When we studied the effects of polyphenols from apple fruits on melanogenesis in B16 mouse melanoma cell lines, phloridzin had dose-dependent progressive effects on melanogenesis between 10 and 500μg/ml without inhibiting cell growth. At a concentration of 500μg/ml, phloridzin increased the melanin content in the cells to 181% of that in control cells. In contrast, phloretin, the aglycon of phloridzin, did not activate melanogenesis in the cell and was cytotoxic at a concentration of 5μg/ml. Phloridzin increased the activity of tyrosinase to 223% of that in control cells. Furthermore, phloridzin inhibited the activity of protein kinase C (PKC), which is recognized to regulate tyrosinase activity. The inhibition of PKC activity continued for 120 min from the addition of phloridzin. Therefore, we estimated that the activation of melanogenesis by phloridzin resulted from the increase of tyrosinase activity caused by the inhibition of PKC activity.

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The antioxidants in Okara Koji (OK), an okara (OC) fermented by Aspergillus oryzae, γ-tocopherol, δ-tocopherol, genistin, daizein, genistein, and 3-hydroxyanthranilic acid were identified by HPLC. OK's extract with 80% methanol strongly inhibited linoleate peroxidation, much more than other OK's extracts with hexane or hot water. The methanol extract accelerated 12-hydroxyeicosatetraenoic acid formation in membrane lipids at 10^-3 concentration, but inhibited the formation at higher concentrations than 10^-3 ex vivo. To confirm the total effect of all components of OK on lipid peroxidation in vivo, rats fed food deficient in vitamin E were put on diets containing OK or OC with oxidized oil. In rats fed the OK diet, no effect of oxidized oil feeding on the body weight gain, of the TBA value in plasma, or of glutathione peroxidase activities of plasma and liver was observed. But in rats fed the OC diet, the effect of oxidized oil feeding was apparently observed on all of those values. These results suggested that OK would scavenge lipid peroxides in vivo.

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We examined linoleic acid hydroperoxide (hydroperoxyoctadecadienoic acid; HPOD) decomposition kinetically with or without various metal ions and at various pHs as effective factors on the stability of hydroperoxides. HPOD decomposition in the reaction system of this experiment was a first-order reaction. Manganese, copper, and especially iron accelerated the decomposition of HPOD, while lithium, sodium, potassium, magnesium, calcium, and aluminium stabilized HPOD. Besides, HPOD was comparatively stable at pH 3, 7, and 8, and unstable at pH 2, 4-6, and 9. According to activation energy, however, it was estimated that only in the reaction system with iron or at pH 2 and 9 the HPOD decomposition mechanism was different from that in water.

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The flavor of glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH) was examined by several sensory evaluations. The measurement of a point of subjective equality (PSE) showed that the peptide increases the flavor characteristics but did not affect the intensity of basic tastes, such as sweetness, saltiness, sourness, and umami. However, the threshold value of GSH decreased significantly in an umami solution containing 0.05% each of monosodium glutamate (MSG) and disodium inosinate (IMP). This suggests that GSH interacts with the umami substance and has a certain effect on the flavor. GSH had a characteristic kokumi flavor, such as continuity, mouthfulness, and thickness in the umami solution as well as in a model beef extract constructed from analyzed components at a concentration of 0.02% w/v. Some foodstuffs, including. meat, were found to contain GSH above its threshold value, which implicates the contribution of GSH to the flavor. The thermal degradation study suggested that a part of GSH have changed into its disulfide, pyroglutamic acid (PCA), and cyclocysteinylglycine in cooked foodstuffs.

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Tea catechins, (-)-epigallocatechin-3-gallate (EGCg) and (-)-epigallocatechin (EGC), have been reported to suppress oxidation of plasma low density lipoprotein (LDL) in vitro. If dietary catechins can be efficiently incorporated into human blood plasma, anti-atherosclerotic effects in preventing oxidative modification of LDL would be expected. In this study, a newly developed chemiluminescence detection-high pressure liquid chromatography (CL-HPLC) method for measuring plasma catechins was used and the incorporation of EGCg and EGC into human plasma was investigated. Healthy subjects orally ingested 3, 5, or 7 capsules of green tea extract (corresponding to 225, 375, and 525 mg EGCg and 7.5, 12.5, and 17.5mg EGC, redpectively). The plasma EGCg and EGC concentrations before the administration were all below the detection limit (< 2 pmol/ml), but 90 min after, significantly and dose-dependently increased to 657, 4300, and 4410 pmol EGCg/ml, and 35, 144, and 255 pmol EGC/ml, in the subjects who received 3, 5, and 7 capsules, respectively. Both EGCg and EGC levels detected in plasma corresponded to 0.2-2.0% of the ingested amount. Catechin intake had no effect on the basal level of endogenous antioxidants (α-tocopherol, β-carotene, and lycopene) or of lipids in plasma. These results suggested that drinking green tea daily would contribute to maintain plasma catechin levels sufficient to exert antioxidant activity against oxidative modification of lipoproteins in blood circulation systems.

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Expression of a membrane-bound hydrogenase of the hydrogen-utilizing bacterium Pseudomonas hydrogenovora was induced specifically in autotrophic condition. Dot blot and primer extension analysis showed that the transcription of the hydrogenase gene started from the region upstream of a hydrogenase structural gene, hupS, which contained three putative σ⁵⁴-type promoter sequences (P_hup1, P_hup2, and P_hup3). The lacZ-fusion analysis of the hupS-upstream region combined with site-directed mutagenesis showed that P_hup1 and P_hup3 would be essential for a transcriptional initiation. It was also found that the region upstream of P_hup sequences was concerned with regulation of transcription.

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1, 2-Di-O-isopropylideneglycerophosphorochloridate prepared from isopropylindene glycerol and phosphorus oxychloride, was allowed to react with Z-serine-N-phthalimidomethyl ester to obtain a derivative of phosphatidylserine. Then, after the isopropylidene group was removed by Amberlite TR-120 (H⁺), and the phosphate group was also blocked as a Ba-salt, this derivative was coupled with docosahexaenoic acid, applying the method of activated ester. Removal of both protective groups of serine was finally done by dry hydrogen chloride in chloroform.

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Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon. Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level. The course of mRNA accumulation paralleled that of PAL enzyme activity. The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity. The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1. In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated. Genomic Southern blot analyses showed that both genes are present in the genome as single copies.

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Bacillus sp. KSM-522 produces three different extracellular endo-1, 4-β-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83-89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser²⁶ through Lys⁶³⁶) was calculated to be 69, 076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50°C, respectively. By ¹H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl β-D-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.

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β-Xylosidase from Aspergillus awamori K4 was purified. The optimum pH and temperature were around pH 4 and 70°C, and the molecular weight was estimated to be 117, 000 on SDS-PAGE analysis. The enzyme has broad acceptor specificity in transxylosylation. Especially, its acceptor accessibility for sorbitol and mannitol of sugar alcohols were higher than that for monosaccharides. Trehalose was a much more effective acceptor than maltose and lactose of other disaccharides. In the reaction with 13-14% xylooligosaccharides (consisting of 3.4% xylose, 67.9% xylobiose, and 28.7% xylotriose) and 9-13% acceptors (sorbitol, mannitol, and trehalose), the amount of transfer products for each acceptor was 7-11%, in 24 h. On ¹H- and ¹³C-NMR analysis, main transfer products with sorbitol and mannitol were 6-O-β-xylosyl sorbitol (77.3%) and 1(6)-O-β-xylosyl mannitol (73.7%), respectively. Two products with trehalose were 6(6')-O-β-xylosyl trehalose (52.1%) and 6, 6'-O-β-di-xylosyl trehalose (47.9%).

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The caries inhibitory effect of the hydrogenated derivative of an isomaltooligosaccharides mixture (IMO-H) was examined in vitro and in vivo experiments. IMO-H could not be used as a substrate for the crude glucosyltransferases (GTases) of Streptococcus sobrinus 6715 to synthesize water-insoluble glucan. Moreover, it not only significantly inhibited the synthesis of water-insoluble glucan from sucrose, but also the sucrose-dependent adherence of these growing cells the glass surfaces. In the in vivo experiment, the addition of IMO-H to a sucrose-containing diet resulted in significant reduction of caries development in specific-pathogen-free (SPF) rats infected with S. sobrinus 6715.

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The BC-ring system of ciguatoxin was stereoselectively synthesized by 12 steps from methylα-D-glucopyranoside.

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5-Aminolevulinic acid (ALA) promoted the growth and yield of several crops and vegetables at concentrations lower than those eliciting herbicidal responses, i.e., less than 1.8 mM by foliar spray and 60μM by root soaking. To evaluate the physiological action of ALA, the effects of ALA on plants were examined by several bioassay systems at 0.0006-600μM. ALA at 0.06-6μM by root soaking increased the growth of rice seedlings in light, but did not affect this in darkness. In horseradish shoot primordia, promotion by ALA was not proportional among total chlorophyll content, chlorophyll concentration, and fresh weight. In the test using pothos, ALA at 0.06μM elicited the accumulation of chlorophyll, but the photosynthesis of the plants was promoted by treatment together with ALA and nutrients. These results suggest that ALA have a variety of plant physiological effects on chlorophyll synthesis, photosynthesis, and plant growth, and ALA acts as a growth regulator in plants at low concentrations. These effects of ALA were also assumed to be linked to light irradiation and an uptake of fertilizer by plants. However, excess ALA suppressed these effects.

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A conjugated enzyme system, alanine dehydrogenase (AlDH) for stereospecific reduction of pyruvate to L-alanine and glucose dehydrogenase (GDH) for regeneration of NADH, were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of L-alanine from pyruvate with NADH regeneration. Since pyruvate was proved to be unstable at neutral pH, it was kept under acidic conditions and supplied to NFMBR separately from the other substrates. As 0.2 M pyruvate in HCl solution (pH 4), 10 mM NAD, 0.2 M glucose, and 0.2 M NH₄Cl in 0.5 M Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized AlDH (100 U/ml) and GDH (140 U/ml) at the retention time of 80 min, the maximum conversion, reactor productivity, and NAD regeneration number were 100%, 320 g/liter/d, and 20, 000, respectively. To avoid the effect of pyruvate instability, a consecutive reaction system, lactate dehydrogenase (L-LDH) and AlDH, was also used. In this system, the L-LDH provides pyruvate, the substrate for the AlDH reaction, from L-lactate regenerating NADH simultaneously, so the pyruvate could be consumed as soon as it was produced. As 0.2 M L-lactate, 10 mM NAD, 0.2 M NH₄Cl in 0.5 M Tris buffer (pH 8) were continuously supplied to. NFMBR with immobilized L-LDH (100 U/ml) and AlDH (100 U/ml) at the retention time of 160 min, the maximum conversion, reactor productivity, and the NAD regeneration number were 100%, 160 g/liter/d, and 20, 000, respectively.

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We measured ubiquinone (CoQ₀) and hydrogen peroxide (H₂O₂) formed in the process of oxidation of ubiquinol (CoQ₀H₂). We found that copper-zinc superoxide dismutase and manganese superoxide dismutase inhibited both the CoQ₀ formation and the H₂O₂ formation only in the presence of chelators such as DTPA (diethylenetriaminepentaacetic acid). The amount of H₂O₂ was almost equal to that of CoQ₀, indicating that the H₂O₂ formation was coupled with the CoQ₀ formation. The lack of inhibitory effects of the corresponding heat-inactivated superoxide dismutase (SOD) confirmed that the inhibition by the original SOD was due to its enzymatic activity. We propose that CoQ₀H₂ oxidation occurs as a chain reaction with superoxide as the chain carrier and that SOD inhibits this reaction by lowering the superoxide concentration.

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Human Cu, Zn-superoxide dismutase (SOD) was incubated with various intermediates of the Maillard reaction and glycolytic pathway (arabinose, glyoxal, glycolaldehyde, glyceraldehyde, glyceraldehyde 3-phosphate, and dihydroxyacetone) and some reducing sugars (sorbose, xylose, and ribose). The change of the activity and the molecular weight were measured and compared with that of SOD incubated with glucose or fructose. Sorbose, xylose, and ribose decreased the activity with a rate comparable to fructose. Site-specific and random fragmentation were observed upon the incubation with them. Arabinose showed a similar inactivation rate as glucose. The intermediates other than arabinose had a high inactivation rate. Especially, glyceraldehyde, glycolaldehyde, and glyoxal most strongly lowered the activity in a concentration-dependent manner and a significant inactivation was recognized even at 1 mM level. SDS-PAGE band patterns indicated that the inactivation by those carbonyl compounds occurred by both crosslinking and site-specific fragmentation of SOD.

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8'-Methylene- and 8'-methylidyneabscisic acids which might act as suicide inhibitors of the 8'-hydroxylase of abscisic acid, were designed, synthesized, and optically resolved. The (+)-isomers showed stronger inhibitory activity in rice elongation and in lettuce seed germination than (+)-abscisic acid. The activity of (+)-8'-methylidyneabscisic acid was the strongest of the analogues synthesized to date, 40-fold stronger than abscisic acid.

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N-Acetylneuraminate (NeuAc) synthase, which catalyzes NeuAc synthesis by condensation of N-acetyl-D-mannosamine (ManNAc) and phosphoenolpyruvate (PEP), was purified from a cell extract of Escherichia coli K1-M12 to electrophoretically homogeneity by serial column chromatographies. The molecular weight of native enzyme was estimated to be 106, 000 by gel filtration. After denaturation in sodium dodecyl sulfate, the molecular weight was reduced to 52, 000, indicating the existence of 2 identical subunits. The optimum pH was 7.5 and the stable pH range was 7.0 to 10.0. The enzyme was thermostable up to 30°C. No metal ion was required for the enzyme activity. SH-inhibitors such as p-chloromercuribenzoic acid and mercury chloride were potent inhibitors. The K_m for ManNAc and PEP were 5.6 mM and 0.04 mM, respectively.

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To study the effects of 13-L-hydroxylinoleic acid (LOH) and food additives on γ-aminobutyric acid (GABA) receptors, ionotropic GABA receptors were expressed in Xenopus oocytes by injecting mRNAs prepared from rat whole brain. LOH, which was prepared by reduction of 13-L-hydroperoxylinoleic acid (LOOH), inhibited the response of GABA receptors in the presence of high concentrations of GABA. LOH also inhibited nicotinic acetylcholine, glycine, and kainate receptors, while it had little effect on NMDA receptors expressed in Xenopus oocytes. However, LOH potentiated the response of GABA receptors as well as LOOH in the presence of low concentrations of GABA, possibly increasing the affinity of GABA for the receptors, while linoleic acid did not. Since some modification of the compounds seemed to change their effects on GABA receptors, the responses of GABA receptors elicited by 10 μM GABA were measured in the presence of compounds with various kinds of functional groups or the structural isomers of pentanol. Potentiation of GABA receptors depended strongly on the species of functional groups and also depended on the structure of the isomers. Then effects of various kinds of food additives on GABA receptors were also examined; perfumes such as alcohols or esters potentiated the responses strongly, while hexylamine, nicotinamide, or caffeine inhibited the responses, mainly in a competitive manner, and vanillin inhibited the responses noncompetitively. These results suggest the possibility that production of LOOH and LOH, or intake of much of some food additives, modulates the neural transmission in the brain, especially through ionotropic GABA receptors and changes the frame of the human mind, as alcohol or tobacco does.

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Styrene oxide isomerase (SOI) [EC 5.3.99.7], most probably located in the cell wall, was partially purified from Corynebacterium sp. AC-5 cells grown in a styrene gas atmospheres. The enzyme catalyzed the isomerization reaction to give phenylacetaldehyde, but did not catalyze its reverse reaction. The optimum pH of the reaction was around 7.0, and the enzyme was unstable below pH 6.0. The K_m toward styrene oxide was very low (7.7×10^-5 M), indicating its high affinity for styrene oxide. The enzyme showed strict substrate specificity, and epoxide compounds other than styrene oxide did not serve as substrates. (S)-Styrene oxide was preferentially converted by the enzyme, compared with the (R)-isomer. The possible application of SOI as a biocatalyst is also discussed.

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The phase behavior was investigated in water/phytoglycogen/SDS and C₁₂EO₂₀ systems. It was found that phytoglycogen was precipitated in the presence of these surfactants. Phytoglycogen in the SDS system was dispersed more homogeneously than in the C₁₂ EO₂₀ system, and a liquid-liquid phase separation region appeared in the SDS system. In the C₁₂EO₂₀ system, the transmittance of a stirred solution in the single-phase region was slightly lower than that of the phytoglycogen controls, whereas the transmittance of the stirred solution was higher than that of the controls in the SDS system. It thus seems that phytoglycogen formed a complex with SDS and that this complex affected the transmittance in the single-phase region. Viscosity measurements of the aqueous solution supported the existence of such a phytoglycogen-SDS complex. These results enabled us to propose a schematic representation of the complex structure. It was clarified that phytoglycogen forms a complex with SDS, and that the structure of the complex modifies the dispersion stability of phytoglycogen.

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The autoxidation reaction of L-ascorbic acid (ASA) in methanol without metal ion catalysis was studied. Besides L-threonolactone (THL) and oxalic acid (OXA), methyl L-threonate, and threonic acid were identified as initial autoxidation products of ASA, which were the C(2)-C(3) fission product via the C(2) oxygen adduct of ASA. This pathway is different from the one via dehydro-L-ASA (DASA), which has long been believed to be the only oxidation pathway of ASA. It was confirmed that this reaction also occurred in both water and other polar solvents, including methanol. It was clarified that mono-dissociated ASA was more reactive than the non-dissociated ASA in this pathway, and that the main reaction products formed from these two forms of ASA were also somewhat different. Determination of the amount of remaining ASA and the yields of THL and OXA, C(2)-C(3) fission products, and of DASA were carried out doing the autoxidation of ASA under various reaction conditions.

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The gene encoding an extracellular levan fructotransferase, designated the lft gene, was cloned from the genomic DNA of Arthrobacter nicotinovorans GS-9, and expressed in Escherichia coli. It was found that a single open reading frame consisted of 1554 base pairs that encoded a polypeptide composed of a signal peptide of 33 amino acids and a mature protein of 484 amino acids (M_r 53, 152), and it was also found that a putative ribosome-binding site was present in the upstream from the ORF. The primary structure had no significant similarity with those of inulin fructotransferases, but had low similarity to the catalytic regions of other fructosylhydrolases. The expression of the lft gene was increased on a plasmid, pLFT-BB1, in which the lft gene was fused with α-peptide of the lacZ gene of pUC18. An E. coli transformant carrying pLFT-BB1 expressed six times as much activity of levan fructotransferase as that of the original strain, A. nicotinovorans GS-9.

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A mixture of purified polyphenol oxidases (PPO), or acetone powders prepared from fruits and vegetables, and polyphenolic compounds (PPs) totally eliminated a methylmercaptan odor. 2-Methyl-thiochlorogenic acid was isolated from the reaction mixture of methylmercaptan and chlorogenic acid with burdock acetone powder. Further, the formation of 5-methylthiochlorogenic acid and 2, 5-bis(methylthio)-chlorogenic acid was suggested. These facts demonstrate that the o-quinone compounds formed from o-diphenols by PPO rapidly reacted with methylmercaptan. The oxidation reaction of PPs by using acetone powder containing PPO or peroxidase is considered to be more effective for removing bad smells from our mouths and from the environment.

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The peroxidative stability of docosahexaenoic acid (DHA)-containing oils (DHA at 10.7 mol% of the total fatty acids), in the form of phospholipids (PL), triacylglycerols (TG), and ethyl esters (EE) with the same constituent fatty acids, was investigated in the dark at 25°C in a bulk phase, and compared with that of control palm oil (supplemented with 20% soybean oil). The oxygen absorption of the DHA-containing oil was significantly lower in the form of PL than in the form of TG and EE during a 10-week oxidation, and the oxygen absorption of PL was almost equivalent to that of the control oil. A gas chromatographic analysis showed that 90% of initial DHA was retained in the form of PL after the 10-week oxidation, while TG and EE respectively more rapidly decayed with the loss of 97% and 64% of DHA. Tocopherol in the form of TG and EE had also completely decayed after the oxidation, while 37% of the initial tocopherol remained in the form of PL. The peroxide and carbonyl values of TG and EE showed large increases after the oxidation, but no such increase was observed for PL. These results show that DHA-containing oil in the form of PL was more resistant to the oxidative degradation of DHA than that in the form of TG and EE in a bulk phase.

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Dietary lipid peroxidation products cause endogenous lipid peroxidation with hepatic dysfunction. In this study, we isolated and cultured hepatocytes of rats that were given secondary autoxidation products of linoleic acid (p.o., 400 mg/rat/day for 3 days), and examined the hormonal responses of these hepatocytes. An increase in thiobarbituric acid reactive substances and a depletion of vitamin E persisted in hepatocytes from treated rats for at least 24 h in culture as compared to those from control rats. As markers for hepatic dysfunction, the activities of six enzymes were measured. In each case, there was an initial decrease in the enzyme activity in hepatocytes from the treated rats, and all activities were restored by 48h in culture. Then, we measured the hormonal responses of these hepatocytes. The responses to insulin or glucagon in hepatocytes from secondary products-treated and control rats were the same. In contrast, the response to dexamethasone was significantly lowered in hepatocytes from secondary products-treated rats as measured by the induction of tryptophan 2, 3-dioxygenase and tyrosine aminotransferase. We conclude that primary cultured hepatocytes from the rats treated in vivo with dietary lipid peroxidation products retained symptoms of oxidative stress and had a low response to glucocorticoids.

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Staphylococcus aureus V8 (ATCC 49775) produces Panton-Valentine leukocidin (PVL) and leukocidin (Luk). PVL and Luk consist of LukF-PV and LukS-PV, and LukF and LukS, respectively. LukF and LukS cooperatively and strongly lyse rabbit erythrocytes besides human and rabbit polymorphonuclear leukocytes. LukF and LukS-PV also cooperatively lysed rabbit erythrocytes, but its activity was only 4% of that in combination [LukF-LukS] after 15 min of incubation. To identify the pivotal region responsible for the hemolytic function of LukS towards rabbit erythrocytes, we created a series of chimeric genes (lukS-PV/lukS) and mutant genes of lukS-P V and had them expressed in Escherichia coli. The chimeric and mutant proteins purified from the sonicated extract from the cells of E. coli were assayed for their hemolytic activities towards rabbit erythrocytes in combination with LukF or LukF-PV. The results indicate that a 2-residue segment (D¹²I¹³) of lukS is the minimum region essential for the hemolytic function of LukS towards rabbit erythrocytes.

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A protease has been purified from sarcocarp of musk melon, Cucumis melo ssp. melo var. reticulatus Naud. Earl's Favourite. The protease was mostly present in the placenta part of the fruit and next in the inside mesocarp. The molecular mass of the enzyme was estimated to be about 62 kDa on SDS-PAGE. The enzyme had a carbohydrate moiety. The optimum pH of the enzyme was 11 at 35°C using casein as a substrate. The enzyme was stable between pH 6 and 11. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but was not inhibited by EDTA or cysteine protease inhibitors. From the digestion of Ala-Ala-Pro-X-pNA (X = Phe, Leu, Val, Ala, Gly, Lys, Glu, Pro, and diaminopropionic acid (Dap) substrates the specificity of the protease was found to be approximately broad, but the preferential cleavage sites were C-terminal sites of hydrophobic or acidic amino acid residues at P₁ position. It was proved that the enzymatic properties of musk melon protease are similar to those of cucumisin [EC 3.4.21.25]. The enzyme was not inhibited by typical proteinous inhibitors such as STI or ovomucoid. Therefore, this enzyme seems to be a useful protease for the food industries.

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Synthesis and fungicidal activities of new 6-alkyl six-membered cyclic phosphates were examined. Ten kinds of 6-alkyl six-membered cyclic thiophosphates were synthesized by reaction with 5-alkyl-2-hydroxybenzyl alcohols and phosphoric agents. Among the prepared compounds, 2-ethoxy-6-ethyl-4H-1, 3, 2-benzodioxaphosphorin 2-sulfide (1) had activity as potent as the commercial fungicide iprobenfos against Pythium sp. and Corticium rofsii at 10 ppm.

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The superoxide-scavenging and the tyrosinase-inhibitory activities of 28 kinds of plants used as Chinese medicines were evaluated. Methanol/water extracts were used for the screening tests, and for those which represented high activities, other kinds of extracts were also studied. The extracts of Mallotus japonicus Muell. Arg. scavenged superoxide strongly; the half-inhibiting concentration (IC₅₀) of its 50% methanol/water extract was 10.57μg of dried material in 1 ml of reaction mixture. The extracts of Fritillalia thunbelgii Miq., Carthamus tinctorius L., and Prunus persica (L.) Batsch had strong tyrosinase-inhibitory activities, and the extracts of Scutellaria baicalensis Georgi represented both kinds of activities. These facts suggested that Chinese medicines may be a treasure house of chemical compounds that have the superoxide-scavenging and the tyrosinase-inhibitory activities.

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Reaction products of 4-methylthio-3-butenyl isothiocyanate (MTBI), the radish pungent principle, in aqueous media were identified and their antimicrobial activities were examined. A rapid degradation of MTBI in aqueous media afforded a mixture of 3-(hydroxy) methylene-2-thioxopyrrolidine (1), (Z)-3-(methylthio)-methylene-2-thioxopyrrolidine (2), its (E-isomer (3), methyl 4-methylthiobutyldithiocarbamate (4), methyl (Z)-4-methylthio-3-butenyldithiocarbamate (5), and its (E)-isomer (6). The products 1, 2, and 3 were detected at all pHs examined, while 4, 5, and 6 were formed at pH over 6.0. The formation of 4 from 6 was accompanied by an oxidation of methanethiol released from MTBI in aqueous media. Antimicrobial activities of 2 and 3 against all microbes examined were much lower than that of 1, which had MICs ranging from 50 to 400μg/ml. As for 4, 5, and 6, antifungal activities were comparable to that of 1, but little antibacterial activities were observed. The antimicrobial activities of the six products were considered to be far lower than that of MTBI.

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The enzyme activity and the messenger RNA level of pineal serotonin N-acetyltransferase were more than 20- and 50-fold higher, respectively, in the dark period than in the light period in normal rats. In hereditary microphthalmic rats, however, the serotonin N-acetyltransferase activity and its mRNA level did not undergo a great diurnal change through the light and dark periods. These results indicate that the diurnal rhythms of the activity and the mRNA level of serotonin n-acetyltransferase are not detected in the pineal gland of hereditary blind rats, suggesting free-running rhythms in individual animals due to desynchronization of their circadian rhythms by a lack of their optic nerve.

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We have investigated the effects of niacin on NAD levels and on DNA repair in human lymphocytes. When lymphocytes were incubated in culture medium with various concentrations of niacin, incubation of lymphocytes with nicotinic acid at 5μM or nicotin-amide at 10 mM caused a 2-3 fold increase in NAD content. Under these conditions lymphocytes were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Interestingly, the rejoining of DNA strand breaks was promoted by nicotinic acid but nicotinamide inhibited the rejoining.

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The regulatory region of Thiobacillus ferrooxidans leuB gene was analyzed. The deletion analysis indicated that the promoter sequences CATCCG and TATTAT, which were similar to the consensus -35 and -10 sequences of Escherichia coli, respectively, existed. The transcriptional initiation site was located at the position of cytosine -26. The deletion analyrsis of the upstream region suggested the existence of a regulatory region by leucine and the region related to transcription of the gene.

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Insulin stimulates tyrosine phosphorylation of 175-195 kDa proteins including insulin receptor substrate-1 (IRS-1) in various tissues and cell types. In intact rat livers, starvation increased the insulin-dependent tyrosine phosphorylation of the insulin receptor and IRS-1 as has been described by others. Surprisingly, starvation greatly increased the tyrosine phosphorylation of the 195-kDa protein induced by insulin, indicating that this protein may be a new substrate of the insulin receptor kinase. The marked increase in tyrosine phosphorylation of the 195-kDa protein may have a physiological role in signal transmission in response to insulin under starvation conditions.

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The cytoplasmic and periplasmic production systems of ovotransferrin N-lobe in Escherichia coli were constructed. The periplasmic, but not cytoplasmic product, was found to have the iron-binding function.

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From methanol extracts of fresh leaves of Psiadia punctulata (DC.) Vatke, lettuce seed radicle growth inhibitors were isolated and identified as E- and Z-eicosanyl p-coumarates by spectroscopic analyses.

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High restriction endonuclease activity was found in a haemolysin-less mutant of the Vibrio parahaemolyticus 1743-1 strain. The endonuclease, named VpaK11BI, recognized the palindromic pentanucleotide sequence of 5'-GGWCC-3' and cleaved double-stranded DNA after the first G, which is exactly the same as the specificity of AvaII. The haemolysin-less mutant of V parahaemolyticus is now available for producing the valuable restriction endonuclease on a commercial scale.

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The 185 nucleotide 5' untranslated region (5'UTR) of potato virus Y ordinary strain (PVY-O) showed translation-enhancing activity on the β-glucuronidase (GUS) gene in tobacco protoplasts. Mutational analysis of the 5'UTR was done to find sequence motifs necessary for the enhancement. Deletions within the 1-130 nucleotide region of 5'UTR stimulated the GUS expression in some cases, while the GUS activity declined with deletions in the 131-185 nucleotide region. The results indicated that the last 55 nucleotides of PVY-O 5'UTR might play the much important role in the translational enhancement in plant cells.

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A cell-free system for converting tryptophan precursor into violacein pigments is reported. Crucial factors were the requirements of the reduced nicotinamides as a cofactor and Zn²⁺ as a metal ion. Optimal pHs were in the range of 8.5-9.5. The effectiveness of NADH was thirty times lower than that of NADPH at a low concentration of 2 mM. The oxygenation mechanism(s) is discussed by using oxygenase inhibitors such as amethopterin, metyrapone, ancymidol, and prohexadione. Metal-chelating agents strongly inhibited the biosynthesis, suggesting that metallo-enzymes are involved in the biosynthesis.

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Vitamin B₁₂ is destroyed by the addition of substantial amounts of vitamin C in the presence of copper. Effects of carnosine and anserine, natural water-soluble antioxidants, on the destruction of vitamin B₁₂, were studied. Addition of carnosine (10mM) effectively repressed the destruction of vitamin B₁₂, but anserine had only weak inhibitory effects.

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Two chitinase isoforms, designated GBC-a and GBC-b, were purified from the bulbs of gladiolus (Gladiolus gandavensis) using CM-cellulose column chromatography followed by Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S FPLC. GBC-a and GBC-b are weakly acidic and weakly basic proteins with molecular masses of 30 kDa, and isoelectric points of 6.0 and 7.5, respectively. GBC-a and GBC-b were found to be homologous proteins with similar amino acid compositions and N-teriminal sequences. The number of half-cystine residues in GBC-a and GBC-b was only one each, which is much lower than those of plant class I (15-17 Cys residues/mol), class II (5-8 Cys residues/mol), and class III (6 Cys residues/mol) chitinases. The N-terminal sequences of GBC-a and GBC-b were completely different from those of plant three classes of chitinases. The optimal pHs of these chitinases toward glycolchitin were pH 5. GBC-a hydrolyzed (GlcNAc)₆ into (GlcNAc)₂, (GlcNAc)₃ and (GlcNAc)₄, and (GlcNAc)₅ into (GlcNAc)₂ and (GlcNAc)₃.

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A cDNA for early light-inducible protein (ELIP) was isolated from Glycine max L. and the nucleotides were sequenced. The cDNA encodes a 192-residue polypeptide of 20.3 kDa. The deduced amino acid sequence included a transit peptide in the amino-terminus and three hydrophobic regions long enough to span the thylakoid membranes, and had a high similarity to those of ELIPs and related polypeptides from other plants.

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All 4 Schizophyllum commune strains among several basidiomycete species were exclusively resistant to flutolanil, which inhibits succinate dehydrogenase complex (SDC) function. Flutolanil resistant strains could be screened from monokaryotic protoplasts of sensitive Coprinus cinereus, by treating them with a cloned genomic SDC iron-sulfur protein (IP) gene from the resistant S. commune. The obtained resistance was stably transferred to the progeny.

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A wild-type soybean contains three major allergenic proteins, Gly m Bd 30K, the α-subunit of conglycinin, and Gly m Bd 28K. A genetically mutated soybean (Tohoku 124), which was originally developed as a cultivar lacking the α- and α'-subunits of conglycinin, was also found to lack Gly m Bd 28K from immunoblot analysis using monoclonal antibodies specific to Gly m Bd 28K. This finding indicates the possibility to prepare soy milk and soy proteins containing none of the three major allergenic soybean proteins from this cultivar. By applying the previous removal procedure [Samoto et al., Biosci. Biotech. Biochem., 60, 1911-1913 (1996)] to Tohoku 124, the substantially complete removal of the three major allergenic proteins from the soy milk was attained. The removal rates of Gly m Bd 30K, α-subunit of conglycinin, and Gly m Bd 28K were 99.8, 100, and 100%, respectively.

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Post-translational modification of core histones is essential in processes requiring chromatin remodeling. We report here a novel modification in histones of the sperm of the starfish, Asterina pectinfera, which involves an ε-(γ-glutamyl)lysine cross-link between the glutamine residue at position 9 of histone H2B and the lysine residue at position 12 of histone H4.

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A new pollen growth inhibitor, named simplicissin, was isolated from Penicillium cf. simplicissimum (Oudemans) Thom No. 410, and its structure was established by spectroscopic methods including 2D NMR. The biological activities of the compound were examined by the bioassay methods involving tea pollen together with lettuce seedlings. The compound inhibited the growth of the tea pollen tube by 45% at a concentration of 3mg/liter and showed complete inhibition at 10 mg/liter.

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