Rats fed with a vitamin B₆-deficient 70% casein diet for 5 weeks were found to have decreased considerably in the content of phosphatidylcholine (PC) in liver microsomes, presumably because of the depressed PC biosynthesis from choline or phosphatidylethanolamine (PE). The activities of choline phosphokinase and choline phosphotransferase in liver decreased, apparently, as compared with the pair-fed control or control rats. The hepatic level of the PE methyltransferase co-substrate, S-adenosylmethionine (SAM), decreased about 1/3, but the level of the inhibitory metabolite, yadenosylhomocysteine (SAH), was elevated due to the marked reduction in the activities of cystathionine β-synthase and γ-cystathionase. The resultant molar ratio of SAM/SAH decreased drastically such that the methylation of PE to PC was decreased in vivo, as confirmed by lowering the activity of PE methyltransferase in vitro in response to a decreased molar ratio of SAM/SAH. A similar effect on the PE methylation was also observed in the pair-fed control rats, but the PC biosynthesis from choline clearly compensated for the drop of PC biosynthesis from PE. Results of this study demonstrate that vitamin B₆ deficiency modified methionine metabolism and decreased choline utilization, and thus indirectly affected the biosynthesis of PC in liver microsomes.

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The biosynthesis of brassinosteroids was investigated by feeding ²H-labeled teasterone (TE), ²H-labeled typhasterol (TY), and a mixture of ²H-labeled and ³H-labeled castasterone (CS) to seedlings of Catharanthus roseus, Nicotiana tabacum, and Oryza sativa. In all the seedlings examined, the conversion of TE to TY, and the conversion of TY to CS and to TE was demonstrated. In the seedlings of C. roseus, the conversion of CS to brassinolide (BL) and to 3-epicastasterone (3-epiCS) was observed, while the conversion of CS to only 3-epiCS was found in the seedlings of N. tabacum and O. sativa. These results demonstrate the presence of the biosynthetic pathway of TE →TY →CS →BL in seedlings of C. roseus, and the biosynthetic pathway of TE→TY→CS in seedlings of N. tabacum and O. sativa. A reversible conversion between TE and TY was also found. This is the first evidence of CS being the biosynthetic origin of 3-epiCS.

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An uncommon heptaketide metabolite, setosol (2, 8-dimethyl-4 methoxy-6, 10, 11-trihydroxy-benzo-oxaonin), was isolated from a liquid culture filtrate of the fungus Pleiochaeta setosa. The biological activity of the molecule was studied by using 12 microbial strains consisting of three bacteria, three yeasts and six fungi. The level of activity was compared with those of known antibiotics and antifungal agents. The metabolite exhibited antifungal and antibiotic activity against Drechslera oryzae, Gerlachia oryzae, Pyricularia oryzae, Cryptococcus neoformans, and Staphylococcus aureus. The acetylated derivative of setosol did not inhibit the growth of any of the target pathogens. Phytotoxicity studies on whole lupine leaves show that setosol is implicated in the pathogenesis of the brown spot disease of lupines since artificial inoculation of the leaves with the metabolite provoked lesions similar to the characteristic brown spots and lesions on lupine leaves infected by the fungus.

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Brassinosteroids (BRs) contained in rice (Oryza sativa L. ) bran were extracted and highly purified. A new BR, (22R, 23R, 24S)-3α, 22, 23-trihydroxy-24-ethyl-5α-cholestan-6-one, which is termed 28-homo-typhasterol, was identified by GC-MS, in addition to 28-homoteasterone and 6-deoxocastasterone.

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α-Linked galactooligosaccharides (α-GOS A from galactose, and α-GOS B from lactose hydrolyzates)were synthesized using the reverse reaction of α-galactosidase from Candida guilliermondii H-404. The α-GOS A and B were isolated and their structures were identified by methylation analysis. The main product of the disaccharides in α-GOS A and α-GOS B was the (1, 6)-isomer. The remaining disaccharides consisted of (1, 3)-, (1, 2)-, and (1, 1)-isomers. Conditions for synthesis of α-GOS B from lactose hydrolyzates were examined. The yield of α-GOS B was approximately 20% when the mixture of heat-treated cells containing α-galactosidase (60 U/g galactose) and 85% lactose hydrolyzates was incubated for 90h at pH 4.5 and 50°C. The α-GOS A and B were available as the donor substrates in transgalactosylation of α-galactosidase in the same manner as melibiose.

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Constitutive overexpression of the biotin operon (type 9 mutation) in a multicopy plasmid resulted in growth inhibition in Escherichia coli. Deletion analysis of the biotin operon indicated that overexpression of the bioB gene alone, the product of which is believed to catalyze the conversion of dethiobiotin to biotin, is sufficient for growth inhibition. This growth inhibition was still observed when the wild-type bioB gene was replaced by several mutant-type bioB genes derived from biotin auxotrophs that have base-pair substitutions creating amino acid substitutions in the bioB gene product. However, the modification of Ala 143 and Gly 99 of the bioB gene product resulted in recovery from growth inhibition. These results suggest that this phenotype of growth inhibition by overexpression of the bioB gene in E. coli is independent of the biotin-forming activity itself, but is caused by some function involving a specific conformation of the bioB gene product.

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For the first time we found that the bacterial strain Gluconobacter oxydans DSM 4025 produced L-ascorbic acid from L-gulono-γ-lactone and therefore we isolated and Purified the enzyme L-gulono-γ-lactone dehydrogenase catalyzing the oxidation reaction. G. oxydans DSM 4025 produced 8.57 and 13.9 mg of L-ascorbic acid Per ml from 70.3 and 89.3 mgof L-gulono-γ-lactone per ml in the growing and resting cell systems, respectively. The enzyme was isolated from the soluble fraction of the cells of G. oxydans DSM 4025 by DEAE-cellulose, Q-Sepharose, hydroxylapatite, and Sephacryl S-300 column chromatographies. The molecular weight of the enzyme was 110, 000 and it consisted of three kinds of subunits of 61, 000, 32, 500, and 16, 5000. L-Gulono-γ-lactone was oxidized to L-ascorbic acid very rapidly in the presence of dyes, such as 2, 6-dichlorophenolindophenol or phenazine methosulfate, but oxygen was not available as an electron acceptor. The absorption spectrum of the reduced form of the native enzyme showed maxima at 416, 521, and 552 nm in the visible region, indicating the presence of cytochrome c. The enzyme isolated from G. oxydans DSM 4025 was entirely different from isofunctional enzymes of eucaryotic organisms.

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The gene (fdm) coding for formaldehyde dismutase (EC 1. 2. 99. 4) from a genomic library of formaldehyde-tolerant Pseudomonas putida F61 was cloned and expressed in Escherichia coli. The nucleotides of the cloned DNA were sequenced ; they included a single open reading frame of 1200 base pairs, coding for a putative protein with a molecular weight of 42, 848. Sequencing of the first 20 N-terminal amino acid residues and of an internal part of the enzyme purified from P. putida F61 established the identity and the start codon of fdm. Comparison of the amino acid sequence predicted from fdm with that of alcohol dehydrogenase from horse liver suggested a putative pyridine-dinucleotide-binding domain in fdm, and also potential ligands for the catalytic domain and the second zinc atom-folding domain. fdm seemed to be expressed in E. coli under control of the promoter of fdm ; there was an E. coli promoter-like sequence upstream from the gene. The enzyme expressed in E. coli was purified to homogeneity. The molecular weight and the sequence of the first 20 N-terminal amino acid residues were identical with those of P. putida formaldehyde dismutase. Each subunit contained 1mol of NAD(H) and 2mol of zinc per mol of protein. The enzyme produced in E. coli catalyzed the dismutation of formaldehyde to form methanol and formic acid at the ratio of 1 : 1 in the absence of the exogenous electron acceptor, NAD(H).

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A (1→3)-β-D-glucan, SSG, from Sclerotinia sclerotiorum IFO 9395 was metabolically labeled using [1-¹³C] and [2-¹³C]glucose, and the fate of the ¹³C-label was examined by ¹³C-NMR spectroscopy. ¹³C-NMR spectra of metabolically labeled SSG (¹³C-SSG) showed that most of the ¹³C-label in glucose residues of ¹³C-SSG were recovered from the originally labeled sites of carbon in glucose residue, and suggested little rearrangement during take-up from the medium. In the case of poor SSG producing culture conditions (reduced shaking rate), ¹³C-glucose incorporation in SSG molecule was similar to that in the case of high SSG-producing culture conditions. In addition, significant amounts of ¹³C-labeled trehalose were found in ¹³C-NMR spectra of the mycelium cultured in both poor and high SSG-producing conditions. These results suggested that different culture conditions affected SSG production, but not the metabolism of glucose and the biosynthetic pathway of SSG.

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An intracellular β-D-fructofuranosidase produced by Aspergillus sydowi IAM 2544 was purified by Q-Sepharose and Alkyl-Sepharose chromatographies. The molecular mass was 50kDa by SDS-PAGE analysis. The optimum pH and temperature of sucrose hydrolyzing activity of the enzyme were 5.5 and 75°C, respectively, but those of fructosyl transferase activity were 5.2 and 55°C, respectively. The enzyme efficiently transferred the fructose residue of sucrose as a donor to trehalose as an acceptor. And the amount of fructosyl and oligofructosyl trehaloses produced was changed by the molar ratio of trehalose as an acceptor to sucrose as a donor used. The most efficient production of the transferred products was achieved at the reaction conditions in the range of molar ratios of 1 : 1 to 3 : 1 (trehalose : sucrose). The chemical structures of these new kinds of resulting series of fructosyl and oligofructosyl trehaloses produced were identified as O-β-D-Fru-(2→6)-α-D-Glc-(1→1)-α-D-glucopyranoside, O-β-D-Fru-(2→1)-O-β-D-Fru-(2→6)-α-D-Glc-(1→1)-α-D-glucopyranoside, and O-β-D-Fru-(2→1)-O-β-D-Fru-(2→1)-O-β-D-Fru-(2→6)-α-D-Glc-(1→1)-α-D-glucopyranoside. These results indicate that β-fructofuranosidase from Aspergillus sydowi specifically transferred the fructose residue of sucrose to the C6-OH position of the glucose residue of trehalose at the early stage of the reaction, following the elongation of the fructose residue by the transfructosylation of the enzyme to form oligofructosyl trehalose of a longer fructose chain.

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We have isolated cyclohexane-tolerant mutants from Escherichia coli strain JA300, which is cyclohexane-sensitive and n-hexane-tolerant. These mutants were resistant to low levels of ampicillin, chloramphenicol, nalidixic acid, and tetracycline, and were sensitive to a low level of kanamycin. Spontaneous clones resistant to low levels of the antibiotics, isolated from JA300, showed altered levels of organic solvent tolerance. The clones resistant to ampicillin and chloramphenicol had cyclohexane tolerance. Some of the resistant clones had cyclohexane and n-pentane tolerances. On the other hand, some kanamycin resistant clones became sensitive to n-hexane. Therefore, mechanisms to improve tolerance levels toward organic solvents are closely correlated with some antibiotic resistant system to low levels of antibiotics.

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Three β-N-acetylglucosaminidase (catalyzing hydrolysis of p-nitrophenyl-β-D-GlcNAc) were purified from the integument tissue of Bombyx mori larvae during metamorphosis into pupae. The largest enzyme (66kDa by SDS-PAGE, 126kDa by gel-filtration chromatography) reacted with chitooligosaccharides to produce GlcNAc. A full-length cDNA encoding this chitooligosaccharidolytic β-GlcNAcase was isolated. Based on the amino acid sequence deduced from the nucleotide sequence, the pre-β-GlcNAcase was found to consist of 596 amino acid residues including a characteristic signal peptide of 23 residues and have an M_r of 68, 212. Homology search and limited proteolytic digestion showed that the enzyme has a C-terminal 58-kDa catalytic domain very similar to that of human lysosomal β-hexosaminidase that is responsible for hydrolyzing gangliosides. Two other enzymes (composed of 58-kDa and 48-kDa polypeptides, respectively) did not hydrolyze chitooligosaccharides, and were not proteolytic fragments from the largest enzyme judged by amino acid sequencing analyses. Natural substrates for the β-GlcNAcases are unknown.

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To improve the acaricidal activity of milbemycins, 8, 9-epoxymilbemycin A₄ (3) and 5-O-acyl-8, 9-epoxymilbemycin A₄ (7) were synthesized by using selective epoxidation of the 8, 9-double bond. The acaricidal activity against the two-spotted spider mite (Tetranychus urticae) was slightly improved by 8, 9-epoxidation of milbemycin A₄ (1b). while acylation of 8, 9-epoxymilbemycin A₄ (3) markedly enhanced its activities. Some 5-O-acylated 8, 9-epoxides totally controlled the mites at a concentration of 3ppm.

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DNA-binding proteins were extracted from both exponentially growing cells of Bacillus cereus ts-4 and cells that were induced to sporulate at different stages of chromosome replication, by using a double-stranded B. cereus ts-4 DNA-cellulose column. Two-dimensional electrophoresis of the proteins found that the amounts of 17 proteins changed drastically after induction of sporulation at all the stages. For 8 of those proteins, the largest or the smallest amount was found in the cells which were induced to sporulate 40min after the initiation of chromosome replication, the sensitive stage for sporulation. The N-terminal amino acids of 6proteins among the selected proteins were sequenced. The sequence obtained from a 59-kDa protein had sequence similarity (> 45%) to GroEL from several bacterial species. In addition, the sequences from 76- and 52-kDa proteins matched deduced amino acid sequences of a Mycobacterium leprae gene showing homology to the bacterial atp operon and the B. subtilis guaB for IMP dehydrogenase, respectively.

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The effects of galactooligosaccharides (GOS), a mixture of galactosyl oligosaccharides formed from lactose by the transgalactosyl reaction of β-D-galactosidase derived from Bacillus circulans, on calcium absorption and prevention of bone loss were examined in ovariectomized (OVX) Wistar rats. Rats fed on a diet containing GOS absorbed calcium more efficiently than those on the control diet after 8-10 days and 18-20 days, and the bone (femur and tibia) ash weight and tibia calcium content of OVX rats fed on the GOS diet were significantly higher than those of the control animals. Although the serum total cholesterol of the ovariectomized rats was significantly elevated, GOS produced a significant hypocholesterolemic effect in the OVX rats. GOS, which is fermented by bacteria in the lower part of the intestine, enhanced volatile fatty acid production, and thus prevented bone loss and lower serum total cholesterol concentration in the ovariectomized rats.

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Soybean 7S and 11S globulins, which are two major components of soybean protein, were mixed in various ratios. Gelation of the protein blend after the addition of glucono-δ-lactone (GDL) or calcium sulfate was studied to analyze the gelation process of tofu (soybean curd). Dynamic viscoelasticity measurements at a constant temperature as a function of time were done. Faster gelation was observed in mixtures containing 11S in a higher proportion for both the GDL and calcium systems. The value of the storage modulus of the 5 : 5 blend system of 7S and 11S globulins was minimum when the GDL concentration was adjusted to 17mM, at which level the gels consisting of 7S globulin alone and 11S globulin alone had a similar storage modulus. Under the same conditions, although the gel of the soybean protein isolate showed almost the same value for the storage modulus as a mixture of 7S : 11S in the same ratio, the gelation was slower than that expected from the 7S and 11S blend. Since the use of calcium sulfate led to a large syneresis for 11S globulin, no homogeneous gels were obtained from this system. The 7S fraction in the mixture prevented the syneresis of 11S globulin gel induced by calcium. Gel networks observed by scanning electron microscopy for 7S globulin with GDL was finer than those for the 11S one. The gel formed by soybean protein isolate involved both the 7S- and 11S-types of networks. The coarser networks were mainly observed in the gel of the 5 : 5 blend. The 11S fraction in the mixed system mainly formed a gel matrix.

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Three new okaramine congeners, okaramines D(1), E(2), and F(3), were isolated from okara fermented with a strain of Penicillium simplicissimum ATCC 90288, and their structures were determined by spectro-scopic methods. Okaramine D exhibited insecticidal activity against silkworms, but the other compounds showed no such activity.

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Many of the putative enzymes mediating oxidative reactions of the citric acid cycle were measured in cell-free extracts of Lactobacillus plantarum ATCC 8014, L. casei ATCC 7469, L. helveticus ATCC 15009, and L. acidophilus ATCC 11506. The activities of citrate synthase and aconitase were demonstrated in all the strains investigated. None of isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase activities were detected in any of the strains tested. Fumarase and malate dehydrogenase activities could be detected in some but not all of the strains. On the other hand, all the strains had the activities of fumarase and malate dehydragenase when measured in the direction of reductive reactions of the cycle. In addition, the activities of fumarate reductase and citrate lyase were found in all the strains and in most strains, respectively. All these results suggest that the oxidative pathway in the citric acid cycle serving both bioenergetic and 2-oxoglutarate biosynthetic functions is completely inoperative in lactobacilli, while a set of reversed reactions leading to succinate synthesis functions in these bacteria. These findings also suggest that the lack of 2-oxoglutarate dehydrogenase activities is closely related to the inability to synthesize glutamate in lactobacilli.

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Escherichia coli excretes acetate during aerobic growth in a rich medium, L-broth containing 0.4% glucose, and growth ceases before depletion of glucose because of the decrease in pH caused by the accumulation of acetate. The addition of sodium phosphate buffer to the medium allows cells to reuse the acetate accumulated. Reuse of the acetate, however, does not occur in the presence of remaining glucose. A gene on a multicopy plasmid was found to significantly decrease the accumulation of acetate by the transformant and the growth did not cease until depletion of both the glucose and acetate in the medium. The gene was tentatively named mlc (making large colonies). The putative Mlc protein has high holology with the NagC protein, which is a regulator ptotein in the nag operon responsible for the use of N-acetylglucosamine. The nagC gene on a multicopy plasmid also decreased the accumulation of acetate. Although the function of the genes in the phenomenon described is still unclear, transformants harboring the mlc gene or nagCgene on a multicopy plasmid will be useful for condensed cultivations involving glucose.

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1, 2-Butanediol (1, 2-BD) dehydrogenase was purified from Rhodococcus sp. strain TB-42. The activity of this enzyme was induced by 1, 2-BD and the highest activity was observed in the cells grown on 1, 2-BD as a carbon source. The native enzyme had a molecular mass of 80 kDa, and was composed of two different subunits with molecular masses of 46kDa and 43kDa. The enzyme showed a pH optimum of 10.5, and displayed the highest activity at 35-40°C. NADP could not replace NAD as an electron acceptor for the reaction. The enzyme oxidized diols with two adjacent hydroxy groups such as 1, 2-propanediol, 1, 2-BD, 2, 3-BD, and 1, 2-pentanediol except for 1, 2-ethanediol. Other diols, primary and secondary alcohols, could not be used as substrates for this enzyme.

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To clarify the mechanism of pressure-induced meat tenderization or acceleration of meat conditioning, the pressure-induced morphological and biochemical changes in sarcoplasmic reticulum (SR), and Ca²⁺ release from SR in the rabbit skeletal muscle treated with high pressure (100-300 MPa, 5min) were investigated in comparison with those of the SR from conditioned muscle. The destruction of the membrane structure of the SR expanded with increasing pressure applied to the muscle. Significant changes in the SDS-PAGE profile were not observed in the SR from the pressurized muscle up to 200MPa, but a marked decrease of the ATPase protein and high-affinity Ca²⁺-binding protein were observed in the SR from the pressurized muscle at 300MPa. The ATPase activities increased in the SR isolated from the muscle exposed to high pressure up to 200MPa. When the muscle was pressurized at 300MPa, the ATPase activity dropped to the same level with that of the SR from the untreated muscle. Ca²⁺ uptake ability of the SR vesicles measured using a fluorescent chelating reagent decreased with increasing pressure applied to the muscle. Ultrastructural studies showed that Ca²⁺, which was mainly localized in the SR region of the untreated fiber bundles, was translocated into myofibrillar space in the pressurized muscle. It is clear that a brief exposure of the muscle to high pressure causes considerable changes in membrane structure and biochemical function of SR as compared with those of SR in the muscle induced by conditioning. The pressure-induced Ca²⁺ release and loss of the structural regularity of myofibrils may be one of the causes for meat tenderization and acceleration of meat conditioning induced by high-pressure treatment.

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The optical resolution of racemic 2, 2, 2-trifluoro-1-(naphthyl)ethanols (TFNEs) was achieved by lipase-catalyzed enantioselective acetylation with vinyl acetate in octane, S-acetates and R-alcohols being obtained. The introduction of a methyl group into the naphthalene ring at the 4-position (2b) or the 2-position (2c) markedly decreased the reactivity, in particular for 2c. 2, 2, 2-Trifluoro-1-(1-naphthyl)ethanol (2a) behaved differently from the regio-isomer, 2, 2, 2-trifluoro-1-(2-naphthyl)ethanol (2d) : The enantioselectivity of lipases [LIP (Pseudomonas), PLC (Alcaligenes), and ALC (Achromobacter)] was high for 2a but low for 2d, whereas lipases [AK and PS (Pseudomonas)] exhibited higher selectivity for 2d than for 2a. Three other derivatives, 2, 2, 2-trifluoro-1-(phenyl)ethanol (2e), 2, 2, 2-trifluoro-1-(indol-3-yl)ethanol (2f), and 1-(1-naphthyl)ethanol (2g), were prepared, and their behaviors with respect to lipase-catalyzed acetylation were compared with 2a.

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DOPA quinone imine conversion factor was partially purified from larval hemolymph of the silkworm, Bombyx mori, using a novel assay. The factor was labile at 4°C below pH 5 and above pH 11, and at pH 8.5 above 55°C. Activity of the factor was maximal at pH 7.5-9. The factor catalyzed the decolorizations of L-DOPA methyl ester and α-methyl DOPA methyl ester chromes besides DOPA quinone imine. HPLC analysis indicated that the factor-catalyzed decolorization reaction of DOPA quinone imine resulted in the accumulation of dihydroxyindole. Based on spectral changes of α-methyl DOPA methyl ester chrome, it was suggested that the factor catalyzed the intramolecular oxidoreduction of DOPA quinone imine. During the oxidation of L-DOPA with endogenous phenoloxidase, the factor facilitated the formation of melanochrome.

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A direct and simple assay method for dimethyl sulfoxide reductase (DMSO reductase) by determining dimethyl sulfoxide (substrate) and dimethyl sulfide (product) in a reaction mixture of the enzyme was developed. Micellar electrokinetic chromatography (MEKC), a kind of capillary electrophoresis, was quite effective for a quantitative determination below the nanogram level without any pretreatment of the reaction mixture.

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The endogenous gibberellins (GAs) of rice were analyzed during seed germination and during the regeneration of plantlets from embryogenic cells. Four 13-hydroxylated (13-OH) GAs (GA₅₃, GA₁₉, GA₂₀, and GA₁) were identified by combined gas chromatography-selected ion monitoring (GC-SIM) from the seeds at every stage of germination examined. A significant increase in their level was observed at the time shoot elongation started. No GA was detected in the embryogenic cells before transferring into a regeneration medium, while the occurrence of 13-OH GAs was confirmed by full-scan GC-MS or GC-SIM during the regeneration of plantlets. These results indicate that the early-13-hydroxylation pathway of GA biosynthesis operated during embryo development and seed germination.

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The genes encoding the extracellular levansucrase and invertase of Zymomonas mobilis have been cloned and sequenced. The levansucrase gene, sucZE2, spans 1269 bp and encodes an M_r 46, 790 polypeptide, and the invertase gene, sucZE3, is of 1239 bp and encodes an M_r 46, 110 polypeptide. The 5'-terminal sequences of both genes corresponded to the N-terminal amino acid sequences of the secreted levansucrase and invertase, implying that the secretion of both enzymes does not involve proteolytic processing of the N-terminals. Both enzyme molecules appear to carry no typical N-terminal secretion signal. Significant homology between sucZE2 and sucZE3 was observed, but both genes showed no homology to the gene encoding an intracellular invertase coexisting in Z. mobilis. Two genes, sucZE2 and sucZE3, are possibly placed in an operon because the expression of two genes were simultaneously controlled by the regulator gene zliE, previously identified.

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We designed three hairpin ribozymes to cleave Escherichia coli β-glucuronidase (GUS) mRNA and tested those activities in vitro. One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites. All ribozymes cleaved the model substrate (100 bases long) at the predicted target site. Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site. Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26°C, a general growth condition of plant cells. Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed.

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Prolylcarboxypeptidase (Angiotensinase C, EC 3. 4. 16. 2) was purified to homogeneity from cell free extracts of Xanthomonas maltophilia by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Sephadex G-150, FPLC-Hiload Superdex 200pg, and FPLC-Hitrap SP columns, with an activity recovery of 15%. The molecular weight of the enzyme was found to be 330, 000 by gelfiltration and 83, 000 by SDS-PAGE, suggesting a tetrameric form for the native enzyme. It had an optimum pH of 8.5 and stability between pH 8.0 and 11.0. The isoelectric point of the enzyme was 6. 6. The enzyme hydrolyzed Pro-X bonds when proline was in the penultimate position from the carboxyl terminal. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), while phenylmethylsulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), iodoacetamide, and metal chelators had no effect.

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Previous studies have shown that there are hematological abnormalities in selenium (Se)-deficient animals. This study examined the effects of Se deficiency on various minerals in serum and other tissues of male Wistar rats. The animals were given free access to either Torula yeast-based Se-deficient (SeD) diet or Se-adequate (SeA) (containing 0.1mg Se/kg diet as sodium selenite) diet. Blood was sampled after 12 and 24 weeks, and the rats were killed after 24 weeks, for the analysis of minerals in serum, liver, kidney, heart, and spleen. Analyses showed that Se deficiency affected the concentrations of magnesium, calcium, iron, copper, and zinc in selected tissues and serum. During the entire feeding period, serum iron concentration was 40-58% greater in SeD rats compared with SeA rats. The transferrin saturation with iron was significantly greater in SeD rats than in SeA rats (57-60% versus 30-31%). Iron concentrations in the tissues ranged from 1.1 to 2.5 times higher in SeD rats than in SeA rats (p<0. 05). Similarly but to a lesser extent, the concentrations of zinc and magnesium were significantly greater in the serum of SeD rats compared with SeA rats, and the concentrations of calcium was significantly higher in kidney and spleen and of copper in liver, while the concentration of magnesium was significantly lower in liver and kidney. These results suggest that Se deficiency may cause a secondary overload of iron and unbalanced distribution of other minerals.

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The formation of 3-{N-(2-formyl-5-hydromethylpyrrolyl)}-propionic acid (3NFHP), 3-{N-(2-methyl-3, 6-dihydro-4, 5, 6-trihydrox-ylpyridinyl)}-propionic acid (3NMDTP) and 3-deoxyglucosone (3DG) was investigated in the reaction system of fructose and β-alanine as a cookie model. 3 NFHP had a caramel-like sweet aroma and seemed to contribute to the favorable flavor of cookies.

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From the pollen of Robinia pseudo-acacia L., bioactive substances in the rice lamina inclination test were extracted and purified by several chromatographic steps to give highly purified fractions. They were derivatized and then analyzed by GC-MS, resulting in identification of castasterone, 6-deoxocastasterone, and typhasterol.

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Anomeric form of the products from chitinase-catalyzed hydrolysis was analyzed by ¹H-NMR spectroscopy. When the chitinase from Streptomyces griseus was added to the solution of substrate, N, N, N, N, N-pentaacetylchitopentaitol, β-anomer was first produced by the enzymatic hydrolysis, and then transformed to α-anomer by mutarotation. In contrast, the chitinase from Dioscorea opposita (yam) produced only α-anomer, which was transformed to β-anomer by mutarotation.

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A catechol 2, 3-dioxygenase (C23O) gene of Pseudomonas aeruginosa was expressed under the Simian virus 40 or Rous sarcoma virus promoter in mammalian cells ; it was found that the gene could be used as a reporter for the study of gene expression. The C23O gene was a more sensitive reporter than the generally used β-galactosidase gene.

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A new lactarane sesquiterpenoid with a β, γ-epoxy-γ-lactone moiety was isolated from the neutral fraction of an extract of Russula emetica, together with three known sesquiterpenoids. The structures of these compounds were identified by chemical transformation of related compounds and by spectroscopic methods.

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In the search for antioxidants from microbial organisms, we found that Penicillium roquefortii IFO 5956 produced an antioxidant. This antioxidant was isolated from a culture broth of the strain, and its structure was identified to be 2, 3-dihydroxy benzoic acid (1). The antioxidative activity of 1 was nearly equal to that of tert-butyl-4-hydroxyanisol(BHA).

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Overproduction of TmrB protein, a 22. 5-kDa protein with an N-terminal ATP-binding region and a C-terminal amphiphilic α-helix, confers resistance to tunicamycin on Bacillus subtilis. TmrB protein was found to bind Sepharose 6B to which tunicamycin was covalently linked. Experiments with mutant proteins found that the C-terminal region of TmrB protein might be involved in the binding to tunicamycin.

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Pseudomonas marginalis N6301 produced pectin lyase (EC 4. 2. 2. 10) in the medium with cell lysis when the culture was treated with mitomycin C. We purified the enzyme by carboxymethyl-cellulose, hydroxylapatite, and gel-filtration column chromatog-raphies. The enzyme had a molecular weight of 34, 000 by SDS polyacrylamide gel electrophoresis and was mostly stable around pH 6.5. The optimum pH and temperature for the enzyme activity were 8.0 and 30°C, respectively. The activity was inhibited severely by 2mM N-ethylmaleimide, maleic anhydride, and p-chloromercuri-phenylsulfonic acid. Further, the pectin lyase produced in a medium containing glycerol was purified. The molecular weight of the enzyme was identical to that of the enzyme produced in the presence of mitomycin C.

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Inhibitory activities toward the endothelin-converting enzyme (ECE) were detected in pepsin digests of bonito pyrolic appendix and beef. After Sep-Pak C₁₈ fractionation, this activity from bonito and beef was recovered in the 50% and 25% ethanol fractions, respectively. Activities were also recovered in the ultrafiltrate (<5000 Da), and disappeared after a pronase treatment. Therefore, these inhibitory activities may have peptidic properties.

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Ribonuclease was purified to homogeneity from Monascus sp. No. 3403 by column chromatography on DEAE-cellulose, DEAE-Toyopearl 650S, 5'-AMP Sepharose 4B, and Sephadex G-75. The molecular weight of the purified enzyme was estimated to be 30, 000by gel filtration and 26, 000 by Sodium dodmlecyl sulfate poly-acrylamide gel electrophoresis. The enzyme contained 5.0% car-bohydrate. Homopolyadenylic acid was a good substrate for the enzyme. The optimum pH of the enzyme was 4.2 and its optimum temperature was 55°C.

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An extracellular lipase from Penicillium roqueforti IAM 7268 was purified by a procedure involving ethanol precipitation, ammonium sulfate precipitation, and DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M, and Toyopearl HW-60 column chromatog-raphies. The purified lipase was homogeneous with 25kDa of molecular mass by SDS-polyacrylamide gel electrophoresis, and had high specificities toward short-chain fatty acid esters.

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We found that Acetobacter xylinum KU-1 produced cellulose from D-mannitol. The optimum culture conditions for cellulose production were 1.5% D-mannitol, 0.5% Polypeptone, 2.0% yeast extract, pH 5, 30°C, and 48h. The amount of cellulose from D-mannitol was more than 3 times as much as that from D-glucose under the same culture conditions [productivity (mg/ml-medium) : from D-mannitol, 4.6 ; from D-glucose, 1. 2].

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We examined the effects of dietary proso-millet protein on plasma concentration of high-density lipoprotein (HDL) cholesterol in mice. The results confirmed also, in this animal, the elevation of plasma concentration of HDL cholesterol without the involvement in raising the concentration of low-density lipoprotein cholesterol like those with rats reported in our previous paper. This would suggest a beneficial effect of millet protein on cholesterol metabolism.

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For the classification of plant chitinases, phylogenetic relationships were analyzed besides the established classification of domain structure and C-terminal extension sequences. Two genetically different subclasses (high and low molecular weight) were found for the main structure of class I and class II chitinases in their phylogenetic trees. The genetic distance of these subclasses showed that the high molecular weight subclass may be an ancestral molecule.

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We found an inducible phenylserine dehydratase [EC 4. 2. 1. -] in a soil bacterium identified as Pseudonlonas pickettii PS22. The enzyme catalyzes the deamination of L-threo-3-phenylserine to yield phenylpyruvate and ammonia. The enzyme had an optimum reactivity at about pH 7.5. The K_m for L-threo-3-phenylserine was 0.21 mM.

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To rapidly establish recombinant protein hyper-producing cell lines we introduced a reporter plasmid into BHK-21 cells that had been`primed' by transfection and amplification of the ras oncogene. The reporter plasmid used carries the human interleukin-6 (hIL-6) gene which is under control of the human cytomegalovirus immediate early promoter. The primed BHK cell lines were shown to produce many stable hIL-6 hyper-producing cells achieving about 15 times higher productivity than the control BHK-21 cells.

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The amplified ras oncogene greatly enhanced the production of recombinant human interleukin-6 (hIL-6) under control of the cytomegalovirus immediate early promoter (CMV promoter) in BHK-21 cells. When the adenovirus E1A oncogene was further transfected into the above mentioned ras-amplified hIL-6 hyper-producing BHK cells, the transfectants had about 10 times higher productivity than non-transfectants. However, the E1A gene alone did not enhance productivity. These results implicate a ras and E1A synergistic ability that acts to enhance hIL-6 production.

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The effect of insulin on polypeptide chain elongation was examined in soleus muscles isolated from 18hour-fasted mice. Treatment with insulin for 1hour increased the elongation rate, which was estimated by the half-transit time. This suggests that insulin stimulated protein synthesis by modifying the elongation rate in addition to the initiation rate.

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New cysteine protease inhibitors, named cathestatin A and B, have been discovered as metabolites of Penicillium citrinum. Cathestatins were found to be decarbamidoyl analogs of estatins and showed a specific inhibition for cysteine proteases. Cathestatins suppressed parathyroid hormone (PTH)-stimulated ⁴⁵Ca release in organ cultures of chick embryonic calvaria.

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A short-route synthesis of an optically active phosphocholine bearing an unsaturated acyl group at the sn-1 position was developed via lipase-catalyzed enantioselective acylation of 2-O-methoxyeth-oxymethylglycerol, removal of the MEM group by employing cat-echol boron bromide, subsequent DCC-mediated acylation of the hydroxy group at the 2-position and final introduction of a choline phosphate moiety.

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