Through the concept of fractals, the new non-integer dimensional world is introduced. Experimental results to show this new world are taken from the author and his colleagues' own works. A few words are given also to the scaling law in the "Critical phenomena" in relation to sol-gel transition, and Chaos, both of which are closely related to the fractal.

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Effects of polyphenolic compounds isolated from green tea (Camellia sinensis) on the growth and adherence of Porphyromonas gingivalis onto human buccal epithelial cells were investigated. Green tea polyphenols, especially (-)-epigallocatechin gallate (EGCg) which is a dominant component of tea polyphenols, completely inhibited the growth and adherence of P. gingivalis onto the buccal epithelial cells at concentrations of 250-500 μg/ml. Among the polyphenolic compounds, (-)-epicatechin gallate (ECg) and (-)-gallocatechin gallate (GCg) were effective next to EGCg in these activities. On the other hand, (+)-catechin (C₍₊₎), (-)-epicatechin (EC), (+)-gallocatechin (GC), and (-)-epigallocatechin (EGC) had very much less activity. These results indicate that the inhibitory effect on the adherence of P. gingivalis onto the buccal epithelial cells is attributed to the presence of the galloyl moiety, which is ester-linked with the 3-OH of the catechin moiety in the polyphenolic compounds.

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The conformation of hydroxy-γ-ionylideneacetic acids produced by Cercospora cruenta was examined by ¹H-NMR analysis. ¹H-¹H COSY and NOE data indicated that (2Z, 4E, 1'R, 4'R)-4'-hydroxy-γ-ionylideneacetic acid has a cyclohexane ring of chair form furnishing a hydroxyl group and a 2, 4-pentadienoic acid moiety, each with equatorial orientation, while (2Z, 4E, 1'S, 3'S)-3'-hydroxy-γ-ionylideneacetic acid has a flexible twisted-boat form of cyclohexane ring. It is suggested that the structure of another (2Z, 4E, 1'S, 4'R)-isomer of 4'-hydroxy-γ-ionylideneacetic acid with an equatorial hydrogen at the 1'-position is the key for further oxygenation to abscisic acid.

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Substituted 4, 4, 4-trifluoro-3-(indole-3-)butyric acids (TFIBAs), novel fluorinated plant growth regulators, were easily synthesized from substituted 2, 2, 2-trifluoro-1-(indole-3-)ethanols. The synthetic substituted TFIBAs showed marked root growth-promoting activity toward Chinese cabbage, lettuce, and rice plants. 5-MeO-2-Me-TFIBA (2d) had especially strong promoting activity toward Chinese cabbage, while 5-Me-TFIBA (2b) markedly promoted the root growth of all three plants. The promoting activity of TFIBAs halogenated at the 5-position in the indole nucleus of the molecule (5-Br- and 5-Cl-TFIBA (2e and 2g)) was also greater than the activity of TFIBA, the original non-substituted compound. 5-NO₂-TFIBA (2h) with a strong electron-withdrawing group showed only very weak root growth-promoting activity.

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An enzyme that catalyzes a reduction of ethyl 2-methyl-3-oxobutanoate (1) to ethyl (2R, 3S) 3-hydroxy-2-methylbutanoate was found in Klebsiella pneumoniae IFO 3319 cells. The enzyme was isolated from the cells and purified 250-fold by ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel filtration. The purified enzyme was found to be a monomer protein with a molecular weight of approximately 31, 000 and an isoelectric point of 6.2. It was NADPH-dependent and had maximum activity at pH 7.0 and 45°C for the reduction and at pH 10.0 and 45°C for oxidation. The K_m's at pH 7.0 were 5.6 mM for 1 and 12.5 mM for benzyl 2-methyl-3-oxobutanoate, respectively. Esters of 2-oxocyclo-alkane carboxylic acids as well as esters of 2-methyl-3-oxobutanoic acid served as substrates, and the corresponding reduced products were obtained with high stereoselectivity.

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The protective effects of chlorogenic acid on paraquat-induced oxidative stress were examined in rats. The activities of erythrocytes and liver glutathione peroxidase, and of both liver catalase and glutathione reductase, which were increased by feeding paraquat, declined to the levels in the control rats by supplementing chlorogenic acid to the paraquat diet. The activity of superoxide dismutase was not changed by dietary paraquat or by supplementing chlorogenic acid to the paraquat diet. Paraquat in the diet markedly decreased the liver triacylglycerol and phospholipid concentrations, as well as the food intake and body weight gain, while chlorogenic acid protected against these decreases. These in vivo results and the in vitro superoxide anion scavenging activity of chlorogenic acid suggest that chlorogenic acid acted preventively against paraquat-induced oxidative stress.

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About 6000 isolates of microorganisms assimilating methylketones (C₃-C₆) were tested for their selective oxidation of 2-methylnaphthalene to 2-methyl-1-naphthol. Strain M192 was the highest 2-methyl-1-naphthol producer and was classified as the genus Rhodococcus. The optimal conditions for the site-specific oxidation were studied using resting Rhodococcus sp. M192. The 2-methyl-1-naphthol productivity was specifically increased using methylethylketone as a carbon source, 1-propanol as a solvent to dissolve the substrate, and ethylxanthate or diethyldithiiocarbamate as an inhibitor of 2-naphthoic acid (side-product) production. In the presence of these compounds, 2-methylnaphthalene was specifically oxidized at the 1-position without the conversion to 2-naphthoic acid. The productivity of 2-methyl-1-naphthol was about 90 μM from 1 mM 2-methylnaphthalene.

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We developed a method that allows rapid isolation of mutant Bacillus subtilis 168 carrying an inversion of a specific DNA segment of the genome. Two incomplete neomycin resistance gene cassettes were integrated at both ends of the 1652-kb segment to be inverted. Reciprocal recombination within the 590-bp homologous region of these two cassettes created an intact neomycin resistance gene with concomitant inversion of the 1652-kb segment flanked by the two cassettes. Structure of the mutant genome was verified by analyzing the physical map for rare cutting endonucleases, SfiI, NotI, I-CeuI, and I-SceI. The inversion rate was estimated to be 6.9±1.4×10^-8/cell/cell division at 37°C. The method should be in principle applicable not only to other regions of the B. subtilis genome but also to other bacterial genomes.

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The inhibitory effects of flavonoids on the activity of two photosynthetic enzymes such as phosphoenolpyruvate carboxylase (PEPCase) and NADP-dependent malic enzyme (NADP-ME) were evaluated. The glycosylation of hydroxyl groups on the flavonoids resulted in compounds that behaved as gradually weaker inhibitors with increased size of the substituent. Quercetin and baicalein showed a competitive inhibition pattern vs. NADP⁺ for NADP-ME, and a similar model for both tlavonoids vs. phosphoenolpyruvate (PEP) was observed when tested on PEPCase. K_i for NADP-ME inhibition at pH 7.0 were 0.83 μM and 1.54 μM for quercetin and baicalein, respectively. K_i for PEPCase inhibition were 0.17 μM and 0.79 μM (quercetin and baicalein, respectively), indicating that these compounds are the most potent inhibitors described for this carboxylase. I₅₀ values for these and other flavonoids were in the micromolar range. A tentative physiological role for the inhibitory effects observed on PEPCase is discussed.

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A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.

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The immune system is composed of various cells with distinct functions. Thus, highly selective immunomodulators are necessary for artificial regulation of immune reactions. We screened microbial products for such immunomodulators and we identified streptonigrin as a selective suppressor of B-cell proliferation induced by lipopolysaccharide. Streptonigrin directly suppressed the late phase of proliferation of B-cells. The inhibition of topoisomerase II was implicated as the mechanism of the B-cell-selective suppression. In cultured cell lines, however, streptonigrin preferentially suppressed the growth of an interleukin-3-dependent myeloid cell line rather than B-cell lines. In addition, the treatment with streptonigrin in vivo suppressed T-cells more significantly than B-cells and dramatically reduced the spleen weight. These results suggest that streptonigrin preferentially suppresses myeloid T-cell precursors in vivo.

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Chemical modification of mold pectinase with dextran- and cellulose-dialdehydes was examined to improve the enzyme characteristics. The modified pectinase with dextran-dialdehyde retained about 50% of the original activity, and more than 80% of the total amino groups were modified. HPLC gel filtration analysis showed an increase in molecular weight of the reaction product. Reaction with Cellulose-dialdehyde provided an immobilized form of pectinase. The immobilized pectinase was resistant to both acidic and alkaline pHs, and also acquired heat stability at 60°C. The optimum pH of the modified enzyme shifted from pH 4.5 to 5.0-5.5, and this enzyme had higher activity at neutral pH regions than the native enzyme. A rather low recovery of immobilized enzyme (14.5%) should be improved by the combination with various methods hitherto established.

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The linear double stranded DNA plasmid pGKL1 encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood. We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex. These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups. The iki2 mutation was mapped to a position near the centromere on chromosome XIII. We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Sccharomyces gene library, and isolated three DNA fragments that complement the iki1, iki3, and iki4 mutations, respectively.

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Three 2-methoxy-3-alkylpyrazines (MPs) in Japanese red wine and grape samples were determined by GC-EIMS, using 2-methyl-3-n-propylpyrazine as an internal standard. MPs in the Cabernet Sauvignon red wines were derived not only from the pulp but also from other parts of the grape berries. All of the Cabernet Sauvignon red wines made annually from 1975 to 1994 contained 2-methoxy-3-isobutylpyrazine (isobutyIMP), although those made from well-ripened grapes had a low isobutyIMP level. It is suggested that the climatic conditions of September might effect the isobutyIMP level of Cabernet Sauvignon grapes and red wines. The mean isobutyIMP level of Cabernet Sauvignon, Cabernet Franc, and Merlot Japanese commercial red wines was significantly higher than the mean isobutyIMP level of Muscat Bailey A and Zweigeltrebe.

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Bovine serum albumin (BSA) added to the reaction medium for the oxidative demethylation of 4-methoxyanisole and its "di-CD₃" isotopomer ([d₆]methoxyanisole), when catalyzed by liver microsomes from untreated rats, decreased the K_m values and increased the V_max/K_m (= V/K) values. The V_max values were not markedly altered. The values for the deuterium isotope effect on V_max and V/K for the reaction with this isotopomer were between 2.2 and 2.8, and that on K_m was close to unity. The magnitude of the isotope effect was not significantly changed by adding BSA. The intramolecular isotope effect with [mono-CD₃]4-methoxyanisole ([d₃]methoxyanisole) in liver microsomes from untreated rats was between 10.3 and 10.8, which was not significantly changed by BSA. Liver microsomes from rats treated with phenobarbital resulted in the intramolecular isotope effect value in the absence of BSA being between 7.2 and 9.1, which was not significantly altered by BSA. Based on these data, the calculated apparent rate constant for the enzyme-substrate complex formation was markedly increased by up to about 1.9- and 3.5-fold by 1% and 2% of BSA added, respectively.

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A ras oncogene-amplified recombinant BHK-21 cell line (ras-rBHK-IgG) has been established, and was shown to hyperproduce the recombinant IgG chimeric human monoclonal antibody (hMAb) AE6F4, which recognizes lung cancer cells. We found that the ras-rBHK-IgG cells could be easily cultured in a protein-free ERDF medium supplemented with iron(III) nitrate, hydroxyethyliminodiacetic acid, and non-protein synthetic attachment factor as well as in a serum-free ERDF medium supplemented with insulin, transferrin, ethanolamine, and sodium selenite. The productivity of recombinant hMAb from the cells cultured in dishes at high cell densities was higher in protein-free medium than in serum-containing medium. True high density culture of the ras-rBHK-IgG cells was done in protein-free medium using the Tecnomouse, which is a novel hollow fiber bioreactor system. After culture for 30 days in protein-free culture, a total amount of about 14 mg of the recombinant hMAb AE6F4 was obtained, and was shown to be reactive against lung cancer cells in tissues.

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The six strains of the Pichia species, once classified in the genus Petasospora, were examined for their 18S (positions 1451-1618, 168 bases) and 26S (positions 1611-1835, 225 bases and 493-622, 130 bases) rRNA partial base sequencings. In the 18S rRNA partial base sequencings, the type species of the genus Petasospora (Petasospora rhodanensis) was found to be closely related to Pichia anomala (≡Hansenula anomala, type species of genus Hansenula) with base differences of two, but not to Pichia membranaefaciens, the type species of the genus Pichia, with base differences of ten. However, the species was not so closely related to P. anomala in the 26S rRNA partial base sequencings with base differences of twelve and 68 percent similarity. The genus Petasospora was known to be a very heterogeneous taxon phylogenetically with base differences of thirty-one to three and ninety-two to eight and with 40 to 83 percent similarities. The sequence data obtained here and the phenotypic features described previously indicate that the type species of the genus Petasospora (Pet. rhodanensis, ≡Pichia rhodanensis) is adequate to be accommodated temporarily in the genus Pichia, a heterogeneous taxon, until the precise taxonomic position of the type species is defined.

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The subsite affinities of the active site of β-amylase from Bacillus cereus var. mycoides were evaluated based on Hiromi's theory, using ¹4C-radiolabeled maltooligosaccharides as substrate. It was estimated that the active site consisted of six subsites, and all subsite affinities could be evaluated. The active site had a common subsite arrangement with those of β-amylases from soybean and wheat bran. The intrinsic breakdown rate constant of α-1, 4 glucosidic linkage (k_int) was five to seven times as large as those of the other enzymes. From the pH dependence of log[k_o/K_m], pK values of two functional ionizable groups were pK₁=4.0 and pK₂=8.4. The pK values were 0.5-0.6 units for pK₁ and 0.2-0.3 units for pK₂ larger than those of the other enzymes. For the affinity-labeling of this enzyme by 2, 3 epoxypropyl α-D-glucopyranoside (α-EPG), the binding affinity of α-EPG was 1-1.6 kcal/mol larger than those of the other β-amylases.

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Oligo-L-methionine ethylester (OMOEt) prepared by the papain-catalyzed oligomerization of L-methionine ethylester (MetOEt) is a mixture of pentamer to dodecamer and has nearly the same supplementary effect as free methionine (Met) for the growth of rats when added to a low casein diet, but its supplementary effect to a low-soy protein isolate (SPI) diet is not consistent and depends on the degree of polymerization. Rats were fed for 2wk with an 8% casein or 10% SPI diet supplemented with 0.3% L-Met, each chemically synthesized Met_nOEt with a polymerization degree (n) of 6, 7, 8, or 9, or with OMOEt prepared by papain-catalyzed polymerization of MetOEt. Met₆OEt, Met₇OEt, and Met₈OEt had nearly the same supplementary effect on the growth of rats, as did free Met, both with the 8% casein and 10% SPI diets. The supplementary effect of Met₉OEt was not significantly lower than that of Met when added to the 8% casein diet, but was significantly lower when added to the 10% SPI diet. The digestibility of Met₉OEt supplemented to the 8% casein and 10% SPI diets was 50.5% and 35.6%, respectively. It appears likely that there is a gap in the bioavailability of oligomethionine between the octamer and nonamer when added to a low-protein diet, probably due to the rigidity of the structure increasing with the polymerization degree by α-helix formation. Although the differences in absorption rate of Met from OMOEt for a short time after feeding has been related to the different effects of supplemented OMOEt, the absorption rate of OMOEt for 30 min after feeding was not considered to be the main cause of the differential effects of OMOEt in this experiment.

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Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105, 000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60 min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80-82% at 30-40°C. The activity was inhibited by Cu²⁺, Hg²⁺, Zn²⁺, and Tris.

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A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31, 000 by SDS-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85°C, and about 50% of the original activity remained after incubation at 90°C for 10 min in the presence of Ca²⁺. The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1, 152 base pairs, coding for a polypeptide of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.

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For the measurement of malate by an enzyme sensor, we did a comparative study using malate dehydrogenase (MDH) alone, MDH and glutamate oxaloacetate transaminase (GOT) together, a malic enzyme (ME) that requires NADP as a cofactor, and MDH and NADH oxidase together. With respect to the response of each reactor to 0.5 mM L-malate, the systems using ME alone and MDH plus NADH oxidase gave high values. The ranges of measurements were 0.05-1.00 mM (MDH alone), 0.01-0.05 mM (MDH plus GOT), 0.01-0.50 mM (ME alone) and 0.02-1.00 mM (MDH plus NADH oxidase). In the system with MDH alone, however, reducing sugars in the sample interfered with measurements and it was impossible to use this system for practical analysis of fruit samples. By contrast, the systems using ME alone or MDH plus NADH oxidase were unaffected by the presence of reducing sugars and were suitable for analysis of samples. Thus, the MDH-NADH oxidase system is recommended for practical analyses of samples.

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The role of endogenous gibberellin (GA) in the flowering of the short-day plant, Pharbitis nil, was investigated by using uniconazole, which is a specific inhibitor of GA biosynthesis. Both the endogenous GA level and flowering response decreased with increasing concentration of uniconazole applied via the roots. The strongest inhibition of flowering was observed when uniconazole was applied one day before a 15-h dark treatment. The inhibition by uniconazole was overcome by an application of GAs to the plumules, the order of effectiveness of the endogenous GAs in P. nil being GA₁≥GA₂₀>GA₁₉≥GA₄₄>GA₅₃»GA₈. This is the first report of the correlation between the endogenous GA level and flowering response in P. nil. It was found that endogenous GAs were required for the flowering of P. nil during or just after the dark period.

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In the microvascular system, pericytes are located at the abdominal side of capillary endothelial cells. To discover the role of pericytes in the microvascular system, we have analyzed the extracellular proteins secreted from pericytes isolated from microvessel fragments of rat epididymal fat pads and found that they synthesize substantial amounts of basement membrane components such as type IV collagen and laminins. Secretion of type IV collagen was markedly stimulated by ascorbic acid phosphate. Reducing and non-reducing sodium dodecyl sulfate gel electrophoresis showed that pericytes produce six laminin chains assembled into different trimeric isoforms. Two of them were similar to laminin variants produced by aortic and pulmonal endothelial cells but others were suggested to be novel variants.

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A number of analogs of lunularic acid varying in the number of methylene carbons between the two benzene rings and in the substituents on their rings were prepared, and their effects on the growth of liverwort gemmaling, watercress, and timothy grass were investigated. Almost all the analogs tested were more inhibitory than lunularic acid, and a correlation between the structure and activity was observed. The differences in the growth-inhibition activity of analogs between higher and lower plants are also discussed.

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Four major molecular species of β-conglycinin, α₃, α₂β, αβ₂, andβ₃, were isolated and purified from seeds of an α' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the α₃ and β₃ components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The α₃ and β₃ components were compared regarding their secondary structures. The secondary structure of the α₃ component deduced from CD measurements showed a higher α-helix content than that of the β₃ component. The β₃ component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm×0.2 mm×0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2₁2₁2₁ and unit cell dimensions of a= 185.1 Å, b = 107.9 Å, and c = 97.6 Å.

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It is known that the anti-tuberculosis drug, isonicotinic acid hydrazide (INH), causes pellagra, a niacin deficiency syndrome, and peripheral neuritis in humans. We investigated the effects of INH on the metabolism of tryptophan to niacin in rats fed on a niacin-free diet. The activity of kynurenine aminotransferase was significantly inhibited by feeding a diet containing INH and by an injection of INH, and the urinary excretion of xanthurenic acid, the side-reaction product of the conversion pathway of tryptophan to niacin, was below the limit of detection. The inhibition of kynurenine aminotransferase and the resulting decreased formation of xanthurenic acid generally mean a higher conversion ratio of tryptophan to niacin. However, the conversion ratio was no different between the control and INH groups.

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A tentative method for measuring brown pigment in food was proposed and applied to 25 items of commercial browned foods. The principle was based on the assumption that any brown pigment with general absorption in the visible wave-length spectrum is quantitatively equivalent to melanoidin prepared from a model system of the Maillard reaction. The spectral curve for general absorption was represented by the equation dE/(dλ)=-kE (E, absorbance; λ=wave length; k=constant>O), of which the value k was found to correlate with the molecular weight and the molar extinction coefficient and to serve as an empirical parameter for the measurement of brown substances in different foods.

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Inhibition of proteases was examined in browned foods including soy pastes, soy sauces, fish sauce, sauces, teas, coffees, and others in association with melanoidin, a Maillard product, which had been found to be a potent inhibitor of trypsin. Most of the foods were effective in restraining trypsin activity based on hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide, while being ineffective on chymotrypsin except for black tea and cola. Especially, teas were noted for their strong inhibitory effect on trypsin. The color intensity of the foods in terms of optical density at 400 nm appeared not to correlate with the extent of inhibition except for soy sauces.

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A naphthalenesulfonate-assimilating bacterium, Pseudomonas sp. TA-2, was found to convert 2-naphthoate to cis-1, 2-dihydroxy-1, 2-dihydronaphthalene-2-carboxylate (DDN2C) and cis-1, 2-dihydroxy-1, 2-dihydronaphthalene-3-carboxylate (DDN3C), and converted 1-naphthoate to cis-1, 2-dihydroxy-1, 2-dihydronaphthalene-1-carboxylate (DDN1C). It was suggested that conversion of naphthoates was done by a dioxygenase with relaxed regioselectivity.

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We studied the immune functions of IgY obtained from hens immunized with a mixture of formalin-treated pathogenic bacteria. The IgY inhibited the growth of Pseudomonas aeruginosa, the production of Staphylococcus aureus enterotoxin-A, and adhesion of Salmonella enteritidis to cultured human intestinal cells (Caco 2). The results indicated that IgY specific for plural bacteria has effects useful toward prevention of bacterial diseases.

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Squash glutamate decarboxylase was purified by DEAE-Cellulose batchwise followed by Blue-Sepharose, Cellulofine GCL-2000, and Toyopearl HW-55F column chromatography. The purified glutamate decarboxylase had a high specific activity (95.0 u/mg). The absorption spectrum of glutamate decarboxylase had an absorption maximum at 420 nm in the range 300-500 nm. A pH change from 5.3 to 7.8 was accompanied by a decrease in absorbancy at 420 nm. One mole of glutamate decarboxylase contained 3.8 and 1.3 mol of pyridoxal 5'-phosphate at pH 5.8 and pH 7.8, respectively.

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The relationship between the concentration of 2, 4-dichlorophenoxyacetic acid (2, 4-D) and the duration of treatment with 2, 4-D on direct somatic embryogenesis from carrot hypocotyl explants was examined. Elevated concentrations of 2, 4-D (up to 100 mg/liter) reduced the duration of pretreatment required for successful embryogenesis.

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Active transport activities of free B-6 vitamers in 35 strains of 8 genera of yeast were measured by isocratic reverse-phase HPLC. Many but not all strains transported pyridoxamine and/or pyridoxine. The active transport activities in some yeast strains tested were completely inhibited by amiloride (0.5 mM). In contrast to cells so far studied, yeast cells showed a novel character in metabolism of accumulated B-6 vitamers: the phosphorylation of the free B-6 vitamers was regulated at a low level. There was no apparent correlation between the presence of the active transport activity in yeasts and the requirement of vitamin B-6 for their growth.

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A bestatin analogue, (2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valine (AHPA-Val), from the culture filtrate of Streptomyces neyagawaensis SL-387 was obtained in a chemically defined medium containing DL-3-amino-3-phenylpropionic acid. AHPA-Val was 6 times (IC₅₀=1.2 μg/ml) as strong as bestatin (IC₅₀=7.0 μg/ml) against porcine kidney microsomal aminiopeptidase N, and 4 times (5.6 μg/ml) as strong as bestatin (IC₅₀=20.7 μg/ml) against amino-peptidase N of human metastatic fibrosarcoma HT1080. To the best of our knowledge, this is the first report on the microbial production of AHPA-Val.

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Uniconazole-P, a gibberellin (GA) biosynthesis inhibitor, did not affect growth of mandarin fruitlets definitely but stimulated abscission markedly. Endogenous abscisic acid concentration in fruitlets treated with uniconazole-P was about 4-fold higher compared with the control. Endogenous indole-3-acetic acid and total GAs levels also changed. The relationships between these changes in endogenous hormone levels and fruit abscission were discussed.

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Two new diterpene glycosides containing 20-hydroxygeranyllinalool were isolated and identified from Nicotiana tabacum. These compounds consisted of five molecules of glucose and/or rhamnose. The locations of the aglycone and glycosides in the molecules were determined by 2D-NMR with the HMBC technique. The structures were (6E, 10E, 14Z)-20-hydroxygeranyllinalyl-3-O-[α-L-rhamnopyranosyl (1→4)]-β-D-glucopyranoside-20-O-[β-D-glucopyranosyl (1→2)]-[α-L-rhamnopyranosyl (1→6)]-β-D-glucopyranoside and (6E, 10E, 14Z)-20-hydroxygeranyllinalyl-3-O-[α-L-rhamnopyranosyl (1→4)]-β-D-glucopyranoside-20-O-[α-L-rhamnopyranosyl (1→4)]-[α-L-rhamnopyranosyl (1→6)]-β-D-glucopyranoside.

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A new anthracycline antibiotic, designated as aclacinomycin X, was isolated from the culture broth of Streptomyces galilaeus ATCC 31133, and was identified as 7-(O-rhodosaminyl-deoxyfucosyl-red-nosyl)-aklavinone. Its in vitro cytotoxicity was tested against several human tumor cell lines.

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Cininamic, p-coumaric and ferulic acids were isolated from pineapple stems (Ananas comosus var. Cayenne). Twenty-four kinds of esters were prepared from these acids, alcohols and the components of Alpinia. Isopropyl 4-hydroxycinnamate (11) and butyl 4-hydroxycinnamate (12) were found to have almost the same effectiveness in antifungal activity against Pythium sp. at 10 ppm as that of the commercial fungicide iprobenfos (Kitazin P).

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A biosensing system based on the response of fungal cells was used for the evaluation of antifungal activity of medicinal plants against Aspergillus niger. This system measured the hyphal growth rate in real time in the presence or absence of Chinese herbal extracts. The sensitivity of this system was 100-fold higher than that of conventional methods, and is advisable for the screening of antifungal compounds.

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An easy preparation method was developed for 3-hydroxy-methylene-2-thioxopyrrolidine (TPC), an antimicrobial degradation product of radish pungent principle. The key intermediate, 4-methoxy-3-butenyl isothiocyanate (MBI), which was prepared from 3-cyanopropionaldehyde dimethyl acetal in 3 reaction steps, was easily converted to TPC in acidic (pH 3.0-4.0) aqueous media. In methanol or ethanol with a few drops of acetic acid, MBI afforded corresponding 3-(α, α-dialkoxy)methyl-2-thioxoprrolidines as the major products.

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A practical synthesis of (R)-3-amino-4-phenylbutyric acid 1, based on Friedel-Crafts acylation with the α-aminocarboxyl group of L-aspartic acid retaining its original α-carbon chirality, is described.

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Schizosaccharomyces pombe has two genes each encoding an isozyme of NADH-dependent glycerol-3-phosphate dehydrogenases (gpd1⁺ and gpd2⁺). To gain an insight into the function of these genes, here we constructed a gpd2 deletion mutant, in addition to the previously constructed gpd1 deletion mutant. We showed that the gpd1⁺ and gpd2⁺ gene-products are both funcional in terms of the de novo glycerol synthesis. Furthermore, the gpd1⁺-mediated glycerol production is primarily responsible for the osmoregulation, but the gpd2⁺ gene is not. Interestingly, however, the gpd2 deletion mutant had histidine- or lysine-auxotrophy for growth on a minimal medium.

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An amylolytic activity, which converts soluble starch to α, α-trehalose (trehalose), was found in the cell homogenate of the hyperthermophilic, acidophilic archaeum Sulfolobus solfataricus KM1. Two enzymes, a glycosyltransferase and an amylase, which are essential for this activity, were purified to homogeneity. A glycosyltransferase catalyzed the conversion of maltooligosaccharides to glycosyltrehaloses. Based on a detailed analysis of the reaction products, kinetic parameters, and an experiment using ³H-labeled substrates, it was verified that glycosyltransferase transferred an oligomer segment of maltooligosaccharide to the C1-OH position of glucose, located at the reducing end of the maltooligosaccharide, to produce a glycosyltrehalose having an α-1, 1 linkage. The reaction appears to be intramolecular. Nine strains of the Sulfolobaceae family were found to have glycosyltransfrases.

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Amylolytic activity, which converts soluble starch to α, α-trehalose (trehalose), was found in the cell homogenate of the hyperthermophilic acidophilic archaeum, Sulfolobus solfataricus KM1. Two enzymes, a glycosyltransferase and an α-amylase, which were essential for this activity were identified. The α-amylase was purified to homogeneity on SDS-PAGE. The α-amylase catalyzed the hydrolysis of glycosyltrehaloses to trehalose. Analysis of the reaction products, kinetic parameters, and experimental findings using ³H-labeled substrates indicated that the α-amylase hydrolyzed only the α-1, 4 glucosidic linkage adjacent to the trehalose unit of the glycosyltrehaloses. Six strains of the Sufolobaceae family examined were observed to have the glycosyltrehalose-hydrolyzing enzyme, the αα-amylase.

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A new glycosidic aroma precursor was isolated from green tea leaves (Camellia sinensis var. sinensis cv. Yabukita) along with the known primeverosides of cis-linalool 3, 6-oxide, linalool and geraniol. These glycosides were separated by chromatographic isolation on Amberlite XAD-2, ODS flash chromatography, and finally HPLC. The chemical structure of the new unknown glycoside was confirmed as geranyl 6-O-α-L-arabinopyranosyl-β-D-glucopyranoside (geranyl β-vicianoside) by spectrometric analyses and by an enzymatic hydrolysis with glycosidase followed by GC-MS and HPLC analyses. Moreover the vicianoside was hydrolyzed with acetone powder obtained from fresh tea leaves to generate the same compounds, suggesting this glycoside to be a tea aroma precursor.

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Digalactosyldiacylglycerol (DGDG) suppressed the inhibition by sulfoquinovosyldiacylglycerol (SQDG) of an α-glucosidase reaction. Suppressing was considered to be an apparent decrease in inhibitory activity of SQDG ascribed to direct interaction between SQDG and DGDG. This suppression was presumed to be caused by less access of SQDG to the enzyme because SQDG and DGDG formed mixed micelles.

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