Noriyoshi Masuoka

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.

Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes.

A new method of hydrogen peroxide determination for the measurement of catalase activity and rates of hydrogen peroxide removal by erythrocytes was described. Hydrogen peroxide was determined by converting it to the indamine dye with a water-soluble ironporphyrin and measuring the absorbance at 590 nm. This method was applied to the assay of catalase in hemolysates from human, rat and mouse blood. The activities obtained were in agreement with those obtained by other methods including UV method. The present method was also applied to the determination of rates of hydrogen peroxide removal by intact erythrocytes from human subjects, rats and mice. Data suggested that normal erythrocytes have substantial capacity to remove extracellular hydrogen peroxide. From the measurement of catalase activity in erythrocytes treated with 3-amino-1,2,4-triazole and rates of hydrogen peroxide removal by the erythrocytes, it is deduced that rate constants related to the hemoglobin content (k/g Hb) for hydrogen peroxide removal by catalase in normal and acatalasemic erythrocytes are 42.0 +/- 6.0 and 8.0 +/- 3.0, respectively.

Low catalase activity in blood is associated with the diabetes caused by alloxan

BACKGROUND Hydrogen peroxide is enzymatically processed by catalase, and catalase deficiency in blood is known as acatalasemia. We examined whether low catalase activity is a risk factor for diabetes mellitus. METHODS Blood glucose, insulin and glucose tolerance test were examined in acatalasemic and normal mice under non-stress and oxidative stress conditions. Alloxan administration was used as oxidative stress. RESULTS Alloxan, which was a drug that caused diabetes mellitus, mostly generated hydrogen peroxide by the reaction of alloxan and reduced glutathione, in vitro. Incidence of hyperglycemia in alloxan-untreated acatalasemic mice was as low as that in the normal mice. However, the incidence of acatalasemia mice treated with alloxan was higher than that in normal mice, and the number of pancreatic beta-cells in the acatalasemic mice was less than that in normal mice. CONCLUSION These results indicate that low catalase activity in the blood is associated with the diabetes mellitus caused by alloxan administration.

Sensitization to alloxan-induced diabetes and pancreatic cell apoptosis in acatalasemic mice

Human acatalasemia may be a risk factor for the development of diabetes mellitus. However, the mechanism by which diabetes is induced is still poorly understood. The impact of catalase deficiency on the onset of diabetes has been studied in homozygous acatalasemic mutant mice or control wild-type mice by intraperitoneal injection of diabetogenic alloxan. The incidence of diabetes was higher in acatalasemic mice treated with a high dose (180 mg/kg body weight) of alloxan. A higher dose of alloxan accelerated severe atrophy of pancreatic islets and induced pancreatic beta cell apoptosis in acatalasemic mice in comparison to wild-type mice. Catalase activity remained low in the acatalasemic pancreas without the significant compensatory up-regulation of glutathione peroxidase or superoxide dismutase. Furthermore, daily intraperitoneal injection of angiotensin II type 1 (AT1) receptor antagonist telmisartan (0.1 mg/kg body weight) prevented the development of alloxan-induced hyperglycemia in acatalasemic mice. This study suggests that catalase plays a crucial role in the defense against oxidative-stress-mediated pancreatic beta cell death in an alloxan-induced diabetes mouse model. Treatment with telmisartan may prevent the onset of alloxan-induced diabetes even under acatalasemic conditions.

Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase

5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C₁₅:₃), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C₁₀:₀) also inhibited superoxide anion generation, but resorcinol and cardol (C₅:₀) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain.

Increased Susceptibility to Oxidant-Mediated Tissue Injury and Peritoneal Fibrosis in Acatalasemic Mice

Background: Peritoneal fibrosis is a major complication leading to the loss of peritoneal function in patients undergoing peritoneal dialysis. However, the effect of catalase depletion on peritoneal fibrosis has not yet been investigated. Methods: The impact of catalase deficiency on progressive peritoneal fibrosis has been studied in homozygous acatalasemic mutant mice or control wild-type mice by intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 14 days. Results: The CG injections resulted in a thicker peritoneal membrane, reflecting peritoneal fibrosis with accumulation of interstitial type I collagen, peritoneal deposition of lipid peroxidation products (4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal), and an elevated level of 8-hydroxy-2′-deoxyguanosine in peritoneal fluid in both mouse groups on day 14. The extent of these changes, however, was significantly higher in acatalasemic mice than in wild-type mice. The level of catalase activity remained low in the acatalasemic peritoneum without the compensatory upregulation of glutathione peroxidase, but with an insufficient upregulation of superoxide dismutase activity in CG-injected mice. Conclusions: Acatalasemia, therefore, exacerbates oxidant tissue injury and induces the peritoneum to develop irreversible fibrosis which is the most important complication of peritoneal dialysis. This study suggests that catalase plays a crucial role in the defense against oxidant-mediated peritoneal injury in a mouse peritoneal fibrosis model.

Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides

Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87+/-0.11 micromol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02+/-0.20 and 1.96+/-0.31 micromol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin.

cDNA cloning and expression of mutant catalase from the hypocatalasemic mouse: comparison with the acatalasemic mutant.

Mutant catalase cDNAs from the hypocatalasemic and acatalasemic mice were cloned and expressed in bacteria. A novel missense mutation, Asp (AAT) to Ser (AGT), was identified at amino acid position 439 of the hypocatalasemic catalase. Analysis of recombinant catalase mutants revealed that the mutation is responsible for the reduced activity of hypocatalasemic catalase and the unstable tetrameric structure of acatalasemic catalase was also suggested.

Synthesis and evaluation of bibenzyl glycosides as potent tyrosinase inhibitors

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.

Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities.

Resveratrol was glucosylated to its 3- and 4′-β-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4′-β-glucoside. P. americana cells converted piceatannol into its 4′-β-glucoside. The 3- and 4′-β-glucosides of resveratrol were further glucosylated to 3- and 4′-β-maltosides of resveratrol, 4′-β-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-β-glucoside and 3-β-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4′-β-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4′-β-glucoside showed high phosphodiesterase inhibitory activity. Graphical Abstract Stilbenes were glucosylated to the corresponding β-glucosides by cultured cells of Phytolacca americana. Stilbene glucosides were converted into β-maltosides by cyclodextrin glucanotransferase.