Effect of hydrogen peroxide on normal and acatalasemic mouse erythrocytes.

OBJECTIVES: Normal and acatalasemic mouse erythrocytes were used to clarify the relationship between oxidative damage in H2O2-treated erythrocytes and catalase activity. DESIGN & METHODS: Generation of hydrolysis-resistant erythrocytes and hemolysis were examined. The osmotic fragility test, the negative charges and the number of membrane-flickering erythrocytes among the H2O2-treated erythrocytes were investigated. RESULTS: Small amounts of hydrolysis-resistant mouse erythrocytes were generated by treatment with 0.1 mM H2O2, and the amount of acatalasemic erythrocytes was larger than untreated controls. Hemolysis in the acatalasemic erythrocytes was observed 30 min after the addition of the H2O2. A drastic increase in hydrolysis-resistant erythrocytes and a loss of membrane proteins in the acatalasemic erythrocytes were found as a result of the addition of 1 mM H2O2. Hemolysis in normal erythrocytes was observed at 3 mM H2O2. CONCLUSIONS: Catalase is a potent H2O2-scavenger even in acatalasemic mouse erythrocytes. It is concluded that the drastic increase of hydrolysis-resistant erythrocytes is induced by a loss of membrane function and is associated with the low catalase activity in these cells.

Glycosylation and Methylation of Quercetin and Myricetin by Cultured Cells of Phytolacca americana.

The glycosylation and methylation of quercetin by cultured plant cells of Phytolacca americana gave quercetin 3-Ο-ß-D-glucoside and isorharnnetin 3-Ο-ß-D- glucoside. Myricetin was glycosylated and methylated to syringetin 3-Ο-ß-D-glucoside by cultured P. americana cells.

Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves.

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world.

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

Glucosylation of taxifolin with cultured plant cells.

Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus.

Effect of vitamin E on alloxan-induced mouse diabetes.

OBJECTIVES: Alloxan generates hydrogen peroxide in the body, and a small amount of alloxan administered to acatalasemic mice results in diabetes. D-α-Tocopherol (vitamin E) is an antioxidant which helps prevent excess oxidation in the body. In this study, we examined the effect of vitamin E on diabetes caused by alloxan administration in mice. METHODS: Mice were maintained on a vitamin E-deprived diet and supplemented diet, respectively, for 14 weeks. Alloxan was then intraperitoneally administered, and blood glucose, glucose tolerance and the insulin level in mouse blood were examined. RESULTS: Hyperglycemia was observed in the mice maintained on the vitamin E-deprived diet. The incidence of hyperglycemia in the mice maintained on the vitamin E-deprived diet was significantly higher than that in the mice maintained on the supplemented diet. The abnormal glucose metabolism caused by alloxan administration was ameliorated by the vitamin E-supplemented diet. CONCLUSIONS: It is deduced that vitamin E can prevent a decrease of insulin concentration in the blood in this mouse model.

Glycosylation of trans-resveratrol by plant-cultured cells.

Plant-cultured cells of Catharanthus roseus converted trans-resveratrol into its 3-O-ß-D-glucopyranoside, 4'-O-ß-D-glucopyranoside, 3-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside, and 3-O-(6-O-α-L-arabinopyranosyl)-ß-D-glucopyranoside. The 3-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside and 3-O-(6-O-α-L-arabinopyranosyl)-ß-D-glucopyranoside compounds of trans-resveratrol are both new. Incubation of plant-cultured cells of Ipomoea batatas and Strophanthus gratus with trans-resveratrol gave trans-resveratrol 3-O-ß-D-glucopyranoside and trans-resveratrol 4'-O-ß-D-glucopyranoside.

Plant-based Paste Fermented by Lactic Acid Bacteria and Yeast: Functional Analysis and Possibility of Application to Functional Foods.

A plant-based paste fermented by lactic acid bacteria and yeast (fermented paste) was made from various plant materials. The paste was made of fermented food by applying traditional food-preservation techniques, that is, fermentation and sugaring. The fermented paste contained major nutrients (carbohydrates, proteins, and lipids), 18 kinds of amino acids, and vitamins (vitamin A, B1, B2, B6, B12, E, K, niacin, biotin, pantothenic acid, and folic acid). It contained five kinds of organic acids, and a large amount of dietary fiber and plant phytochemicals. Sucrose from brown sugar, used as a material, was completely resolved into glucose and fructose. Some physiological functions of the fermented paste were examined in vitro. It was demonstrated that the paste possessed antioxidant, antihypertensive, antibacterial, anti-inflammatory, anti-allergy and anti-tyrosinase activities in vitro. It was thought that the fermented paste would be a helpful functional food with various nutrients to help prevent lifestyle diseases.

Enzymatic preparation of arbutin derivatives: lipase-catalyzed direct acylation without the need of vinyl ester as an acyl donor.

Direct and regioselective acylation of arbutin with aromatic or aliphatic acid using a lipase obtained from Candida antarctica in an organic solvent was investigated. We achieved the enzymatic synthesis of feruloyl arbutin and lipoyl arbutin without the need of vinyl ferulate and vinyl lipoate as acyl donors, respectively.

One-procedure synthesis of capsiate from capsaicin by lipase-catalyzed dynamic transacylation.

Capsiate has a structure similar to capsaicin but no oral pungency. Furthermore, capsiate displayed antioxidant activity and inhibited angiogenesis and vascular permeability, and therefore, showed potential as a medicine and food supplement. Capsaicin is now commercially available, however capsiate is scarcely present in natural foods. We investigated the direct enzymatic conversion of a capsaicinoid to a capsinoid. It was observed that the rate of lipase-catalyzed acylation of vanillyl alcohol with nonanoic acid was faster than that of hydrolysis of N-vanillylnonanamide to vanillyl amide and nonanoic acid in n-hexane at 70°C. As a result, we performed a one-procedure synthesis of capsiate from capsaicin via lipase-catalyzed transacylation.

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.

Stereoselective reduction of carbonyl compounds with Actinomycete: purification and characterization of three alpha-keto ester reductases from Streptomyces avermitilis.

We achieved the purification of three alpha-keto ester reductases (Streptomyces avermitilis keto ester reductase, SAKERs-I, -II, and -III) from Streptomyces avermitilis NBRC14893 whole cells. The molecular masses of the native SAKERs-I, -II, and -III were estimated to be 72, 38, and 36 kDa, respectively, by gel filtration chromatography. The subunit molecular masses of SAKERs-I, -II, and -III were also estimated to be 32, 32, and 34 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The purified SAKERs-II and -III showed a reducing activity for alpha-keto esters (in particular, for ethyl pyruvate). SAKER-I showed a high reducing activity not only toward the alpha- and beta-keto esters, but also toward alpha-keto acid. The N-terminal region amino acid sequences of SAKERs-I, -II, and -III were identical to that of a putative oxidoreductase, SAV2750, a putative oxidoreductase, SAV1849, and a putative oxidoreductase, SAV4117, respectively, hypothetical proteins coded on the S. avermitilis genome.

Glucosylation of phenols and phenylalkyl alcohols using cultured plant cells.

Cultured plant cells of Eucalyptus perriniana can convert phenol and phenylalkyl alcohols [C(6)H(5)(CH(2))(n)OH, n=0-3] into the corresponding beta-D-glucopyranosides in a good yield. The cells preferentially glucosylated phenylmethanol (n=1, 59% yield) rather than phenol (n=0, 49%), 2-phenylethanol (n=2, 38%), and 3-phenylpropan-1-ol (n=3, 20%). On the other hand, 2-, 3-, and 4-hydroxyphenylmethanols were also glucosylated to (hydroxymethyl)phenyl beta-D-glucopyranosides and (hydroxyphenyl)methyl beta-D-glucopyranosides by cultured E. perriniana cells.

Stereoselective reduction of carbonyl compounds using the cell-free extract of the earthworm, Lumbricus rubellus, as a novel source of biocatalyst.

We found the reducing activity toward carbonyl compounds in the cell-free extract of the earthworm, Lumbricus rubellus. The earthworm extract had a reducing activity for keto esters in the presence of NADH or NADPH as a coenzyme. The earthworm extract reduced ethyl 3-methyl-2-oxobutanoate to the corresponding alcohol with a high enantiomeric excess (91%, R-form) at 50 degrees C in the presence of NADH. In particular, ethyl 2-oxoheptanoate was exclusively reduced to the corresponding (R)-hydroxyl ester with a high enantiomeric excess (>99%).

Enhancement of water-solubility and bioactivity of paclitaxel using modified cyclodextrins.

We investigated the aqueous solubility of paclitaxel using 11 kinds of cyclodextrins (CDs) and the bioactivity of the paclitaxel-CD inclusion complex. 2,6-Dimethyl beta-cyclodextrin was the most effective and its solubility was 2.3 mM in a 0.1 M 2,6-dimethyl beta-cyclodextrin aqueous solution. The inclusion complex of paclitaxel and 2,6-dimethyl beta-cyclodextrin had a 1.23-fold polymerization activity as paclitaxel in a tubulin assay.

An isozyme of earthworm serine proteases acts on hydrolysis of triacylglycerol.

An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.

Analysis of isoflavone content in tempeh, a fermented soybean, and preparation of a new isoflavone-enriched tempeh.

To produce a tempeh-like functional food containing a high level of isoflavone with a high absorptivity, we analyzed changes in the composition of isoflavone during tempeh fermentation and the difference in isoflavone content depending on the soybean variety and particular tissue. By adding soybean germ (hypocotyl) that contained a large amount of isoflavone, we prepared a new isoflavone-enriched tempeh in the form of a granular fermented soybean-based food, which can serve as a nutritious supplement for the elderly.

Purification and characterization of alpha-keto amide reductase from Saccharomyces cerevisiae.

An NADPH-dependent alpha-keto amide reductase was purified from Saccharomyces cerevisiae. The molecular mass of the native enzyme was estimated to be 33 and 36 kDa by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The purified enzyme showed a reducing activity not only for aromatic alpha-keto amides but also for aliphatic and aromatic alpha-keto esters. The internal sequence of the enzyme was identical with that of a hypothetical protein (ORF YDL 124w) coded by yeast chromosome IV.

A novel zinc-containing alpha-keto ester reductase from actinomycete: an approach based on protein chemistry and bioinformatics.

We have achieved the purification of an alpha-keto ester reductase (SCKER) from S. coelicolor A3(2) whole cells. SCKER proved to be a homotetramer of 132 kDa containing one equivalent of zinc ion per subunit. The enzyme differed from other alpha-keto ester reductases from microorganisms with regard to subunit structure and metal ion dependency. From a computer search using the protein data banks, the N-terminal amino acid sequence of SCKER was consistent with that of a possible zinc containing alcohol dehydrogenase in S. coelicolor A3(2). None of three hypothetical proteins of S. coelocor A3(2) having a high homology sequence with those of already purified alpha-keto ester reductases from S. thermocyaneoviolaceus [Yamaguchi, H., et al., Biosci. Biotechnol. Biochem., 66, 588-597 (2002)] was identical with that of SCKER.