A single form of exo-type cellulase (Exo I ; M_W, 65, 000), purified from a Trichoderma viride proteasedepressed mutant, HK-75, digested Avicel to cellobiose exowise, and hydrolyzed cellotriose, cellotetraose, and cellopentaose in the strict manner of splitting off by cellobiose units. Exo I, however, hydrolyzed cellohexaose by both cellobiose and cellotriose units. Exo I was proteolyzed by papain into two fragments ; GPExo (M_W, 9, 000) and Exo I' (M_W, 56, 000). The GPExo intensively adsorbed onto Avicel but did not hydrolyzed it. Exo I' had nearly identical activity to that of intact Exo I toward cellooligosaccharides but was almost inert to Avicel in digestion and adsorption. Sequence analysis of N-terminal and C-terminal amino acids showed that GPExo was between Gly⁴³⁵ and Leu⁴⁹⁶ and Exo I' between Glu¹ and Gly⁴³⁴ in Exo I. Exo I therefore consists of two domains, one for adsorption to Avicel, as demonstrated by the Avicel-affinity site, GPExo and the other for the cleavage of glycosidic linkages as demonstrated in Exo I'.

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Retinyl acetate suppressed the free radical-induced oxidation of methyl linoleate. Retinyl acetate was reacted with an alkylperoxyl radical in two solvent systems, methanol and benzene. The alkylperoxyl radical was generated by the thermal decomposition of a free radical initiator, 2, 2'-azobis (2, 4-dimthylvaleronitrile), at 37°C. The reaction products were isolated by HPLC and their structures were identified. The main product in methanol was 14-hydroxy-13-methoxyretinyl acetate in addition to some minor products. The reaction in benzene gave some products with low yields. They were 5, 6-epoxyretinyl acetate, 11, 14-epoxyretinyl acetate, and 5, 6, 11, 14-diepoxyretinyl acetate. The results indicate that retinyl acetate is capable of scavenging peroxyl radicals by its conjugated polyene structure.

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The Acidic and neutral fractions of an extract of the marine alga, Eisenia bicyclis (Japanese name "Arame"), showed strong antioxidative activity when tested by the ferric thiocyanate and TBA methods. Chromatographic purification of the neutral fraction gave an active oily substance identified as pyropheophytin a, one of the chlorophyll a-related compounds. This compound showed higher antioxidative activity than a-tocopherol and could be expected to act as an antioxidant in foods. The porphyrin ring system seems to be important for the antioxidative activity in the dark.

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A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60, 000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M_r : 15, 000). The enzyme hydrolyzed predominately acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles resembles carboxypeptidase G₁ or G₂ in its glutamate-releasing activity. However, it acts not noly on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G₁ or G₂, it was named carboxypeptidase G₃.

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Dried bonito (Katsuobusi), a Japanese traditional seasoning made of bonito muscle was hydrolyzed by various proteases and the inhibitory activity of the hydrolyzates for angiotensin I-converting enzyme (ACE)【EC 3.4.15.1】 was measured. Among the digests, thermolysin digest showed the most potent inhibitory activity. Eight inhibitory peptides were isolated from the digest using HPLC. The amino acid sequences of inhibitory peptides were Ile-Lys-Pro-Leu-Asn-Tyr, Ile-Val-Gly-Arg-Pro-Arg-His-Gln-Gly, Ile-Trp-His-His-Thr, Ala-Leu-Pro-His-Ala, Phe-Gln-Pro, Leu-Lys-Pro-Asn-Met, Ile-Tyr, and Asp-Tyr-Gly-Leu-Tyr-Pro. By searching for the sequence homology in many proteins, four of them were found in the primary structure of actin. Asp-Met-Ile-Pro-Ala-Gln-Lys was obtained from the boiling water extract of dried bonito and this peptide was found in the primary structure of creatine kinase. Fragments of these peptides were prepared by further enzymatic digestion or chemical synthesis and their ACE-inhibitory activities were measured. Among them, Ile-Lys-Pro, Ile-Trp, Leu-Lys-Pro, and Leu-Tyr-Pro had higher inhibitory activity than their parental peptides. Ile-Lys-Pro suppressed the hypertensive activity of angiotensin I.

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Chitosanolytic enzymes from Enterobacter sp.G-1 were immobilized on various carriers to continuously hydrolyze chitosan. Four different carriers were tested : FE-3901 (strong basic anion exchange resin, ionic binding), glutaraldehyde-treated FE-4612 (weak basic anion exchange resin, cross-linking), Chitopearl (chitosan beads), and alginate calcium. Glutaraldehyde-treated FE-4612 and Chitopearl immobilized more protein than the others. The enzyme immobilized on FE-3901 had the greatest activity. The activity of enzyme immobilized on FE-3901 decreased rapidly when exposed to a continuous flow of 1% chitosan. The enzyme immobilized with Chitopearl retained more than 50% of its original activity after 17 days, and the activity was fully restored by re-immobilization.

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A transglycosylation reaction with 2-chloro-4-nitrophenyl β-maltoside as an acceptor was done with 4, 6-O-3-ketobutylidene maltopentaose and Bacillus macerans cyclodextrin glucanotransferase in an aqueous solution containing 50% n-propanol, and there were two main transglycosylation products. They were identified as 2-chloro-4-nitrophenyl 4, 6-O-3-ketobutylidene β-maltopentaoside and 2-chloro-4-nitrophenyl 4, 6-O-3-ketobutylidene β-maltohexaoside, and their yields were 30% and 21% respectively on the basis of the decrease of 4, 6-O-3-ketobutylidene maltopentaose. For the production of 2-chloro-4-nitrophenyl 4, 6-O-3-ketobutylidene β-maltopentaoside at high substrates concentrations, the addition of n-propanol in this reaction not noly increased the solubility of 2-chloro-4-nitrophenyl β-maltoside sufficiently but also suppressed side reactions.

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Two sialyllactose isomers, NeuAcα2→6Galβ1→4(NeuAcα2→6)Glc, were prepared by incubation of a concentrated solution of N-acetylneuraminic acid and lactose in the presence of a neuraminidase from Arthrobacter ureafaciens. Each sialyllactose was isolated by a combination of ion-exchange chromatography and high performance liquid chromatography. The structure of each sialyllactose was identified by mass spectrometry, nuclear magnetic resonance spectrometry, and enzymatic analysis.

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Yacon (Polymnia sonchifolia) leaves possess glandular trichomes on the surface. The exudate from the glandular trichome and the leaf are itself rich in ent-kaurenic acid (ent-kaur-16-en-19-oic acid). A kaurene derivative, 15-α-angeloyloxy-ent-kauren-19-oic acid 16-epoxide, was isolated from the leaves, together with two known angeloyloxykaurenic acids. The high content of ent-kaurenic acid in the leaf suggests that these diterpenes play a certain physiological role, since the glandular trichome exudates of other species function in their defensive mechanism.

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The surface lipids of Nicotiana benthamiana contained novel glycerolipids and several varieties of glycolipids. As glycerolipids, the triacylglycerol, 1, 3-diacylglycerol, and 1, 2-diacylglycerol types of glycerolipids were isolated and identified. Each lipid contained acetyl, 16-methylheptadecanoyl, and 18-methylnonadecanoyl moieties. The acetylated position of each lipid was determined by 2D-NMR, using the HMBC technique. The structures were 1, 3-di-O-acetyl-2-O-acylglycerol, 1-O-acetyl-3-O-acylglycerol, and 1-O-acetyl-2-O-acylglycerol. As glycolipids, one glucose ester and four types of sucrose esters were isolated and identified. These glycolipids contained acetic acid and such branched short-chain fatty acids as 5-methylhexanoic, 4-methylhexanoic, 6-methyheptanoic, and 5-methylhexanoic acids. The structure of the glucose ester was 3, 4-di-O-acyl-α-D-glucopyranose. The structures of the sucrose esters were 6-O-acetyl-4-O-acyl-α-D-glucopyranosyl-(3-O-acyl)-β-D-fructofuranoside, 4-O-acyl-α-D-glucopyranosyl-(3-O-acyl)-β-D-fructofuranoside, 3, 4-di-O-acyl-α-D-glucopyranosyl-β-D-fructofuranoside, and 6-O-acetyl-α-D-glucopyranosyl-β-D-fructofuranoside.

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Six novel phloroglucinol dialdehyde diterpene derivatives (macrocarpals B-G), which have antibacterial activity, were isolated from leaves of Eucalyptus macrocarpa. These compounds have closely related structures, the molecular formula for B-F being C₂₈H₄₀O₆, and that of G being C₂₈H₃₈O₅. The structures of macrocarpals B, D, and G were analyzed by means of NMR analyses.

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Sulfated primary alcohols and methyl α-D-galactopyranoside 6-sulfate were converted into desulfated and trimethylsilylated alcohols and galactoside, respectively, by treating their pyridinium salts with N, O-bis (trimethylsilyl) acetamide (BTSA) or N, O-bis (trimethylsilyl) trifluoroacetamide (BTSTFA) in pyridine. Sulfated secondary alcohols and methyl galactoside 2-, 3-, and 4-sulfates did not have their sulfates eliminated under similar conditions, indicating that the reaction was specific to the primary hydroxyl groups. Methyl α-galactoside 2-sulfate was preparatively obtained by the BTSA treatment of methyl α-galactoside 2, 6-disulfate, indicating that the method is applicable to regioselective desulfation on a preparative scale.

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New fluorinated pyrethroids were designed to block microsomal oxidation at the C-10 methyl group of biophenothrin and at the C-7' methylene of bioallethrin. By using diethylaminosulfur trifluoride and/or hexafluoropropene diethylamine, 10, 10-difluorobiophenothrin and 7'-fluorobioallethrin were synthesized from the oxidized derivatives of the parent insecticides, which have been recognized as microsomal metabolites. While 10-hydroxybiophenothrin was not successfully converted into the corresponding 10-fluoro derivative by either fluorination reagent, other monofluorinated compounds were formed and their structures were elucidated.

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Pseudomonas testosteroni ATCC 11996 was found to produce a novel bile acid sulfate sulfatase that hydrolyzes the sulfate ester bond in lithocholic acid sulfate (LCA-S). The enzyme synthesis was induced by several kinds of bile acids including LCA-S. Mn²⁺ functioned as an essential component for the enzyme synthesis and SO^2-₄ suppressed it. This sulfatase hydrolyzes LCA-S to isolithocholic acid and sulfuric acid with inversion of α- to β-configuration of the hydroxyl group at the third position of lithocholic acid.

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(+)-Juvabione 1 was synthesized by employing (1R, 4S, 6S)-6-hydroxybicyclo[2.2.2] octan-2-one 2 as a chiral source. (+)-Juvabione 1 shows juvenile hormone activity, and its racemate has been repeatedly synthesized.¹⁾Optically active 1 was synthesized by Pawson et al. from (+)-limonene, ²⁾ and by Trost et al. from (+)-perillaldehyde.³⁾ However, their syntheses were not at all efficient for providing a suitable smount of optically active 1.

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The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that ; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met¹-Ala³³ leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, ¹⁾ (3) 3rd letters of codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mcmA) while the flanking regions had little homology with the corresponding ones of mcmA, except some homology at the -10th and -35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.

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Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for pectate lyase II (pelII) of E.carotovora Er was cloned and expressed both in Escherichia coli and E.carotovora Er. Localization experiments in E.coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38, 169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.

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Dissimilatory nitrate reductase activity in Pseudomonas denitrificans grown under denitrifving conditions was markedly decreased upon incubation of the cells with Cys or GSH. The activity of the enzyme was also low in the membrane fraction isolated from the Cys-treated cells, indicating that the decrease in enzyme activity was due to the inactivation of the enzyme. Cys appeared to exert its effect, at least in part, after conversion to GSH as judged by the stimulation of the Cys effect by concomitant addition of Glu, although even a trace of GSH could not be detected in the cells presumably owing to the high activity of GSH consumption. The inactivating effect of Cys on the enzyme was found to compete with the activating effect of Na⁺ that had been reported previously, suggesting the presence of a natural mechanism controlling nitrite accumulation in the environment.

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Z-Val-, Z-Pro-, Z-Leu-Leu-, and Z-Leu-Pro-lysinals and Bz-DL-lysinal were chemically synthesized and tested as novel inhibitors for Achromobacter protease I (API), a lysine-specific serine protease. Among the lysinal derivatives tested, Z-Val-lysinal was the most potent competitive inhibitor, its K_i being estimated as 6.5 nM in an esterolytic assay with Tos-Lys-OMe. In an amidolytic assay, Z-Leu-Leu-lysinal was the most potent inhibitor and the apparent mode of inhibition was non-competitive. The K_is of the other lysinal derivatives in both esterolytic and amidolytic assays were more than 10³ times lower than that of leupeptin. Z-Val-lysinol, lacking the aldehyde group, was a poor competitive inhibitor. These results suggest that acyl-, aclyaminoacyl-, and acylpeptidyllysinals function as a transition-state inhibitor for Achromobacter protease I.

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Two kinds of α-glucuronidases, CM-I and CM-II, were purified from a cell-free extract of Aspergillus niger 5-16. Molecular weights of both CM-I and CM-II were 130, 000 by SDS-PAGE, and 150, 000 by gel filtration. The optimum temperature and pH for the both enzyme reactions were 60°C and 4.8. Both enzymes were stable up to 50°C, and between pHs 4.5 to 7.0 for 1 hr. Both enzymes were strongly inhibited by Ag⁺, Hg²⁺, Pb²⁺, Sn²⁺, Fe²⁺, Fe³⁺, Al³⁺, sodium dodecyl sulfate, monoiodoacetic acid, and N-bromosuccinimide. The K_m of CM-I toward glucuronosyl-xylotriose and 4-O-methyl-glucuronosyl-xylotriose were 0.77 and 0.37, and those of CM-II were 0.82 and 0.47 mM, respectively. The V_max of CM-I toward glucuronosyl-xylotriose and 4-O-methyl-glucuronosyl-xylotriose were 5.20 and 1.42, and those of CM-II were 17.1 and 4.68 μmol of glucuronic acid formed/min/mg of protein, respectively.

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α_s1-Casein can elicit a proliferative response in responding T cell clone 3D20 cells (specific for I-A^b plus fragment 136-151), even when using fixed splenic antigen-presenting cells (APC) not carrying antigen processing ability. The order of potency of each tested antigen for fixed APC was the determinant peptide (136-151)>the long peptide (136-195)>the intact protein (199 residues), indicating that regions outside the determinant area negatively affected the stimulatory potency of the antigens. On the other hand, the order for normal splenic APC was the short peptide>the intact protein>the long peptide. This shows that negative effects by regions outside the determinant area were strongly influenced by the antigen processing.

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Several chemicals were evaluated for their efficacy to induce sterility in rice (Oryza sativa L., Yamabiko) in 1990. Two chemicals, kasugamycin and aminooxyacetic acid (AOA), indicated a remarkable effect to induce sterility in rice when treated at meiosis. In 1991, kasugamycin and AOA were applied to rice plants at two different growth stages to find the optimum application period and dosage for the induction of sterility. Kasugamycin affected sterility in rice only at meiosis, but AOA affected it from the panicle formation stage to meiosis at a low concentration of 100 mg/liter. Rice plants treated with kasugamycin were similar to control for the number of seeds, spike length, culm length and weight of ripe seeds, but rice plants treated with AOA were inferior to the control in these respects. Interaction between kasugamycin and the plant hormone for the restoration of fertility was shown in rice when α-naphthylacetic acid (NAA) was applied simultaneously with kasugamycin.

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A variety of derivatives of 3- and 4-(dimethylaminomethyl)-1, 2-dithiolanes were prepared as their biological equivalents in an analogous way to that for the nereistoxin derivatives. Most of them showed insecticidal and acaricidal activity. High potency against Chilo suppressalis was displayed by S, S'-[2-(dimethylaminomethyl)trimethylene] bis(thiobenzoate), and a broad spectrum of activity was observed for 5-(dimethylaminomethyl)-1, 2, 3-trithiane. The stereochemistry of the 1, 3-dithianes is also discussed.

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The cell wall of Fusarium oxysporum f.sp.lycopersici was digested with chitinase to analyze the structure of its chitinous components. In spite of a similar acetylation degree of the cell wall components to that of 25-35% acetylated chitosan, only N-acetylglucosamine disaccharide [(GlcNAc)₂] was obtained from chitinase hydrolyzate of the fungal cell wall by CM-Sephadex C-25 column chromatography, while (GlcNAc)₂ and several types of deacetylated chitooligosaccharides were separated from that of 25-35% acetylated chitosan. The results indicate that N-acetylglucosamine residues in the polysaccharide chains of the fungal cell wall are most likely condensed into some region, while acetylated residues are more scattered in 25-35% acetylated chitosan.

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The effect of exercise on the protein metabolism in skeletal muscles (gastrocnemius and soleus), liver and small intestine was investigated in rats. Treadmill treatment for 7 d resulted in atrophy of the liver and small intestine, which was associated with a reduction in protein content. The rates of protein synthesis in the liver and small intestine were significantly suppressed in rats subjected to exercise. The change in protein synthesis in the visceral organs was mediated by the change in RNA activity (protein synthesis per unit RNA) but not by the change in RNA concentration. The tissue weight and the rate of protein synthesis in the gastrocnemius and soleus muscles were not affected by exercise. The results suggest that these changes in protein synthesis in the liver and small intestine may explain, at least partly, the atrophy of these organs which was observed after 7 d of exercise.

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Metabolism of glucose and L-amino acids in an obligately aerobic marine bacterium isolated from Pacific mackerel intestines was investigated for the mechanism and pathway of eicosapentaenoic acid (EPA) biosynthesis. This bacterium could not uptake glucose but the cell-free extract of this bacterium had the enzymatic activities of L-alanine oxidase (EC 1.4.3.2), L-alanine dehydrogenase (EC 1.4.1.1). L-serine dehydratase (EC 4.2.1.13), and malate dehydrogenase (EC 1.1.1.40), and of seven enzymes involved in the TCA cycle of the usual aerobes. On the other hand, the carbon-13 concentration in cellular fatty acids of the bacterium, especially that in their methyl carbon atoms in contrast to their carbonyl carbons, increased drastically when the bacterium was grown in the presence of ¹³CH₃COONa. These results indicate that : (i) the TCA cycle works in this bacterium, (ii) glucose is not utilized and pyruvic acid is in vivo synthesized from L-alanine, L-serine, and malic acid, and (iii) EPA and other cellular fatty acids are in vivo synthesized from acetyl coenzyme A by the usual de novo synthesis route.

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The activities of RNA polymerases from Pseudomonas putida and Pseudomonas aeruginosa were compared with that of Escherichia coli RNA polymerase in an in vitro transcription system. All three enzymes initiated transcription at the tac promoter and the RNA I promoter of E.coli. We measured the rate of open complex formation between the RNA polymerases and the promoters, and the saturation level of open complex formation at equilibrium in single-round transcription. The relative rates of open complex formation were P.putida>E.coli>P. aeruginosa and the relative saturation levels of open complex formation at equilibrium were E.coli>P.putida>P. aeruginosa for the tac and RNA I promoters. The interaction of the RNA polymerases with the promoters was also studied by DNase I footprinting. The patterns of protection of the Pseudomonas RNA polymerases on the tac promoter were similar to that of E.coli RNA polymerase. However, the protection patterns of the Pseudomonas RNA polymerases on the RNA I promoter were slightly different from that of E.coli RNA polymerase.

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To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S.pombe posttranslationally, and glycosylated to the same extent as in the ER of S.cerevisiae. This sugested that the size of N-linked core-oligosaccharide in the ER of S.pombe is similar to that in S.cerevisiae. This translocation into the ER of S.pombe was inhibited by puromycin, but the translocation in the P-cells of S.cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S.pombe was sensitive to puromycin and different from that of S.cerevisiae.

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In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156, 602, a C5a receptor antagonist. L-156, 602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156, 602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hindfootpads, indicating that the efferent phase of DTH was affected by L-156, 602. The results demonstrated that L-156, 602 preferentially suppressed the DTH response.

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