The hydrolysis of glucosidic linkage catalyzed by every carbohydrate-hydrolase is a reaction in which the product retains (α→α or β→β) or inverts (α→β or β→α) the anomeric configuration of the substrate. α-Glucosidase and glucoamylase are essentially distinguished by releasing α-glucose and β-glucose, respectively, from the common substrates having α-glucosidic linkage. The distinction in the substrate specificities of the two enzymes was explained by the subsite affinities in their active sites. The amino acid sequences of the regions containing the catalytic sites were compared in α-glucosidases and glucoamylases from various sources. α-Glucosidases were suggested to be grouped into two families by their primary structures. The catalytic reaction mechanisms of carbohydrate-hydrolases were discussed in the two significant models of a nucleophilic displacement mechanism and an oxocarbenium ion intermediate mechanism.

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During streptokinase (SK) recovery by cross-flow microfiltration (CFMF), a loss of activity of 14.4% was observed, after the initial volume was concentrated 8-fold (VCF=8.0); 51.5% of the activity was recovered in the filtrate and 34.1% remained in the retentate. Immunological experiments using polyclonal antibodies against SK have demonstrated that SK activity loss during CFMF processes could be related to denaturation of SK, forming molecules of lower or no activity. Accumulation of denatured SK in the retentate suggests that denaturation could be the results of an aggregation phenomenon (as has been demonstrated by light scattering and chromatographic studies), leading to the formation of protein aggregates that are retained by the microfiltration (MF) membrane, affecting the fouling phenomenon and the concentration in the retentate of permeable molecules.

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Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q₁₀-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q₉-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9-6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q₁0-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q₁0-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (≡Acetobacter methanolicus), the type species of the genus Acidomonas, had 16-9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.

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Extensive screening for cell-bound lipase-producing filamentous fungi suitable for immobilization in biomass support particles (BSPs) was done. A total of 91 fungi were assayed for their lipolytic activity after cultivation and immobilization in BSPs. In the cultivation, polypepton and oleic acid were used as an organic nitrogen source and a lipase inducer, respectively. Several species, such as Rhizopus species and Rhizomucor miehei, showed higher cell-bound lipolytic activity than that of Rhizopus chinensis, which is known to have considerable high cell-bound lipolytic activity from previous work. For two strains of fungi (Rhizopus stolonfer, Rhizomucor miehei), the cell-bound lipolytic activities of cells immobilized in BSPs did not show strong dependence on polypepton concentration (10-60 g/liter), while the extracellular lipolytic activities in the culture liquid increased with the increase in the polypepton concentration. Thirteen fungi that showed high cell-bound lipolytic activities were examined with respect to their acidolytic activities using fish oil and caprylic acid in n-hexane. There was a roughly proportional relationship between lipolytic and acidolytic activities (the linear correlation coeflicient, R, was 0.63). As the most potent fungus having the highest cell-bound acidolytic activity in BSP, Rhizomucor miehei IFO 9740 was selected.

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The transglycosylation reaction was done with a β-galactanase from Penicillium citrinum. The regioselectivity in the transglycosylation reaction was studied using soy bean arabinogalactan as a donor and mono- or disaccharide derivatives containing β-galactosyl residue as acceptors. We also synthesized oligosaccharides containing Galβ1→4Gal sequence such as Galβ1→4Galβ1→4Glc, Galβ1→4Galβ1→3GlcNAc, Galβ1→4Galβ1→4GlcNAc, Galβ1→4Galβ1→6GlcNAc, and Galβ1→4Galβ1→3GalNAc for use in the total synthesis of complex sugar chains.

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Partially purified lipoxygenase (LOX) extracts were obtained from Fusarium proliferatum, Fusarium oxysporum, Chlorella pyrenoidosa, and Saccharomyces cerevisiae; the enzymatic extract of F. proliferatum showed the highest LOX activity while those of F. oxysporum and S. cerevisiae demonstrated only 27.8 and 16.5% of the activity at pH 8.0, and 61.2 and 9.7% of the enzyme activity at pH 10.0, respectively. The lowest LOX activity was that in the C. pyrenoidosa extract. The microbial enzymatic preparations were assayed with linoleic acid as substrate, which was bioconverted into 9- and 13-hydroperoxides (HPODEs) by all four extracts; in additon, the LOX activity in the F. oxysporum extract produced the 10- and 12-HPODEs from linoleic acid while that of the C. pyrenoidosa extract produced only the 10-HPODE. When assayed with the 9- and 13-HPODEs as substrates, the selected microbial extracts had secondary enzyme activities, one of which produced hexanal. The highest hexanal-producing activity was 1.51 and 1.39 nmol hexanol/mg protein/min in the F. proliferatum and C. pyrenoidosa extracts, respectively, while those of F. oxysporum and S. cerevisiae had approximately 15% of the HPODE-cleaving enzyme activity. The C. pyrenoidosa extract had the highest proportion of pentanone, which was produced at only one-fourth the concentration by the HPODE-cleaving enzyme activity in the three other microbial enzymatic extracts.

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A novel β-galactosidase (β-gal) gene was cloned from Bacillus circulans ATCC 31382. The coding region was 1, 758 bp and encoded a polypeptide of 586 amino acids with a deduced molecular mass of 66, 888. Active staining for β-gal showed that B. circulans ATCC 31382 produced three β-gal isozymes. Two of these were detected in Biolacta N5 (Daiwakasei Co.), but the product of this novel gene corresponded to the one not contained in Biolacta N5. The novel β-gal showed the highest amino acid sequence identity (43.3% ) with a β1→3 > 1→4 galactosidase from Xanthomonas manihotis and was also highly similar to β-gals from animals, plants, and fungi. This suggests an evolutionary relationship between this novel gene and those of eukaryotic origins. One of the two B. circulans β-gals, the nucleotide sequences of which are available in the GenBank, was 20% identical to the novel β-gal. Other bacterial β-gals showed little or no similarity. We propose that this novel β-gal be called B. circulans β-gal-3, and the gene be called bgaC.

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The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)_1-3-p-nitroanilide (Suc-(Ala)_1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P₂ positions were not cleaved. On the other hand, the non-polar dmino acid or prolitie at P₂ were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.

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A novel sulfated β-1, 3-xylan product was synthesized from algal cell wall microfibril homoxylan by the N, N-dimethylformamide (DMF)-SO₃ complex sulfation method. Antithrombin activity appeared in this product was 6.5 times higher than that of standard heparin. From the results of ¹H- and ¹³C-NMR spectroscopic analyses by DQF-COSY and HMQC and an infrared spectroscopic analysis, it was revealed that the ordered structure of β-1, 3-xylan as a triple helix had decayed and the resulting conformational changes had been caused by the sulfation reaction. The sulfated positions on the C-4 hydroxyl groups of the xylose residues were determined from ¹³C-NMR chemical shifts, and it was found that regioselective sulfation had occurred predominantly with the C-4 secondary hydroxyl groups to produce a mono-substituent. Another type of sulfation of β-1, 4-xylan that showed no regioselectivity is considered to have been due to the different conformation of both xylans chains such as the triple helix in β-1, 3-xylan and the double straight chain like cellulose in β-1, 4-xylan. Therefore, the different type of regioselective sulfation of β-1, 3- and β-1, 4-xylan was caused by the difference in steric hindrance due to these conformations. These different types of regioselective sulfation with different linkage positions are also discussed for the secondary hydroxyl groups in β-1, 3- and β-1, 4-glucan after chemoselective sulfation of the C-6 primary hydroxyl groups.

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Whey proteins from soybean seeds of Japanese varieties were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Among 11 varieties of soybean, three green and one black soybeans lacked a 26-kDa band that was found in all yellow soybeans. In this paper, the 26-kDa protein was named AS26k (acid soluble 26-kDa protein) temporarily. The AS26k protein was purified from Glycine max cv. Nattosyoryu, which is yellow soybean, through four purification steps: 30-35% saturated ammonium sulfate fractionation, ion exchange chromatography on S Sepharose Fast Flow, gel filtration on Sephadex G-100, and hydrophobic chromatography on phenyl Sepharose CL-4B. Purified AS26k was cleaved with V8 proteinase from Staphylococcus aureus or CNBr. The cleaved polypeptide contained two typical dehydrin motif sequences: DEYGNPV and (M)DKIKEKLPG, and a 19 amino acids sequence similar to a pea dehydrin. Native AS26k had a molecular mass of 32 kDa on gel filtration and a pI of 7.2 on two-dimensional PAGE. Similarly to other dehydrins and late embryogenesis abundant (LEA) proteins, AS26k was rich in hydrophilic amino acids, and highly heat stable. These results showed that AS26k was a dehydrin, a group II LEA protein in soybean seeds.

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The overproduction of tumor necrosis factor-α(TNF-α) was suppressed by orally administering a perilla leaf extract (PLE). When mice were successively injected with OK-432, severe TNF-α was induced in the serum, but this elevated TNF-α level was reduced after an oral administration of PLE (400 μl/mouse). Oral administration of PLE also inhibited TNF-α production that was induced by muramyl dipeptide (500 μg/mouse) and OK-432 (3 KE/mouse). These characteristics were obtained from all strains of perilla. The inhibitory activity against TNF-α production was heat-stable, and the existence of several active molecules was suggested. When PLE was passed through an ultrafilter, the inhibitory activity against TNF-α production was collected in those fractions with a mass of 0.5 to 1 kDa and more than 10 kDa. When PLE was solvent-extracted, the strongest activity was recognized with aqueous preparation, although significant activity was also detected in preparations extracted with n-hexane and ethyl acetate. These findings suggest that the daily use of certain functional foods may be useful for controlling the host defense system.

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The distribution coefficients of glucose, maltose, and maltotriose on to a cation-exchange resin in sodium-ion form were measured by using methanol- or ethanol-water mixtures of various content as eluents. With increasing methanol or ethanol content, the resin shrank and the coefficients increased. This increase in the coefficients could not be explained by only the effect of the swelling pressure of the resin on the distribution, and it seems that the complex formation of Na⁺ and the saccharides played an important role. The binding constants between some saccharides and Na⁺ were evaluated, and the electronegativity of each saccharide relative to that of glucose was estimated.

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Age-associated changes in the induction of heme oxygenase (HO-1) and heat shock protein 70 (HSP70) after the administration of paraquat were investigated in the liver of senescence-accelerated mice (SAMs). The extent of HO-1 and HSP70 induced in response to paraquat decreased significantly with age, and the level of oxidized proteins increased with age. Moreover, the extent of induced HSP70 was lower in mice that were prone to accelerated senescence (SAMP1//Fky) than in mice that were resistant to accelerated senescencd (SAMR1/Fky) of the same age. These results suggest that an age-associated decline in the levels of HO-1 and HSP70 enhanced oxidative damage during the aging process. Age-dependent changes in HO-1 and in the levels of oxidized proteins were examined in SAMP1//Fky. The accumulation of oxidized proteins was suppressed when HO-1 was induced, but increased markedly after the induction of HO-1 decreased. Free iron, the residuum from heme degradation, might mediate free radical production. The role of HO-1 is discussed in relation to the oxidative damage associated with age.

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Water sorption isotherms of native and denatured ovalbumin samples were measured, and their affinity for water was quantitatively analyzed by solution thermodynamics. The structural change of the proteins was evaluated by several methods. Water activity above 0.9 was measured by adding a specific amount of water to a sample, while that below 0.9 was measured with apparatus for water sorption isotherms. Thus, water sorption isotherms for native and denatured ovalbumin samples were obtained up to a water activity of 0.99. The water sorption behavior of ovalbumin was affected not by its hydrophobicity but by its prominent conformational change. It was confirmed that parameter ΔG^s calculated by solution thermodynamics was more suitable for evaluating the amnity for water from water sorption isotherms than ΔG^s_w calculated by conventional adsorption thermodynamics.

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Protein phosphorylation may be required for plant cell response to phytohormones and other extracellular signals. Protein phosphorylation and protein kinase activity in the culm of heading time of rice (Oryza sativa L.) were studied. Before heading, protein kinase activity was increased by Ca^<2+> in the membrane fraction of the panicle and culm. The protein kinases with M_r of 51, 900, 49, 200, and 45, 500 isolated from the membrane fraction of culm increased the protein phosphorylation of M_r and pI of 40, 000/7.5 and 40, 000/7.6 in the culm extract. The activation of protein kinases, associated with membrane and subsequent protein phosphorylation, thus appears to be involved in the regulation of heading time in rice.

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The caries-inducing activity of the hydrogenated derivative of an isomaltooligosaccharide mixture (IMO-H) was evaluated in vitro for its acidogenicity and in vivo an experimental caries system with specific-pathogen-free (SPF) rats. Streptococcus sobrinus 6715 (serotype g) did not produce a significant amount of acid from IMO-H, whereas Streptococcus mutans MT8148 (serotype c) gradually produced a small amount of acid, although the degree was less than that of sucrose. In vivo experiments were conducted on rats which were provided with the test sugars at two different times: at the time of organism inoculation, and after the organisms had become conpletely established. IMO-H did not induce significant dental caries in rats infected with the S. sobrinus 6715 or S. mutans MT8148R strain.

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The gene encoding renin-binding protein was isolated from a Sprague-Dawley rat genomic DNA library and the exon-intron boundaries and transcription initiation sites were identified. The gene was found to span about 9.0 kilobase pairs and to be composed of eleven exons. The exon size ranged 76 to 225 base pairs. All the exon-intron junctional sequences conformed to the canonical GT-AG rule. The first exon encoded a 5'-untranslated region with the ATG translation start codon being in exon 2. The promoter region did not contain a TATA box, but it had two GC boxes in the 5'-untranslated sequence. The overall exon pattern of the rat RnBP gene correlated with the human RnBP gene [S. Takahashi, H. Inoue, and Y. Miyake, J. Biol. Chem., 267, 13007-13013 (1992)]. Moreover, the sizes of exons 2 to 10 in the rat RnBP gene were the same as those of the human RnBP gene. These results indicate that the structure of the RnBP gene is conserved among animals.

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Purification of onion alliin lyase gave two fractions by cation exchange chromatography. Both fractions showed the comparable high catalytic activity of cysteine-S-conjugate β-lyase with that of alliin lyase using S-(2-chloro-6-nitrophenyl)-L-cysteine and alliin, S-allyl-L-cysteine sulfoxide as substrates. All the active substrates tested with onion alliin lyase were also active to the cysteine-S-conjugate β-lyase of Mucor javanicus, but the catalytic activity of the Mucor enzyme was lower for all the substrates. The pyridoxal phosphate binding site of the onion alliin lyase was identified as Lys 285 in the amino acid sequence deduced from cDNA which has been reported. This lysine was conserved in all the sequences from the alliin lyase cDNAs, while similarity was not found between the sequences around pyridoxal phosphate binding sites of both the onion alliin lyase and the Mucor cysteine-S-conjugate β-lyase.

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To investigate the effects of vitamin B₆ (B₆) deficiency on an antibody production in BALB/c mice, the production of specific immunoglobulin (Ig) E antibody against dinitrophenylated ovalbumin (DNP-OVA) were measured by the methods of enzyme linked immunosorbent assay. The mice fed on on a B₆ deficient diet for 4 weeks were immunized intraperitoneally with DNP-OVA absorbed to aluminum hydroxide gel. The contents of anti DNP-IgE antibodies in sera of B₆ deficient mice significantly increased compared to that of control mice fed on a diet containing B₆. In addition, Interleukin-4, which was known to induce IgE production in allergic reactions from splenocytes of B₆ deficient mice, was approximately four-fold higher than that in control mice. According to the recovery test to the B₆ deficient mice, that is feeding the control diet for 21 days, all values in terms of the body, thymus, and spleen weight, total serum protein, IgG, and anti DNP-IgE content, regained almost the same levels as those of control. These results suggest that B₆ deficiency in mice would have relation to the stimulation of specific IgE antibody production against DNP-OVA.

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We investigated the effect of dietary wheat bran (Wb) on colonic tumorigenesis soon after a single administration of 1, 2-dimethylhydrazine (DMH). Rats that had been fed on either a fiber-free diet or a 20% Wb diet were injected with 1, 2-dimethylhydrazine (20 mg/kg body weight). At 6h, 12h, 1d, 3d, or 7d after the injection, the colorectum was excised for histological analyses. The number of crypt cells more rapidly recovered in the 20% Wb group than in the fiber-free group after its temporary reduction by injection of DMH. At 6 h after the DMH treatment, the apoptotic cells were signiticantly greater in number in the fiber-free group than in the 20% Wb group. In contrast, those in distal colon were significantly fewer in the fiber-free group than in the 20% Wb group at 7d after the treatment. These results suggest that the ingestion of Wb affected the turnover of colonic epithelial cells and would thereby bring about a protective effect against DMH-induced tumorigenesis.

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As a model for the synthesis of optically active 6-aryl-2-aryloxy-1-hydroxy-3, 7-dioxabicyclo-[3.3.0]octanes, which are 1, 2-dioxygenated furofuran lignans, the most important intermediate, (+)-(1S, 2S, 5R, 6S)-1-hydroxysamin (1), was synthesized from L-(+)-arabinose.

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A 7.2-kb genomic DNA fragment containing N-acetylglucosamine-6-phosphate deacetylase gene (nagA) was cloned from the chitinase-producing bacterium Vibrio cholerae non-O1 strain 1148A (IFO 15429). Sequence analysis of the DNA fragment found three other complete open reading frames (ORFs) and the 5' end of an ORF. Amino acid sequences of two ORFs, ORF2 and ORF4, showed similarity with that of NagC, the repressor of nag operons and that of NagE, N-acetylglucosamine-specific transporter II^Nag of phosphoenolpyruvate transport system of Escherichia coli, respectively. In the presence of N-acetylglucosamine, nagA and ORF2 (nagC) were co-transcribed. ORF4 (nagC), which is upstream from nagAC but is expressed in the opposite direction was also transcriptionally induced in the presence of N-acetylglucosamine. These results indicated that nagE-nagAC existed as divergent operons in V. cholerae non-O1, Unlike E. coli, nagB and nagD were not in the operons.

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The role of cysteine residues for structure and function of formaldehyde dehydrogenase from Pseudomonas putida was analysed by amino acid sequence comparison, homology-based structure modeling, site-directed mutagenesis, and chemical modification. Five out of seven cysteine residues found in the enzyme were concluded to coordinate with an active site zinc (Cys-46) and structural zinc atoms (Cys-97, -100, -103, and -111) from the sequence comparison with other Zn-containing medium-chain alcohol dehydrogenase homologues. The three-dimensional structure model based on the known structure of the horse liver E-type alcohol dehydrogenase (ADH) indicated that Cys-257 is located very far from the active site Zn and NAD⁺ binding region, suggesting that Cys-257 does not participate in the enzyme reaction. The structure also suggested that Cys-166 does not coordinate to active site Zn, but Asp-169 functions as a Zn-ligand, instead.

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We examined the effect of soluble corn bran hemicellulose (CBH, 10g/day) on glucose control and serum insulin in three groups: patients with impaired glucose tolerance (IGT) with (20 subjects) or without (8 subjects) obesity and with healthy non-obese controls (10 subjects). Long-term supplementation (6 months) with CBH decreased the post oGTT curve for patients with impaired mild Type II diabetes, but not that for the controls. Hemoglobin A_1c decreased significantly during CBH supplementation in the obese patients, while the fasting glucose level decreased in all three groups, although not significantly. A decrbased serum insulin response by oGTT was found in those patients with IGT. The improved oGTT result was associated with improved insulin release and perhaps with peripheral insulin sensitivity. These findings suggest that CBH at a low dose might contribute to glycemic control and would play a useful role in treating Type II diabetes patients.

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Role of endogenous gibberellins (GAs) in the thermoperiodic regulation of stem elongation in Dendranthema grandiflorum was investigated by gas chromatography-mass spectrometry with deuterated GAs as internal standards. GA₁, GA₂₀, GA_{19%gt;, GA<44}, and GA₅₃, which all belong to the early 13-hydroxylation pathway of GA-biosynthesis, and GA₉, which belongs to the early nonhydroxylation pathway, were identified from the stem. The thermoperiodic treatments employed were a higher day temperature than night temperature (DT > NT) and several hours of temperature drop from beginning of the day (TD). TD markedly decreased the stem elongation rate when compared to the effect of DT > NT. Further, TD decreased the concentrations of stem GA₁, GA₂₀, and GA₁₉ more than DT > NT, while TD increased the concentration of stem GA₄₄ more than DT > NT. Thus, TD probably retarded the conversion of GA₄₄ to GA?<19>, resulting in a low concentration of GA₁, the probable biologically-active GA. These changes in endogenous GA concentration are probably an important physiological factor for the thermoperiodic regulation of stem elongation in D. grandflorum.

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We developed a transformation system for Aspergillus oryzae using the Aspergillus nidulans sC gene encoding ATP sulfurylase as a selectable marker. The sC^- mutants can be readily isolated by positive selection for selenate resistance, thereby the niaD^- mutant strain of A. oryzae was bestowed with the sC^- mutation. Transformation of the A. oryzae host (niaD^-, sC^-) with the plasmid carrying A. nidulans sC gave random and multi-copy integrants, while that with the A. oryzae niaD-carrying plasmid occurred mainly by single-copy and homologous integration events (more than 50% frequency), indicating that with this transformation system, the transformation marker could be selected according to the integration pattern one desires.

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We evaluated the catalytic ability of 29 yeast strains to reduce ethyl acetoacetate (EA) in the presence of ethanol or glucose. In 18 yeast strains, the reduction in the presence of ethanol proceeded as well as in the presence of glucose. Among them, Kloeckera magna (AKU 4704) effectively catalyzed the NADPH-dependent reduction of EA in the presence of ethanol. In this reduction, 1 mol of EA was reduced by consuming 1 mol of ethanol. We found that the NADPH regeneration system responsible for EA reduction in K. magna was coupled with the oxidation of acetaldehyde to acetic acid catalyzed by an NADP⁺ -dependent aldehyde dehydrogenase.

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It is of great importance to find ways to protect concrete from corrosion, to maintain the concrete structure for a long time. A sulfur-oxidizing bacterium, Thiobacillus thiooxidans, has been known to play a crucial role in concrete corrosion and the concrete supplemented with nickel was resistant to corrosion. To obtain biological bases of this Ni protection, the effects of Ni on sulfur dioxygenase and sulfate oxidase of T. thiooxidans NB1-3 isolated from corroded concrete were studied. Nickel sulfate strongly inhibited a sulfur dioxygenase that catalyzes the oxidation of elemental sulfur to shlfite and a sulfite oxidase that catalyzes oxidation of sulfite to sulfate. Nickel sulfate, antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide, and myxothiazol inhibited both sulfite oxidase and ubiquinol oxidase. Reduced mammalian cytochrome c was not oxidized by a cell extract of strain NB1-3. The b and a-type cytochromes in the plasma membrane was reduced by sulfite and ubiquinol-2 and these reductions were inhibited by NiSO₄. The amounts of Ni in the plasma membrane with or without 0.1 mM nickel treatment were 32.6 and 2.8 nmol/mg protein, respectively. These results indicate that nickel binds to the plasma membrane and inhibits sulfur dioxygenase and sulfite oxidase, and as a result, inhibits cell growth.

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The inclusion complex between DHA oil containing tridocosahexaenoin to 45% and cyclodextrin (CD) was prepared by a twin-screw kneader. The powder from of DHA oil was examined for its stability against oxidation as such or in the form of a mixture with fish meal paste ("kamaboko"). The powdery tridocosahexaenoin exhibited marked resistance against autoxidation for a long period, during which the peroxide value remained constant at a marginal level. Above all, POV for the powdery tridocosahexaenoin with α-CD remained virtually unchanged for 20 days without using any antioxidant during storage at 4°C and seeiried to be effective for fortifying fish meal products.

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A cDNA encoding spinach putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) was cloned and sequenced. The cDNA included an open reading frame that encoded a polypeptide of 171 amino acid residues. The deduced amino acid sequence showed about 77 and 50% similarity to plant putative PHGPXs and mammalian PHGPXs, respectively. PCR products with the same size as that of the spinach putative PHGPX were obtained from maize, soybeans, and Arabidopsis, suggesting the expression of putative PHGPX genes in other plants.

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The rate parameters for maltooligosaccharides of two α-glucosidases from spinach seeds were examined and subsite affinities were evaluated. The subsite affinities for subsites 1, 2, 3, 4, 5, 6, and 7 in the active site of α-glucosidase A and B were 1.73, 2.91, 1.10, 0.25, 0.94, 0.03, and -0.01 kcal/mol, and 0.33, 4.94, 0.26, 0.14, -0.24, -0.52, and 0.14 kcal/mol, respectively. The six and four subsites exist in α-glucosidase A and B, respectively, which maltooligosaccharides or soluble starch could be bound.

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Uniconazole applied to cotyledons inhibited the flowering response in Pharbitis nil. The application of gibberellin A₁ (GA₁) to cotyledons overcame the effect of uniconazole and restored the flowering response. The transport of GA₁ from cotyledons to plumules was confirmed by GC-MS analyses. The results indicate that GAs transported from cotyledons are concerned with regulating the endogenous level of GAs in plumules.

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Eleven strains of Aspergillus species, isolated from Katsuobushi (dried bonito), were grown in a liquid medium containing 2, 6-dimethoxy-4-methylphenol, to examine the possibility of production of 1, 2, 3-trinlethoxy-5-methylbenzene, which has a musty odor, during the molding process in Katsuobushi production. In the liquid medium, 2, 6-dimethoxy-4-methylphenol was O-methylated by 4 strains of A. glaucus.

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We have identified 2 DnaK and 3 GroEL homologues from moderately halophilic Acinetobacter, Pseudomonas, and Planococcus species by partial purification using an ATP-agarose column and by the analysis and similarity search of these NH₂-terminal amino acid sequences. Although these bacteria required 1 to 2M NaCl for growth, these DnaK and GroEL homologues did not require high salt to bind to the ATP column, thus suggesting that these chaperones did not require high salts for their biochemically activities.

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A water-soluble ester, methyl formate, was detected as a metabolite in the culture medium of methylotrophic yeasts. Methyl formate synthase, which catalyses NAD⁺-dependent dehydrogenation of the hemiacetal adduct of methanol and formaldehyde, catalyses the ester synthesis. The enzyme activity was induced on a methanol medium and was increased further by the addition of formaldehyde. In the reaction system using intact cells of Pichia methanolica AKU 4262, 135 mM (8.1 g/liter) methyl formate was produced from 2M methanol. This is a new biological process for ester synthesis that couples spontaneous formation of hemiacetal and alcohol dehydrogenase.

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(E)-and(Z)-Vinylsilanes for the Still-Wittig rearrangement were easily prepared from propargyl alcohol 4. The Still-Wittig rearrangement of (Z)-vinylsilane 1d afforded (E)-allylsilane 3d stereoselectively. The stereoselectivity of (E)-vinylsilane, however, was unexpectedly low.

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Two derivatives of shikimic acid were isolated from croziers of the dwarf tree fern, Dicksonia antarctica, and their structures were elucidated as 4-O-caffeoylshikimic acid and 4-O-(p-coumaroyl)-shikimic acid on the basis of mass spectrometric and NMR spectroscopic evidence.

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Various samples of fish meat were examined for the formation of the hepatotoxic aldehyde, 4-hydroxy-trans-2-hexenal (HHE). HHE was detected in all samples analyzed at the concentration of 1.5-39.3 nmol/g. Yellowtail meat contained more HHE than 4-hydroxy-trans-2-nonenal (HNE). The HHE and malonaldehyde concentration increased during 13 days of storage at 0°C in the meat of yellowtail. On the other hand, no HNE was detected during storage.

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The microfibrillar structure of bacterial cellulose was investigated. Bacterial cellulose cultured in a jar fermentor was hydrolyzed in 1 N hydrochloric acid for 2 h at 100°C. The residual bacterial cellulose consisted of short strands after hydrolysis. The lengths of 529 short strands on transmission electron micrographs were measured with a ruler, and the length distribution of the short strands was then evaluated by a computer-aided line shape analysis. The length distribution was deconvoluted into five peaks with Gaussian distribution, and the center positions of the peaks occurred at regular intervals. This result demonstrates that the cellulose microfibrils consisted of regularly repeating units of constant length (ca. 0.8μm) and suggests that each unit consisted of an acid hydrolysis-susceptible region (ca. 0.2μm) and an unsusceptible region (ca. 0.6μm).

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Miltpain (EC 3.4.22.-) is a new cysteine proteinase that has been isolated from the milt of chum salmon (Oncorhynchus keta), and is strongly inhibited by cysteine proteinase inhibitors such as E-64, iodoacetamide, NEM, and PCMB. This paper deals with a unique feature, that the enzyme becomes inhibited by o-phenanthroline, phenanthrenequinone, phenanthrene, phenazine, and acridine which have some structural resemblance to a hydrophobic planar shape. Dixon plots showed that the inhibitions of the enzyme by o-phenanthroline and phenanthrenequinone were noncompetitive.

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Pradimicin (PRM), a mannose-binding antifungal antibiotic, recognizes a D-mannoside in the presence of calcium. We demonstrated that BMY-28864, a semi-synthetic analog of PRM, induced apoptosis in U937 cells which had been incubated with 1-deoxymannojirimycin (DMJ). Characteristic morphological changes such as formation of apoptotic bodies and DNA fragmentation were observed in apoptotic cells.

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In the post-source decay (PSD) fragment spectrum of a reduced xyloglucan heptasaccharide (XXXGol) from tamarind seeds, eleven sodium-adduct fragment ions and a precursor ion [M+Na]⁺ were clearly observed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Each fragment ion interval corresponded to the absence of unhydroxylose, unhydroglucose, and glucitol residues, indicating that PSD fragmentation cleavage in the sugar compound occurred only at glycosidic linkages close to the oxygen atom of saccharide ring members, and not in inner sugar ring bonds. The PSD fragment ions were classified into two series, one involving the reducing end and the other involving the non-reducing end. Structural information from both the reducing and non-reducing ends could therefore be simultaneously obtained from the measurement of the positive ion mode. Almost all the fragment ions from species larger than trisaccharide residues could be detected in this PSD fragment experiment. Such fragmentation information will enable the structural determination of xyloglucan oligosaccharides.

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The tumor promoter (-)-indolactam-V (1) exists in two stable conformers (twist and sofa) due to isomerization of the amide group. Indolactone-V (2), the lactone analogue of 1, has been synthesized to investigate the effects of the amide group on its conformation and biological actirities. Indolactone-V (2) existed only as the inactive sofa-like conformer, indicating that the amide group of 1 plays a critical role in formation of the active twist conformation.

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