A new enzyme, 2-methylisocitrate dehydratase, isolated from Yallowia lipolytica, functioning in the methylcitric acid cycle for propionate metabolism, had a pI of 4.4 and a M_r of 69, 500. The enzyme was composed of 624 residues of amino acids per molecule. No cofactor was required for full enzyme activity. The enzyme was competitively inhibited by threo-D_s-isocitrate (K_i=68mM), but not by any other tested metabolites. The enzyme was weakly inhibited by some thiol reagents, but not by any metal-chelating reagents, differing from aconitase, which dehydrates 2-methylisocitrate. This difference between the enzymes made it possible to estimate the activity of the new enzyme even in crude cell-free extracts. The enzyme was constitutively synthesized, but had no regulatory function in the methylcitric acid cycle. The enzyme was supposed to have evolutionarily developed from a hypothetical and prototypical isocitrate dehydratase.

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Nonenzymatic glycation of bovine serum albumin (BSA) was inhibited in vitro by some extracts of 34 kinds of spices. The methanol extract of thyme (Thymus vulgaris) had the most potent inhibitory activity among them. Chromatographic purification yielded four flavonoids, quercetin (1), eriodictyol (2), 5, 6, 4'-trihydroxy-7, 8, 3'-trimethoxyflavone (3), and cirsilineol (4). These known flavonoids suppressed the levels of advanced glycation end products (AGEs) and fructosamines, shown by the measurement of specific fluorescent groups and the reduction of nitroblue tetrazolium (NBT), respectively. The inhibitory activities were compared with those of other structure-related flavonoids and aminoguanidine.

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Fast protein liquid and ion-exchange chromatography, fluorescence, FT-IR spectra, and elemental and electrophoretic analyses were used to characterize proteins from seven species of Cactaceae, which can be divided into three groups based on their chemical and biochemical properties. Some cactus juice proteins precipitated by ammonium sulfate yielded complex electrophoretic patterns where the major bands cor- respond to approximately 24, 000 Da and 32, 000 Da in the presence of sodium dodecyl sulfate (SDS). The SDS-polyacrylamide gel electrophoretic (PAGE) patterns did not differ by the year of sample collection (1986 and 1992). Chromatographic analysis showed that protein characterization of cactus juices may be useful in cactus taxonomy at the family level. Fluorescence emission and FT-IR spectra of studied cacti species were measured to compare protein structure. Differences in the emission peak response and fluorescence intensity, as well as the changes in amide band content were found.

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The actinomycete strain Streptomyces sp. H37 produces a novel glycosphingolipid-degrading enzyme. This strain was capable of converting ganglioside GM1 to lyso-GM1. After cultivation for 5 days in medium containing GM1, peptone, and detergent, GM1 was found to be almost completely converted to lyso-GM1. The product was purified on a DEAE-Sephadex A-25 column and thin layer chromatographies. The purified lyso-GM1 was hydrolyzed by endoglycoceramidase, and the released oligosaccharide moiety was identified as that of GM1 by HPLC using the pyridylaminoderivative method. The counterpart sphingosine moiety was confirmed with TLC. Moreover, the structure of lyso-GM1 was ascertained by ¹H-NMR analysis. The maximum formation of lyso-GM1 was found in 50mM potassium phosphate buffer (pH 7.5) containing O.1% glycodeoxycholate. Various lyso-glycoshingolipids, including those of ganglio-, neolacto-, and globo-types, were formed from their parent glycosphingolipids using this strain.

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Eleven flavones, including cochliophilin A and a chromone, were chemically prepared to determine the attracting activity for zoospores of Aphanomyces cochlioides, a causative fungus of spinach root rot. Analyses of the structure-activity relationship of each revealed a significant correlation between the zoospore attracting activity and the A-ring oxygenation at C-5 and C-7 in the flavone skeleton. The relative attractancy of four regio-isomers of Ntrans-feruloyl 4-O-methyldopamine, which was identitied as a zoospore attractant specific to another host plant Chenopodium album, was also examined.

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We measured the electroantennography (EAG) activities of the geometrical isomers of several pheromone-related compounds towards male eri-silk moths (Samia cynthia richini) using EAG-gas chromatography (GC). C₁₆-aldehyde and formyl ester, with the same chain length as C₁₆-aldehyde, were found to be much more active than other compounds with a shorter chain. For the compounds with (1Z, 6Z)- or (1E, 6E)-contiguration, the activity in decreasing order was C₁₄-formyl ester > C ₁₆-aldehyde > C ₁₄-aldehyde>C₁₂-formyl ester, while that for the compounds with (1Z, 6E)- or (1E, 6Z)-configuration is C₁₆-aldehyde > C₁₄-formyl ester > other compounds with a shorter C₁₄ chain.

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Ten standard strains of hiochi bacteria were selected based on the SDS-PAGE patterns of their cellular proteins. We then obtained ten hybridoma systems that secreted highly reactive monoclonal antibodies (MAbs) to the whole cells of each strain. It was apparent that these MAbs were highly reactive and specific for hiochi bacterial whole cells when using an enzyme-linked immunosorbent assay (ELISA). Three MAbs (two against the homo-fermentative hiochi lactobacilli and an MAb against the hetero-fermentative true hiochi bacilli) showed cross-reactivity to some of the other strains of lactobacilli tested. However, the other MAbs did not react with strains other than the immunogen. A sensitive ELISA method for the detection of hiochi bacteria was examined. It was possible to detect the order of 10³ cells of the ten standard strains. Using this procedure and a mixture of the ten MAbs, a detection limit of 10⁴ cells or less could be obtained for 98.2% of the hiochi bacteria isolated from sake brewing factories. Thus, this immunological technique using MAbs specific for hiochi bacteria is a sensitive and rapid detection method for hiochi bacteria, which can be used in the quality control of sake.

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Chattonella is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide and hydroxyl radicals, which may be responsible for the toxicity of this plankton. However, little is known about the mechanism of the production of oxygen radicals by Chattonella, and the role of oxygen radicals in Chattonella themselves is also unclear. In this study, we found that superoxide dismutase (SOD) and catalase inhibited the growth of Chattonella marina concomitant with their morphological changes. In the presence of these enzymes, the shape of vegetative C. marina cells changed from spindle to round. Furthermore, the generation of oxygen radicals by C. marina depended on the growth phase ; the rate of superoxide and hydrogen peroxide generation was the highest during exponentially growing phase and subsequently decreased to one-fifth of the maximal level in the stationary growth phase. These results suggest that oxygen radicals generated by C. marina play an essential role in their own survival, especially in cell division.

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A new antifungal substance, 11-hydroxy-9(Z)-undecenoic acid, was isolated from the leaves of wild rice (Oryza officinalis, W-0002) which is resistant to the rice blight fungus (Pyricularia oryzae P₂), together with the known 12-hydroxy-9(Z)-dodecenoic acid and azelaic acid monomethyl ester, and its structure was established on the basis of spectroscopic data and chemical synthesis.

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Reinvestigation of the phenolic Ferrier reaction is described. Ferrier reaction between acetylglycals and phenols in toluene in the presence of a catalytic amount of Lewis acid at low temperature gave aryl O-Δ²-glycosides in moderate to good yields, but the reaction in dichloromethane resulted in the formation of aryl C-Δ²-glycosides predominantly.

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We efficiently produced a small peptide by the host-vector system using Bacillus brevis as a host. DNA encoding the physiologically functional casoxin D, composed of seven amino acids, was ligated in tandem. An expression-secretion vector containing DNA, which codes for a fusion protein of epidermal growth factor-casoxin D pentamer, was constructed. B. brevis transformed with this plasmid produced about 0.5g/liter of the fusion protein in the culture supernatant. The fusion protein was puritied with ammonium sulfate fractionation from the supernatant and digested with two kinds of proteinases. A peptide well separated by high pressure liquid chromatography was identified as biologically active casoxin D.

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To investigate how vitamin B₆ (B₆) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets ; a complete 20% casein diet (control diet), the control diet without B₆, the control diet without nicotinic acid, and the control diet without nicotinic acid and B₆, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B₆-deficient groups, when compared with the B₆-containing groups. These results indicate that the rats fed with the B₆-free diets were in the vitamin-deticient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B₆-free diet than in the B₆-containing diet under the niacin-free conditions.

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Costunolide and dehydrocostus lactone were isolated from an extract of mokko (Saussurea lappa Clarke) as inhibitors of killing activity of cytotoxic T lymphocytes (CTL). Mokko lactone was also isolated as an inactive compound from the extract. The structure-activity relationship indicated that α-methylene-γ-butyrolactone is required for the inhibitory effect. Costunolide markedly inhibited the granule exocytosis and the production of inositol phosphates in response to anti-CD3 monoclonal antibody (mAb) stimulation at a concentration that did not affect the binding of anti-CD3 mAb. Tyrosine phosphorylation induced by crosslinking of CD3 molecules was significantly inhibited by costunolide in a dose-dependent manner. These results suggest that costunolide inhibits the killing activity of CTL through preventing the increase in tyrosine phosphorylation in response to the crosslinking of T-cell receptors.

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Cytophaga sp. strain L43-1 secretes a collagenase [Y. Sasagawa et al., Biosci. Biotech. Biochem., 57, 1894-1898 (1993)]. A cog gene encoding the collagenase from this strain was cloned, and the nucleotides were sequenced. The structural gene of cog consisted of 3846 base pairs, which encoded a polypeptide consisting of 1282 amino acid residues with a predicted molecular mass of 130kDa which was synthesized as a pre-matured enzyme. The deduced N-terminal 14 amino acids sequence, molecular mass of 120kDa, and pI of 4.96 of the predicted matured enzyme were consistent with those previously found for the collagenase purified from the strain. The cog gene was expressed in Escherichia coli using the lac promoter and ribosomal binding sequence in plasmid vector pUC119 or pKK223-3, but not its own putative promoter and Shine-Dalgarno sequence. The consensus amino acid sequence (His-Glu-Xaa-Xaa-His) of an active site of the metal proteases including the collagenase from Vibrio arginolyticus and a series of human MMPs was found in the Cog protein of the strain.

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A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been puritied to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL. ProB is a zinc-enzyme with a mass of 18kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1, 10-phenanthroline, and Zn²⁺-depleted ProB can regain the activity with Zn²⁺, Co²⁺, or Mn²⁺. Specitic cleavage of Pro₂₉-Lys₃₀ in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the α-amino group of lysyl residues. ProC is a metalloendopeptidase of a mass of 42.5KDa, and Zn²⁺ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.

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A new cytolytic toxin, designated as S-Hemolysin, was found in the culture filtrate of Streptomyces sp. strain No. A-6288, isolated from a soil sample. The molecular weight of S-Hemolysin was estimated to be 10, 000 by SDS-polyacrylamide gel electrophoresis and to be 20, 000 by Sephadex G-100. S-Hemolysin is a glycoprotein that is composed of 102 amino acid residues with 11.6% glucose, and the isoelectric point is around pH 5.8. The phospholipase C activity of S-Hemolysin was specific for the following substrates in this order : sphingomyelin > lysophosphatidylethanolamine > lysophosphatidylcholine > phosphatidylethanolamine > phosphatidylcholine. S-Hemolysin had hemolytic activity against rabbit, human, and sheep erythrocytes, but did not cause aggregation of human platelets. These activities were accelerated with Mg²⁺, Mn²⁺, and Co²⁺ ions and inhibited by the addition of Ca<2+>, Cu²⁺, and Zn²⁺ ions. This enzyme was shown to be different from the known bacterial phospholipase C.

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Prolidase (iminodipeptidase, EC 3.4.13.9) was purified from an extract of Xanthomonas maltophilia, by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Toyopearl HW65C, FPLC-Hiload Superdex 200pg, and FPLC-Hitrap Q columns, which an activity recovery of 2.3%. The enzyme was the most active at pH 7.5 with Leu-Pro as substrate. It was stable between pH 6.0 and 8.5 for 60min at 37°C and retained half of activity after 60min at 37°C. The isoelectric point of the enzyme was 3.7. Its molecular weight was estimated to be 100, 000 by gel filtration on FPLC-Hiload Superdex 200 and 51, 000 by SDS-PAGE, suggesting that it is a dimer. It hydrolyzed dipeptides only if proline is located at the carboxyl terminal position. The enzyme was inhibited by PCMB and o-phenanthroline, and was activated by Mn²⁺ .

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The reaction of bicyclo[3.1.0]hexane 1, possessing a doubly activated cyclopropane ring, with acetic acid and potassium acetate in DMSO proceeded smoothly to give the adduct 2 in good yield. A formal total synthesis of the potent anti-HIV agent (±)-carbovir (9) was done by converting 2 into a known precursor 8 in 8 steps via allyl alcohol 7 including the regioselective introduction of a double bond (4 to 5) and attachment of the nucleobase using the Mitsunobu reaction (7 to 8).

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It takes about 2 months for the molding process of Katsuobushi (dried bonito) production. A model of a short-term system for the molding process was desirable for the efficient selection of useful fungi for Katsuobushi production by evaluating the flavor change in the model system. A liquid culture system using Nijiru (waste fluid of Katsuobushi sterilization), which is obtained from the Kezuribushi (sliced Katsuobushi) manufacturing process and contains abundant phenolic compounds of smoke tar, was found to be suitable for the short-term evaluation of a 1O-day culture.

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Two base non-specific acid RNases (RNase Irp₁ and RNase Irp₂) were purified from a commercial enzyme, "Driselase" (Irpex lacteus) in a homogeneous state on SDS-PAGE by several steps of chromatographic separations. RNAse Irp₂ was a simple polypeptide with 235 amino acid residues and RNase Irp₁ was a glycopeptide with 248 amino acid residues. The amino acid sequences of both RNases were identified by Edman degradation of the peptides derived from these RNAses. RNase Irp₁ was composed of the RNase Irp₂ and extra C-terminal 13 residues of peptide. The phylogenetic relation of these RNases with the other fungal RNases already known was discussed. The sequence of RNase Irp₂ was very highly homologous (67.5%) with that of RNase Le₂ from Lentinus edodes.

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Strategies for the synthesis of a dialkylmaleic anhydride segment of tautomycin and tautomycetin are presented. The most difficult point was the stereocontrolled construction of the tetrasubstituted oletin. The maleate-type olefin construction through dehydration proved not to be adoptable because the corresponding β-hydroxyimine or aldol precursors were extremely unstable even under neutral conditions, suffering the retro-aldol-type decomposition. The Horner-Wadsworth-Emmons reaction on the α-ketoester with an appropriate protecting group had acceptable levels of diastereoselectivity giving rise to the dialkylmaleate diester, which was further led to the segment used in the synthesis of tautomycin and tautomycetin. In such the strategy, functional group protections were important in the olefination reaction and the final anhydride formation.

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Low molecular weight chitosan (LMWC) (a mixture of chitooligosaccharides with high degrees of polymerization ; an average degree of polymerization is 6.8) stimulated mitogenic response to platelet-derived growth factor (PDGF) in a dose-dependent manner in cultured rat vascular smooth muscle cells, and a maximum effect was observed at 100μg/ml. However, the mitogenic response was not induced when cells were incubated with LMWC alone. This stimulatory effect of LMWC on the mitogenic response to PDGF (a competence factor) appeared to resemble the effect of insulin as a progression factor. Chitooligosaccharides with higher degrees of polymerization were more effective, but D-glucosamine, chitobiose, and chitotriose were barely active. LMWC as well as PDGF induced protein tyrosine phosphorylation in vascular smooth muscle cells.

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The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6(Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51, 918. This value agreed well with the apparent molecular weight of 52, 000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH₂-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine, and humans.

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An adenosine (phosphate) deaminase from the squid liver had much lower activity for 5'-deoxyadenosine than that for adenosine, 2'-, or 3'-deoxyadenosine. 3'-IMP and inosine as well as purine riboside and adenine competitively inhibited the deamination of adenosine 3'-phenylphosphonate by the enzyme, but 5'-AMP and 5'-IMP did not. The enzyme deaminated the 5'-hydroxyl terminal adenosine residue in dinucleotides and trinucleotide, but not the 3'-hydroxyl terminal one in dinucleotides. The 5'-hydroxyl group of the ribose moiety was necessary for the substrate binding and catalytic activity of the squid enzyme. These results indicated that the recognition of ribose moiety in the substrate by the squid enzyme might be intermediate between those by adenosine deaminase and adenosine (phosphate) deaminase from microorganisms.

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Bacillus subtilis No. 428 is a cytidine-producing mutant strain derived from wild-type strain No. 122. To identify the rate-determining factor for production of cytidine by strain No. 428, we examined the properties of the strain. We found that carbamyl phosphate synthetase P (CPSase P) from strain No. 428 was sensitive to feedback inhibition by uridine 5'-monophosphate (UMP), and therefore, this enzyme reaction was the rate-determining step for cytidine production. Thus, we introduced a mutation causing lack of feedback inhibition of CPSase P into the CPSase P-deficient mutant of strain No. 428 by homologous recombination. Some of the transformants had higher productivity of cytidine. Among them, strain No. 515 produced 18. 8 mg/ml cytidine in the culture. CPSase P from strain No. 515 was freed from feedback inhibition by UMP, having obtained the characteristic of the donor.

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The administration of glycemia-affecting chemicals such as alloxan, streptozotocin, and 6-aminonicotinamide decreases the conversion ratio of tryptophan to niacin. Adrenalin is also known to increase the glucose level. For this reason, the effects of adrenalin on the conversion ratio were investigated. We found that the conversion ratio of tryptophan to niacin was reduced to half by the intraperitoneal injection of adrenalin at 75μg/day/rat (body weight, about 250g) every day for 7 days. Niacin decreases adrenalin-stimulated glycogenolysis via stimulating phosphodiesterase activity or depressing adenyl cyclase activity. Accordingly, in urgent need of energy, animals would have to decrease the concentration of niacin within the body.

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Hypocholesterolemic activities and other properties of three different molecular weight pectin were examined. The low-molecular-weight pectin (M_r≒66, 000) obtained by decomposition of original pectin (M_r≒750, 000) had the properties of low viscosity and high solubility, but it lost hypocholesterolemic activities in rats. On the other hand, the medium-molecular-weight pectin (M_r ≒185, 000) had characteristics of both low viscosity and hypocholesterolemic activities.

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The substrate specificity of α-L-arabinofuranosidase (EC 3.2.1.55)from Bacillus subtilis 3-6 was explored using methyl 2-O-, methyl 3-O-, and methyl 15-O-α-L-arabinofuranosyl-α-L-arabinofuranosides, and methyl 3, 5-di-0-α-L-arabinofuranosyl-α-L-arabinofuranoside. The enzyme hydrolyzed the methyl α-L-arabinofuranobiosides to arabinose and methyl α-L-arabinofuranoside in the order of (1→2)- > (1→3)- > (1→5)-linkages. The enzyme hydrolyzed the (1→3)-linkage more than the (1→5)-linkage of the methyl α-L-arabinofuranotrioside.

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The effects of a component of green tea on the proliferation of smooth muscle cells were measured in terms of [³H]thymidine uptake. When green tea tannin mixture was added to the medium of cultured smooth muscle cells, it suppressed the proliferation of the cells dose-dependently. Similarly to the effects of the green tea tannin mixture, (-)-epigallocatechin 3-O-gallate, its main ingredient, had an inhibitory effect on smooth muscle cell proliferation at a low concentration. (-)-Epicatechin 3-O-gallate was also an effective component. Among four types of gallate-free tannin, (-)-epigallocatechin, (-)-epicatechin, and (+)-catechin showed significant dose-dependent inhibition of smooth muscle cell proliferation. However, caffeine and theanine were found to have no such action.

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Enterobacter cloacae GAO, a bacterium isolated from water in which apples have been steeped, is also found in traditional fermented wheat foods. One important characteristic of this bacterium is that it produces hydrogen gas during inflation as these foods are being prepared. The effects of various nutrient sources and culture conditions on the production of hydrogen gas by the bacterium were examined for the improvement of these traditional foods. Glucose was found to be an effective carbon source and casamino acids were found to be effective nitrogen sources. E. cloacae GAO was used to make mantou. When wheat flour with casamino acids was used to make mantou, a better internal structure and taste were obtained.

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The ultrastructure of protein bodies in embryonic cells of the Amaranthus cruentus seed was examined by transmission electron microscopy. Most of the protein bodies of 2-5μm in diameter were densely electron-stained, containing phytin globoid inclusions but not protein crystalloid. Numerous small lipid bodies (sphero-somes) of various sizes and shapes surrounded these protein bodies and filled the cytoplasm. No ER-bound polysomes were detectable around these protein bodies, suggesting that Amaranthus seed protein bodies are morphologically similar to the globulin type of legumes, quinoa (Chenopodium quinoa Willd. ), and buckwheat (Fagopyrum esculentum Moench), the last two of which are taxonomically close to Amaranthaceae.

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Two types of trehalase activities different in the optimum reaction pH (4.3 and 6.7) were found in a freeze-tolerant yeast, Torulaspora delbrueckii D2-4, and three types of the activities at pH 4.3, 5.7, and 6.7 were found in its freeze-sensitive mutant (60B3). The pH 4.3 activities in the cell extracts of the two strains were on the same level, but the pH 6.7 activity in the strain D2-4 was about half of that in the strain 60B3. The enzyme with the pH 4.3 activity and the other two enzymes were analogous to acid and neutral trehalases, respectively, in the sensitivity to metal ions and in response to phosphorylation and dephosphorylation. The trehalose content in the strain D2-4 was three times higher than that in the strain 60B3. The change of trehalose content during growth of the strain 60B3 correlated only to the change of the pH 5.7 activity.

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An α-glucosidase was purified in an electrophoretically pure state from an extract of koji culture of Aspergillus sp. KT-11. This enzyme was found to have a transferring activity when the reaction was done in a high concentration of leucrose at pH 4. 5. Two kinds of transfer products, fractions I and II, were obtained from leucrose by the enzyme and they were identified as [(α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→5)-D-fructopyra-nose] and [α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→5)-D-fructopyranose], respectively. These are considered to be novel oligosaccharides.

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Highly homologous 170-bp sequences were found to be carried in the same orientation by the chromosomes of Bacillus subtilis GSY908(a 168 derivative), B. subtilis R, and B. subtilis var. natto. These sequences were in 5'- and 3'-flanking regions of a tetracycline-resistance determinant in B. subtilis GSY908 and B. subtilis R.

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We examined the mutagenicity of extracts (juice and ethanol extract) from a transgenic tomato that was established by transfection of a gene encoding the coat protein of tobacco mosaic virus (TMV) to the F1 hybrid between Lycopersicon esculentum LA1000 and L. peruvianum PI128650, by the umu-test with Salmonella typhimurium TA1535/pSK1002 as the test organism. The extracts showed no detectable mutagenicity. The extracts from the above-mentioned F1 hybrids and wild tomatoes and cultivars (L. peruvianum PI128650, L. peruvianum PI126944, L. pimpinellifolium LS1524, L. pimpinellifolium LA722, L. hirsutum LS503, Mini-carol, Sun-cherry, Momotaro, Odoriko, Kagome77, and Ponderosa) also showed no detectable mutagenicity.

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The oxidation of lycopene by hydrogen peroxide afforded lycopene-1, 2-epoxide and a novel carotenoid with an epoxyiridane skeleton.

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Both free and conjugated brassinosteroids (BRs) in the pollen and anthers of Erythronium japonicum Decne. were investigated. As a free form of BRs, typhasterol was identified by GC-MS. Polar conjugated BRs occurred only in the anther, and non-polar conjugated BRs occurred both in the pollen and mainly in the anther. BR parts of acid hydrolysis of the former were identified to be teasterone (major) and castasterone (minor). Those of alkaline hydrolysis of the latter were identitied to be typhasterol (major) and teasterone (minor).

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Microorganisms that degrade heavy oil C (HOC) were screened by enrichment in alumina columns containing HOC and a slowreleasing fertilizer. The alumina columns were immersed in a bath in which natural sea water was periodically ebbed and flowed through a siphon. After two months of enrichment, 153 HOC degraders were isolated from the cultures, and three of them were demonstrated to degrade about 50% of HOC at 30°C for l0 days. A taxonomic examination of these three strains indicated that two of them belonged to the genus Rhodococcus while the third the genus Agrobacterium. The degradability of these strains was 1.7 to 4.8 times higher than that of other known oil-degrading microorganisms.

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Six chitinases were purified from a culture supernatant of Aeromonas sp. no. 10S-24 by ammonium sulfate precipitation, DEAE-Sephadex A-50, Butyl-Toyopearl 650M, and chromatofocusing. These enzymes were most active at pH 3.5-4.5 and the optimum temperature were 50°C. The molecular weights of the enzymes were 89, 000 to 120, 000 from SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid sequences of the enzymes were similar to that of chitinase I.

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The intermediate of the aromatization of 4-oxocyclohexanecarboxylic acid (OHA) to 4-hydroxybenzoic acid (HA) by Corynebacterium cyclohexanicum was identified as (+)-4-oxocyclohex-2-enecarboxylic acid (O2A) using a combined system of gas-liquid chromatography (GLC) and a mass spectrometer and polarimeter.

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Sucrose phosphorylase from Leuconostoc mesenteriodes catalyzed transglucosylation from sucrose to (-)-epigallocatechin gallate. Two main transfer products were obtained, and their structures were identified as (-)-epigallocatechin gallate 4'-O-α-D-glucopyranoside and (-)-epigallocatechin gallate 4', 4"-O-α-D-diglucopyranoside on the basis of secondary ion mass spectrometry analysis, component analyses of their enzymatic hydrolyzates, and nuclear magnetic resonance analysis. The stability of (-)-epigallocatechin gallate was greatly increased by the glucosylation.

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Photosystem II inhibition by s-triazines having hydrophilic amino groups was examined in isolated spinach chloroplasts. Among them, the hydroxyalkyl- and alkoxyalkylamino derivatives were potent inhibitors. The hydroxyalkylamino-s-triazines seemed to interact with the binding site in a manner different from that of other triazines, since they needed a time to build up to constant inhibition and showed a different thermoluminescence glow peak.

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A 0.4-kb ScaI-HpaI fragment, 199bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp. KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells. We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5' region of the endoglucanase that contained a possible promoter region. Using recombinant B. subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.

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Occurrence of free D-alanine was verified in various marine macroalgae by reversed-phase HPLC analysis. The presence of free D-alanine was detected in 15 species (63%) out of the 24 species of algae examined. The free D-alanine content was high in Hizikia fusiformis, Heterochordaria abietina, and Sargassum nigrifolium of Phaeophyta (96, 83, and 78nmol/g wet weight, respectively). The D-alanine contents in various parts of H. fusiformis were measured and found to be highest in the blade followed by the main branch, and was lowest in the adhesive root.

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Enantioselective syntheses of both enantiomers of 1-arylethanols with a condensed aromatic ring have been done through acetylation of the racemic alcohols with vinyl acetate in the presence of a lipase from Pseudomonas aeruginosa (Toyobo, LIP). The lipase LIP showed high enantioselectivity and reactivity for the title compounds, reacted acetates, and remaining alcohols were obtained with high optical purity.

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α-Glucosidase (EC 3.2.1.20) from Trichoderma viride was purified by a procedure including Sephacryl S-200 HR column chromatography, preparative isoelectric focusing, and Superdex 200 HR column chromatography. The enzyme was found to have an apparent molecular weight of 67, 000 on SDS/PAGE and 65, 000 on gelfiltration, showing that the enzyme is a single peptide. The isoelectric point of the enzyme was 5.0. The enzyme hydrolyzed α-1, 4-glucosidic linkage more rapidly than α-1, 6-glucosidic and α-1, 3-glucosidic link-ages. The enzyme was weakly inhibited by castanospermine.

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A ferredoxin-nitrite reductase (EC 1.7.7.1) cDNA was isolated and sequenced from a λgt 11 cDNA library constructed from nitrate-induced greening shoots of rice (Oryza sativa L.) seedlings. The nucleotide sequence of the cDNA clone contains an open reading frame of 1788 nucleotides. There exists a strong bias for the third codon usage of G/C (95.5%) as in the case of the maize enzyme. The deduced amino acid sequence shows an overall homology to the maize (81%) and the dicot enzymes (70-74%), suggesting that the primary structure of ferredoxin-nitrite reductase is highly conserved in higher plants.

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The seasonal variation of fatty acid composition in lipids of various organs and stomach contents of bonito (Euthynnus pelamis) was investigated. Although docosahexaenoic acid in the lipids of the stomach contents originating from their prey organisms varied from about 13% to 31%, it was the dominant unsaturated fatty acid and ccounted for more than 25% of the total fatty acids in the lipids of every organ of bonito in and out of season, and its seasonal variation was comparatively small.

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A bacterium, Pimelobacter sp. R48, isolated from soil, showed the ability to produce trehalose from maltose. The partially purified enzyme from a cell-free extract catalyzed the conversion of maltose into trehalose without requiring phosphate. The enzyme was considered to be a new intramolecular glucosyltransferase. The enzyme was also tentatively found to exist in Pseudomonas putida H262 isolated from soil and in some Thermus strains.

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We had shown that production of quinolinic acid is high in the brain of epilepsy-prone El mice and that this is due to an increase in the activity of 3-hydroxyanthranilate 3, 4-dioxygenase (3-HAO, EC 1.13.11.6). We demonstrated here that the level of mRNA for 3-HAO was markedly increased in the brain of El mice.

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