Two novel highly water-soluble tetrazolium salts, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) and WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) were applied to the assay of superoxide dismutase (SOD). The superoxide anion generated by xanthine/xanthine oxidase (XO) reduced WST-1 and WST-8 to water-soluble formazans which exhibited absorbance maxima at 438 and 460 nm, respectively. The rates of reduction were linearly related to the XO activity, and reduction was inhibited by SOD. Complete inhibition by SOD of the reduction of both WST-1 and WST-8 was achieved, suggesting that these WSTs were not reduced with XO. WST-1 was found more useful than WST-8 because it had shown higher sensitivity which was apparently not dependent on the assay pH value in the range pH 8.0-10.2. These properties of WST-1 are ideal for the spectrophotometric assay of SOD in an aqueous system.

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  An improved bioassay for measurement of peroxyl radical scavenging activity^1,2) is described. The modifications included the use of (1) Enterococcus faecium strain S, (2) 25% dimethyl sulfoxide, (3) L-ascorbic acid for the termination of the bactericidal reaction, and (4) the concentration of antioxidants that gives 75% of the log (colony forming unit/ml) of the control reaction (without methemoglobin or tert-butyl hydroperoxide), for the expression of antioxidant activity.

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  4,5-Dihydro-7H-pyrano[3,4-c]isoxazoles (II and III) with an o-chlorophenyl or p-chlorophenyl group at C-7 were synthesized and the effect of substitution at C-3 of II and III on fungicidal activity was investigated in vivo. When the substituent at C-3 of II and III was CH₂Br, CH=NOMe, CH=NOEt or CH=NO-allyl, the fungicidal effect was significant and selectively high on wheat leaf rust and barley powdery mildew at 250 ppm. Compound IId with the CH₂Br substituent at C-7 showed high fungicidal activity against rice blast, providing more than 90% control of the disease at 2 ppm.

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  From the seeds of Ginkgo biloba, a glycoprotein, which is a major component that reacts with an antiserum against β1→2 xylose-containing N-glycans, has been purified and characterized. The N-terminal amino acid sequence of the purified glycoprotein was H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-L-L-F-G-Q-. The molecular mass was estimated to be 17 kDa and 16 kDa by SDS-PAGE under reducing conditions, however, the molecular mass of this glycoprotein in the native state was 30,762 by MALDI-TOF MS, suggesting that this glycoprotein consists of two subunits; one is glycosylated and the other is not. The structure of N-glycan linked to this glycoprotein (designated 30 kDa GBGP) was identified as Man₃Fuc₁Xyl₁GlcNAc₂, which is the predominant N-glycan linked to the storage glycoproteins in the same seeds (Kimura, Y et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261). From the peptic digest of the carboxymethylated glycosylated subunit, one glycopeptide was purified by RP-HPLC and the amino acid sequence was identified as H-K-A-N-N(Man₃Fuc₁Xyl₁GlcNAc₂)-V-T-V-A-F, which corresponded to the N-terminal amino acid sequence.

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  To obtain strains of Lactobacillus delbrueckii subsp. bulgaricus with high immunopotentiating activity, we screened 90 strains of this bacterial species for the proliferative response of murine spleen and β-lactoglobulin-primed lymph node cells. In this screening, certain strains showed strong immunopotentiating activity. Among them, strain 1023 had the strongest mitogenic activity for murine Peyer’s patch (PP) cells. Furthermore, strain 1023 induced IgA antibody production by PP cells as strongly as Bifidobacterium longum 6001, which had adjuvant activity when orally administered. Also in the assays using immune cells from human-flora-associated mice a few strains including 1023 showed strong immunopotentiating activity comparable to B. longum 6001. These results suggest that L. delbrueckii subsp. bulgaricus strains such as 1023 may be useful for the production of fermented milk with a more beneficial effect on the systemic and mucosal immune system.

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  The effects of reducing agents on solubilization and activation of the debranching enzyme (pullulanase) were examined using rice flour. The activity of the debranching enzyme was observed in a buffer solution (pH 7.5) in which rice flour was incubated together with thiol-reducing reagents, (dithiothreitol, 2-mercaptoethanol etc.), but there was only low activity in the absence of reducing agents. Immunochemical measurement and the specific activity of the enzyme showed that the activation caused by the reductant was due to solubilization of the enzyme protein besides the enzyme activation.

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  The biosynthetic relationship between acutumine 1 and dechloroacutumine 2 was studied using ¹³C-labeled tyrosine and ³H-labeled 2 as tracers. ¹³C-NMR spectra of ¹³C-labeled 1 and 2 showed that the alkaloids, each composed of two molecules of tyrosine, are derived from the same biosynthetic pathway. Feeding Menispermum dauricum (Menispermaceae) roots, cultured in a chloride-enriched medium, with ³H-labeled 2 demonstrated that 1 is the only alkaloid metabolite of 2. Conversion (5%) of the exogenously applied 2, taken up by the roots, into 1 showed that 2 is the precursor of 1. Incomplete conversion of 2 into 1 suggests accumulation of the exogenously applied 2 in cell organelles and/or compartmentation of the enzymes involved in the biosynthesis of 1.

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  Several genes that may be involved in embryogenesis have been isolated from somatic embryos of carrot by many workers. However, the function of these genes has not been discovered yet. As the first step toward finding the function of these genes, we established a rapid and efficient method for transformation of carrot by using direct embryogenesis from hypocotyl segments treated with 2,4-dichlorophenoxyacetic acid (2,4-D) for a short period.

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  1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M_r 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the K_m for S-adenosyl-L-methionine of 1.74 mM and k_cat of 0.56 s^-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M_r 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a K_m for ACC of 4.8 mM and k_cat of 3.52 s^-1. The presence of 7 mM Cu²⁺ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.

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  Recombinant human prorenin was activated by incubation with anti-prorenin prosegment (L^1PPTDTTTFKRIFLKR^15P) antiserum at 4°C. This activation was dependent on the concentration of the antiserum and incubation time. After the activation no molecular weight alteration of prorenin was observed by immunoblotting analysis. A peptide of L^1PPTDTTTF^8P as well as L^1PPTDTTTFKRIFLKR^15P potently interfered with the activation. Most of the activated prorenin bound to Protein A Sepharose CL 4B. The K_m and V_max values of the activated prorenin were 0.2 μM and 23.7 μg Ang I/ml/h, respectively, which were similar in level to those of mature renin obtained by trypsinization.

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  Polyphenolic compounds derived from tea catechins were examined for apoptosis-inducing activity in human histiolytic lymphoma U937 cells. (−)-Epigallocatechin gallate, theasinensin D, compound OH-5, theaflavin, and theaflavin digallate induced apoptosis as evidenced by DNA ladder formation, its inhibition by a caspase inhibitor, and chromatin condensation. Theasinensin D was the most potent inducer and the data suggest the importance of the number and three dimensional localization of their phenolic groups in this activity. These apoptosis-inducible compounds may be useful as a cancer chemopreventive and chemotherapeutic agent.

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  The Bacillus subtilis 168 genome is being used to clone DNA segments, particularly unmarked DNA segments between the two available clones. To facilitate this cloning, a counter selection method was developed in which loss of a cI repressor gene rendered the host strain resistant to neomycin. This method is promoted to use the B. subtilis genome as a general cloning vehicle.

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  Brachyury (T) is involved in mesoderm induction during early mouse development. We analyzed the region regulating expression of T in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro. Transfection of plasmids encoding reporter genes under the control of the 5′-flanking region showed positive regulatory elements between -298 and -129 bp are responsible for driving T expression in mesodermal cells.

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  Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.

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  A lowered subjective evaluation of the taste and flavor of beer due to staleness or to the addition of an unpleasant taste and flavor was found to be closely correlated with the urination rate. Beer in the same lot was compared immediately after shipment from the brewery and after leaving at room temperature for 1 month or 5 months. Each beer sample was given to volunteers at the rate of 3 ml/kg/15 min for 2 hours, and the urine volume was measured every 30 minutes. The urination rate was highest from the volunteers who drank fresh beer and lowest from those who drank 5-month-old beer. The subjective evaluation of both the taste and drinkability of 5-month-old beer was significantly lower than that of fresh beer. Beer samples with various unpleasant taste and flavor substances added lowered the urination rate. The results suggest that the perception of an unpleasant taste and off-flavor would lower the urination rate.

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  Monosodium glutamate and nucleotides are umami taste substances in animals and have a synergistic effect on each other. We studied the ligand-binding properties of the glutamate receptors in taste epithelial cells isolated from bovine tongue. Specific glutamate binding was observed in an enriched suspension of taste receptor cells in Hanks’ balanced salt solution, while no specific glutamate binding was apparent in the absence of divalent ions or when the cells had been depolarized by a high content of potassium in Hanks’ balanced salt solution. There was no significant difference between the release of glutamate under depolarized or divalent ion-free conditions and under normal conditions. However, glutamate was easily released from the depolarized cells in the absence of divalent ions. These data suggest that the binding of glutamate to receptors depends on divalent ions, which also have an effect on maintaining binding between glutamate and receptors.

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  A methanol extract of red pepper showed potent acetyl-CoA carboxylase inhibitory activity. The active principles were isolated and identified as (E, E)- and (E, Z)-9-oxooctadeca-10,12-dienoic acids by instrumental analyses. The IC₅₀ values of the compounds were 1.4×10^-6 and 1.5×10^-6 M, respectively, their activity being nearly sixty-times higher than that of the common fatty acids themselves. A comparative study of the structure-activity relationship among their related compounds showed that the inhibitory activity was influenced neither by the position and species of the oxygen functional group in the middle of the alkyl chain nor by the configurations of the double bonds. However, it was found that the presence of double bonds between the terminal carboxyl and the mid-chain oxygen functional group lowered the inhibitory activity which could be recovered by hydrogenation of the double bonds.

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  We determined whether the synthesis and degradation of N-acetylglutamate would regulate urea synthesis when the ornithine status was manipulated. Experiments were done on two groups of rats, each being treated with ornithine or saline (control). The plasma concentration of urea and the liver concentration of N-acetylglutamate in rats given ornithine were each significantly higher than in the control rats. Compared with the control rats, the liver N-acetylglutamate degradation was significantly lower in those rats treated with ornithine. Treatment of the rats with ornithine did not affect N-acetylglutamate synthesis in the liver. An inverse correlation between the liver N-acetylglutamate degradation and liver concentration of N-acetylglutamate was found. These results suggest that the lower degradation of N-acetylglutamate in the ornithine treatment group would be likely to increase the hepatic concentration of this compound and stimulate urea synthesis.

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  The new bitter diterpenes, rabdosianone I (C₂₀H₂₄O₅) and II (C₂₂H₂₈O₆), were isolated from Isodon japonicus (Japanese name, enmeiso), and their structures were elucidated by spectroscopic methods. Electrophysiological experiments were performed to compare rabdosianone I with quinine. The taste responses of chorda tympani nerves to rabdosianone I were smaller than those to quinine in Wistar rats.

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  Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweetpotato, 3-(6,6′-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.

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  Soybean protein, casein, bonito protein and chicken protein, each as foodstuff protein, were hydrolyzed with four proteinases; namely, pepsin, trypsin, α-chymotrypsin and bromelain. Since the chicken protein hydrolysate with bromelain possessed the most favorable umami taste, eleven peptides were isolated from the chicken protein hydrolysate by successive chromatography on ODS, Amberlite IR-120B, Amberlite IRA-410 and AG-50W; their structures were Asp-Ala, Asp-Val, Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Asp-Glu-Glu, Asp-Glu-Ser, Glu-Glu-Asn, Ser-Pro-Glu, and Glu-Pro-Ala-Asp. Many of them did not show any umami taste by themselves, but Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Asp-Glu-Glu, and Ser-Pro-Glu were recognized to enhance the umami taste of 0.02% 5′-inosine monophosphate (IMP). A combination of these peptides, especially 0.5% each of Glu-Glu, Glu-Val, Asp-Glu-Glu and Glu-Glu-Asn, with 0.02% IMP produced a delicious “full” umami taste.

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  The major wheat allergens contain a number of glutamine residues, suggesting that deamidation would be a promising method to produce hypoallergenic wheat proteins. Gluten was deamidated by acid under various conditions. The 30%-, 50%- and 90%-deamidated glutens were reacted with sera of patients allergic to wheat proteins. The results indicate that the reactivity was dramatically decreased according to the degree of deamidation.

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  Tea constituents that had a preventive effect on D-galactosamine-induced liver injury in rats were partially purified by column chromatography from a n-butanol-soluble fraction of green tea. The fraction containing glycosidic flavonoids was found to suppress the D-galactosamine-induced increase of plasma alanine aminotransferase and aspartate aminotransferase activities. These results indicate that glycosidic flavonoids contribute, at least in part, to the liver injury-preventive effect of green tea.

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  Sprague-Dawley rats were fed α-tocopherol, tocotrienol, or quercetin to examine their dietary effects on serum lipid contents and immunoglobulin productivity. In tocotrienol or quercetin groups, serum triglyceride was lower than in the none group. Moreover, in the α-tocopherol group, serum IgA level and IgA productivity of MLN lymphocytes were high, while in the tocotrienol group, IgM productivity of spleen lymphocytes and IgA, IgG, and IgM productivity of MLN lymphocytes were high. Thus, we suggested each antioxidant had different effects in rats.

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  An organosulfur compound was isolated from oil-macerated garlic extract by silica gel column chromatography and preparative TLC. From the results of NMR, IR, and MS analyses, its structure was determined as E-4,5,9-trithiadeca-1,7-diene-9-oxide (iso-E-10-devinylajoene, iso-E-10-DA). This compound was different from E-4,5,9-trithiadeca-1,6-diene-9-oxide (E-10-devinylajoene, E-10-DA) only in the position of a double bond. Iso-E-10-DA had antimicrobial activity against Gram-positive bacteria, such as Bacillus cereus, B. subtilis, and Staphylococcus aureus, and yeasts at the concentration lower than 100 μg/ml, but Gram-negative bacteria were not inhibited at the same concentration. The antimicrobial activity of iso-E-10-DA was inferior to those of similar oil-macerated garlic extract compounds such as E-ajoene, Z-ajoene, and Z-10-DA. From these results, it was suggested that trans structure and/or the position of double bond of iso-E-10-DA reduce the antimicrobial activity.

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  Three thiosulfinates were isolated from oil-macerated garlic extract, and their structures were identified as 2-propene-1-sulfinothioic acid S-(Z,E)-1-propenyl ester [AllS(O)SPn-(Z,E)], 2-propenesulfinothioic acid S-methyl ester [AllS(O)SMe], and methanesulfinothioic acid S-(Z,E)-1-propenyl ester [MeS(O)SPn-(Z,E)]. This is the first report of isolating these thiosulfinates from oil-macerated garlic extract. Antimicrobial activities of AllS(O)SPn-(Z,E) and AllS(O)SMe against Gram-positive and negative bacteria and yeasts were compared with 2-propene-1-sulfinothioic acid S-2-propenyl ester [AllS(O)SAll, allicin] which is well-known as the major thiosulfinate in garlic. Antimicrobial activity of AllS(O)SMe and AllS(O)SPn-(Z,E) were comparable and inferior to that of allicin, respectively. This result suggested that the antimicrobial activity of 2-propene sulfinothioic acid S-alk(en)yl esters were affected by alk(en)yl groups. The order for antimicrobial activity was: allyl≥methyl>propenyl.

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  Weanling rats were given diets contained castor oil (CAO-diet), coconut oil (CO-diet), or high-oleic safflower oil (HO-diet) each 10% (wt). No growth retardations were observed on the CAO-diets. The CAO-diet group showed significantly lower serum cholesterol and hepatic triacylglycerols than the HO-diet group. Ricinoleic acid was found at an extremely low level in perirenal adipose tissue.

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  The radical scavenging activity of Japanese edible seaweeds was screened by the DPPH (1-diphenyl-2-picrylhydrazyl) assay to evaluate the DPPH radical scavenging activity in organic extracts. The fresh brown alga Hijikia fusiformis showed the strongest DPPH radical scavenging activity, followed by Undaria pinnatifida and Sargassum fulvellum. The major active compound from Hijikia fusiformis in its acetone extract was identified as fucoxanthin by ¹³C-NMR spectroscopy.

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  The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H₂O₂) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H₂O₂ resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H₂O₂. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H₂O₂. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested.

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  A microbial process for removing cadmium from a homogenate of hepatopancreas, a waste of scallop processing, was devised to use this waste for value-added protein resources. Microorganisms were screened on the basis of the ability to remove cadmium from a medium with the initial concentration of 10 mg/l of cadmium. One soil isolate, identified as Xanthomonas sp. UR No. 2 by its taxonomical characteristics, removed 98% of the cadmium in the medium in 2 d. During cultivation of this strain in the homogenates of hepatopancreas digested by endopeptidases, 90% of cadmium was removed, while this strain had little effect on the simple non-digested homogenates. The mass balance of cadmium during homogenizations of the hepatopancreas tissues and cultivations in the protease-treated homogenate were examined. The content of crude proteins of culture supernatant treated by Xanthomonas sp. UR No. 2 was equivalent to those of various feedstuffs on the market.

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  Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by Ω-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.

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  Saccharomyces cerevisiae strain ②-39/10A is able to ferment alcohol at 42°C. The ability of various yeast strains, including ②-39/10A, to grow at high temperatures was compared. The strain ②-39/10A was able to grow at 42°C and the high temperature growth was found to be governed by more than one gene. The yeast strains that can grow at 42°C were bred by crossing the haploid strains, which are inherently unable to grow at high temperatures.

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  Bacillus megaterium strain NK84-0218 produces a potent antiviral antibiotic, oxetanocin A, which has an oxetanosyl-N-glycoside linkage to an adenine moiety. However, the oxetanocin A productivity of the original strain was unstable and low. In this study, oxetanocin A productivity and resistance was shown to be lost simultaneously when a 51.5-kb plasmid, pOXT1, was cured during cultivation. The deficiency of oxetanocin A productivity and resistance was restored by re-introduction of the pOXT1 plasmid into the cured strain. By a cloning experiment it was shown that a 6.8-kb BglII-D fragment of the pOXT1 plasmid was responsible for oxetanocin A productivity and resistance.

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  Sorbitol dehydrogenase (EC 1.1.1.14), which catalyzes the NAD⁺-linked interconversion of D-sorbitol and D-fructose, was purified and crystallized from cell-free extracts of Bacillus fructosus grown on D-sorbitol as a sole carbon source. The crystalline enzyme was homogeneous on disc electrophoresis and ultracentrifugation. The molecular weight was 102,000 by the sedimentation equilibrium method. The enzyme acted specifically on D-sorbitol, and showed an optimum pH at 9.0. The K_m values for D-sorbitol and NAD⁺ were 1.1×10^-2 M and 2.2×10^-4 M, respectively. The enzyme activity was inhibited by p-chloromercuribenzoate, Ag⁺, Hg²⁺, and Cu²⁺.

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  A koji-based medicine composed of powder of Aspergillus oryzae NK koji, dried yeast, and lactobacilli koji had high antioxidant activity measured by a modified t-butyl peroxyl radical scavenging assay. This activity was mainly derived from A. oryzae NK koji. Digestion of koji-making grain germ medium with several commercial enzymes also increased antioxidant activity. By two weeks of oral administration of A. oryzae NK koji, the serum lipid peroxide levels elevated in STZ-induced diabetic rats could be decreased significantly.

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  Chlorella pyrenoidosa Chick reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the diastereomer (anti/syn) ratio of 53/47. The enantiomer excesses of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters were 89 and >99ee% respectively. C. vulgaris and C. regularis afforded predominantly the syn-isomer, contrary to C. pyrenoidosa. The differences in the activity of reducing ethyl 2-methyl 3-oxobutanoate were observed among three strains of Chlorella. Addition of 2% metal salts slightly increased the chemical yield of the hydroxy ester.

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