Rhizoctonia sp. D-90 decolorized molasses melanoidin medium and a synthetic melanoidin medium by 87.5% and 84.5%, respectively, under experimental growth conditions. Mycelia grown in solutions of melanoidin turned dark brown. However, the melanoidin (dark brown in color) could be eluted from the mycelia by washing in a solution of NaOH, and the maximum yield of melanoidin from mycelia reached 96. 1%. Mycelia grown in potato dextrose medium did not have any electron-dense materials in the cytoplasm or around the cell membrane, but when such mycelia were transferred to melanoidin media, abundant electron-dense material appeared in the cytoplasm and around cell membranes. Subsequently, the electron-dense materials disappeared when the mycelia were returned to the potato dextrose medium for further growth. The mechanism of decolorization of melanoidin by Rhizoctonia sp. D-90 involved absorption of the melanoidin pigment by the cells as a macromolecule and its intracellular accumulation in the cytoplasm and around the cell membrane as a melanoidin complex, which was then gradually decolorized by intracellular enzymes.

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Isomaltooligosaccharides (IMO) are a mixture of isomaltose, isomaltotriose, panose, isomaltotetraose, etc. IMO and its hydrogenated derivative (IMH) were characterized for their luminal clearance from rat jujunum loops as the indication of their digestibility. They were compared with a disaccharide fraction (IM2) and a higher oligosaccharide fraction (IM3) prepared from IMO, typical digestible saccharides (maltose, maltotriose, and sucrose), and typical nondigestible saccharides (maltitol, raffinose, and fructooligosaccharides (FO)). The clearance rate of IMO was significantly smaller than that of IM2, which was mainly composed of isomaltose (64.3%), and digestible saccharides, and significantly larger than that of nondigestible saccharides. That of IM2 was almost the same as that of sucrose or maltotriose but significantly smaller than that of maltose. That of IM3 tended to be smaller than that of IMO, and larger than that of nondigestible saccharides. That of IMH was significantly smaller than that of IMO and similar to that of maltitol. These results seem to indicate that IMO is slowly digested in the jejunum, that the components having higher degree of polymerization of IMO are less digestible, and that IMH is nondigestible.

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A dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus sp. strain SY1 could also use dimethyl sulfide (DMS), dimethyl sulfoxide (DMSO), and several alkylsulfonates as sole sources of sulfur. Strain SY1 grown on DMSO accumulated DMS, dimethyl sulfone (DMSO₂), and methanol in the culture medium and methane in the gas phase, and methane was the product of methanesulfonate desulfonation. These results indicated that strain SY1 could finally degrade DMS in the oxidative pathway via DMSO, DMSO₂, and methanesulfonate to methane and sulfate, reducing a part of DMSO back to DMS. From desulfonation patterns of alkyl and aromatic sulfonates, different types of desulfonation are proposed. Sulfate was suggested to repress the expression of the enzymes involved in DBT desulfurization. Removal of the sulfate produced by the addition of BaCl₂ enhanced the degradation rate of DBT about 14%.

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A lipase from Aspergillus oryzae was purified by ammonium sulfate fractionation, anion exchange chromatography, hydrophobic interaction chromatography, and anion exchange chromatography. The purified enzyme was a monomeric protein with a molecular mass of 41 kDa estimated by SDS-PAGE and 39 kDa by gel filtration. The optimum pH at 30°C and optimum temperature at pH 7.0 were 7.0 and 30°C, respectively. The enzyme was stable over a pH range of 6-9 at 25°C for 18 h, and up to 30°C at pH 7.0 for 3 h. Ag⁺, Fe³⁺, Hg²⁺, Cu²⁺, and Zn²⁺ inhibited the enzyme activity severely. The enzyme was a lipase that hydrolyzed monoacylglycerols and diacylglycerols, but did not hydrolyze triacylglycerols. The N-terminal amino acid sequence of the enzyme was highly homologous with that of the mono- and diacylglycerol lipase from Penicillium camembertii U-150.

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The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.

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A new approach to the pre-column derivatization and analysis of histamine by HPLC is described, the method being based upon the formation of a diazo-coupled derivative of histamine. This derivatization method is simple, efficient, sensitive and specific for histamine and other imidazole compounds without a preliminary clean-up step. The liquid chromatographic system allows for rapid, bonded-phase separation with visible-light detection of these compounds within 20 min at a 10-pmol sensitivity. By using this method, the production of a large amount of histamine was confirmed in the muscle of mackerel Scomber japonicus incubated with the intestinal contents of the mackerel.

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We investigated the effects of continuous and massive intake of chitosan with sodium ascorbate (AsN) on the mineral and the fat-soluble vitamin status in male Sprague-Dawley rats fed on a high-fat diet. The apparent fat digestibility in the chitosan-receiving group was significantly lower than that in the cellulose- or glucosamine-receiving group. Chitosan feeding for 2 weeks caused a decrease in mineral absorption and bone mineral content, and it was necessary to administer twice the amount of Ca in the AIN-76 formula, which was supplemented with AsN, to prevent such a decrease in the bone mineral content. Moreover, the ingestion of chitosan along with AsN led to a marked and rapid decrease in the serum vitamin E level, while such a loss in vitamin E was not observed for rats given glucosamine monomer instead of chitosan.

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The 4993-bp cryptic plasmid, pMA2, from a cyanobacterium, Microcystis aeruginosa f. aeruginosa Kutzing, originally derived from Kasumigaura lake, was completely sequenced and analyzed. Plasmid pMA2 had a unique sequence motif CTTGATT, which was proposed to be a nicking site on the smaller plasmid pMA1 (2287-bp) by the presence of single-stranded DNA susceptible to S1 nuclease. We had detected the occurrence of the single-stranded pMA1 in the living M. aeruginosa cells. This was also the case of pMA2. Thus, we suggest that both plasmids, pMA1 and pMA2, replicate through the rolling circle mechanism. By computer analysis of the pMA2 sequence, two open reading frames were found : one had a predicted molecular size of 30, 440 Da and the other on a complementary one had a predicted molecular size of 10, 852 Da. No rep protein was detected. Replication mechanisms of the plasmids are discussed.

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To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A. ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them. The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identitified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.

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Poly(vinyl alcohol) dehydrogenase (PVADH-S) purified from Pseudomonas sp. 113P3 dehydrogenized poly(vinyl alcohol) (PVA), but the enzyme reaction was significantly affected by changing the properties of PVA, in terms of saponification degree, ethylene content, polymerization degree, tacticity, and 1, 2-glycol content. The apparent V_max/K_m value of PVA containing 10 mol% ethylene was over 10-fold greater than that of PVA. The hydrophobicity of PVA-related polymers might be important in the affinity for the PVADH-S.

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The deodorizing mechanism of (-)-epigallocatechin gallate (EGCg), the main constituent of a green tea extract, against methyl mercaptan (CH₃SH) was investigated. EGCg showed deodorizing activity against CH₃SH by a chemical reaction between EGCg and CH₃SH. The non-volatile reaction products were identified to be compounds introducing a methylthio and/or a methylsulfinyl group into the B ring of EGCg, and gaseous oxygen was necessary for deodorizing activity. From these results, it was assumed that the deodorizing mechanism of EGCg was due to the addition of a methylthio group to the ortho-quinone generated by atmospheric oxygen. It was also found that secondary compounds produced by the reaction between EGCg and CH₃SH had a stronger deodorizing activity than that of EGCg itself.

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We have examined the effects of dietary level of protein (5% or 30% in casein) on enzyme activities and mRNA levels of two xenobiotic-inducible UDPglucuronosyltransferase (UDPGT) enzymes, such as chloramphenicol-UDPGT (CP-UDPGT) and 4-nitrophenol-UDPGT (4NP-UDPGT), in the livers of rats treated or not treated with polychlorinated biphenyls (PCB). In animals fed 5% casein and not treated with PCB, CP-UDPGT activity tended to be lower than in rats fed 30% casein. In contrast, 4NP-UDPGT activity was higher in rats fed 5% casein. In rats treated with PCB, the activities of both enzymes were increased. CP-UDPGT mRNA level was higher (1.4-fold) in rats fed 30% dietary casein than in those fed 5% casein. In contrast, 4NP-UDPGT mRNA level was higher (2.8-fold) in rats fed 5% casein than in those fed 30% casein. These changes in mRNA levels paralleled those in the activities of the two enzymes. The data indicate that the dietary protein is an important factor controlling the expression of hepatic xenobiotic-inducible UDPGT genes and modulates the capacity of the liver to metabolize foreign compounds.

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Pectinase (polygalacturonase) was purified from a commercial pectinase preparation from a mold. Substrate binding of pectinase was measured by centrifugal affinity chromatography using an immobilized substrate, pectic acid. Desorption of pectinase from the affinity matrix with the substrate pectin and pectic acid gave K_d values of 5.3 and 8.5mg/ml, respectively. Chemical modification of pectinase by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and diethyl pyrocarbonate (DEP) caused a loss of most of the enzyme activity, but the substrate binding ability was not impaired. Thus, the pectinase preparation was digested with lysyl endopeptidase and the resulting peptides were treated with pectic acid-affinity gel. Three peptide fragments, which were recovered from the affinity column and sequenced, were identical to sequences in the second pectinase gene from Aspergillus niger. The first peptide contained 17 amino acids, Asp101-Ser117, and the second and third peptides corresponded to 18 amino acids of Asn152-Asp169. These results indicate that the inactivated pectinase retained substrate binding ability and would function as an acidic polysaccharide recognizing protein.

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During the course of purifying the androgenic gland hormone of the terrestrial isopod, Armadillidium vulgare, that induces post-embryonic sex differentiation, four structurally related peptides were obtained and their structures determined by a combination of microsequence and mass spectral analyses. These peptides were found to exist speciffically in the seminal vesicle and vas deferens by a Western blot analysis, therefore being designated as seminal vesicle-specific peptides (SVSPs). They had essentially the same amino acid sequences but differed from one another in the truncation of several residues at the N-terminus and of one residue at the C-terminus, and in the modification of glutamine to pyroglutamate at the N-terminus. The longest peptides, SVSP-4, consisted of 60 amino acid residues with two intramolecular disulfide bridges. There is no significant homology with any other vertebrate or invertebrate peptides.

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Enantioselective syntheses of all the possible stereoisomers of 1-phenyl-2, 3-butanediol (erythro isomer 1 and threo isomer 2) and of 3-hydroxy-4-phenyl-2-butanone 3, the odor components of wisteria flowers, was accomplished via Sharpless asymmetric epoxydation. The absolute configurations of 1-3 were determined by an HPLC analysis of the corresponding MTPA esters of synthetic samples.

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The conformational change of hen egg-white lysozyme (EC 3.2.1.17) induced by the interaction with tri-N-acetyl-D-glucosamine were investigated by steady state and time-resolved fluorescence spectroscopy. To identify more clearly the conformation of hen egg-white lysozyme interacting with the ligand, the fluorescence decay kinetics of the lysozyme and its complex with the ligand were precisely measured at their full spectral regions. The spectral analysis based on the time-resolved studies showed that the binding of the ligand affected not only the Trp62 directly linked to the ligand but its influence was extended to the vicinity of Trp108 and further to the hydrophobic matrix box region. Near the binding site, the intramolecular distance between Trp108 and Glu35 was expanded or contracted depending on the pH of the buffer solution. On the other hand, the interaction of Trp28 and/or Trp111 with their surroundings was reduced by restriction of fluctuational motions at the hydrophobic matrix box region.

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The optimal hydrolytic conditions for the hydrolysis of castor oil using the lipase from Pseudomonas sp. f-B-24 (lipase PC) were identified. The optimal amount of lipase for hydrolysis was found to be about 45μg per g of substrate. The optimal temperature was 40°C. The apparent K_m and V_max for castor oil hydrolysis were 416 g·l^-1 and 110 μmol·mg^-1·min^-1, respectively. The addition of isooctane, Triton X-100, or PEG-6000 to the reaction mixture slightly improved the hydrolysis of castor oil using the enzyme, but CaCl₂ inhibited it. The hydrolysis catalyzed by lipase PC was 1, 3-specific by TLC analysis of the enzymatic hydrolyzates of triolein, however the estolide, the intermolecular ester compound of liberated ricinoleic acid, was produced in the hydrolyzates of castor oil using lipase PC. Results of the hydrolyzability of lipase PC toward castor oil derivatives and the several methyl esters of C18-fatty acid showed that the hydrolytic activity of lipase PC was not affected either by hydroxyl groups of substituent or hydrophobic groups such as the O-acetyl group, though other lipases were affected by both.

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To investigate the effects of dietary nucleotides on lipid metabolism and learning ability, male Sprague-Dawley rats were fed with a nucleotides-supplemented diet or a nucleotides-free diet for 5 weeks. The content of nucleotides in the diet was 1.0% and their composition resembled that in human milk. The content of phosphatidylcholine (PC) and the ratio of PC to phosphatidylethanolamine (PE) in the cerebral cortex of rats fed the nucleotides-supplemented diet were significantly higher than that of rats fed the nucleotides-free diet. However, there was no difference in the content of PC and the ratio of PC to PE in the liver between the two groups. The levels of docosahexaenoic acid (C22:6n-3) and arachidonic acid (C20:4n-6) in the cerebral PC fraction were higher in rats fed the nucleotides-supplemented diet. The learning ability of rats fed the nucleotides-supplemented diet, which was evaluated by the water-filled multiple T-maze test and passive avoidance test, was superior to that of rats fed the nucleotides-free diet. The results presented here suggest that dietary nucleotides may influence lipid metabolism of the cerebral cortex and contribute to the rise in learning ability of rats.

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The allergenicity of a Kunitz-type soybean trypsin inhibitor (KSTI) was investigated by a micro-assay of β-N-acetylhexosaminidase released from RBL-2H3 cells primed with the anti-KSTI serum. KSTI stimulated the release of β-N-acetylhexosaminidase from RBL-2H3 cells primed with the antiserum. The response of RBL-2H3 cells to the reaginic activity of the mouse anti-KSTI serum correlates fairly well with that by the passive cutaneous anaphylaxis (PCA) test, the sensitivity of both assays appearing to be similar. These results suggest that measuring the β-N-acetylhexosaminidase released from RBL-2H3 is a convenient way for studying the allergen or the reaginic activity of a murine serum in place of the PCA test.

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An enzyme that catalyzed the deamination of adenosine 3'-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60, 000 by SDS-PAGE and 140, 000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2'-deoxyadenosine, 3'-AMP, and 2', 3'-cyclic AMP, but not adenine, 5'-AMP, 3', 5'-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34 mM and 179-295μmol min^-1 mg^-1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3'-phenylphosphonate, at pH 5.5 for adenosine and 2'-deoxyadenosine, and at pH 4.0 for 2', 3'-cyclic AMP and 3'-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the V_max were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.

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PS-990, which is a novel microbial metabolite, induced neurite formation in a murine neuroblastoma cell line, Neuro2A. In the presence of PS-990 at 30μg/ml, significant neurite outgrowth was observed. Cultures maintained for 12 h in the presence of PS-990 resulted in the maximal number of neurite-bearing cells, and then the neurites formed were gradually retracted. The retracted cells again yielded the neurite formation when the cells were exposed again to PS-990. PS-990 inhibited both the cell growth and thymidine incorporation into the cells at the same concentration range. Although the type of neurite formation with PS-990 is similar to that with a cyclic AMP analog and indeed PS-990 has an inhibitory potency against calcium and calmodulin-dependent cyclic nucleotide phosphodiesterase, the intracellular cyclic AMP level was not elevated when treated with PS-990. These results suggest that PS-990 reversibly induces neurite formation with arrest of the cell growth through a mechanism distinct from an increase in the intracellular cyclic AMP concentration.

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The enzymatic optical resolution of racemic N-benzyl-3-pyrrolidinol (2) was investigated. It was found that only (R)-2 was acetylated by the lipase Amano P in yields of 50% and 100%e.e. Remaining (S)-alcohol 2 could be chemically inverted to (R)-acetate 3. A continuous reaction in a column reactor could be also applied to this optical resolution. After concentrating the eluted solution, the residue was subjected to the inversion reaction to provide (R)-acetate 3 in an 83% yield and 91%e.e. from the racemate.

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Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), an active phorbol diester, to quiescent cultured Madin-Darby canine kidney (MDCK) cells caused a maximal stimulation of phosphatidylethanol formation within 1-2 h in the presence of 1% ethanol, indicating the activation of phospholipase D (PLD). The specificity of phorbol diesters for the activation of PLD activation was confirmed by the fact that phorbol 12, 13-dibutyrate (PDBu) was effective, whereas 4α-phorbol 12, 13-didecanoate (4α-PDD) was without effect. Down-regulation caused by the long-term pretreatment of the cells with active phorbol diesters significantly decreased the production of phosphatidylethanol. Staurosporine, a well known protein kinase (PK)C inhibitor at 1μM, decreased the activation of PLD. Taken together, these observations suggested the involvement of PKC in the activation of PLD. The cellular PLD activity was found to be selectively localized in the particulate fraction by centrifugation at 12, 000×g. The particulate PLD showed the selective substrate specificity for phosphatidylcholine rather than phosphatidyl-ethanolamine. In response to the addition of 100 nM PMA, 1, 2-diacylglycerol (DG) increased in a biphasic fashion. In view of the time course of the activation of PLD, the second increase in the 1, 2-DG around 20 min was contributed by the activation of PLD. In reponse to the simultaneous addition of 100 nM PMA and 100 nM A23187, the cultured MDCK cells activated the arachidonate cascades to form prostaglandin (PG)E₂ and PGF_2α as major products, requiring slower 24h to reach maximal levels. The pretreatment of the cells with 1% ethanol caused a significant drop in the synthesis of PGE₂ rather than PGF_2α. The results indicated that either phosphatidic acid (PA) or 1, 2-DG from the hydrolysis of PA served as an activator of cellular response or a direct source of free arachidonic acid as substrates for phospholipases.

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The absolute stereochemistry of yeast reduction products 2s and 2a from 2-(3', 4'-dimethoxy-cinnamyl)-2-methylcyclopentane-1, 3-dione (1) was investigated. The results from several spectral studies and chemical correlations enabled their absolute configurations to be concluded as (2S, 3S) and (2R, 3S), respectively.

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Four rice seed proteins encoded by cDNAs belonging to the α-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

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The component monounsaturated 2-hydroxy fatty acids with carbon numbers of 22, 24, 25, and 26 in wheat leaf cerebroside were found to be Z-forms of the n-9 series. From liquid chromatography-mass spectrometry of the cerebroside, an exclusive combination of the unsaturated hydroxy fatty acids with 4-hydroxysphingenine was confirmed, one of the principal ceramide moieties being 2'-hydroxy-16'-Z-tetracosenoyl-4-hydroxy-8-sphingenine. The distribution of the unsaturated hydroxy fatty acids in the cerebrosides from the leaves of gramineous plants was analyzed. Cerebroside species having unsaturated hydroxy fatty acids were detected in the leaves of chilling-resistant rye and oats as well as wheat. These cerebroside species, however, were not detected in the leaves of chilling-sensitive plants such as rice and maize. The unsaturated hydroxy fatty acids were found only in 3 species of chilling-resistant plants (broccoli, chrysanthemum, and dandelion) among the 21 plant species analyzed. It was noteworthy that no species of chilling-sensitive plants analyzed included significant amounts of cerebroside species containing these fatty acids.

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The complete amino acid sequence of a Ca²⁺-dependent lectin, CEL-IV, from the marine invertebrate Cucumaria echinata was analyzed. The established sequence showed that CEL-IV comprises 157 amino acid residues with a molecular mass of 17, 098 Da (without disulfide bonds). From comparison with other proteins, CEL-IV was apparently homologous with the C-type lectin family. The identity was relatively high with a sea cucumber (Stichopus japonicus) lectin SJL-I (40.0%) and a sea urchin (Anthocidaris crassispina) lectin echinoidin (32.6%). In CEL-IV, one interchain and two intrachain disulfide bonds were identified. Interestingly, one of the two intrachain disulfide bonds that were highly conserved among the other C-type lectins was missing, suggesting that this might be a characteristic feature of C-type lectins in the Holothuroidea.

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With the aim to ascertain the possibility of diffusion of fenoxycarb in the agro-ecosystem, investigations were done in a greenhouse of about 40 m³ in which two-year-old mulberry plants were growing. A water solution of the commercial product Insegar (2.0 gl^-1) was poured in Petri dishes for a total area of 6, 104 cm². The air inside the greenhouse was collected by a suction pump when 1/3 of the solution evaporated. It contained 3.56 μg m^-3 of fenoxycarb. As soon as the solution evaporated completely, the mulberry leaves were collected and washed with water. This water contained 390 μg of fenoxycarb kg^-1 of leaves. The leaves, dried in the shade and analyzed 15 months after collection, contained 13 μg kg^-1 of fenoxycarb.

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We purified a β-glucosidase from the mutant strain Candida molischiana 35M5N. Analysis of the kinetic properties of this enzyme did not show any differences between the previously purified wild-type enzyme and that of the mutant. Nevertheless, a study of the stability of the enzyme at different pH levels and temperatures showed the increased resistance of this protein. This enzyme was found to be stable at pH 5 for 145 h and retained 78% of its initial activity after the same time at pH 3.5 (optimal pH) and 30°C. This difference between the wild-type and the mutant enzyme could be explained by differences in the quantity or quality of glycosylation. This glycoprotein showed different forms after deglycosylation. Some peptides from this protein were also sequenced. An homology analysis found similarities between this β-glucosidase and β-glucosidases of Candida pelliculosa and Schizophyllum commune.

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Escherichia coli strain JA300 grows in the presence of n-hexane, but not in the presence of cyclohexane. We isolated a 10.5-kb DNA fragment that provided cyclohexane tolerance on a multi-copy plasmid, from the chromosomal DNA of JA300. In this fragment, there were found C-terminal 10-amino acids truncated soxR (soxR') and soxS which control the superoxide response regulon genes. Characterization of subclones found that both the soxR' and overexpression of the soxS increased the levels of organic solvent tolerance in several E. coli strains.

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Irradiation of mulberry leaves with UV reduced the feeding response of Bombyx mori silkworm larvae. An organic extract of UV-irradiated leaves was fractionated and, in the acidic fraction which showed antifeedant activity, moracins C and N, known as phytoalexins, were identified. These compounds were induced by UV irradiation and affected the insect's food consumption by acting as feeding deterrents.

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A gene coding for NADPH-cytochrome P-450 reductase of an n-alkane-assimilating yeast, Candida maltosa, was isolated and sequenced. Northern analysis and assay of the expression of the reporter gene under the control of the promoter of this gene showed that the transcriptional level was induced 4 to 8-fold in cells grown on n-alkane relative to cells grown on glucose.

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The gene coding creatinine amidohydrolase (EC 3.5.2.10) was isolated from Pseudomonas sp. PS-7. The primary structure of creatinine amidohydrolase deduced from the nucleotide sequence showed that the protein is composed of 259 amino acids and has a molecular weight of 28, 437. The enzymatic property of creatinine amidohydrolase produced by recombinant E. coli was identical with those by Pseudomonas sp. PS-7.

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Dehydroaltenusin, cyclooctasulfur, atrovenetinone, and altenusin were isolated from the culture broths of Penicillium verruculosum IAM-13756, Streptomyces verticillus subsp. tsukushiensis ATCC-21633, Penicillium sp. SPC-16375, and Penicillium sp. SPC-16524, respectively, as new myosin light chain kinase (MLCK) inhibitors. These compounds inhibited the calmodulin-dependent activity of MLCK with IC₅₀ values of 0.69, 0.86, 3.7, and 340 μM, respectively. Among them, dehydroaltenusin was the best MLCK inhibitor in terms of potency and selectivity examined in the purified enzyme systems.

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A major metabolite of niacin in an extract from cultured tobacco cells when cultured with niacin at 1 mM was analyzed by various spectroscopic and chromatographic methods, and the structure was assigned as N-(beta-D-glucopyranosyl)nicotinic acid. It may be a detoxified form of excess niacin in cultured tobacco cells.

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The activity of aryl-alcohol oxidase was detected in the mycelial extracts of a lignin-degrading basidiomycete, Phanerochaete chrysosporium. The induction of production of the enzyme by aryl-alcohols was suggested. The enzyme was purified to homogeneity. The molecular weight was estimated to be about 78, 000. The prosthetic group was found to be FAD. Several aryl alcohols can serve as substrates but aliphatic alcohols are inert.

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A 2, 5-dimethylhexane (2, 5-DMH) assimilating bacterial strain, identified as Rhodococcus sp. and some Mycobacterium species, catalyzed the asymmetric oxidation of the isopropyl moiety of hydrocarbons. The 2, 5-DMH-grown Rhodococcus sp. strain 11B produced (-)-2, 5-dimethylhexanoic acid from 2, 5-DMH, and (S)-2-phenylpropionic acid and (R)-2-phenyl-1-propanol from cumene. The biodegradability of isoalkanes, which have various branching patterns, using this strain is also discussed.

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To analyze the primary structure of Mortierella vinacea α-galactosidase, a cDNA library of M. vinacea mRNA in λgt10 was constructed. A clone, which has an insert size of about 1.4 kilobase pairs, was found to contain the coding region of the mature enzyme. The deduced amino acid sequence showed that the mature enzyme consisted of 397 amino acid residues with a molecular mass of 44, 350Da. The sequence identity of the mature enzyme with α-galactosidases from Saccharomyces carlsbergensis, Cyamopsis tetragonoloba (guar), and human were 47%, 43%, and 34%, respectively.

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In the process of the purification of a dibenzothiophene (DBT)-degrading enzyme system from cell-free extracts of Rhodococcus erythropolis D-1, flavin coenzymes, FMN and FAD, were found to be involved in the enzymatic degradation of DBT in addition to NADH. Under these experimental conditions, the optimal concentrations of FMN and FAD were both 10 μM and the activity was completely inhibited by adding 1 mM FMN or FAD. DBT was converted to DBT sulfone stoichiometrically and 2-hydroxybiphenyl formation was not observed when the reaction was done using the enzyme preparation purified by DEAE-Sepharose column chromatography.

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When n-alcohols were added to the β-glucosidase-catalyzed hydrolysis of arbutin, the rate was decreased almost linearly with their elevating concentrations. The inhibition was shown in two modes, competitive for higher n-alcohols and noncompetitive for lower n-alcohols than n-butanol. The inhibition constants (K_i) obtained, moreover, clearly demonstrated classifying the modes in two groups according to physicochemical parameters, logP, Hildebrand solubility parameter (δ), and dielectric constant (ε), of n-alcohols. n-Butanol being between two modes of inhibition showed a mixed-type inhibition. The data suggested that the inhibition of n-alcohols for β-glucosidase may be related with their chain length. An imaging model in this case of inhibition is presented.

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A new class of synthetic pyrethroid, 2-halo-3-arylcyclopropyl-methyl 3-phenoxybenzyl ethers (1a-h), exhibited significant insecticidal activity against tobacco cutworm and housefly.

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An Enterococcus faecalis plasmid, pAM373, has a high frequency of transfer in a liquid medium when induced by a recipient-produced sex pheromone, cAM373. The sex pheromone inhibitor against cAM373, termed iAM373, was isolated from a culture supernatant of E. faecalis harboring pAM377 (=pAM373::Tn917), and its structure was identified as a heptapeptide, H-Ser-Ile-Phe-Thr-Leu-Val-Ala-OH.

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By genomic Southern blot analysis, the DNA sequences homologous to the gene cluster responsible for biosynthesis of 6-demethyl-chlortetracycline in Streptomyces aureofaciens NRRL3203 were shown to be highly conserved in independent chlortetracycline- or tetracycline producing Streptomyces strains. By contrast, oxytetracycline-producing Streptomyces strains had no hybridization with the cluster DNA.

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The stereochemistry of the tryptophan residue of N-malonyl-tryptophan has been believed to be of D-form. We reinvestigated the stereochemistry by using HPLC with a chiral column. Both forms were detected; however, the L-form was a major component. The ratio of D:L was determined to be 1:32 in pea leaves and 1:3 in wheat roots.

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An alkali-sensitive mutant, 18224, of the alkaliphilic Bacillus sp. strain C-125 was characterized. The nucleotide sequence of the Pvul-NlaIV DNA fragment that recovers the alkaliphily of 18224 has been cloned from the mutant and sequenced. Comparison of the nucleotide sequences of the corresponding regions found a G to A substitution in the mutant. The mutation resulted in an amino acid substitution from ⁸²Gly to Glu of the putative ORF3 product, which consisted a gene cluster of at least four tandemly located open reading frames. The ORF3 product was deduced to be an 112 amino acid polypeptide with hydrophobic properties, which was expressed using an in vitro translation system.

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Non-reducing oligosaccharides having an isomaltosyl residue, various alkyl α-isomaltosides, 6_G-α-isomaltosyl sucrose, and 6-α-isomaltosyl trehalose, were prepared in more than 30% yields using the transfer action of isomaltodextranase (EC 3.2.1.94) from Arthrobacter globiformis T6. 6_G-α-Isomaltosyl sucrose and 6-α-isomaltosyl trehalose were hydrolyzed by isomaltodextranase more easily than alkyl α-isomaltosides. The structure of these transfer products were identified by the hydrolysis of isomaltodextranase, reducing end analysis, methylation analysis, and ¹H-NMR analysis.

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The hydrogen-transfer stereospecificity of cabbage histidinol dehydrogenase at the C-4 position of NAD⁺ was determined by means of ¹H-NMR. A dehydrogenase reaction with enzymatically prepared [4-²H]NAD⁺ was performed. The NMR spectrum of the reaction mixture showed a peak at about 2.8 ppm, indicating the production of [(4S)-²H]NADH, indicating that the stereospecificity of the enzyme was pro-R-specific.

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Hydrolytic reactions of maltooligosaccharides (degree of polymerization n=3-7) and isomaltooligosaccharides (n=3-7) by Rhizopus glucoamylase (at pH 4.5, 25°C) were observed by HPLC. It was confirmed that initial rates of the decrease of n-mer substrate and the increase of the products, glucose and (n-1)-mer oligosaccharide, are the same for all the substrates, indicating that the reaction mode of the enzyme is "random attack, " and no "multiple attack" or "single chain attack" is operating.

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New syringopeptins SP(SC)-1 and -2 were isolated from culture filtrates of phytopathogenic bacterium strain SC1 of Pseudomonas syringae pv. syringae. These syringopeptins were composed of a β-hydroxy fatty acid, a long sequence of aliphatic amino acids, and a lactone moiety of eight amino acids. The amino acid sequences were deduced from a comparison of their tandem mass sepctra with those of known syringopeptins SP-22a and SP-25a. SP(SC)-1 and SP(SC)-2 resembled SP-22a, but differed from the latter by 3 amino acids.

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Most rice contains the 16-kDa allergenic protein belonging to the α-amylase/trypsin inhibitor family. The amount of this protein in more than 150 rice strains was examined by using the antibody raised against the 16-kDa allergenic protein. While all of Japanese cultivars tested contained nearly same amount of the 16-kDa allergen, some strains in other Asian countries contained little or none of this protein.

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