This review covers recent examples of synthetic transformation of nitriles and amides by microbial enzyme systems. A variety of substrates and products involving enantiomerically enriched forms of chiral substances are referred to. The stereochemical course of enzyme-catalyzed hydrolysis is briefly commented on. Special emphasis is placed upon the range of functional groups that are acceptable by enzymes and/or survive under the transformation, as well as the advantages as a synthetic tool for conversion under mild conditions.

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In the course of our screening for free radical scavengers, (1'E)-erythro-4-(3', 4'-dihydroxypentenyl)oxazole (1) (1'E, 4'S)-4-(3'-oxo-4'-hydroxypentenyl)oxazole (2) and 6-pentyl-α-pyrone (3) were isolated from an unidentified fungal metabolite. These compounds, especially novel oxazolyl compound 2, inhibited the bactericidal effect of the Fenton reagent toward Bacillus subtilis. They and their acetylated compounds (diAc-1 and Ac-2) also showed inhibitory activity against linoleate autoxidation. Furthermore, 1-3 inhibited oxidative enzymes (soybean lipoxygenase and mushroom tyrosinase). To investigate the radical scavenging mechanism of 3, two oxidized products (4 and 5) were isolated from the reaction mixture of 3 and the Fenton reagent. Compounds 4 and 5 seemed to be derived from 3 by scavenging the hydroxyl radical.

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Products from tea catechins ((+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epigallocatechin gallate) after heat treatment were individually isolated by preparative HPLC and subsequent crysiallization. FAB-MS, elemental, ¹H- and ¹³C-NMR, and optical rotation analyses indicated these products to be the C-2 epimers of the original catechins. The effects of temperature, time, pH, and concentration on this conversion of tea catechins to their epimers were examined. It was determined that the epimerization reaction should be conducted with 1% each of the tea catechin solution (pH 5) at 120°C for 30 min.

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New antimicrobial substances against Streptomyces scabies, rosmic acid and rosmanol-related compounds, were isolated from leaves of rosemary (Rosmarinus officinalis L.). The active compounds were separated in pure form by a combination of gel permeation chromatography and reverse-phase HPLC. Their structures were determined by two-dimensional NMR experiments and HR-MS analyses.

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We cloned the putative polyketide synthase genes (pms genes) for pradimicin A biosynthesis from Actinomadura hibisca using an oligonucleotide probe designed on the basis of conserved amino acid sequences of other polyketide synthases (PKSs). By DNA sequencing of an 8.2-kb SacI fragment that hybridized with the oligonucleotide probe, 11 open reading frames (ORFs) were found. All of the ORFs except for ORF10 were predicted to be translated in the same direction. Each of the deduced ORFs has significant sequence similarity to the protein responsible for polyketide biosynthesis or spore pigmentation. In particular, ORF1, ORF2, and ORF3 were 50%-70% identical with genes coding for PKSs for actinorhodin biosynthesis. Specific DNA regions similar in sequence to pms genes were found with genomic Southern hybridization in all of the pradimicin producers examined, but were not found in pradimicin nonproducers, suggesting that the genes cloned in this study encode polyketide synthase for pradimicin biosynthesis.

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In order to identify an adequate intake level of docosahexaenoic acid (DHA), changes in various parameters related to health benefits were studied in rats fed on diets containing 10% test lipids at different n-3(DHA)/n-6 ratios for two weeks. An evaluation of the critical level of the dietary n-3/n-6 ratio which had a significant effect on the parameters of several tissues indicated that the response to the dietary ratios differed according to the parameter, the variation in ratio ranging approximately from 0.20 to 1.77 with either a positive or negative effect on the health benefit. These results suggest that a suitable intake level of DHA would be within this range. In view of safety, however, the critical level for the dietary n-3/n-6 ratio may be around 0.56, as shown by a detailed analysis on the lower limit level of the harmful parameters. We thus propose that the dietary intake of DHA should not be more than 0.56 in terms of the n-3/n-6 ratio.

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The gene from Bacillus brevis TT02-8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31, 891, which was consistent with that previously calculated for arginase purified from this bacterium. Comparison of the deduced amino acid sequence of the B. brevis TT02-8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Babillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33-66% of the sequences to be similar; there were several highly conserved regions. Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.

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Separation of acetic acid from palm oil mill effluent (POME) to increase its concentration by an anion exchange resin was examined as a preliminary study for its recovery from POME that had been anaerobically treated by sludge from a palm oil mill. This paper concerns the acetic acid thus separated for producing bacterial polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus. It was found that sludge particles in POME strongly inhibited the adsorption of acetic acid on the anion exchange resin. Removing the sludge particles from the POME facilitated the separation of acetic acid from the POME efficiently. The concentrated acetic acid thus obtained from anaerobically treated POME could be used as a substrate in the fed-batch production of polyhydroxyalkanoate by Alcaligenes eutrophus.

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Plasmid pSLL of Streptomyces laurentii ATCC31255 (wild-type strain P0) is a 93-kilobase linear DNA plasmid that carries a protein bound to each 5' end of the DNA. It was self-transmitted to the pSLL-cured strain by conjugation in solid culture. The pSLL-cured strain carried a circular plasmid, pSLS, and showed a marked decrease in spore formation and thiostrepton productivity, owing to the pSLS. However, by retransmission of pSLL, these things reverted to levels seen in strain P0. Thus, plasmid pSLL suppressed the injurious effects of pSLS on the host mycelia.

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We prepared corn peptide (CP), a vegetable oligopeptide and tried to discover the effects of its ingestion on facilitating alcohol metabolism in healthy adult men. Ten healthy male volunteers ingested 5 g of CP, wheat peptide (WP), pea peptide (PP), alanine, or leucine 30 min before alcohol intake at a dose of 0.5 g/kg, and blood ethanol and plasma amino acid concentrations were measured during a 2-h observation period after alcohol intake. In subjects who ingested CP, the blood ethanol level was lower than that in the WP, alanine and leucine ingestion groups, but did not decrease as compared to the control when they ingested PP. Similarly there was a difference in the blood ethanol level between alanine and leucine ingestion groups, and leucine ingestion was more effective than alanine against the reduction of the increase in blood ethanol level. On the other hand, there was no significant difference in the plasma concentrations of individual amino acids except alanine, leucine, or lysine after alcohol intake among experimental groups as compared to the control. CP ingestion significantly elevated plasma alanine and leucine rather than other groups during a 2-h observation period. These results suggested that CP may have the effect on the reduction of increase in blood ethanol level after alcohol intake by the marked elevation of plasma alanine and leucine, especially leucine, but neither by the delay of ethanol release from the stomach nor malabsorption of ethanol in the gastrointestinal tracts.

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The antioxidative activity of garlic homogenates blended with distilled water and different amounts of soybean oil was examined. The antioxidative activity of garlic aroma concentrate and that of the oil layer from the garlic homogenate was strong. The stability of the three sulfur compounds in garlic, allicin, diallyl disulfide, and diallyl trisulfide, was analyzed by HPLC, diallyl disulfide being the most stable. It was found that the effectiveness and strength of diallyl disulfide as an antioxidant could be increased by additing α-tocopherol and L-ascorbyl palmitate in a lard system.

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Ferredoxin-sulfite reductase (Fd-SiR) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from turnip leaves (SiR-L) has been purified to homogeneity and its enzymatic properties compared with that from turnip roots (SiR-R). Each enzyme had a molecular mass of 64.5±0.5 kDa by SDS-PAGE and an isoelectric point of 5.15±0.05. Although each had a pH optimum around 7.8 with the same effects of inhibitors, SiR-L had higher heat stability at 60°C than SiR-R. Moreover, SiR-R had a lower K_m and a higher specificity constant (k_cat/K_m) for turnip leaf ferredoxin than SiR-L. The N-terminal amino acid sequence of SiR-L was different from that of SiR-R. The results of amino acid analysis and peptide mapping suggested that SiR-L and SiR-R have different primary structures.

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The molecular characteristics of polysaccharide obtained by extracting with water from jelly fig (Ficus awkeotsang Makino) seeds were elucidated by measuring the weight-average molecular weight (M_w) and radius of gyration (R_G), indicating the molecular expanse, by the light-scattering method. The M_w and R_G values of the water-soluble jelly fig polysaccharide were 84-130×10⁴ and 240-450 mm, respectively, these values being larger than those of commercial LM pectin and HM pectin. In addition, the M_w and R_G values of the water-soluble jelly fig polysaccharide initially increased after being extracted, indicating the highest values 5 hours after the extraction, and thereafter decreased. These changes in the time-course of the molecule reflected well the changes with time in the mechanical characteristics and network structure of the water-soluble jelly fig polysaccharide gel. Exponent α in the expression R_G ≤prop M_w^α was found to be 0.37. From these results, the conformation of the water-soluble jelly fig polysaccharide molecule after association by the contained inorganic elements proved to be of globular form rather than a random coil shape as a result of contraction of the molecule.

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The location of polyphenol oxidase (PPO) in cells of apple fruit was examined by immunohistochemistry and subcellular fractionation. In mature apple fruits, where vacuoles occupy most of the cells, PPO was detected immunochemically near the cell walls with use of anti-apple PPO antibodies. In cells of immature fruits and tissue culture, PPO was detected in organelles other than the vacuoles, probably in plastids. The plastid fraction was purified by density gradient ultracentrifugation, and the activities of PPO and marker enzymes of plastids were the highest in the plastids. Most apple PPO was in plastids, as are other plant PPOS, and Some of the protein was solubilized and proteolyzed during ripening and storage.

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The cDNA fragment coding for mung bean (Vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01. After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-β-galactoside, high level expression of the recombinant enzyme was obtained. The enzyme had a tetrameric form that conserved the activity of sucrose synthase. Although the K_m and V_max of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the K_m for sucrose was higher by a factor of 10 for the recombinant enzyme. This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction.

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Theaflavin and theaflavin digallate, which are components of black tea were examined by in vitro invasion assay with mouse Lewis lung carcinoma LL2-Lu3 cells, which are highly metastatic. The compounds inhibited invasion by the tumor cells. Gelatin zymography showed that the cells secreted matrix metalloproteinases (MMPs), probably including MMP-2 and MMP-9, which may be involved in tumor cell invasion and metastasis. Theaflavin and theaflavin digallate also inhibited MMPs from the culture medium of these tumor cells, as did (-)-epigallocatechin gallate. These results suggest that theaflavin, theaflavin digallate, and (-)-epigallocatechin gallate inhibit tumor cell invasion by inhibiting type IV collagenases of the LL2-Lu3 cells.

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To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.

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We had analyzed the detailed structures of the phosphoryl oligosaccharide-1 (PO-1) fraction that was the main component of phosphoryl oligosaccharides (POs) prepared from a potato starch hydrolysate. PO-1 fraction was made up of 3-phosphoryl oligosaccharides and 6-phosphoryl oligosaccharides. Aspergillus niger strain KU-8 produced two types of intracellular acid phosphatase (EC 3.1.3.2, ACPase); ACPase I and II. ACPase II preferentially dephosphorylated 6-phosphoryl oligosaccharides rather than 3-phosphoryl oligosaccharides. The molecular weight of the enzyme was estimated as 66kDa by SDS-polyacrylamide gel electrophoresis and about 260kDa by gel filtration, implying the active form to be a tetramer. The optimum pH and temperature of the enzyme were 2.0-2.5 and 60°C, respectively. ACPase II was stable below 50°C for 30 min and pH 2.0-10.0 for 60 min. In spite of the strict specificity toward 6-phosphoryl oligosaccharides in the PO-1 fraction, ACPase II was able to hydrolyze Fru-1, 6-di-P, ATP, pyrophosphate, and polyphosphate as well as pNPP and Glc-6-P, a broad substrate specificity.

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Adults of the turnip sawfly, Athalia rosae ruficornis, are strongly attracted to the leaves of Ajuga decumbens (Labiatae). Specific feeding stimulants of the sawfly were examined in the leaf surface extracts of A. decumbens. Among seven neoclerodane diterpenoids isolated from the extracts, two new analogs, ajugatakasins A and B, were characterized as 6α, 18-diacetoxy-1β, 12-ditigloyloxy-4α, 17-epoxyneoclerod-13-en-15, 16-olide and its 1β, 12-di-(2-methylbutanoyl)-oxy analog, respectively. Among these analogs, clerodendrin D, a very minor constituent which possesses a tetrahydrofurofuran moiety, was identified as the feeding stimulant for A. rosae ruficornis, while the other analogs with an α, β-unsaturated γ-lactone moiety were found to be completely inactive in the sawfly feeding test.

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Among seventy five strains of iron-oxidizing bacteria obtained from natural environments, only one strain, Thiobacillus ferrooxidans Funis 2-1, grew on Fe²⁺-medium with 1.25mM of sodium molybdate (Mo⁶⁺). In contrast, T. ferrooxidans AP19-3, the representative of Mo sensitive strains, could not grow on Fe²⁺ -medium with 1.0 mM of sodium molybdate. By comparing the levels of inhibition of iron oxidase and cytochrome c oxidase by Mo⁶⁺ or Mo⁵⁺, it was found that Mo⁵⁺ but not Mo⁶⁺ is an actual inhibitor for the iron oxidation enzyme system, especially for cytochrome c oxidase. Cytochrome c oxidase of Funis 2-1 was more resistant to Mo⁵⁺ than AP19-3. Mo⁵⁺, compared to Mo⁶⁺, strongly binds to both cells and the plasma membrane of T. ferrooxidans. Funis 2-1 cells showed a lower binding activity to Mo⁶⁺ or Mo⁵⁺ compared to AP19-3. Cytochrome c oxidase of T. ferrooxidans has been known to catalyze the oxidation of not only reduced mammalian cytochrome c but also Mo⁵⁺. Mo⁶⁺ -oxidizing activities medsured with intact cells and a purified cytochrome c oxidase from Funis 2-1 cells were higher than those of AP19-3, suggesting that Funis 2-1 cells can oxidize toxic Mo⁵⁺ more rapidly to harmless Mo⁶⁺ than AP19-3 does. Since Mo⁶⁺ is known to be chemically reduced by Fe²⁺ to give Mo⁵⁺ and Fe³⁺, the growth inhibition by sodium molybdate(Mo⁶⁺) observed in T. ferrooxidans is explained as follows: Mo⁶⁺ added to Fe⁶⁺ -medium is chemically reduced by Fe²⁺, and Mo⁵⁺ thus produced binds to the plasma membrane and inhibits iron oxidase, as a result, growth of the bacterium is stopped.

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The aspergillic acid degrading enzyme (ADE) that catalyzes the cleavage of the pyrazine ring in aspergillic acid (AA, 1-hydroxy-3-isobutyl-6-sec-butyl-2-pyrazinone) was purified to electrophoretic homogeneity from extracts of Trichoderma koningii ATCC 76666. ADE was a homodimeric protein with a molecular mass of 112 kDa, contained 1 mol of FAD per mol of subunit, and required NAD(P)H and molecular oxygen for its activity. ADE had an isoelectric point of around 5.3, and an optimum pH of 7.0-8.0. p-Chloromercuribenzoate and HgCl₂ completely inhibited ADE activity, while metal chelating reagents, α, α'-dipyridyl and o-phenanthroline, were not inhibitors. The substrate specificity among AA-related compounds was that hydroxyaspergillic acid was a poor substrate (16% of the activity for AA) and deoxyaspergillic acid did not serve as a substrate.

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Glycosides were screened for their lowering effect on the postprandial blood glucose rise in vivo. The effect of phlorizin and other phenolic glycosides on the postprandial blood glucose response to glucose ingestion was evaluated in Std ddY mice. When phlorizin was simultaneously added, the peak blood glucose level was significantly decreased by 51% (p<0.01) compared to vehicles following glucose ingestion by mice, while the blood insulin responses were generally similar. Screening experiments were conducted with different classes of phenolic glycosides added to a glucose solution. Reductions of 40-52% (p<0.05) were observed in vehicles containing arbutin, 4-hydroxyphenyl-α-D-glucopyranoside (hydroquinone-α-glucoside) or glycyrrhizin, and of only 15-31% (not significant) in vehicles containing neohesperidin dihydrochalcone, glycyrrhetinic acid monoglucuronide, or 3, 4-dimethoxyphenyl-β-D-glucopyranoside. No lowering effect was observed in vehicles containing salicin. Since glycyrrhizin, arbutin, and hydroquinone-α-glucoside blunted to varying degrees the postprandial blood glucose rise following glucose ingestion, they may be useful adjuvants for the treatment of diabetic subjects.

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The effect of the thyroid hormone on the rate of brain protein synthesis in rats was studied. Experiments were conducted on three groups of rats given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without a triiodothyronine (T₃) treatment, those treated with PTU + T₃, and those treated with neither PTU nor T₃ (control). The fractional rates of protein synthesis in the brain, liver, and kidney of rats given PTU + T₃ were significantly greater than those in rats given PTU alone. In the brain and kidney, the RNA activity [g of protein synthesized/(g of RNA.d)] were significantly correlated with the fractional rates of protein synthesis. In the liver and kidney, the RNA concentration (mg of RNA/g of protein) was related to the fractional rate of protein synthesis. These results suggest that the thyroid hormone treatment would be likely to increase the rate of protein synthesis in the brain of rats, and that the RNA activity is, at least partly, related to the fractional rate of brain protein synthesis.

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The bacterial cellulose produced in an agitated culture (Ag-BC) showed the highest emulsion-stabilizing effect among the examined cellulosic materials. It was clarified that a mechanical barrier and a scaffolding structure composed of tine fibrils of bacterial cellulose interrupted the coalescence of oil droplets to stabilize the emulsion without reducing the interfacial tension as occurred with sorbitan monolaurate. Since Ag-BC consists of thinner fibrils and smaller flocs than any other cellulodic material, Ag-BC would cover a larger surface area of the oil droplet as a mechanical barrier. The emulsion containing Ag-BC was stable against the addition of salt, and changes in pH and temperature in comparison with xanthan gum and sorbitan monolaurate. This stability would have been due to the stability of the mechanical barrier and a scaffolding structure composed of stable crystalline cellulose. In contrast, instability in the conformation of xanthan gum and a reduction in the interfacial tension of the surfactant would lead to instability of the emulsion.

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2-Hydroxy-6-methylbenzaldehyde (5) is a component of astigmatid mites, functioning as the alarm and sex pheromones, and the establishment of its convenient synthesis is prerequisite and of vital importance to develop applications of these pheromones for practical use. The target compound (5) was effectively synthesized from m-cresol (1) by the following four steps in a 44% overall yield: protection of the hydroxyl group by a tetrahydropyranyl group, blocking the active site (6-position of 1) by a trimethylsilyl group via lithiation and subsequent silylation, formylation via lithiation and successive quenching by dimethylformamide, and deprotection by a trifluoroacetic acid treatment.

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The effects of various (1→3)-β-D-glucans on hematopoietic responses of mice were investigated by measuring colony stimulating activity in sera and ascites of the mice administered glucan. We have demonstrated that the hematopoietic response was increased by various structures of (1→3)-β-D-glucans, i.e. soluble glucans (linear, branched, single helix, triple helix) and particulate glucans. From the viewpoint of structure and activity relationships, we found several characteristic features: i) hematopoietic response induced by the particulate glucan disappeared faster than that by the soluble glucans, ii) conformation of the glucans, single vs. triple helix, are relatively independent of the response, iii) linear glucan had a weaker response, and iv) there is a strong strain-dependency of the response. These results corresponded well with the fact that branched (1→3)-β-D-glucans, but not linear and not particulate, are often used as biological response modifiers for cancer patients.

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A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26, 000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P'₁ positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.

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The β-ray induced scission of puC18 plasmid DNA from E. coli in tritiated water was examined in the presence or absence of a green tea percolate (TP) and the main constituent, (-)-epigallocatechin gallate (EGCg). An analysis of the ratio of the original closed-circular to the open-circular form of DNA, which was formed by the strand scission of DNA, revealed that TP and EGCg showed a protective effect on DNA scission depending on their concentrations. A new technique, named solid state spin trapping, was applied to examine this scavenging ability toward the hydroxyl (OH) radical generated in tritiated water. The result was kinetically analyzed to reveal that TP and EGCg showed the scavenging effect, suggesting that the protective effect on DNA scission was attributable to the scavenging effect on the OH radical.

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Hiburipyranone / a cytotoxic metabolite of the marine sponge / Mycale adhaerens / was synthesized as a racemate

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A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.20%. The results of reductive hydrolysis, Nglycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.

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Lignostilbene-α, β-dioxygenase isozyme IV was purified from ultrasonic extracts of Pseudomonas paucimobilis TMY1009 through four steps of column chromatography. The fraction obtained gave a single band on SDS-PAGE and a single peak on reversed-phase HPLC and DEAE-HPLC. The specific activity and K_m of purified isozyme IV were 110μkat/g and 4.2μM for 4, 4'-dihydroxy-3, 3'-dimethoxystilbene, 150μkat/g and 3.3μM for 4, 2'-dihydroxy-3, 3'-dimethoxy-5'-(2"-carboxyvinyl)stilbene. The molecular mass of intact isozyme IV was estimated to be 94 kDa by gel permeation chromatography, and that of its subunits was 52 kDa by SDS-PAGE under denaturing conditions. The N-terminal amino acid sequence of isozyme IV differed slightly from that of other isozymes. Isozyme IV seemed to be composed of two identical subunits, γγ.

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The photosynthetic reaction center from the photosynthetic bacterium Rhodobacter sphaeroides was reconstituted into neutral, positively charged, or negatively charged liposomes. About 70% of photosynthtetic reaction centers were reconstituted in the proteoliposomes exposing their H-subunit outside with positively charged lipids while only 30-40% of them were in the same topological orientation with neutral or negatively charged lipids.

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We developed a sensitive, accurate, and fast method to detect foreign gene expression using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) in poplar tree (Populus alba L.) protoplasts. Template total RNA was purified by removing the transfected foreign gene vector completely with DNase I treatment before the RT reaction. Expression of cDNA that encodes goat growth hormone was confirmed at the mRNA level 24h after electroporation mediated DNA transfer.

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Soybeans, tea leaves, and mushrooms were conspicuously rich in spermidine, while oranges contained a large amount of putrescine. Among the fermented foods, soy sauces were rich in putrescine and histamine, while Japanese sake contained plenty of agmatine. These polyamines are thought to be produced from amino acids during fermentation with amino acid decarboxylases formed by the microorganisms.

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For the industrial production of bacterial cellulose from sucrose in static cultures, the possibility of a high rate of cellulose production was investigated. An Acetobacter strain, S-35, which had been isolated from a grape, was selected from 1500 isolates. This strain was found to produce a large amount of cellulose from either glucose ot fructose. Using this strain, high cellulose production rates of 3.3 g/liter/d or 40 g/m²/d from sucrose were seen in static culture.

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The Arg-Gly-Asp (RGD) motif-containing fragment of high-molecular-weight cell-adhesion protein MFBA derived from Lentinus edodes caused a significant aggregation of the fragmented hyphal cells of Schizophyllum commune. This fungal cell-aggregation was inhibited by a previous treatment of the cells with the Gly-Arg-Gly-Asp-Ser-Pro peptide, but not with the Gly-Arg-Gly-Glu-Ser-Pro peptide, showing that the RGD motif is essential for the cell-aggregation activity.

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The induction of malic enzyme gene expression by triiodothyronine and insulin was severely blunted in rat monolayer hepatocytes cultured on type I collagen compared with that in spherical hepatocytes cultured on a reconstituted basement membrane gel (EHS-gel). Although the mRNA level of thyroid hormone receptor β (TRβ) gradually decreased in the monolayer hepatocytes during culture, the mRNA level in the hepatocytes on EHS-gel was maintained at around the in vivo level. Our results suggest that the maintenance of TRβ mRNA on EHS-gel is responsible for the high responsiveness to thyroid hormone in a hepatocyte culture.

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We have isolated a genomic clone of a putative xylanase gene (xyn1) from Humicola grisea by using the DNA fragment encoding a cellulose-binding domain of H grisea cellobiohydrolase 1 as a probe. The translation product of the xynl gene predicts a xylanase of 429 amino acids in length, with a cellulose-binding domain in the C-terminus.

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The ggt encoding γ-glutamyltranspeptidase (GGT) from Bacillus subtilis (natto) was cloned and sequenced. The DNA sequence contains a single open reading frame of 1761bp that might be translated to a protein of 587 amino acid residues, and indicates that B. subtilis (natto) GGT is synthesized as prepro-GGT and processed later into large and small subunits. The putative catabolite responsive element (CRE) was located upstream of the ggt coding region.

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Ribonuclease T₁ was nitroguanidinated with N-methyl-N'-nitro-N-nitrosoguanidine to investigate the effects of modification of the amino groups on the activity and at the same time to shed some light on its usefulness in specific chemical modification of proteins. Under the conditions used (at pH 7.0 and 37°C for 72h), the amino group of the NH₂-terminal Ala 1 was modified completely, and the ε-amino group of Lys 41 about 90%. No other residue was modified. The modified enzyme had approximately 84% of the original activity toward RNA, and showed a characteristic absorption maximum at 270 nm.

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We studied the effects of benadrostin, an inhibitor of poly (ADP-ribose) polymerase, on apoptosis of HL-60 cells induced by 5-azacytidine. Benadrostin interfered with the apoptosis and inhibited the cell death in a dose- and time-dependent manner.

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It had been suggested that cyanidin 3-O-(6"-O-monomalonyl-β-glucopyranoside) and another unknown compound occur as major pigments in the purplish-red flower of chrysanthemum (Dendranthema grandflorum). We determined the structure of the unknown compound as cyanidin 3-O-(3", 6"-O-dimalonyl-β-glucopyranoside) by FAB-MS and ¹H-NMR. This is the first rebort of the identification of the cyanidin 3-dimalonyl glucoside as a flower pigment.

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Radiolabeled putrescine incorporation into dimethyl casein is widely measured in transglutaminase assays. We assessed the hypothesis that proteases can catalyze this reaction. Acceleration of putrescine incorporation by chymotrypsin, subtilisin, and papain, all purified to homogeneity, was investigated, and all of the samples tested accelerated the reaction. These effects disappeared when the proteases were first boiled. The rate of incorporation was proportional to the enzyme concentration. The amount incorporated when chymotrypsin was tested was proportional to the reaction time. On the basis of these findins, we concluded that some proteases catalyze reactions with putrescine incorporation.

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An antiviral protein purified from the leaves of Amaranthus viridis was named amaranthin. The ih vivo antiviral activity of amaranthin was confirmed in tobacco mosaic virus (TMV) infection test on Nicotiana glutinosa leaves. The molecular mass of the amaranthin was estimated about 30 kDa by SDS-PAGE and the pI was measured as 9.8 by isoelectric focusing (IEF) dnalysis. Cytotoxicity of the amaranthin using in vitro translation inhibition assay was similar to that of pokeweed antiviral protein (PAP) with IC₅₀ of 25 pM. Depurination activity (N-glycosidase activity) against animal rRNA was also confirmed.

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A new derivative of sulfatide, 2-O-α-L-fucopyranosyl sulfatide, was synthesized. The compound inhibited the binding of HL-60 cells, which express sialyl Lewis X, to P- and L-selectin more than the corresponding non fucosylated compound.

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Acid-stable (KAA) and neutral (KNA) α-amylases from shochu koji (A. kawachii) were purified and their actions towards maltooligosaccharides were studied. KAA could be distinguished from KNA by the following actions: with KAA, maltopentaose (G5) was preferentially hydrolyzed at the third glycoside bond, and the addition of potassium thiocyanate (KSCN) decreased the rate of CNP-release from 2-chloro-4-nitrophenyl-α-maltotrioside (CNP-G3).

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