A gene encoding an inulin fructotransferase (DFA I-producing) [EC 2. 4. 1. 200] from Arthrobacter globformis S14-3 was cloned and the nucleotides sequenced, for the first time. The sequence indicated that the native enzyme protein is composed of 392 amino acid residues. The native enzyme is an extracellar enzyme produced in the culture supernatant of A. globformis S14-3, but the nucleotide sequence of the gene lacks a sequence for signal peptide for secretion. The 1. 5-kb DNA fragment encoding the gene was found to produce the active enzyme in the culture supernatant of an E. coli clone, under the control of the lac promoter of pUC119.

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Pseudomonas sp. 113P3, when cultured on a medium containing poly(vinyl alcohol) (PVA) in the presence of pyroroquinoline quinone (PQQ), produced a high level of a PVA dehydrogenase (PVADH). This enzyme was purified to a homogeneous state by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from the cell-free extract and found to be a hemoquino-protein. 1, 3-Diols, especially PVA, were the most favorable substrate ; primary alcohols and secondary alcohols and other diols could not act as substrates. PVADH showed an activity in the presence of PQQ. The molecular weight of the enzyme was found to be about 67, 000 by SDS-PAGE and high pressure liquid chromatography using a TSK gel G-3000SW column. Our PVADH catalyzed dehydrogenation of PVA with an artificial electron acceptor, like the PQQ-dependent PVADH reported by Shimao et al. The molecular weight, PQQ dependency, requirement for metal ion, and absorption spectra of the PVADH were very similar to those of the hemoquino-protein primary alcohol dehydrogenase (ADH) isolated from Comamonas testosteroni ATCC 15667 (formerly called Pseudomonas testosteroni), but differed in the substrate spectra.

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During shotgun cloning of an amylase gene, we found a transformant of Escherichia coli with a reddish color. The transformant produced highly water-soluble red pigments the molecular masses of which were less than 3000. The plasmid harbored by the transformant contained a DNA fragment derived from a strain of Bacillus stearothermophilus. Truncation of the insert DNA showed that an 1. 1-kbp Sau 3A-SalI fragment was responsible for the reddish colony. An open reading frame was found in the nucleotide sequence of the 1. 1-kbp DNA fragment. The production of the red pigment was accompanied by a colorless 28-kDa protein. The sequence of the 28-kDa protein was highly homologous to bacterial uroporphyrinogen III methylases participating in corrinoid biosynthesis. The 28-kDa protein was found to be a thermostable uroporphyrinogen III methylase.

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2-Methylisocitrate dehydratase (EC 4. 2. 1. - ; (2S, 3R)-3-hydroxybutane-1, 2, 3-tricarboxylate hydrolyase), a new enzyme functioning in the methylcitric acid cycle of propionate metabolism, was found in cells of Yallowia lipolytica, separated from the usual aconitase (EC 4. 2. 1. 3), and purified about 120-fold to homogeneity as judged electrophoretically. The new enzyme catalyzed a reversible reaction between 2-methylisocitrate and 2-methyl-cis-aconitate, but was inactive toward 2-methylcitrate, citrate, cis- or trans-aconitate, isocitrate, or the other hydroxy-acids tested. Equilibrium was reached at about 92% of 2-methylisocitrate, with the rest as 2-methyl-cis-aconitate. The isolated aconitase also catalyzed the reversible reaction and its K_m for 2-methylisocitrate was 120μM, but that of the new enzyme was 18μM.

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A trehalose-producing bacterium, Arthrobacter sp. strain Q36, was isolated from soil. From a supernatant of the culture broth, two novel enzymes related to trehalose synthesis were partially purified by Sepabeads FP-DA column chromatography. One enzyme catalyzed the conversion of maltopentaose into maltotriosyl trehalose by intramolecular transglycosylation, showing it to be maltooligosyl trehalose synthase. The other hydrolyzed the product transferred by the former into maltotriose and trehalose specifically, showing it to be maltooligosyl trehalose trehalohydrolase. In addition to the bacterial strain isolated, several bacteria kept in our laboratory were found to produce these enzymes. The enzymatic system was proposed to be a novel biosynthesis of trehalose in bacteria involving the following reactions : maltodextrin→maltooligosyl trehalose, maltooligosyl trehalose→maltodextrin + trehalose. When these enzymes acted on amylose simultaneously, the trehalose in the reaction mixture reached more than 80% in content.

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By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis. The analysis of the nucleotide sequence of the 2. 8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.

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The promotive effects of poly-cations on immunoglobulin production was investigated using human hybridoma cells. Among poly-cations tested, ε-poly-L-lysine with hydrochloride (approximately 4kDa), which has been used as an antibacterial food additive, had the greatest activity in enhancing IgM production of human-human hybridoma HB4C5 cells without stimulating cell proliferation. Immunoglobulin production stimulatory (IPS) activity of ε-poly-lysine was not affected by trypsin digestion. It was stable below 60°C but completely inactivated with heating at 1000°C for 30min. ε-Poly-lysine also enhanced interferon-β(IFN-β) production of human osteosarcoma MG-63 cells, but this stimulatory effect was reduced by the trypsin digestion.

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Two cDNA clones, cLRG5 and cLRG11, that respond to gibberellin (GA) were isolated from seeds of photoblastic lettuce (Lectuca sativa L. cv. Grand Rapids) by differential screening. Northern blot analysis indicated that the levels of LRG5 and LRG11 mRNAs were raised to slightly higher levels 10h after the start of GA treatment and the levels were maintained at least for further 8h, while those in the control seeds gradually decreased. Red light irradiation had effects similar to GA treatment. The cLRG5 insert encodes a putative polypeptide of 380 amino acids that is highly homologous to alcohol dehydrogenases from several higher plants. With regard to the cLRG11 insert, no homologous gene has been reported.

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The gene encoding dimethyl sulfoxide (DMSO) reductase, which contains a molybdenum cofactor, of the phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans was isolated using an oligonucleotide probe, which was synthesized based on a internal amino acid sequence of the purified enzyme. The DMSO reductase gene coded for 822 amino acids (2466 base pairs, Mr = 89, 206) as a precursor form having a signal peptide of 42 amino acids. The deduced amino acid sequence had high homology with those of some enzymes containing a molybdenum cofactor : trimethyl amine N-oxide reductase (48%), biotin sulfoxide reductase (44%), and DMSO reductase (29%) of Escherichia coli.

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A soil bacterium capable of growing on a polysaccharide containing β(1→6)galactofuranoside residues derived from the acidic polysaccharide of Fusarium sp. as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as a Bacillus sp. The bacterium produced β-galactofuranosidase inductively in the culture media. The most effective inducer for the β-galacto-furanosidase production was a polysaccharide containing β(1→5) or β(1→6)-linked galactofuranoside residues, but gum arabic, gum guar, gum ghati, arabinogalactam, araban, and pectic acid did not induce the enzyme. The enzyme had three different molecular weight forms. The low molecular-weight form was purified by a combination of Toyopearl HW-55 and DEAE-Toyopearl 650S column chromatographies, and preparative polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 67, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6 and 37oC, and was stable between pH 4 to 8 at 5oC. The action of the enzyme was inhibited by the addition of Cd2 +, Co2+, Hg2+, Zn2+, iodoacetic acid, and EDTA. The purified enzyme cleaved β(1→5) and β(1→6)-linked galactofuranosyl chains. Based upon the mode of liberation of galactofuranosyl residues from pyridylamino-β(1→6)-linked galactofuranoside oligomers, the enzyme can be classified as an endo-β-galactofuranosidase that randomly hydrolyzes the linkage.

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Novel cyclic isomaltooligosaccharides, cyclodextran, strongly inhibited the dextransucrase reaction. The inhibition was dependent on the cyclodextran concentration and greatly enhanced by the first incubation at 30°C for 30min. Cyclodextran-heptaose and -octaose were competitive inhibitors for sucrose yielding K_i's of 0. 25 and 0. 64mM, respectively. Both reducing sugar and dextran producing activities of dextransucrase were almost equally inhibited by the cyclodextrans. Although γ-cyclodextrin, palatinose, sucrose-monocaprate, and maltitol gave 5-35% inhibition, cyclodextran-heptaose gave 95% inhibition. Moreover, water-insoluble glucan (mutan) synthesis by the glucosyltransferase from Streptococcus mutans was significantly repressed by the addition of cyclodextran.

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A genomic DNA library of Pseudomonas paucimobilis TMY1009 was constructed using a cosmid vector pWE15. Screening of the library for lignostilbene-α, β-dioxygenase (LSD) isozyme genes was done with a common probe for the α, β, and γ subunits, which composed LSD isozymes. The positive clones obtained by colony hybridization were further confirmed by Southern hybridization. A 4. 2-kb BamHI-HindIII fragment hybridized with the probe was subcloned into pUC118 to yield the plasmid pKHE1700. Escherichia coli MV1184 carrying pKHE1700 produced one of the LSD isozymes. The cloned LSD was purified and compared with LSD isozymes from P. paucimobilis TMY1009. The behavior on column chromatographies through all purification steps accorded with that of LSD-III. Furthermore' the mobility on polyacrylamide gel electrophoresis, the elution profile on reversed-phase HPLC, and the partial amino acid sequence were common to the cloned LSD and the native LSD-III. Thus the cloned LSD could be identified as LSD-III. It was found that the gene of LSD-III (lsdB) was composed of 489 codons.

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A genomic DNA encoding ribonuclease (RNase) T₁ from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe. The cloned gene (designated rntA) encoded functional RNase T₁, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T_l activity (over 200 times) than a transformant with a vector. A cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) with primers corresponding to the 5' terminus and 3' terminus of the reading frame of the rntA gene. Nucleotide sequencing analysis of both DNAs found that RNase T₁ had a prepro-sequence consisting of 26 amino acids and the rntA gene had only one intron (114 bp) in the region encoding the signal sequence. The A. oryzae transformant with cDNA controlled by the amyB promoter also showed higher activity (over 300 times), indicating that the cloned cDNA encoded functional RNase T₁. On the other hand, the Saccharomyces cerevisiae transformant with cDNA controlled by the GAL1 promoter could not grow on a medium containing galactose. These results suggests that A. oryzae may have a protection mechanism from RNase T₁.

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The cell growth-stimulating activity of lactoferrin (LF) in combination with epidermal growth factor (EGF) was evaluated by using a rat intestinal epithelial cell line, IEC-18. LF was found to be more effective than EGF for inducing an increase in cell numbers when cultured for over 6days using a medium containing 0. 2% fetal calf serum (FCS), although the 3H-thymidine incorporation-stimulating activity of EGF was more potent than that of LF. A synergistic effect of LF and EGF was observed in both cell proliferation and DNA synthesis assays. The increase in cell numbers when stimulated with LF plus EGF corresponded to about 5 times that of the control. Iron was not required for manifestation of these effects of LF. On the other hand, iron-saturated transferrin (TF) had cell-growth-stimulating activity, but iron-free TF did not, either in the presence or absence of EGF. These results indicate that LF induces cell proliferation by a mechanism distinct from that of TF. A pepsin-generated hydrolysate of LF (LFH) had an activity similar to that of undigested LF, and a peptide with cell-growth-stimulating activity from bovine LFH was isolated by monitoring its effects in combination with EGF on DNA synthesis in IEC-18 cells. Sequence analysis indicated that the peptide has the structure Ala-Glu-Ile-Tyr-Gly-Thr-Lys-Glu-Ser-Pro-Gln-Thr-His-Tyr-Tyr, corresponding to residues 79-93 of bovine LF.

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The activity of cerebroside synthase (UDP-glucose : ceramide glucosyltransferase) was found in the microsomal fraction of radish hypocotyls, and was studied. One % of the radioactivity due to UDP-[3H]glucose added to the membrane fraction was incorporated into cerebroside within 60min. Optimum pH and temperature of the activity were pH 7. 8 and 30°C, respectively. No metal ions enhanced the cerebroside synthase activity, dissimilar to that in animal tissues. The apparent Km for UDP-glucose was approximately 200μM. The same activity was also observed in radish roots and cotyledons, but proportions of UDP-[3H]glucose incorporation were slightly lower than that in hypocotyls. Exogenous ceramide species having trihydroxy sphingoid bases, which were major ceramide components of radish cerebrosides, usually stimulated cerebroside formation in microsomal fractions from radish seedlings, while any ceramide types having dihydroxy sphingoid bases were ineffective on the glucosylation reaction. It was assumed, therefore, that cerebroside synthase in radish seedlings would have substrate selectivity for ceramide species.

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Hydroxycinnamic acid derivatives of coffee beans were analyzed by three-dimensional HPLC. Thirteen peaks were detected at 320nm by a reversed-phase column and were categorized into 4 groups by UV-absorption sepectra. Eleven chlorogenic acids, caffeoyltryptophan, and a novel compound, p-coumaroyl-(L)-tryptophan, were identified by instrumental analyses.

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Prodigiosin 25-C had little effect on DNA, RNA, and protein synthesis, and cellular ATP content, but the drug markedly inhibited the incorporation of acetate into lipid fractions. Under the same conditions, the incorporation of other lipid precursors including glycerol, mevalonate, palmitate, and oleate was not affected. A decrease in the incorporation of acetate was not due to the inhibition of fatty acid biosynthesis, because prodigiosin 25-C did not affect the activity of acetyl-CoA synthetase, acetyl-CoA carboxylase or fatty acid synthase in cell-free assay systems prepared from rat liver cytosol. In contrast, prodigiosin 25-C strongly inhibited the rapid uptake of acetate into acid-soluble fraction in intact cells. The results suggest that prodigiosin 25-C specifically perturbs the permeation of acetate through plasma membranes.

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We have previously demonstrated that low-casein diets supplemented with cystine and threonine reduced hyperlipidemia and proteinuria in nephritic rats without noticeable protein malnutrition. In the present study, we examined whether or not a low-casein diet supplemented with methionine, sulfur amino acid other than cystine, and threonine would ameliorate the symptoms without protein malnutrition in rats with nephrotoxic serum nephritis by feeding experimental diets for 10 days. A methionine-threonine-supplemented 8. 5% casein diet (8. 5 CMT), when compared with a basal 20% casein diet, improved hypoalbuminemia as well as hyperlipidemia and proteinuria without noticeable growth retardation and fatty liver induction in nephritic rats. Fecal bile acid excretion and microsomal cholesterol 7α-hydroxylase activity were enhanced by 8. 5CMT feeding. These results suggest that amino acid-balanced low protein diet would have a beneficial effect on the symptoms of nephritis. They also suggest that the hypocholesterolemic action of 8. 5CMT may be, at least in part, due to increased fecal bile acid excretion accompanied by elevated microsomal cholesterol 7α-hydroxylase activity.

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Chitosan derivatives, sulfated N-acyl-chitosan (S-Cn-chitosan) possessing various lengths of alkyl chain, were prepared, and the properties of their aqueous solutions were examined. The 1H-NMR spectrum of D2O solutions of S-C12-chitosan showed broadening of the proton signals caused by aggregation of the alkyl chain. The solubility of a hydrophobic compounds, azobenzene, was small in the aqueous solutions of S-Cn-chitosan with shorter alkyl chains, but increased with increasing length of the chains above C 1o, showing that micelles had been formed. The ESR spectrum of a spin probe, TEMPO, in an S-C 14-chitosan solution showed the existence of a hydrophobic region in the solution, but this region did not exist in the S-C2-chitosan solution. The rigidity of this region was examined by using a spin probe, 16-doxyl-stearic acid. From these results, it was revealed that S-Cn-chitosan with longer alkyl chains formed a novel type of micelle called a "polymer micelle, "which was more stable than the ordinary micelles formed from low-molecular-weight surfactants.

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Asymmetric trans-esterification of meso-2, 5-dibromoadipate to ( - )-benzyl methyl 2, 5-dibromoadipate by lipase with subsequent chemical reactions afforded optically active cis-2, 5-disubstituted pyrrolidines. An equivalent asymmetric transformation was performed by selectively hydrolyzing a cis-2, 5-disubstituted pyrrolidine having a chiral N-substituent.

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Trehalose phosphorylase (EC 2. 4. 1. 64), which catalyzes the reversible reaction of phosphorolysis and synthesis of trehalose, was purified to homogeneity from a cell-free extract of Micrococcus varians strain No. 39. The enzyme was shown to have a molecular weight of 570, 000 to 580, 000 by gel filtration, and to have a subunit of molecular weight of 105, 000 by SDS-polyacrylamide gel electrophoresis. The stoichiometry of the reaction between trehalose, Pi, glucose, and β-glucose 1-phosphate was 1 : 1 : 1 : 1 (molar ratio). The enzyme had high specificity for trehalose, glucose, and β-glucose 1-phosphate. The Kms for trehalose, Pi, glucose, and β-glucose 1-phosphate were 10, 3. 1, 23, and 38mM, respectively. The Kcats were 200s-1 for trehalose phosphorolysis and 660 s - 1 for trehalose synthesis. The enzyme was inhibited by validamycin A, validoxylamine A, 1-deoxynojirimycin, and Cu2+ during trehalose phosphorolysis, and by Cu2+, Zn2+, and Ni2+ during trehalose synthesis. Inhibition competitive against trehalose was noted with validamycin A, validoxylamide A, and 1-deoxynojirimycin. Initial velocity, product inhibition, and dead-end inhibition studies suggested that both trehalose phosphorolysis and trehalose synthesis proceeded through an ordered Bi Bi mechanism.

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Previous studies have shown that selenium (Se) deficiency is associated with hematological abnormalities, which may result in an increased distribution of iron in various tissues. This report describes histological studies of the location of excess iron deposits in tissues. Male Wistar rats were fed a Torula yeast-based Se-deficient [Se( - )] or Se-adequate [Se( + ) ; containing 0. 1 mg Se/kg as sodium selenite] diet for 8 or 82weeks. Excised tissues were embedded in either paraffin or epoxy resin. A dramatic increase was observed in iron deposition in the liver and kidneys of rats on the Se( - ) diet. Prussian blue-stained sections under the light microscope showed iron deposits in the parenchymal cells and Kupffer cells of liver and in the proximal tubules of kidneys. The liver and kidneys of Se( - ) rats had considerably altered morphology : lysosomes were enlarged and contained electron-dense areas. X-Ray microanalysis showed that the areas that corresponded to the lysosomes contained iron. No iron deposits were observed in sections of kidney and liver from rats fed the Se( + ) diet. Thus, these studies identified subcellular sites of iron deposition in the liver and kidneys of Se( - ) rats. These iron deposits may be an important factor in the pathogenesis of Se deficiency.

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Aspyrone (1) was elaborated in an optically pure form by a key reaction involving the highly diastereoselective addition of tetrahydropyranone enolate to 2-tosyloxy-aldehyde and the subsequent in situ formation of an epoxide.

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A new immunohistochemical method for analyzing the localization of gibberellins was developed and applied to rice anthers. This method uses 1, 3-diisopropylcarbodiimide gas fixation to prevent the drift of gibberellins during the fixation procedure. Spikelets before anthesis were frozen in liquid nitrogen and lyophilized. They were then fixed with diisopropylcarbodiimide gas in a desiccator, which catalyzed amide formation between the 7-carboxyl groups of gibberellins and the amino groups of proteins in the tissue. The fixed spikelets were embedded in paraffin and sectioned, the sections being stained with the anti-GA 1-Me antibody and visualized by using an immunoperoxidase system. The anthers just before anthesis were stained well, while those 4-5days before heading were not stained. We analyzed the gibberellins in an extract of the rice anthers by an immunoassay, using the anti-GA 1-Me antibody. On the basis of the results, we conclude that the major substances stained in this immunohistochemical study should be 16α, 17-dihydroxy-16, 17-dihydrogibberellin A4-17-O-β-D-glucoside and 16α, 17-dihydroxy-16, 17-dihydrogibberellin A7-17-O-β- D-glucoside.

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A well-reproducible method for determining five common chlorophenols in soils is described. Chlorophenols were extracted from a soil with sodium hydroxide and, after being acidified to pH 6. 5, the extract was cleaned by partitioning between chloroform and sodium hydroxide. After adjusting the pH value to 5. 5, the sample was concentrated in a C-18 minicolumn. The chlorophenols were desorbed with methanol, the extract was basified, the solvent volume was reduced to 100μl, and the concentrated extract was acidified. An aliquot was analyzed by HPLC with UV detection (225 nm). Recovery experiments were performed at concentration levels of 1 ppm, 0. 1 ppm, and 0. 01 ppm. Recovery levels between 65% and 83% with a standard deviation of ±3. 75 were achieved at the concentration level of 0. 1 ppm. Detection limits between 2 ppb (2, 4, 6-trichlorophenol) and 2. 5 ppb (4-chloro-3-methylphenol) were determined.

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The polysaccharide fraction from Porphyra yezoensis (PASF) has already been shown to stimulate murine phagocytic functions in vivo and in vitro [Y. Yoshizawa et al, Biosci. Biotech. Biochem., 57, 1862-1866 (1993)]. In this study, various treatments were applied to PASF to assess its structure-function relationships. Desulfation of PASF decreased in vitro macrophage-stimulation activity, while further sulfation of PASF did not change the activity. Among 7 fractions obtained by anion-exchange chromatography of PASF, stronger activity was found in the fractions having a lower or higher sulfate content than in those having a medium sulfate content. Digests of PASF with β-agarase showed higher activity and solubility, and lower viscosity, than undigested PASF. These results indicate that the sulfate groups in PASF, probably porphyran, contributed to the macrophage stimulating activity, although a larger number of sulfate groups did not always cause stronger activity.

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We constructed two plasmids that have a strong tac promoter and a structural gene for tryptophanase of Enterobacter aerogenes SM-18 (pKT901EA) or Escherichia coli K-12 (pKT951EC). The tryptophanase activity of E. coli JM109 transformed with pKT901EA (JM109/pKT901EA) was inducible with isopropyl-β-D-thiogalactopyranoside, and 3. 6 times higher than that of E. aerogenes SM-18. Cells of JM109/pKT901EA induced for tryptophanase synthesized L-tryptophan from indole, ammonia, and Pyruvate more efficiently than E. aerogenes SM-18. Although JM109/pKT951EC expressed a similar level of tryptophanase activity to that of JM109/pKT901EA, the synthesis of L-tryptophan by the cells of JM109/pKT951EC did not proceed well compared with JM109/pKT901EA. Tryptophanases from E. aerogenes and E. coli K-12 were purified, and their properties were investigated. The purified E. aerogenes tryptophanase showed higher stability against heat inactivation than E. coli tryptophanase.

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A glycosidic fraction was extracted from fresh cherry leaves and treated with commercial β-glucosidase and the acetone powder from cherry leaves. After enzymatic hydrolysis, the formation of mod-erately antifungal benzaldehyde, benzyl alcohol, 2-phenylethanol and coumarin was confirmed by GLC and GC-MS. Thus, it was expected that the precursors of antifungal substances existed as glycosides in the leaves and would be hydrolyzed by the endogenous hydrolytic enzymes when the leaves were damaged. A survey of the constituents in the glycosidic fraction revealed the presence of benzyl β-D-glucoside and 2-phenylethyl β-D-glucoside, and of mandelonitrile β-D-glucosides, sambunigrin and prunasin.

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A proteinaceous component eluted at the concentration of 0. 8-1. 0 M NaCl was obtained from hen egg white by heparin affinity chromatography. On SDS-PAGE, the component was resolved into three bands of 18. 5, 16. 5, and 16. 0kDa. The isoelectric point was estimated to be pH 8. 8. This stimulated the DNA synthesis and proliferation of the cultured cells from chichen embryos. The amino acid composition and N-terminal analyses of the main 18. 5 kDa component showed that this is a novel minor protein in egg white, and therefore was named "Ovofactor-1".

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Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25MPa and 35oC.

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The tight junction (TJ) of the human intestinal Caco-2-SF monolayer was shown not to have been stably formed. TJ was stabilized by incubating the cell monolayer with β-lactoglobulin (β-Lg) and bovine serum albumin (BSA). Thus, the Caco-2-SF cell-culture system will provide a useful model for studying the factors which stabilize TJ and for investigating the mechanism of TJ regulation.

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Phosphoinositol kinase was purified from suspension-cultured rice (Oryza sativa L. ) cells. The apparent molecular mass of the rice enzyme was estimated to be 58kDa by SDS-PAGE and 234kDa by Toyopearl HW-55S gel filtration, indicating that it is a tetrameric enzyme. The enzyme absolutely required Mg2+ for the activity, but Mn2 + and Ca2 + did not affect it. In addition, this kinase phosphorylated inositol monophosphate to phytate. Judging from these data, rice phosphoinositol kinase was concluded to be a different enzyme distinguished from the other plant phosphoinositol kinases.

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The sweetness-suppressing polypeptide gurmarin has been isolated from the leaves of Gyntnenla sylvestre and consists of 35 amino acid residues including three intramolecular disulfide bonds. The primary structure has already been determined. The positions of the disulfide bonds were located, by a combination of mass spectrometric analysis and sequencing of cystine-containing peptides obtained by thermolysin-catalyzed hydrolysis of gurmarin, to be at CysFCys18, Cys10-Cys23, and Cys17-Cys33.

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A sandwich chemiluminescence enzyme immunoassay for measuring the level of VEGF/VPF in serum was constructed. The detectability of the assay is very low (1. O pg/ml) and the measurable range of the assay was very wide (1-1000 pg/ml). The assay showed that the average level of VEGF/VPF in human sera from healthy blood donors was approximately 19 pg/ml.

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Two cyclic ureide compound-hydrolyzing enzymes were found in Blastobacter sp. A17p-4, and partially purified. One hydrolyzed 5-substituted hydantoins D-stereospecifically and had dihydropyrimidinase activity. The other was a novel enzyme which should be called an imidase. The imidase preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds, and produced monoamidated dicarboxylates.

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An aquatic filamentous fungus, Saprolegnia sp. 28YTF-1, was found to accumulate odd chain polyunsaturated fatty acids (PUFAs)of 17 to 19 carbon chain length, when grown with fatty acids with 13, 15, or 17 carbons. A novel ω2 PUFA identified as 5, 8, 11, 14, 17-cis-nonadecapentaenoic acid (19 : 5ω2) was detected as one of the major PUFAs, together with 5, 8, 11, 14-cis-nona-decatetraenoic acid (19 : 4ω5), in both nonpolar and polar lipids from the mycelia. The biosynthetic route to 19 : 5ω2 was presumed to mimic the ω6 route to arachidonic acid (20 : 4ω6) and ω3 de-saturation of 20 : 4ω6 to 5, 8, 11, 14, 17-cis-eicosapentaenoic acid (20 : 5ω3), as follows : 15 : 0→17 : 0→17 : 1→17 : 2ω5→17 : 3ω5→19 : 4ω5 → 19 : 5ω2.

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A facultative anaerobe isolated from Korean domestic soil produced an antibacterial substance under strict anaerobic conditions. Based on the morphological and biochemical tests, and cellular fatty acid profiles, the anaerobe was identified as Streptococcus faecium. An antimicrobial compound produced from the S. faecium was identified as 3, 7, 12-trihydroxy-24-cholanic acid methylester on the basis of its physico-chemical analysis. This substance had potent antibacterial activities against a test organism harboring multiple antibiotic resistance markers, and a variety of pathogenic bacteria. The isolated S. faecium produced lactic acid as well as the antibiotic compound under the anaerobic conditions.

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( + )-Abscisyl β-D-glucopyranoside was isolated from mature Citrus unshu peelings as a germination inhibitor component. This is the first report of the direct isolation of ( + )-abscisyl β-D-gluco-pyranoside from this source.

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The endogenous levels of gibberellins (GAs) and abscisic acid (ABA) were investigated during the germination of barley seeds. The level of GA1 was highest on the second day of germination, while that of ABA was lowest on the first day. These results con-firm the hypothesis that GA and ABA regulate the induction of α-amylase in barley seeds during germination.

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Immunochemical relationships between salt-soluble proteins (albumins plus globulins) from buckwheat and indigo seeds were shown by immunoblot and immunodiffusion analyses using rabbit antisera raised against buckwheat globulins. These antigenic crossreactivities were roughly consistent with their polypeptide components judged by two-dimensional electrophoresis and SDS-PAGE analyses.

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Biological activity of intermediate steroids in the biosynthesis of brassinolide was examined by the rice-lamina inclination test and wheat-leaf unrolling test. In both bioassays, biological activity of intermediates increased according to the order in the biosynthetic pathway. The introduction of a hydroxyl group in the side chain (C-22), the step of 6-oxocampestanol to cathasterone, strikingly enhanced the activity. The activities of teasterone, 3-dehydrotea-sterone, and typhasterol were almost the same. Relative activities in the rice-lamina inclination test were as follows : 6-oxocampesta-nol (0. 002), cathasterone (1), teasterone (20), castasterone (240), and brassinolide (1200).

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Expression of a gene encoding the extracellular domain of the human growth hormone receptor (hGHR-ED) inserted into the genome of Autographa californica nuclear polyhedrosis virus was done using a desktop-scale spinner culture. Spodoptera frugiperda 9 (Sf9) cells infected with the recombinant virus secreted a protein with hGH-binding activity into the medium. Oxygen supplementation was required for high level secretion of the product. The highest cell production capability was estimated at more than 15 mg hGHR-ED/liter of culture. A protein-free medium supported the production similar to that obtained in traditional serum-containing media. This spinner culture system is simple to operate, and does not require expert knowledge of culture techniques.

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A new natural mosquito repellent was isolated from fresh leaves of Vitex rotundifolia. Its structure was elucidated by an extensive NMR spectral analysis to be a cyclopentene dialdehyde named rotundial. This compound possessed potent repelling activity against Aedes aegypti.

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Extracellular chitinase from marine Alteromonas sp. strain 0-7is unique because of the activation by four major cations contained in sea water, such as Na +, K +, Mg 2 +, and Ca 2 + . The positions of S-S bonds of Alteromonas chitinase were identified. Alteromonas chitinase was fragmented by TPCK-trypsin and Staphylococcus aureus V8 protease. The amino acid and sequence analyses of three peptides showed that the positions of disulfide bonds are Cys(94FCys(99), Cys(174/Cys(196), and Cys(386/Cys(395).

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L-Galactono-γ-lactone dehydrogenase, which catalyzes the final step of the biosynthesis of L-ascorbate, is bound to spinach mitochondrial membrane, as confirmed by linear sucrose density gradient centrifugation. The solubilized enzyme was very labile, but stabilized in the presence of L-galactono-γ-lactone under anaerobic conditions. The enzyme reduced cytochrome c and phenazine methosulfate in the presence of L-galactono-γ-lactone, but not when L-gulono-γ-lactone was used as an electron donor. The Kms of the enzyme for L-galactono-γ-lactone and cytochrome c were 192 μM and 180 μM, respectively.

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( - )-trans-Chrysanthenol (1) and ( + )-trans-chrysanthenyl acetate (2), which had been isolated from the dried kougiku flower, and their enantiomers were synthesized via optical resolution step starting from ( + )-verbenone.

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Klebsiella sp. LF 1202 was isolated as a bacterium that can assimilate adenosine as a sole source of carbon and nitrogen [F. Ling et al., Agric. Biol. Chem., 55, 573-575 (1991)] from a soil sample. Both the purine nucleoside phosphorylase (PNPase) and uridine phosphorylase (UPase) of this bacterium were induced simultaneously when the bacterium was cultured in a medium containing adenosine or uridine as a sole source of carbon and nitrogen. This induction profile is different from that of Escherichia coli. Here we cloned and sequenced the gene corresponding to each enzyme. The open reading frame (ORF) of the PNPase gene consisted of 717 bp that encoded a polypeptide of 239 amino acids with a molecular weight of 26, 198. The ORF of the UPase gene consisted of 8Mbp that encoded a polypetide of 278 amino acids with a molecular weight of 28, 912.

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A structural gene encoding the β-amylase that is abundant in the starchy endosperm of ungerminated barley seeds was isolated and characterized. It was 3825bp in length. In the sequence of the structural gene, a sequence identical to that of the cDNA was found to contain seven exons and six introns.

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The nucleotide sequence of the pdh gene coding for PheDH from Bacillus badius IAM 11059 was analyzed. The gene consists of an ORF of 1140 nucleotides which specifies a protein of 380 codons. The primary structure of PheDH is similar to PheDH from B. sphaericus, PheDH from Themoactinomyces intermedius, and leucine dehydrogenase from B. stearothemophilus, etc.

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An ice nucleation-active strain of Erwinia uredovora shed vesicles when cultured at low temperature (10°C). We isolated an ice nucleation-active vesicle fraction from the culture medium by ultrafiltration, ultracentrifugation, and gel filtration. Western blot analysis showed that this cell-free vesicle fraction contained an ice nucleation-active protein (InaU). The process of the InaU transport to a shed vesicle was examined by immunohistochemical analysis using electron microscopy. The examination showed the following successive processes : InaU molecules first assemble around the inner membrane, then the assembly enters a vesicle just forming on the surface of the outer membrane, and finally the vesicle, 100-400 nm in diameter, leaves the surface to be shed with InaU molecules occluded .

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