The asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine, was prepared, and the absolute configurations of the enantiomers were determined by an X-ray analysis. The insecticidal activity against the housefly measured with metabolic inhibitors showed the (S) enantiomer to be slightly more active than the (R) isomer. Electrophysiological measurements on the American cockroach central nerve cord showed the compounds to elicite the impulses and subsequently blocked them. The neuroblocking potency of the (S) isomer was 5.9 μM, while that of the (R) isomer was as high as 73 μM. The molar concentrations required for 50% inhibition of the specific binding of [³H]imidacloprid to the housefly head membrane preparation were respectively 0.19 μM and 0.95 μM for the (S) and (R) isomers. This enatioselectivity ratio was smaller than 35 for nicotine isomers but greater than 2 for epibatidine isomers.

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  Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-(17-hydroxy-linoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.

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  The novel Diels-Alder adduct, dicaffeoyl quinone as its hydrate, was formed from the oxidation of 3,4-dihydroxycinnamic acid (caffeic acid) with NaIO₄. The structure of this hydrate was determined by spectroscopic methods.

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  A sensitive method to examine the brassinolide (BL) response of barley (Hordeum vulgare L.) using dark-grown leaf segments was established based on the known method for wheat. BL responses of 53 dwarf isogenic lines of barley were examined, and two lines were found having a uzu gene that doesn't respond significantly. These results indicate that uzu dwarfism may be caused by the non-responding character to BL.

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  A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.

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  Although expression of natriuretic peptides in cardiac tissues is up-regulated in response to pressure overload, no significant change in cGMP level in hypertrophied ventricles was observed. Activities of two cyclic nucleotide phosphodiesterase (PDE) isoforms, Ca²⁺/calmodulin-stimulated PDE (PDE1) and cGMP-stimulated PDE (PDE2), were significantly higher in rat left ventricles 14 days after aortic banding. The absence of significant changes in PDE1A and PDE2A mRNA levels indicated that the two PDE activities were post-transcriptionally up-regulated. These results suggested that the increased cGMP-PDE activity in response to pressure overload plays an important role in neutralizing cGMP action in cardiac tissue.

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  Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2′-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2′-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.

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  3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.

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  Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl α-D-glucopyranoside (Glcα-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glcα-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl β-D-thioglucopyranoside (Glcβ-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger α-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH₃CN (1: 1 v/v) at 37°C. High activity of the enzyme was observed in the reaction between 2D-Glcα-O-pNP and Glcβ-S-Et to afford the monodeoxy analogs of ethyl β-thiomaltoside and ethyl β-thioisomaltoside that contain a 2-deoxy α-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,4)-β-D-thioglucopyranoside and ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 6.72% and 46.6% isolated yields (based on 2D-Glcα-O-pNP), respectively. Moreover, from 3D-Glcα-O-pNP and Glcβ-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl β-thioisomaltoside that was modified at the glycon α-D-glucopyranose moiety, namely ethyl 3-deoxy-α-D-ribo-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 23.0% isolated yield (based on 3D-Glcα-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glcα-O-pNP and Glcβ-S-Et.

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  Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a K_m of 165 μM and a V_max of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a K_d of 0.52 μM by BiP, lower than that for RCMLA (K_d=1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (K_d=21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.

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  We identified four new family 19 chitinases in Oryza sativa L. cv. Nipponbare: one class I (OsChia1d), two class II (OsChia2a and OsChia2b), and one class IV (OsChia4a). OsChia2a resembled (about 60% identity) the catalytic domains of class I chitinases, but OsChia2b was almost identical (95% identity) to that of the class IV enzyme. OsChia1c, OsChia1cΔCBD (a deletion of OsChia1c lacking a chitin-binding domain, CBD), and OsChia2b were separately expressed and purified in Pichia pastoris. OsChia1c inhibited fungal growth significantly more than OsChia1cΔCBD or OsChia2b. The activities of these enzymes on chitin polymers were similar, but they acted differently on N-acetylchitooligosaccharides, (GlcNAC)_n. OsChia1c slowly hydrolyzed (GlcNAC)₆ and very poorly hydrolyzed (GlcNAC)₄ and (GlcNAC)₅. In contrast, OsChia2b efficiently hydrolyzed these oligosaccharides. The high antifungal activity and low hydrolytic activity of the class I enzyme towards (GlcNAC)_n imply that it participates in the generation of N-acetylchitooligosaccharide elicitors from the cell walls of infecting fungi.

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  Two α-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a β-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl₂, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): k_cat, 71.8 and 55.5 s⁻¹, K_m, 0.113 and 0.097 mg/ml; for blocked 4-nitrophenyl α-D-maltoheptaoside: k_cat, 62.4 and 85.3 s⁻¹, K_m, 0.22 and 0.37 mM, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards β-cyclodextrin Sepharose was due to some modification on/near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.

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  Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as4-mono-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, and sachharide D was 4,4′-di-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.

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  Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.

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  The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP⁺ for the pentose phosphate cycle.

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  The denitrifying fungus Cylindrocarpon tonkinense was thought to be able to denitrify only nitrite (NO₂⁻) but not nitrate (NO₃⁻) to form nitrous oxide (N₂O). Here we found, however, that C. tonkinense can denitrify NO₃⁻ under certain conditions. Presence of ammonium (NH₃+) in addition to NO₃⁻ and the use of a fermentable sugar as an electron donor were key conditions for inducing the denitrifying activity. Such induction accompanied a remarkable increase in the intracellular level of the enzyme activities related to NO₃⁻ metabolism. These activities contained assimilatory type NADPH (or NADH)-dependent NO₃⁻ reductase (aNar), dissimilatory nitrite reductase (dNir), and nitric oxide reductase (P450nor), but did not contain ubiquinol-dependent, dissimilatory NO₃⁻ reductase (dNar). The denitrification was inhibited by tungstate, an inhibitor of Nar. These results demonstrated occurrence of a novel type of denitrification in C. tonkinense, in which assimilatory type Nar is possibly involved.

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  Water-soluble vitamin, niacin, and its related compounds were examined for their differentiation-inducing activity in human promyelocytic leukemia cells (HL-60). Among the compounds, which inhibited cell proliferation measured by MTT assay, isonicotinic acid, nicotinamide N-oxide, and nicotinamide induced NBT reducing activity. HL-60 cells were differentiated into granulocyte-like cells by these compounds, judging from morphological changes and loss of nonspecific esterase activity. The differentiation-inducing activity of water-soluble vitamin and its related compounds suggest that these compounds may be applicable for medical use.

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  Aspergillus niger ATCC 9642 isopullulanase (IPU) was heterologously expressed by Pichia pastoris GS115 under three different signal sequences of Saccharomyces cerevisiae acid phosphatase, S. cerevisiae α-factor prepro peptide, and A. niger isopullulanase. One-step purification using lectin Con A affinity chromatography yielded recombinant IPU (IPU-PP) with high purity. IPU-PP had a higher carbohydrate content than native IPU and IPU-AO expressed in A. oryzae M-2-3. IPU-PP hydrolyzed various substrates containing the structure of panose, which indicated a strict subsite recognition of the panose motif.

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  Taste bud cells have elongated shape and are anchored to the basement membrane. Here we analyzed the subunits of laminin, a major component of the basement membrane, in circumvallate papillae of rat tongue. Two β subunits, β1 and β2, were detected by RT-PCR, of which the β2 subunit was immunohistochemically identified as a major component of the basement membrane. The γ1 subunit was also immunohistochemically identified as a major component. Profiles of these two laminin subunits were nearly the same as that obtained by an anti-laminin antibody, indicating that laminins of the basement membrane in the papillae contain β2 and γ1 as major β and γ subunits, respectively. As potential receptors for these laminin ligands, integrin subunits were analyzed by RT-PCR. Three integrin β subunit species, β1, β4, and β5, were shown to be expressed in the epithelium of the circumvallate papillae by RT-PCR among the five species examined.

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  Myrothecium verrucaria bilirubin oxidase expressed in Aspergillus oryzae is in a resting form different from that of the authentic bilirubin oxidase, but reaches the resting form of the authentic enzyme after one cycle of reduction and reoxidation with dioxygen as shown by the absorption and electron paramagnetic resonance spectra.

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  Transglucosylation activities of spinach α-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, α-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and α-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-α-D-glucosyl-glucose. Transglucosylation to sucrose by α-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach α-glucosidase I and IV are useful to synthesize the α-1,6-glucosylated and α-1,2- and 1,4-glucosylated products, respectively.

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  Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.

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  Transformation of Escherichia coli is a basic technique for genetic engineering. We used a liposome-mediated lipofection method to transform electrocompetent E. coli cells which has little natural competence of foreign DNA without electroporation treatment, and got transformants with simple and quick treatment by a plasmid or a transposon and transposase complex.

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  Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→} (CTS). Structural analysis showed that the transfer product was3-O-β-N-acetylglucosaminyl CTS, cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[β-GlcNAc-(1→3)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.

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  In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P₂. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P₂ is required for sporulation, diploid cells defective in production of PI 3,5-P₂ were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P₂ but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P₂ although the forespore membrane was found to be less dense in these cells.

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  The effect of methanol extract and protocatechuic acid from the leaves of Zanthoxylum piperitum on lipid peroxidation and drug metabolizing enzymes were investigated in the liver of bromobenzene-treated rats. The methanol extract and protocatechuic acid reduced the level of lipid peroxide induced by bromobenzene. The methanol extract and protocatechuic acid reduced the activity of aniline hydroxylase that had been increased by bromobenzene, while did not affect the activities of aminopyrine N-demethylase and glutathione S-transferase. The methanol extract and compound effectively restored the activity of epoxide hydrolase which had been decreased by bromobenzene. These results may suggest that the methanol extract of Z. piperitum and protocatechuic acid prevented lipid peroxidation by reducing the activity of aniline hydroxylase, an epoxide-producing enzyme, and by enhancing the activity of epoxide hydrolase, an epoxide-removing enzyme, in rats that had been intoxicated with bromobenzene.

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  Different kinds of lactobacilli and Bifidobacteria fermented milk were fed to ovalbumin-specific IgE-elevated mice for 3 days, and after the final administration, changes in the ovalbumin-specific IgE values for each sample were compared to the value for non-fermented milk. Seven of the Lactobacillus-fermented milks caused a significant decrease in the serum ovalbumin-specific IgE levels. Above all, Lactobacillus acidophilus L92, Lactobacillus acidophilus CP1613, and Lactobacillus fermentum CP34 fermented milk had the most significant effects of decreasing the serum ovalbumin-specific IgE levels compared to a control group. The L. acidophilus L92 and L. fermentum CP34 cells also showed significant ovalbumin-specific IgE lowering activities. From these results, an active component seems to exist in the cells of L. acidophilus L92 and L. fermentum CP34 strains. Recovery of the radiolabeled L. acidophilus L92 and L. fermentum CP34 cells from the small intestine and the large intestine of the mouse 13 h after oral administration were higher than the recovery of any other strain.

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  The effects of dietary eritadenine on the concentration of plasma lipoprotein lipids and the molecular species profile of plasma lipoprotein phosphatidylcholine (PC) were investigated in rats fed cholesterol-free and cholesterol-enriched diets to obtain insights into the relationship between the changes in PC molecular species profile and the hypocholesterolemic action of eritadenine. The effect of eritadenine on the secretion rate of very low density lipoprotein (VLDL) from the liver was also estimated. Rats were fed the control or eritadenine-supplemented (50 mg/kg) diets with or without exogenous cholesterol for 14 d. Eritadenine supplementation significantly decreased the cholesterol of major plasma lipoproteins, high density lipoprotein and VLDL, in rats fed cholesterol-free and cholesterol-enriched diets, respectively. The ratio of PC to phosphatidylethanolamine, Δ6-desaturase activity, and the ratio of arachidonic acid to linoleic acid in liver microsomes were markedly decreased by eritadenine irrespective of the presence or absence of exogenous cholesterol. Dietary eritadenine increased the proportion of 16:0-18:2 molecular species with a decrease in 18:0-20:4 in plasma lipoprotein PC in both rats fed cholesterol-free and cholesterol-enriched diets. Eritadenine did not depress the secretion rate of VLDL in rats fed a cholesterol-free diet containing a high level of choline. The results indicate that dietary eritadenine elicits its hypocholesterolemic action with modulations of the fatty acid and molecular species profiles of PC irrespective of the presence or absence of exogenous cholesterol. The eritadenine-induced alteration of PC molcular species profile is discussed in relation to the hypocholesterolemic action of eritadenine.

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  Combinatorial chemopreventive strategies, in contrast to those with individual agents, show potential in terms of potentially lower toxicity and higher efficacy. In this study, we combined several agents and examined their suppressive effects on the combined lipopolysaccharide (LPS)- and interferon(IFN)-γ-induced formation of proinflammatory mediators, including prostaglandin (PG) E₂ and tumor necrosis factor (TNF)-α, in RAW264.7 murine macrophages. The combinatorial effects of indomethacin/genistein (GEN) and aspirin/GEN were found to be synergistic for PGE₂ suppression, while the nimesulide/GEN combination was antagonistic. Further, while (−)-epigallocatechin gallate (EGCG) alone increased LPS/IFM-γ-induced production of PGE₂ and TNF-α as well as cyclooxygenase-2 expression, the EGCG/GEN combination markedly suppressed these parameters. Our results suggest that certain chemopreventive agents act complexly and that, when used in combination, they affect the intracellular signaling pathways of the paired agents to exert additive, synergistic, or antagonistic effects.

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  Significant antiviral activity against respiratory illness viruses has been found in a hot-water extract of black soybean. This black soybean extract showed significant antiviral activity against human adenovirus type 1 and coxsackievirus B1 in a dose-dependent manner, while the hot-water extract from common yellowish soybean showed only weak activity. The antiviral activity could not be extracted from the black soybean by 70% aqueous ethanol, suggesting that saponin in the seed did not contribute to this activity. The antiviral activity was only recovered from cotyledons and not from seed coats with the hot water, showing that the activity was distributed in the cotyledons and that antocyanins in the black soybean seed coats did not contribute to the antiviral activity. The antiviral compound(s) in the black soybean was partially purified by up to 166 times by a combination of gel filtration, reversed phase HPLC, and ion-exchange HPLC. The partially purified antiviral compound showed hydrophilic and anionic properties, and a maximum absorption at 260 nm, suggesting that this antiviral fraction may contain a phenyl group(s). On the other hand, an amino acid analysis with the acid hydrolyzate and a neutral sugar analysis showed that the antiviral compound from black soybean might not be a polypeptide or glycoconjugate bearing neutral sugar(s).

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  The effect on the IgE content induced by concanavalin A in spleen lymphocytes of the presence wogonin, ganhuangenin, wogonoside and 3,5,7,2′,6′-pentahydroxyl flavanone was investigated. These flavonoid components markedly inhibited the histamine released from cells stimulated with the calcium ionophore, A23187. However, the magnitude of the inhibitory effect on the degree of lipid peroxidation by ConA of these components was in order of PHF> GHG>WG>WGS. Interestingly, WG, GHG and WGS, with a methoxyl group in the A and B rings, strongly inhibited histamine and IgE production, whereas PHF without a methoxyl group was much stronger than that for lipid peroxidation. We suggest that WG, GHG and WGS might block the pathway for the release of histamine, and that the IgE level in spleen lymphocytes is responsible for the lipid peroxidation.

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  Paraquat causes lung injury by oxidative stress. After 48 h of intraperitoneal administration of paraquat (50 mg/kg of body weight) to rats, the vitamin C concentration in the lungs was significantly decreased, while lung vitamin E content was increased after 12 h. These results indicate that vitamin C directly reflected the oxidative stress in the lungs.

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  We have developed an in vitro assay system for the evaluation of the inhibitory effects of phenolic antioxidants on myeloperoxidase (MPO) activity. The formation of dityrosine from the MPO/H₂O₂/L-tyrosine system was used as an indicator of the MPO activity. Because the buffer system used does not include chloride ion, this assay has the advantage of exclusion of direct reaction between an antioxidant and HOCl. In this assay, ferulic acid, gallic acid, and quercetin strongly inhibited the dityrosine formation, and curcumin and caffeic acid were also effective.

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  To confirm the absorption of proanthocyanidin (PA) into the human body, four healthy adults were administered 2.0 g of PA-rich grape seed extract (GSE). Blood were drawn before intake and 2 h after intake. Through the enzymatic treatment of sulfatase and β-glucuronidase, blood samples were analyzed by HPLC coupled with mass-spectrometry (LC/MS). Procyanidin B1 [epicatechin-(4β→8)-catechin] was detected in human serum 2 h after intake. Its concentration was 10.6±2.5 nmol/l.

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  Anti-rat Δ6-desaturase serum was produced by immunizing rabbits with the 14 N-terminal amino acids (2-15) of rat Δ6-desaturase. The antiserum prevented the enzymatic activity of Δ6-desaturase in microsomes. Subsequently, the antiserum was used to demonstrate that gemfibrozil, a ligand for peroxisome proliferator-activated receptor α, is involved in activating Δ6-desaturase gene expression, thereby elevating the protein level and the activity.

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  Acrylamide in potato chips made from tubers stored at 2 or 20°C for two weeks after harvest was analyzed by GC-MS. The acrylamide level in the former chips was higher than ten times of that in the latter, which was highly correlated with both glucose and fructose levels in the tubers.

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  High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membrane-bound, quinoprotein, ADH-deficient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and purified from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl₂ in the column buffer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the long-chain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear difference in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.

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  A cultivation procedure for the preparation of a new tempeh-like fermented soybean containing a high level of γ-aminobutyric acid was developed. Steamed soybeans were incubated aerobically with Rhizopus microsporus var. oligosporus IFO 8631 for 20 h, and then anaerobically incubated for 5 h by replacement of the atmosphere with nitrogen. The GABA content in the aerobically fermented soybeans was about 30 mg per 100 g dry fermented soybeans, while the anaerobically cultivation was about 370 mg/100 g dry fermented soybeans. The incubation with several strains of Rhizopus species showed that all of R. microsporus var. oligosporus and R. oryzae examined accumulated GABA in the anaerobically fermented soybeans. In particular, R. microsporus var. oligosporus IFO 32002 and IFO 32003 showed the highest content of GABA (1,740 mg/100 g dry fermented soybeans and 1,500 mg/100 g dry fermented soybeans, respectively). Moreover, the free protein amino acids increased greatly in the fermented soybeans during the anaerobic cultivation.

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  Among the members of the copper protein superfamily, the type I enzyme rusticyanin, which is found as an electron carrier in the oxidative respiratory chain of Acidithiobacillus ferrooxidans, is the only one to have both a high redox potential and acid stability. Here we report that two forms of the rusticyanin gene (rus) are present in the genomes of some strains of A. ferrooxidans. The more common form of rus (type-A) was found to be present in all six strains studied, including those harboring only a single copy of the gene. In addition a less common form (type-B) occurred in strains harboring multiple copies of the gene. The two genes were expressed as rusticyanin isozymes with differing surface charges due to differences in their amino acid composition. Still, the copper coordination sites were completely conserved, thereby maintaining the high redox potential necessary for an electron carrier.

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  Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.

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  Marine bacterial strains (BP-PH, CAR-SF, and DBF-MAK) were isolated using biphenyl, carbazole (CAR), or dibenzofuran (DF) respectively as substrates for growth. Their 16S ribosomal DNA sequences showed that the species closest to strain BP-PH, strain CAR-SF, and strain DBF-MAK are Alteromonas macleodii (96.3% identity), Neptunomonas naphthovorans (93.1% identity), and Cycloclasticus pugetii (97.3% identity), respectively. The metabolites produced suggested that strain CAR-SF degrades CAR via dioxygenation in the angular position and by the meta-cleavage pathway, and that strain DBF-MAK degrades DF via both lateral and angular dioxygenation. Polychlorinated biphenyl (KC-300) and 2,3-dichlorodibenzo-p-dioxin were partially degraded by strain BP-PH and strain DBF-MAK, while 2,7-dichlorodibenzo-p-dioxin and 2,4,8-trichlorodibenzofuran remained virtually unchanged.

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  A recombinant Rhizobium strain, PBK3-IS, that constitutively expressed the oxygenase component of carbazole 1,9a-dioxygenase from Sphingomonas sp. strain KA1, was constructed. In the water-cultured siratro rhizospheres inoculated with strain PBK3-IS, 48% of the dibenzofuran was removed within 3 days (initial substrate, 25 μg). Similar results were obtained in soil-cultured siratro rhizospheres using sterile vermiculite. When non-sterile field soils were used instead of sterile vermiculite, the inoculated recombinant strain could grow on the siratro root in all soils tested, except for wet paddy field.

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  The mono trans geometrical isomer of eicosapentaenoic acid, 5c,8c,11c,14c,17t-eicosapenntaenoic acid (20:5Δ5c,8c,11c,14c,17t), was synthesized by fatty acid microbial conversion using a Δ12-desaturase defective mutant of an arachidonic acid (AA)-producing fungus, Mortierella alpina 1S-4. The substrate for the bioconversion, a geometrical isomer of linolenic acid, was prepared by isomerization of linseed oil methyl ester by the nitrous acid method, followed by purification on a AgNO₃-silica gel column. The structure and double bond geometry were identified after hydrazine reduction followed by permanganate oxidation to 20:5Δ5c, 8c,11c,14c,17t. The biosynthetic route from 18:3Δ6c, 9c,12t to 20:5Δ5c,8c,11c,14c,17t was presumed to mimic the route from linoleic acid to arachidonic acid.

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