the uric acid formation and O2(-) generation catalyzed by xanthine oxidase were analyzed. The scavenging activities of 1,1-diphenyl-2-picryhydrazyl (DPPH) radical and O2(-) generated with phenazine methosulfate (PMS) and NADH were examined. Caffeic acid derivatives equally suppressed O2(-) generation, and the suppression is stronger than inhibition of xanthine oxidase. Scavenging activity of O2(-) is low compared to the suppression of O2(-) generation. Suppression of O2(-) generation catalyzed by xanthine oxidase with caffeic acid derivatives was not due to enzyme inhibition or O2(-) scavenging but due to the reduction of xanthine oxidase molecules. Alkyl caffeates are effective inhibitors of uric acid and O2(-) cataly...
Anacardic acid C15:3 and cardol C15:3 sigmoidally suppressed superoxide anion (O2-) generation us... more Anacardic acid C15:3 and cardol C15:3 sigmoidally suppressed superoxide anion (O2-) generation using xanthine oxidase. To study this suppression activity, anacardic acids, cardanols and cardols having different numbers of double bonds in alk(en)yl chains were prepared. The O2- scavenging activity and H2O2 formation from O2- using PMS-NADH were examined. Anacardic acids and cardols indicated sigmoidal O2- scavenging activity but cardanols did not. The O2- scavenging activity of anacardic acid C15:3 was weaker than the suppression activity using xanthine oxidase, but the scavenging activity of cardol C15:3 was quite similar to the suppression using xanthine oxidase. The H2O2 formation from O2- decreased by the addition of anacardic acids, cardanols and cardols but increased by the addition of gallic and caffeic acids. From these results, we deduced that the O2- suppression activity of xanthine oxidase reaction with cardols is the O2- scavenging activity and that anacardic acids and ca...
ABSTRACT More and more chemicals are synthesized for industrial and consumer use, it is impossibl... more ABSTRACT More and more chemicals are synthesized for industrial and consumer use, it is impossible to finish long-term rodent bioassay for identification of environmental hazards in all chemicals because it involves large numbers of animals and is extremely expensive. Therefore, simple and efficient pre-screening alternatives to animal experimentation are desirable. This study attempted to develop a risk assessment method to accurately identify environmental oxidative chemicals and assess their potential toxic effect at low doses on biological systems. Primary cultured hepatocytes from catalase-mutant mice (Csb) and wild type (Csa) were isolated using collagenase perfusion techniques. The catalase level of Csb was 73% of Csa. We preliminarily examined hydroquinone, a widely used chemical in clinical situations and cosmetics industry because of its inhibitive effect of melanin formation. We found that even low dose of hydroquinone exposure reduced the cell viability significantly and increased cytochrome P450 CYP2E1 mRNA expression with negative correlation to catalase levels. The mRNA level of CYP2E1 was increased about 1.5-fold after 24hr exposure to 0.4mM hydroquinone in Csb relative to Csa. Pretreatment of resveratrol markedly suppressed hydroquinone toxicity, particularly in Csb. Oxidized glutathione (GSSG) levels in Csb was significantly increased by hydroquinone in comparison with Csa. Hydroquinone-induced cell apoptosis was also observed in both Csa and Csb. The present results suggest a possibility of this test system for environmental hazard assessment of oxidative chemicals.
Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargy... more Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.
Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among t... more Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among these metabolites, cystathionine ketimine significantly enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in parallel with tyrosyl phosphorylation of 45 kDa protein in human neutrophils. We investigated the effect of various sulfur amino acids on fMLP-, phorbol-12-myristate-13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in human neutrophils. In addition, the effects of these sulfur amino acids on the membrane translocation of cytosolic compounds p47(phox) and p67(phox) and on the scavenging of superoxide anions were investigated. When the cells were preincubated with various sulfur amino acids, fMLP-induced superoxide generation was enhanced by D,L-homocysteine and D,L-homocysteine-thiolactone but was inhibited by other sulfur amino acids in a concentration-dependent manner. The AA-induced superoxide was enhanced by L-cysteine, N-acetyl-L-cysteine and D,L-homocysteine. The strength of enhancing effect was: L-cysteine>N-acetyl-L-cysteine>D,L-homocysteine. On the other hand, the superoxide generation was weakly inhibited by L-cystathionine. The superoxide generation induced by PMA was weakly inhibited by L-cysteine, N-acetyl-L-cysteine and L-cystathionine. Homocysteine and D,L-homocysteine-thiolactone had no effect. In addition, D,L-homocysteine also enhanced translocation to the cell membrane of cytosolic compounds p47(phox) and p67(phox). Conversely, L-cystathionine and N-acetyl-L-cysteine inhibited the translocation to membrane of p47(phox) and p67(phox) in a concentration-dependent manner. N-acetyl-L-cysteine and L-cysteine revealed scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The sulfur amino acids tested also indicated radical scavenging activity on superoxide anion generated by phenazine methoxysulfate (PMS)-NADH system. D,L-homocysteine and D,L-homocysteine-thiolactone enhanced fMLP-induced superoxide generation by the increment of translocation to membrane of p47(phox) and p67(phox). L-cystathionine and N-acetyl-L-cysteine suppressed fMLP- and PMA-induced superoxide generation by the inhibition of translocation to membrane of p47(phox) and p67(phox). N-acetyl-L-cysteine also had scavenging activity against DPPH radicals and superoxide anion.
A new method for the determination of glutathione peroxidase activity in erythrocytes was develop... more A new method for the determination of glutathione peroxidase activity in erythrocytes was developed. The present method was applied to the measurement of hydrogen peroxide removal rates by glutathione peroxidase in erythrocytes at 70 microM hydrogen peroxide under simulated in vivo conditions. The removal rates by glutathione peroxidase in mouse erythrocytes were twenty-times faster than those in human ones and were 5.2 mumol/sec/g of Hb. The removal rates in acatalasemic mouse erythrocytes indicate that glutathione peroxidase is the main means of hydrogen peroxide removal in acatalasemic mouse erythrocytes. Based on these results, we concluded that glutathione peroxidase in mouse erythrocytes had sufficient ability to remove hydrogen peroxide at even relatively high concentrations. This may be one of the reasons why acatalasemic mice suffer no health problems while Japanese acatalasemic patients suffer from Takahara disease when infected with hydrogen peroxide-generating bacteria.
Uploads
Papers by noriyoshi masuoka