Inhibition of platelet aggregation was found in the essential oils extracted from the various parts of upland wasabi (Wasabia japonica forma terrestris Hisauti). The antiplatelet activity increased in the order of leaves, rhizomes, petioles, and roots. To examine the inhibitory principles in upland wasabi oil, components in each part of the essential oil were identified and measured. Most of the volatile components in upland wasabi oils were ω-alkenyl isothiocyanates and ω-methylthioalkyl isothiocyanates, allyl isothiocyanate being the main component in every oil. Essential oils from roots and petioles contained rather large amounts of ω-methylthioalkyl isothiocyanates. Although every authentic isothiocyanate inhibited platelet aggregation, ω-methylthioalkyl isothiocyanates were more inhibitory, which explained the stronger inhibitory effects of essential oils from roots and petioles on platelet aggregation.

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Phospholipase D (PLD) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoyl-phosphatidyl residue of 1, 2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to dihydroxyacetone (DHA) in a biphasic system, to afford 1, 2-dipalmitoyl-3-sn-phosphatidyldihydroxyacetone (DPP-DHA). The structure of DPP-DHA was identified by NMR, FAB-mass, IR, and UV spectra. PLD also catalyzed the transphosphatidylation reaction of DPP-DHA to choline, and DPPC was reproduced. In the presence of phospholipase C (PLC) from Bacillus cereus, DPP-DHA was hydrolyzed to 1, 2-dipalmitoylglycerol (DPG). DPP-DHA might be another source of DHA phosphate.

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Phospholipase D (PLD) from Streptomyces sp. was found to catalyze the transfer reaction of the dipalmitoylphosphatidyl residue from 1, 2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to an aromatic hydroxy group in a biphasic system of an organic solvent with low water solubility and acetate buffer. The following phosphatidyl aromatic compounds were synthesized : 1, 2-dipalmitoyl-3-sn-phosphatidylphenol(1), 1, 2-dipalmitoyl-3-sn-phosphatidyl-4-methoxyphenol(2, DPP-PMP), 1, 2-dipalmitoyl-3-sn-phosphatidyl-4-methylphenol (3), 1, 2-dipalmitoyl-3-sn-phosphatidylhydroquinone (4), 1, 2-dipalmitoyl-3-sn-phosphatidyl-4-chlorophenol (5), 1, 2-dipalmitoyl-3-sn-phosphatidylcatechol (6), 1, 2-dipalmitoyl-3-sn-phosphatidyl-3-methoxyphenol (7), 1, 2-dipalmitoyl-3-sn-phosphatidyl-2-naphthol (8), and 1, 2-dipalmitoyl-3-sn-phosphatidyl-5-hydroxyindole (9). The transphosphatidylation reaction of DPPC by PLD was accompanied by hydrolysis of DPPC, and the ratios were significantly affected by both organic solvents and water contents in a biphasic system. PLD also catalyzed the transphosphatidylation reaction of DPP-PMP (2) to choline, to reproduce DPPC and liberate 4-methoxyphenol. This is the first report on the enzymatic transfer of phosphatidyl residue to aromatic hydroxy compounds.

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Delipidated cell walls from Aureobasidium pullulans were fractionated systematically. The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5 : 3.9 : 1.0 : 1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose. This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues. It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan. The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1-2×10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5-4.5×10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.

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Feeding xenobiotics such as polychlorinated biphenyls (PCB) causes hypercholesterolemia and fatty liver in rats. The hypercholesterolemia was characterized by high levels of high density lipoproteins (HDL)and apolipoprotein A-I (apo A-I), and by very low density lipoproteins (VLDL) rich in cholesterol and apo E (designated "PCB-VLDL"). The mechanisms for the generation of "PCB-VLDL" and fatty liver, and for hyper-α-lipoproteinemia in rats fed PCB were investigated. The secretion rate of VLDL-lipids was increased by PCB on day 3, while the secretion rate of only VLDL-cholesterol and phospholipid were increased by PCB on days 8 and 57. Although all liver lipids were accumulated by PCB, the accumulation of esterified cholesterol was the most drastic. These results suggested that PCB stimulated the secretion of VLDL at the early period of PCB feeding (on day 3), and that cholesterol-rich VLDL, "PCB-VLDL", was not generated in the circulation, but was originally secreted from the liver. In spite of the stimulation of VLDL secretion, liver lipids accumulated within 8 days on the PCB diet. On days 3 and 8, serum levels of free fatty acids were not changed by PCB feeding. These data and our previous findings that PCB induced hepatic lipogenic enzymes lead us to speculate that fatty liver induced by PCB may be attributed to a stimulation of de novo synthesis of liver lipids. Even when hepatic secretion of VLDL was blocked by orotic acid, HDL-cholesterol was increased by PCB feeding, suggesting that the increase in serum level of HDL by PCB was not due to stimulation of cholesterol transport into HDL from VLDL.

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Egg yolk oil was formed from a hen's egg without using the traditional charring method and organic solvents. By treating a spray-dried egg yolk suspension with a commercial crude enzyme preparation having protease and lipase activities, the lipids (egg yolk oil) could be easily separated, and a transparent solution of soluble polypeptides was obtained. When the enzyme preparation (4 mg) was added to a 5% spray-dried egg yolk (100mg) suspension, the reaction mixture became transparent, and the egg yolk oil floated to the surface of the reaction mixture within 3 h. In the case of a 10% spray-dried egg yolk suspension, a transparent solution and egg yolk oil could be successfully obtained within 4 to 5 h by using a larger reaction vessel and a 0.2 M lactate buffer. About 87% of the total polypeptides in the initial reaction mixture was recovered from the transparent solution, while about 83% of the total phospholipids was recovered from the floating egg yolk oil.

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An F₁-ATPase-defective mutant, TBLA-1, was constructed by the transduction of a defective gene for the α subunit of F₁-ATPase, atpA401, into Escherichia coli W1485lip2, a lipoic acid-requiring pyruvic acid producer. The pyruvic acid production of the strain TBLA-1 was found to be improved markedly compared with that of strain W1485lip2. In cultures using a jar fermentor, the strain W1485lip2 consumed 50g/liter of glucose and produced 25g/liter of pyruvic acid after culture for 32 h, while strain TBLA-1 consumed the same amount of glucose, and produced more than 30g/liter of pyruvic acid in a 24-h culture. A revertant, No. 63-1, derived from the strain TBLA-1, had a normal level of F₁-ATPase activity, and showed a similar pattern of pyruvic acid production to that of strain W₁485lip2.

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A new enzyme, D-tagatose 3-epimerase, was found in Pseudomonas sp. ST-24 during the course of studies on D-sorbose fermentation. This new enzyme catalyzes epimerization of keto-sugars, for example between D-tagatose and D-sorbose, and between D-fructose and D-psicose. It was shown that this enzyme epimerizes the configuration at the C-3 position of these substrates. This epimerase didn't act on D-fructose 6-phosphate and D-ribulose 5-phosphate. The enzyme has been purified from cells grown on a medium containing 1% D-glucose and 0.05% D-tagatose, and it appeared homogeneous on electrophoresis. The enzyme has a molecular weight of about 68, 000 by gel filtration and consists of two subunits identical in molecular weight (about 33, 000 by SDS-PAGE). The maximum activity at 30°C was obtained at pH 7-9, and the enzyme was stable from pH 7-11. The optimum temperature was around 60°C, and it was stable up to 60°C for 10 min.

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Photosystem (PS) II core complexes that had been solubilized by the use of octyl-β-D-glucopyranoside were centrifuged in a sucrose density gradient in the presence of the detergent for determination of M_r of the oxygen-evolving PS II unit. From its sedimentation velocity relative to those of M_r standards, the oxygen-evolving PS II core complexes appeared to have an M_r of 590, 000, which is twice the sum of the M_r of the individual subunit proteins and prosthetic small molecules of PS II. The sedimentation velocity of the PS II core complex from CaCl₂-treated PS II membranes was markedly lower than that of the oxygen-evolving PS II core complex and corresponded to an M_r of 370, 000. Sedimentation velocities of the isolated extrinsic proteins of 33 kDa, 24 kDa, and 18 kDa corresponded to their respective M_r's. The absence of the dimeric 33-kDa protein in the OGP-containing solution ruled out the cross-linking of PS II core complexes through self-association between 33-kDa extrinsic proteins. Dissociation of the dimeric PS II reaction center complexes may arise from the structure of the complex destabilized upon the dissociation of the extrinsic 33-kDa protein from the PS II complex.

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A potent inhibitor of larval settlement by the barnacle, Balanus amphitrite, was isolated as 2, 5, 6-tribromo-1-methylgramine from a marine invertebrate. In comparative tests on the activity of related compounds, such compounds as 2-methylgramine and 2-methyl-3-(morpholinomethyl)-indole exhibited potent inhibitory activity. The inhibitory activity toward larval settlement was found not to be due to toxicity but to a repellent effect.

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Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-β, γ-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.

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Aibellin is a 20-residue peptide antibiotic that has been isolated from the fungus Verticimonosporium ellipticum. Sequence-specific assignment of the ¹H- and ¹³C-NMR signals of aibellin in a methanol solution was achieved by using the two-dimensional NMR technique. Furthermore, its secondary structure was characterized by circular dichroism (CD) and NOESY spectra. The observed NOEs, ³J_NHCαH coupling constants and amide hydrogen-deuterium (H-D) exchange rates show that the peptide consisted of two α-helices and a bent structure around a Pro-14 residue.

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A levoglucosan (1, 6-anhydro-β-D-glucopyranose)-using bacterium, isolated from soil, was identified. It was shown to belong to the genus Arthrobacter and tentatively named Arthrobacter sp. I-552. A novel enzyme catalyzed the dehydrogenation of levoglucosan to form 1, 6-anhydro-β-D-ribo-hexopyranos-3-ulose (3-keto levoglucosan), using NAD⁺ as an electron acceptor, i. e. NAD⁺ : 1, 6-anhydro-β-D-glucopyranose oxidoreductase (trivial name : levoglucosan dehydrogenase). This enzyme was purified and characterized. A possible reaction scheme for the glucose formation was proposed. This pathway for levoglucosan use is distinct from those in yeast and fungi.

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Water extracts of leaves of black tea, oolong tea, and green tea, were examined for antimutagenic activity with Salmonella typhimurium test strains, TA 98 and TA 100. These water extracts significantly decreased the reverse mutation induced by crude dimethyl sulfoxide extracts of grilled beef and, by Trp-P-1, Glu-P-1, and B[a]P in the presence of a rat liver microsomal activation system. They also decreased the mutagenicity of AF-2 and 4-NQO requiring no metabolic activation to cause the mutation. Removal of caffeine from tea extracts did not influence the DEGB-induced mutation, but ethyl acetate extraction abolished the effect.

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Polyamines have been shown to be necessary for the activity of the extracellular ice-nucleating matter (EIM) from the ice-nucleating bacterium, Erwinia uredovora KUIN-3. When this bacterium was cultured in the presence of methylglyoxal bis(guanylhydrazone), MGBG (2 mM), the ice-nucleating activity of the EIM significantly decreased. Further, the thermal (25-40°C) and pH (alkaline region) stabilities of the activity were stimulated by the addition of spermidine. This phenomenon only occurred in the class A and B structures, and it showed that the hydrophobicities of the class A and B structures in the EIM increased with the addition of spermidine as judged by the freezing difference spectra. We then found by using fluorescent reagents that the physiological roles of spermidine in the EIM controlled the charge, free-amino groups, and hydrophobicities on the surface of the EIM. In conclusion, one could predict that spermidine took part in the charge of the surface, the control of hydrophobicity, and the stability of protein conformation in the class A and B structures in the EIM, and is a critical component in the class A and B nucleating structures.

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Calcium-binding properties of some chemical substances known as inhibitors against calcium phosphate insolubilization were investigated using a calcium-ion-selective electrode. First, the calcium binding properties of citrate, a well-known inhibitor, were evaluated by both the formation constant K_f and two parameters N and K in the Langmuir equation. It was confirmed that the parameters N and K were superior to the formation constant K_f to describe the calcium-binding property. Second, the calcium-binding properties of several inhibitors such as poly-L-aspartate, poly-L-glutamate, and alginates were analyzed by the Langmuir equation. They were well-described by the Langmuir equation. The tested inhibitors were classified into two groups : inhibitors of which parameters were independent of their concentrations (Group 1) and those whose parameters were dependent on their concentrations (Group 2). The values of the affinity parameter K for the Group 1 inhibitors were larger than those for the Group 2 inhibitors.

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Immunoglobulin M productivity of a human-human hybridoma line, HB4C5, was enhanced by the addition of histones H1, H2A, and H2B in serum-free medium, which are lysine-rich histones. On the contrary arginine-rich histones, histone H3 and H4, did not stimulate immunoglobulin production at all. Another arginine-rich protein, protamine also did not significantly stimulate immunoglobulin production. These results suggested that there is a correlation between amino acid residues of basic proteins and stimulation of immunoglobulin production. In this connection, immunoglobulin production stimulating activities of poly-lysine and poly-arginine were investigated. Both poly-L-lysine and poly-D-lysine stimulated IgM productivity of the human-human hybridomas. These results show that the enhancing effect of lysine residues was derived from its physiochemical features. On the other hand, poly-L-arginine did not enhance the productivity.

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3-Deoxyglucosone (3DG), the main intermediate compound in the Maillard reaction of proteins with glucose, suppressed the proliferation of various cell lines by inhibition of DNA synthesis. We investigated the mechanism of the suppression of cell proliferation from the standpoint of the progression of cell cycle. When 3DG was added to the culture of 3Y1 cells, rat fibroblasts, growing in exponential phase, the addition of 300 or 600μg/ml of 3DG increased the numbers of the cells apparently arrested at the G1 or G2/M phase, respectively. We observed that 3DG specifically inhibited the time-dependent progression during the S phase of a synchronous culture released from the early S phase in 3Y1 cells. 3DG influenced the cells released from the G0 phase but not the G0-arrested cells. When an intracellular concentration of reduced glutathione (GSH) in 3Y1 cells was decreased by using a GSH synthetase inhibitor, the inhibition of [³H]thymidine incorporation by 3DG was enhanced. Therefore, we assumed that the cells proliferating actively, in which the intracellular GSH concentrations have been reported to be lower, were more susceptible to the inhibitory effects of 3DG on the cell-cycle progression during the S phase.

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N-Phenyl-N'-alkoxyformamidines were found to have flower-inducing activity in dioecious Asparagus officinalis L. seedlings. Compounds that induced flowering in 95-100% of the seedlings at 40-100μM were developed by optimizing the structure, providing a new class of reagents useful for the selection of commercially preferable males that give a greater yield than the females. In the microsome preparation, the compounds inhibited t-cinnamic acid 4-hydroxylase (C4H) considerably less than the previously reported flower-inducing compounds, n-propyl N-(3, 4-dichlorophenyl)carbamate and piperonyl butoxide, a broad-spectrum P-450 inhibitor. These findings provide evidence that the inhibition of C4H is not the cause of flower induction. The amidines are considered to be an entity that acts on microsomes differently from the previous compounds, but that interacts commonly with the site that is responsible for flowering.

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A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1. 2 colonies per μg of integrative vector and 100 colonies per μg of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

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Saccharomyces cerevisiae wy2 segregated to 2 mater and 2 non-mater in relation to mating ability. The non-mater segregants behaved as the normal type of homothallic life cycle. On the other hand, the mater segregants gradually formed spores during successive subcultures, indicating that slow interconversion of mating-type happened to occur during subcultures. We termed this novel type of life cycle "delayed homothallism". The results of complementation tests with standard ho strains and introduction of a wild type HO gene showed that delayed homothallism was caused by a defective HO gene. The amino acid sequence deduced from the nucleotide sequence of the wy2 HO gene differed from the wild type HO gene in three amino acid residues. In the carboxy terminus of HO protein, there are three repeats of cysteine and histidine that are postulated to play a role in binding of HO protein to DNA. However, wy2 HO protein lacked one such repeat at residues Cys470-His475, where His was replaced by Leu.

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Escherichia coli grown in a rich medium excreted acetate and reused the acetate. Using cloned genes and a plasmid with a temperature-sensitive replication origin, three kinds of Pta-Ack pathway deletion mutants were constructed. Acetate production and reuse by wild-type cells grown in the rich medium was confirmed to largely occur through the Pta-Ack pathway. The deletion mutants of the gene encoding phosphotransacetylase secreted pyruvate before the secretion of acetate into the medium. A deletion mutant of the gene endocing acetate kinase grew at a slow rate, but its secretion and use of acetate were rapid. These results indicated that a pathway(s), other than the Pta-Ack pathway, functions in the control of excess carbon flow in the mutants.

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Three α-keto ester reductases (yeast keto ester reductase, YKER-II, -IV, -V) were purified from bakers'yeast. YKER-II, -IV, and -V are dimeric, monomeric, and dimeric enzymes, respectively, and molecular masses are estimated to be 58, 31-39, and 83kDa, respectively, based on gel filtration and SDS-polyacrylamide electrophoresis. Kinetic parameters and stereoselectivities in reduction of α-keto esters have been measured. YKER-IV contributes mainly to reduction by bakers' yeast at low substrate concentrations, and is useful for synthetic purposes.

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Castasterone (1), teasterone (2), and 6-deoxocastasterone (3), as well as monoolein, monolinolein, and monopalmitin, have been identified in immature seeds of rice (Oryza sativa) as active principles in the rice lamina inclination bioassay. Castasterone, as well as monoolein and monopalmitin, were likewise identified in immature seeds of Perilla frutescens, while monopalmitin was identified as a major active principle in cultured cells of Nicotiana tabacum. It seems that the occurrence of monoglycerides in higher plants as biologically active principles might be a general feature.

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The ACE inhibitory activity of an alkaline protease hydrolyzate from sardine muscle did not change after being treated by gastrointestinal proteases (IC₅0 = 0. 082 mg protein/ml). Eleven new ACE inhibitory peptides, constructed with 2 to 4 amino acid residues, were isolated from the hydrolyzate. The ACE inhibitory activity of each was mostly below 100μM of IC₅0 value ; the maximal inhibitory activity was observed for Lys-Trp (IC₅0 = 1. 63 μM). The isolated peptides inhibited ACE competitively, except for Met-Tyr with non-competitive inhibition. As the result of sequence homology, Arg-Val-Tyr isolated from the hydrolyzate was found in the primary structure of angiotensins I, II, and III, and of des Asp[1]-angiotensin I.

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A new method is reported for analyzing the medium- and high-boiling-point volatiles in beer, using CCI₃F for extracting the whole volatiles and then the headspace trapping method with Tenax TA. This method seems to be effective for analyzing the flavor not only of beer but also of other beverages, especially those containing a large amount of ethanol.

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A DDMP (2, 3-dihydro-2, 5-dihydroxy-6-methyl-4H-pyran-4-one)-saponin, named soyasaponin βg, was isolated from rootstock of the American groundnut (Apios americana). The structure was identified by ¹H-NMR and ¹3C-NMR spectroscopy and by chemical techniques. The distribution of this DDMP-saponin in the rootstock was detected as the brown color produced by the reaction with FeCl₃. A high concentration of DDMP-saponin was observed around the cells in fibrovascular bundle connecting the stem to plumule.

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Oxidation of grayanotoxin (GTX) II with lead (IV) acetate in methanol gave a new derivative, the 1(R)-spiro-3, 6(S), 14, 16-tetra-hydroxy-5-keto derivative. Treatment of GTX-II tetraacetate in acetic acid by using Pb(IV) acetate as an oxidizing agent gave a novel 1, 5-seco-GTX derivative, Δ¹⁽¹⁰⁾-1, 5-seco-GTX-pentaacetate, together with the l, 5-seco-GTX-1(R) derivative. Oxidation of GTX-II-tetraacetate with Tl(III) acetate in acetic acid or benzene gave the 1, 5-seco-GTX-1 (S) derivative.

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We isolated a class I chitin synthase gene (chsC) from Aspergillus nidulans. Expression of this gene was confirmed by Northern analysis and by sequencing of the PCR-amplified DNA fragments from cDNA. chsC disruptants showed no difference of morphology in the asexual cycle and no difference of growth rate compared to a wild-type strain.

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Two types of protein isolates have been obtained from defatted Lupinus mutabilis meal. Both, isolate-A and -B, were obtained by alkaline extraction with 0. 2% NaOH, and 0. 25% bisulfite, followed by precipitation at the isoelectric point (pH = 4. 8). Total lipids were extracted with 86% ethanol, and neutral lipids were separated using a Florisil column. The lipids found in the isolates were similar to those found in the original meal, and the following types of compounds were separated, identified, and measured : hydrocarbons, waxes, methylesters, triglycerides, free fatty acids, diglycerides, and free sterols.

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We isolated a yeast from air, strain S-2, which produces raw starch-digesting α-amylase, xylanase, and polygalacturonase. The fermentation ability was negative, and D. B. B., urease, and DNase tests were positive. The major ubiquinone was Q-10 and the mol% G + C content of nuclear DNA was 67. The cells were oval to ellipsoidal ; no true mycelium, pseudomycelium, or teliospores were observed ; and it contained a large amount of xylose (19. 7 mol%) in the whole-cell hydrolyzates. From these characters, strain S-2 was assigned to the genus Cryptococcus.

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Superoxide dismutase (SOD)-like compounds and activators of SOD were screened for in the extracts of fruits, vegetables, and mushrooms by measuring their effects on pyrogallol autoxidation, which is catalyzed by superoxide anion. SOD-like activity was high in aqueous extract of nameko, garlic, broccoli, and oriental lettuce. Ethanolic extracts of onion and watermelon could enhance human SOD activity more than 40%.

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Tumor necrosis factor α inhibitor (TNF-INH) was purified from human urine and it was composed of 161 amino acid residues. The complete amino acid sequence of TNF-INH found by sequence analysis agreed with that predicted from the cDNA structure for the extracellular domain (1-161 portion) of 55-kDa TNF receptor and its processing site at the C-terminal was Asn-161.

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Cultivation of an ice nucleation-active strain of Xanthomonas campestris in the presence (1 ppm) of 4-hydroxy-3-nitrophenylacetic acid resulted in enhancement of its ice-nucleation activity. Both the ice-nucleation-active protein, InaX, and its mRNA were effectively expressed in the bacterial cells cultured in the presence of this compound. This indicates that this compound stimulated the biosynthesis of the ice-nucleation-active protein.

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Bacillus sp. YUF-4 did not produce acetylacetoin with general culture media, such as bouillon medium containing glucose or acetoin. When diacetyl was added to a medium cultured for 18-20 h in the presence of glucose or acetoin, AAC was produced as culture continued. AAC was assayed by GLC with a Carbowax 20M capillary column. The AAC produced was purified by several steps : the final HPLC using a Shodex E411 column was effective. The yield of AAC was 346mg per liter of the medium (7. 5% recovery)and the purity was 97%. AAC was identified by ¹H-NMR and ¹³C-NMR.

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The extracellular ice nuclei of Fusarium avenaceum IFO 7158(FEIN) were stable at pH levels from 2 to 12 and tolerated temperatures up to 40°C. In an activity of the FEIN, proteins were important, however, saccharide and lipid which were proved to be a part of components of bacterial ice nuclei did not participate.

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Cell walls of facultative alkaliphilic Bacillus sp. C-125 are composed of peptidoglycan, teichuronic acid, and teichuronopeptide. A mutant lacking teichuronic acid, or defective in both teichuronic acid and teichuronopeptide has been isolated from the organism. We now constructed another type of a cell-wall defective mutant that was defective in teichuronopeptide but had teichuronic acid, by cell fusion using protoplasts prepared from a wild-type strain and a mutant defective in teichuronopeptide and teichuronic acid. This mutant grew more poorly than wild type or a teichuronic acid-defective strain of C-125. The growth, however, was faster than that of the parental strain defective in both teichuronic acid and teichuronopeptide.

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The effects of the flower lipid-saccharide complex (lipopolysaccharide analogue) and unsaponifiable matter (1 g/kg diet) of the sunflower on various lipid parameters was studied in rats given cholesterol-enriched diets. After six weeks of feeding, there were no significant differences in the concentrations of serum cholesterol and triacylglycerol, although they tended to decrease with dietary sunflower consumption. The liver cholesterol concentration was significantly reduced with the sunflower diet. The linoleate de-saturation index, an arachidonic acid/linoleic acid ratio, in tissue phosphatidylcholine significantly increased in the unsaponifiable matter group. The fecal coprostanol and bile acid concentrations increased significantly in the lipid-saccharide complex and unsaponifiable matter groups. The fecal cholesterol concentrations also tended to increase with the dietary sunflower consumption.

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The gene coding for L-fucose dehydrogenase (FDH) which is specific to NADP⁺ as a hydrogen acceptor, was cloned, sequenced, and expressed in Escherichia coli from Pseudomonas sp. No. 1143. FDH was shown to be composed of 329 amino acid residues.

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The contribution of the Asp-197 and Asp-202 residues in chitinase A1 of Bacillus circulans WL-12 to the catalytic reaction was studied by site-directed mutagenesis of these residues. A kinetic analysis of the purified mutant chitinases suggests the involvement of both the Asp-197 and Asp-202 residues in the catalytic events of this enzyme, although the effects of mutations of Asp-197 were less severe than those of the other mutations.

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Peptidylarginine deiminase catalyzes the conversion of arginyl residues in proteins to citrullyl residues in the presence of Ca²⁺. We described the preparation of monoclonal antibody (subclass type IgG₁) specific to mouse peptidylarginine deiminase type II. The antibody had no effect on the enzyme activity and its specific epitope was localized in the eight-residue segment at the amino-terminal portion of the enzyme.

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IM0, a commercially available mixture of isomaltooligosaccharides, was fractionated by preparative HPLC, and two fractions (IMO2 and IMO3) were obtained. IMO2 contained mainly disaccharides (86. 4%), which IMO3 contained tri- and higher oligosaccharides (89. 9%). The administration of IMO2 or IMO3, ranging in amount from 5 to 20g/day, increased human intestinal bifidobacteria in dose-dependent manner. An IMO2 intake of 10 g/day and IMO3 of 5 g/day each produced a significant increase of bifidobacterial number in feces and the ratio in fecal microflora within 12 days.

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The accumulation of γ-aminobutyric acid (Gaba), a well-known blood pressure-lowering compound, in the rice germ during water soaking was investigated by using ten different cultivars. The amount and pattern of the Gaba accumulation varied considerably depending on the cultivar. Hokkai 269 exhibited a remarkable accumulation of Gaba in the germ and is thus suggested to be a very promising source for Gaba

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The fungus, Pyricularia oryzae Cavara, was found to convert phenylpropenes into phenylpropanediols. The stereochemistry of the diol products was dependent on the geometry of the double bonds of the phenylpropenes.

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Xyloglucan oligosaccharides, which were derived from tamarind xyloglucan by cellulase digestion, were measured by a competitive ELISA method using antibodies raised against a complex of BSA and xyloglucan nonasaccharide. By this method, tamarind xyloglucan oligosaccharides could be measured in the range of O. 1 to 40 nmol/well.

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We constructed deletion mutant clones of a pectin lyase gene, and measured their pectin lyase activities in Escherichia coli. Pectin lyase activities were detected only in a recA⁺ strain but not in a recA^- strain of E. coli. We also cloned and sequenced recA from Pseudomonas marginalis N6301. The recA from P. marginalis N6301 can complement recA^- to form recA⁺ in the phenotype of E. coli. Highly conserved sequences of recA are observed among E. coli, P. fluorescens, and P. marginalis. From these results, we presume that recA is required for the expression of the pectin lyase gene in P. marginalis N6301.

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We report cloning and sequencing of a gene encoding a putative aldehyde dehydrogenase in Synechococcus sp. PCC7942. It was found that this phototrophic microorganism has a protein very similar to mammalian class-3 aldehyde dehydrogenases. A mutant strain lacking this gene was hypersensitive in growth to an aromatic aldehyde.

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A 3, 6-di-O-benzylated demethylallosamizoline derivative was glycosylated at the 4-position with an N, N'-diphthaloylchitobiosyl moiety by using the thioglycoside method. After de-protections, the resulting demethylallosamidin-like pseudotrisaccharide was evaluated as an inhibitor against a couple of chitinases.

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A monooxygenase specific for theobromine demethylation was purified from the cell-free extract of Pseudomonas putida No. 352to homogeneity. Caffeine appears to be demethylated to 7-methyl-xanthine at least by two enzymes, caffeine demethylase and theobromine demethylase. The latter is sensitive to Zn²⁺ and possibly caused an accumulation of theobromine in P. putida No. 352.

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The Escherichia coli pgsA3 mutation, which causes acidic-phospholipid deficiency, was found to repress the flagellar master operon, as assessed by the β-galactosidase activities of flhD-lacZ fusions. This explained the impairment of flagellar formation and motility by the mutation. A series of deletion analysis indicated that a 40-bp region, at the 5' end of the flhD locus examined, was responsible for the repression of a downstream transcription initiation that was catabolite-repression sensitive. This novel regulatory region was 200 bp upstream of the first possible translation initiation site.

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