The theanine (THE : γ-glutamylethylamide) content and the growth rate of cultured cells of tea (Camellia sinensis L.) were increased greatly to 22.3% in dry wt. with a medium containing 60 mM nitrate and 25 mM ethylamine as a nitrogen source. The optimum concentrations of nitrate, Mg²⁺, and K⁺ for the growth and formation of THE in suspension cells were 40 mM, 3 mM, and 104 mM, respectively. The yield of THE accumulated in the cultured cells with the medium modified for THE formation was increased greatly due to a great increase of the growth rate.

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A microbe of Streptomyces werraensis TT 14, which was newly isolated from soil, decolorized the model melanoidin prepared from glucose and glycine, the decolorization rate being 64% in the optimal medium of pH 5.5 (2.0% starch, 1.0% yeast, 0.3% NaCl, and 0.3% CaCO₃) and 45% in a synthetic medium. There was virtually no difference in the UV-VIS absorption spectra of the microbially treated melanoidin and the control. The peaks of the gel permeation chromatogram for the treated melanoidin and for the control showed the same retention times, but lower molecular weight compounds increased in the decolorized melanoidin. The Cu(II)-chelating activity of the decolorized melanoidin was reduced to about half that of the control. The melanoidin component of pI 2.5 was increased and that of pI 3.5 was reduced by the microbial treatment.

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The non-Newtonian behavior and dynamic modulus of a series of aqueous mixtures of xanthan (native, deacetylated, depyruvated, and deacylated) and konjac glucomannan were measured with a rheogoniometer. The flow curves, at 55°C, of a mixed solution of xanthan and glucomannan showed plastic behavior at 0.1% total gums. At a concentration of 0.1% total gums, gelation occurred at room temperature. A much stronger gel was observed in a mixture with deacetylated xanthan, about twice as strong as that of a mixture with depyruvated xanthan. The dynamic modulus of a mixture of deacylated xanthan and glucomannan stayed at very small value in the presence of CaCl₂. (6.8 mM) and urea (4.0 M). The side chains of xanthan may be dominant in the interaction with konjac glucomannan molecules.

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The haedoxan analog, (±)-2-(2, 6-dimethoxyphenoxy)-1-hydroxy-6-(6-methoxy-1, 4-benzodioxan-7-yl)-3, 7-dioxabicyclo[3.3.0]octane, and its congeners with 2-alkoxymethyl, 2-hydroxymethyl, 2-chloro-methyl and 3-(3, 4-methylenedioxyphenyl) substituents on the 1, 4-benzodioxanyl group were synthesized from 6-methoxy-1, 4-benzodioxan-7-carbaldehyde and its (±)-2-and 3-substituted derivatives, respectively. Some analogs were considerably insecticidal, although much less active than natural haedoxan A. The assay results suggest that 2, 3-disubstitution on the 1, 4-benzodioxanyl group was necessary to intensify the insecticidal activity.

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A subunit with carboxymethyl cellulase (CMCase) activity was isolated from the cellulosomes of Clostridium thermocellum after dissociation of the cellulosomes by a mild sodium dodecyl sulfate (SDS) treatment. The subunit displayed only one protein band of 51kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but after boiling with SDS it had 3 bands of 60, 56, and 48 kDa. Prolonged incubation with SDS changed the subunit to display exclusively the 48-kDa band after boiling. The 51-kDa subunit was presumably a partially denatured form, and differentiated into 3 species with apparent M_r of 60, 56, and 48k through deglycosylation in SDS solution. Enzymatic properties of the 51-kDa subunit resembled those of the endoglucanase A which was purified from the culture fluid and from a E. coli clone with exceptions of temperature and pH optima.

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β-N-Acetylglucosaminidase (EC 3.2.1.30) was purified from a cell-free extract of Aeromonas sp. 10S-24 by precipitation with ammonium sulfate and column chromatographies with DEAE-Toyopearl, Sephacryl S-200 HR, chromatofocusing, and Q-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was about 103, 000 by SDS-PAGE, and 100, 000 by gel filtration. The pI was 5.05 by chromatofocusing. The optimum pH was 7.0 and the stable pH was around 7.0. The enzyme was potently inhibited by various metal ions and SH reagents. The K_m was 0.27 mM for p-nitrophenyl-β-D-N-acetylglucosaminide. The enzyme hydrolyzed N-acetylchitooligosaccharides, di-N-acetylchitobiose through hexa-N-acetylchitohexaose.

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Many compounds with attaching repellent activity in the blue mussel assay also showed antifouling activity in the conventional submerged assay. Only those attaching repellents with antimicrobial activity against marine Gram-positive bacteria isolated from slime, as well as Staphylococcus aureus, Bacillus subtilis, Trichophyton mentagrophytes, and Candida albicans exhibited antifouling activity. A combination of the blue mussel and antimicrobial assays is suggested to be a simple and efficient screening procedure for antifouling substances.

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Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS-PAGE. The molecular weight was estimated to be 5200 by SDS-PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PW_XL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.

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Microorganisms that converted racemic 1-phenyl-1, 2-ethanediol((RS)-1) to optically active mandelic acid (2) were screened for, and Bordetella parapertussis KPPA 20005 was selected as the best strain. This bacterium accumulated 2.0g/liter of (R)-2 (95% e.e.), along with a small amount of benzoylformic (3) and benzoic acid (4) starting from 5.0g/liter of (RS)-1. Production of 3 and 4 were inhibited by adding the substrate together with 0.3 to 3.0% ethanol after growth for 2 days.

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The antimicrobial mechanism of altersolanol A was studied by using Pseudomonas aeruginosa IFO 3080. This reduced anthraquinone antibiotic inhibited the incorporation of radioactive precursors into macromolecules (DNA, RNA, and Protein) in whole cells nonspecifically. It increased respiration of P.aeruginosa cells, though it had no effect on proton condution. Oxidation of NADH in the membrane fraction isolated from P.aeruginosa was enhanced by altersolanol A. Furthermore, altersolanol A stimulated the oxidation of NADH by the enzyme preparation of cytochrome c reductase in the absence of cytochrome c. Altersolanol A seemed to interfere with the respiratory chain in the bacterial membrane as an electron acceptor.

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Interactions between auxin-binding protein-I (ABP-I), purified from etiolated mumg bean seedlings, and nuclear components from mung bean tissues were investigated. When NaCl-solubilized components of chromatin were put on an affinity column of ABP-I-linked Sepharose 4B, a small amount of the material was retained on the affinity column and was eluted with 1 M NaCl. RNA polymerase II activity was detected in the eluted fraction. Partially purified RNA polymerase II from mung bean nuclei and purified RNA polymerase II from wheat germ also bound to ABP-I. Indole-3-acetic acid was not necessary for the binding of RNA polymerase II to ABP-I. Acid-denatured ABP-I did not bind to RNA polymerase II from wheat germ. The addition of ABP-I to the reaction mixture for RNA synthesis in vitro caused a stimulation of the activity of wheat germ RNA polymerase.

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A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35, 698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.

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Methyl linoleate (ML), linoleic acid (LA), and ethyl eicosapentaenoate (EE) were entrapped in saccharide and protein matrixes, and then stored at 37°C in a desiccator controlled at 75% relative humidity. ML entrapped with α-cyclodextrin, maltodextrin, and pullulan was extremely resistant to autoxidation, but LA entrapped with maltodextrin and pullulan rapidly oxidized. LA entrapped with α-cyclodextrin was the most stable against oxidation. ML entrapped with gelatin or gum arabic was less resistant to autoxidation than that that entrapped with pullulan. there was little difference in the susceptibility to oxidation between ML and LA entrapped with gelatin or gum arabic. Egg albumin protected ML more effectively against oxidation than LA, while sodium caseinate protected LA more in ML. Ethyl eicosapentaenoate entrapped with pullulan was highly resistant to oxidation, 90% of the total lipid remaining after 35 days. The effect on the oxidation of diffusion of oxygen through the matrix was estimated. It is suggested that the retardation of oxidation of the entrapped lipid can not be explained only by the effect of diffusion.

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Three inhibitors (CMCTI-I, II, and III) were isolated from oriental pickling melon (Cucumis melo L.var. Conomon MAKINO) seeds by acetone precipitation, gel filtration, and reversed phase chromatography. The amino acid sequences of these inhibitors were : [table] The reactive sites (P1 and P1' sites) of these inhibitors are presumed to be the Lys-Ile indicated by an arrow, comparing them with other squash family inhibitors. All three inhibitors can inhibit lysyl endopeptidase and trypsin at the enzyme-inhibitor ratio of 1 : 1.

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Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50, 000 by both Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitrophenylphosphorylcholine. V_max and K_m values were 5.0 μmol/min/mg protein and 0.3 mM, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca²⁺-dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250μM Ca²⁺ and higher Ca²⁺ concentrations than 1 mM had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca²⁺ concentrations in the rice cells is discussed.

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Microorganisms producing D-β-acetylthioisobutyric acid from methyl DL-β-acetylthioisobutyrate were screened from stock cultures. The D-β-acetylthioisobutyric acid-producing ability was found in 15 strains belonging to the genera Pseudomonas, Agrobacterium, Enterobacter, Cellulomonas, Rhodococcus, Brevibacterium, and Torulopsis. A strain of Pseudomonas fluorescens, IFO 3081, was selected as the best microorganism. The cells having activity (558units/g of dry cells) could be easily prepared by cultivation at 25°C at pH 6.6 for 24hr in a glucose-containing medium. The D-form of methyl DL-β-acetylthioisobutyrate was selectively hydrolyzed with the cells so that D-β-acetylthioisobutyric acid (97.2% enantiomeric excess) was produced in a high yield.

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Ten 7-hydroxycoumarin derivatives (4-13), containing two newly identified compounds (8 and 9), were isolated from the juice oil of fruits of Citrus hassaku, along with aurapten (1), (+)-epoxyaurapten (2), and (+)-marmin (3), and their structures were absolutely determined by spectroscopic and chemical methods. The spasmolytic activity of the isolated compounds was measured by the Magnus method, using guinea pig ileums. Three compounds (4, 7, and 11) showed higher activity than that of aurapten (1). The other compounds showed no activity under these conditions. The structure and activity relationship is discussed.

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We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Y1, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.

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An extracellular phytase from Bacillus subtilis (natto) N-77 was purified 322-fold to homogeneity with the specific activity of 8.7 units per mg protein by ultrafiltration, and a combination of Sephadex G-100 and DEAE-Sepharose CL-6B column chromatographies. The molecular weight of the purified enzyme was estimated to be 36 kDa on gel filtration and 38 kDa on SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a monomeric protein. The enzyme had the isoelectric point of pH 6.25, and Ca²⁺ requirement for the production and activity, the K_m value of 0.5 mM, and the activation energy of 9.87 kcal/mol for sodium phytate. The enzyme proved to be fairly specific for phytate and was most active at pH 6.0-6.5 and 60°C. Its activity was greatly inhibited by reagents and metal ions such as EDTA, Zn²⁺, Cd²⁺, Ba²⁺, Cu²⁺, Fe²⁺, and Al³⁺.

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The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.

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The zinc-containing neutral endopeptidase (neutral protease : BANP) from Bacillus subtilis var. amlosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.

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The melting temperature of gelatin gel increased with increasing concentration of polyols and sugars added at any gelatin concentation. The enthalpy of gel melting measured by Eldridge-Ferry plots and calorimetric measurements was decreased by addition of these polyhydric compounds, indicating that the gel stabilization by them is predominantly due to the large decrease in entropy of gel melting. Circular dichroism analysis showed that the helix formation of gelatin molecules was enhanced with increasing concentrations of these additives, but there was no positive correlation between their helix-forming ability and gel-stabilizing ability. The viscosity of gelatin solution was less in these mixed solvents than in water. Based on these results, a possible stabilization mechanism of gelatin gel by polyhydric compounds was discussed, in comparison with that of native collagen, in terms of the thermodynamics of protein-solvent interactions.

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High pressure at 101.3 to 506.6MPa was applied to bovine liver and fractioned lysosomes to examine its effects on the lysosomes and the enzymes involved. Carboxypeptidase was completely inactivated at 405.3 MPa. The activities of about 60, 70, 30, and 5O% of acid phosphatase and cathepsins B, D, and H, respectively, remained after pressurization at 506.6 MPa. Pressurization of the liver at 506.6 MPa or lower caused changes in the subcellular distribution of lysosomal enzymes. Acid phosphatase and cathepsins B and H were released from fractioned lysosome by treatment higher than 202.7 MPa and cathepsin D at 101.3MPa. These findings indicate that high pressure at 101.3 to 202.7MPa induces destruction of the lysosome membranes and leakage of the lysosomal enzymes.

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Field studies on the control of potato common scab were done with non-antibiotic Actinomycete biofertilizer, which was manufactured from swine feces with coprophilous actinomycetes and acidified to pH 5.0 with sulfuric acid. The field soil at pH 5.0 was mixed with 0.56 kg of the acidified biofertilizer per subplot (1.0×1.2 m) to a depth of the 15 cm. The scab severity, in terms of the percent coverage of the total surface area of potato tubers, was l0.7% in the control, while the lesions were slight in the biofertilizer subplot, scab severity being only 2.3%, and the potato production was increased 3 times with the biofertilizer. The viable counts of Streptomyces scabies in the biofertilizer subplot decreased to 1×10³ per gram soil from 5×10⁵ by 12 weeks, while the inhabitant fluorescent pseudomonads increased to 2.4×10⁷ per gram soil from 3×10⁴, and the increased fluorescent pseudomonads were antagonistic to the pathogenic strains.

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The structures of the insecticidal alkaloids, guinesines (4-hydroxy-3-(1-methyl-2-pyrrolidinyl)-1, 2-dithiolanes), were simplified in several ways while retaining the dithiolane ring. Most of the compounds synthesized showed activity against Chilo suppressalis and Tetranychus urticae. High potency against Chilo suppressalis was displayed by 3-(dimethylaminomethyl)-1, 2-dithiolane, and a broad pesticidal spectrum was recognized in the case of 4-(dimethylaminomethyl)-1, 2-dithiolane.

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