Contents and Abstracts of BBB

(Vol.68 No.2 2004)

Protective Effects of Quercetin and Its Metabolites on H2O2-Induced Chromosomal Damage to WIL2-NS Cells
Akiko Saito,1,† Ayako Sugisawa,2 Keizo Umegaki,2 and Hiroyuki Sunagawa1 p.271

Cloning and Sequencing of the ura3 and ura5 Genes, and Isolation and Characterization of Uracil Auxotrophs of the Fungus Mortierella alpina 1S-4
Seiki Takeno,1 Eiji Sakuradani,1 Shoichi Murata,1 Misa Inohara-Ochiai,2 Hiroshi Kawashima,3 Toshihiko Ashikari,2 and Sakayu Shimizu1,† p.277

Isolation of Thermophilic Ammonium-tolerant Bacterium and Its Application to Reduce Ammonia Emission during Composting of Animal Wastes
Kazutaka Kuroda,1,† Dai Hanajima,1 Yasuyuki Fukumoto,1 Kazuyoshi Suzuki,1 Shinichi Kawamoto,2 Jun Shima,2 and Kiyonori Haga1 p.286

Identification of Nucleotide Binding Sites in V-Type Na+-ATPase from Enterococcus hirae
Toshiaki Hosaka,1 Takeshi Murata,2 Yoshimi Kakinuma,3 and Ichiro Yamato1,† p.293

A Novel Method for Producing a Foodstuff from Defatted Black Sesame Seed That Inhibits Allergen Absorption
Shoko Kobayashi,1,† Jun Watanabe,2 Jun Kawabata,3 Eri Fukushi,3 and Hiroshi Shinmoto2 p.300

Characterization of Two Genes Encoding Putative Cysteine Synthase Required for Cysteine Biosynthesis in Schizosaccharomyces pombe
Yasuko Fujita1,2 and Kaoru Takegawa1,† p.306

MS Fragment Isotope Ratio Analysis for Evaluation of Citrus Essential Oils by HRGC-MS
Atsushi Satake,1,2,† Kiyoshi Furukawa,2 Takao Ueno,1 Hiroyuki Ukeda,1 and Masayoshi Sawamura1 p.312

Pseudomonas putida NCTC 10936 Balances Membrane Fluidity in Response to Physical and Chemical Stress by Changing the Saturation Degree and the Trans/cis Ratio of Fatty Acids
Norbert Loffhagen,† Claus Hartig, and Wolfgang Babel p.317

Molecular Cloning, Functional Expression, and Mutagenesis of cDNA Encoding Class I Chitinase from Rye (Secale cereale) Seeds
Takayuki Ohnuma,1 Toki Taira,2 Takeshi Yamagami,1 Yoichi Aso,1 and Masatsune Ishiguro1,† p.324

Cytotoxicity and Oxidative Stress Induced by the Glyceraldehyde-related Maillard Reaction Products for HL-60 Cells
Teruyuki Usui,1 Satomi Shizuuchi,1 Hirohito Watanabe,2 and Fumitaka Hayase1,† p.333

Molecular Cloning and Expression of the Gene Encoding Family 19 Chitinase from Streptomyces sp. J-13-3*
Katsuichiro Okazaki,1,† Yousuke Yamashita,1 Minoru Noda,1 Noriyuki Sueyoshi,1 Isamu Kameshita,1 and Shigeru Hayakawa2 p.341

Soybean β-Conglycinin Diet Suppresses Serum Triglyceride Levels in Normal and Genetically Obese Mice by Induction of β-Oxidation, Downregulation of Fatty Acid Synthase, and Inhibition of Triglyceride Absorption
Tatsuya Moriyama,† Keiko Kishimoto, Kiyoko Nagai, Reiko Urade, Tadashi Ogawa, Shigeru Utsumi, Nobuyuki Maruyama, and Motohiro Maebuchi p.352

Identification of Single Base-pair Mutation on uidA Gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) Mediated PCR Clamping
Toshiyuki Takiya,1,4 Satoshi Futo,4 Mika Tsuna,4 Takanori Namimatsu,5 Tetsuya Sakano,5 Keiichi Kawai,1,2 and Tohru Suzuki1,3,† p.360

Structure-activity Relationships for α-Glucosidase Inhibition of Baicalein, 5,6,7-Trihydroxyflavone: the Effect of A-Ring Substitution
Hong Gao, Tetsuo Nishioka, Jun Kawabata,† and Takanori Kasai p.369

Structural and Functional Characterization of Ovotransferrin Produced by Pichia pastoris
Kimihiko Mizutani,1 Ikuko Okamoto,1 Kiyotaka Fujita,2 Kenji Yamamoto,2 and Masaaki Hirose1,† p.376

Ingestion of Raffinose Promotes Calcium Absorption in the Large Intestine of Rats
Rieko Mitamura, Hiroshi Hara,† and Yoritaka Aoyama p.384

Biotransformation of Cystothiazole A, a Myxobacterial Antibiotic, into Novel Derivatives by the Mother Producer, Cystobacter fuscus
Yoshihiro Suzuki, Makoto Ojika,† and Youji Sakagami p.390

Ureases of Extreme Halophiles of the Genus Haloarcula with a Unique Structure of Gene Cluster
Toru Mizuki,1 Masahiro Kamekura,2,† Shiladitya DasSarma,3 Tadamasa Fukushima,1 Ron Usami,1 Yasuhiko Yoshida,1 and Koki Horikoshi1 p.397

Absolute Configuration of a Polyol Fragment of Blasticidin A, a Specific Inhibitor of Aflatoxin Production
Shohei Sakuda,† Hiroyuki Ikeda, Takefumi Nakamura, and Hiromichi Nagasawa p.407

Purification, Characterization, and Subsite Affinities of Thermoactinomyces vulgaris R-47 Maltooligosaccharide-metabolizing Enzyme Homologous to Glucoamylases
Kazuhiro Ichikawa, Takashi Tonozuka, Rie Uotsu-Tomita,* Hiromi Akeboshi, Atsushi Nishikawa, and Yoshiyuki Sakano† p.413

Note
Inhibition of the Bacterial Surface Protein Anchoring Transpeptidase Sortase by Isoquinoline Alkaloids
Soo-Hwan Kim,1 Dong-Sun Shin,1 Mi-Na Oh,2 Soon-Chun Chung,2 Jang-Suk Lee,2 and Ki-Bong Oh1,2,† p.421

Note
New Trifluoromethanesulfonanilide Compounds Having High Miticidal Activity against House Dust Mites
Tatsuya Mori,† Yoji Takada, Makoto Hatakoshi, and Noritada Matsuo p.425

Note
Isoflavones Inhibit 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase in Vitro
Jang Hoon Sung,1 Sung-Joon Lee,2 Kwan Hwa Park,1 and Tae Wha Moon1,† p.428

Note
Effects of Ionized Cyclodextrin on Decomposition of Allyl Isothiocyanate in Alkaline Solutions
Yoshio Ohta,1,† Kenichi Takatani,1,* and Shunro Kawakishi2 p.433

Note
Suppressive Effects of Natural and Synthetic Agents on Dextran Sulfate Sodium-induced Interleukin-1β Release from Murine Peritoneal Macrophages
Ki Han Kwon, Akira Murakami, and Hajime Ohigashi† p.436

Note
Specification of Amino Acid Residues Essential for the Catalytic Reaction of Cold-active Protein-tyrosine Phosphatase of a Psychrophile, Shewanella sp.
Hiroki Tsuruta,2,† Jun Tamura,1 Hiroshi Yamagata,1 and Yasuo Aizono1 p.440

Note
The Effect of Pre-germinated Brown Rice Intake on Blood Glucose and PAI-1 Levels in Streptozotocin-induced Diabetic Rats
Hiromi Hagiwara, Taiichiro Seki,† and Toyohiko Ariga p.444

Note
Dehydrotrametenonic Acid and Dehydroeburiconic Acid from Poria cocos and Their Inhibitory Effects on Eukaryotic DNA Polymerase α and β
Toshihiro Akihisa,1,† Yoshiyuki Mizushina,2 Motohiko Ukiya,1 Manabu Oshikubo,1 Seizo Kondo,3 Yumiko Kimura,4 Takashi Suzuki,4 and Takaaki Tai5 p.448

Note
Reduction in Photostability by the Esterification of β -Cryptoxanthin
Shin Arita,1 Kojiro Otsuki,2 Ken-ichi Osaki,1 Yoshiyuki Murata,2 Yasuaki Shimoishi,2 and Mikiro Tada1,† p.451

Note
“Aqua-space®”, a New Headspace Method for Isolation of Natural Floral Aromas Using Humidified Air as a Carrier Gas
Masashi Ishikawa,1,† Tsutomu Honda,1 Akira Fujita,1 Yoshiko Kurobayashi,1 and Takeshi Kitahara2 p.454

Note
Transcriptional Analysis of the ostA/imp Gene Involved in Organic Solvent Sensitivity in Escherichia coli
Iwao Ohtsu,1 Nobuto Kakuda,1 Norihiko Tsukagoshi,1 Noriyuki Dokyu,1 Hiroshi Takagi,2 Masaaki Wachi,3,† and Rikizo Aono1 p.458

Note
Comparative Studies of the AHP Histidine-containing Phosphotransmitters Implicated in His-to-Asp Phosphorelay in Arabidopsis thaliana
Yasushi Tanaka, Tomomi Suzuki, Takafumi Yamashino, and Takeshi Mizuno† p.462

Note
Hydrogen Gas Generation from Refuse-derived Fuel (RDF) under Wet Conditions
Makiko Sakka,† Tetsuya Kimura, Kazuo Sakka, and Kunio Ohmiya p.466

Communication
Isolation of Genes Specifically Expressed in the Fruit Body of the Edible Basidiomycete Lentinula edodes
Tatsuya Hirano,1,† Toshitsugu Sato,2 and Hitoshi Enei2 p.468

Communication
Nitrous Oxide-forming Codenitrification Catalyzed by Cytochrome P450nor
Fei Su,1 Naoki Takaya,1 and Hirofumi Shoun2,† p.473

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Protective Effects of Quercetin and Its Metabolites on H2O2-Induced Chromosomal Damage to WIL2-NS Cells

Akiko Saito,1,† Ayako Sugisawa,2 Keizo Umegaki,2 and Hiroyuki Sunagawa1

1Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo, Hokkaido 060-0819, Japan
2National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan

We investigated chromosomal damage caused by a typical flavonoid, quercetin, and its two conjugates, quercetin-3-O-sulfate and isorhamnetin, and their protective effects against chromosomal damage induced by H2O2. The chromosomal damage was detected by the cytokinesis-block micronucleus (CBMN) assay using a lymphoblastoid cell line, WIL2-NS. We found that quercetin itself induced chromosomal damage at 10 μ M, but quercetin-3-O-sulfate and isorhamnetin did not induce damage up to 30 μ M. In the medium used for the CBMN assay, quercetin (at 100 μ M) generated a high concentration of H2O2, but the two conjugates did not at the same concentration. On the other hand, pretreatment with quercetin (at 1 μ M), quercetin-3-O-sulfate (at 10 μ M), and isorhamnetin (at 5 μ M) prevented H2O2-induced chromosomal damage to WIL2-NS cells. These findings suggest that the induction and prevention of H2O2-induced chromosomal damage are different between quercetin and its metabolites.

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Cloning and Sequencing of the ura3 and ura5 Genes, and Isolation and Characterization of Uracil Auxotrophs of the Fungus Mortierella alpina 1S-4

Seiki Takeno,1 Eiji Sakuradani,1 Shoichi Murata,1 Misa Inohara-Ochiai,2 Hiroshi Kawashima,3 Toshihiko Ashikari,2 and Sakayu Shimizu1,†

1Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Institute for Advanced Technology, Suntory Ltd., Osaka 618-0001, Japan
3Institute for Food & Beverage Health Care Science Laboratory, Suntory Ltd., Osaka 618-0001, Japan

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5’-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.

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Isolation of Thermophilic Ammonium-tolerant Bacterium and Its Application to Reduce Ammonia Emission during Composting of Animal Wastes

Kazutaka Kuroda,1,† Dai Hanajima,1 Yasuyuki Fukumoto,1 Kazuyoshi Suzuki,1 Shinichi Kawamoto,2 Jun Shima,2 and Kiyonori Haga1

1National Institute of Livestock and Grassland Science, National Agriculture and Bio-oriented Research Organization, Ikenodai 2, Tukuba, Ibaraki 305-0901, Japan
2National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan

A thermophilic bacterium, strain TAT105, was isolated from compost made of animal wastes. TAT105 had high tolerance to ammonium nitrogen up to 1200 mM, and highly assimilated nitrogen during the growth on swine feces. The strain was classified into Bacillus, close to Bacillus pallidus. To evaluate the effect of adding TAT105 to ammonia (NH3) emission during the composting process of animal wastes, laboratory scale composting was done. NH3 emission tended to be lower and nitrogen loss was smaller in the TAT105-added material than in the control material to which TAT105 was not added. Thermophilic ammonium-tolerant bacteria in the TAT105-added material increased to about 8 × 109 CFU/g of dry matter on the average during the tests, and most of them were judged to be TAT105 from morphological colony discrimination. These results suggested the possibility of reducing NH3 emission from composting of animal wastes by adding TAT105.

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Identification of Nucleotide Binding Sites in V-Type Na+-ATPase from Enterococcus hirae

Toshiaki Hosaka,1 Takeshi Murata,2 Yoshimi Kakinuma,3 and Ichiro Yamato1,†

1Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan
2The Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK
3Department of Applied Chemistry, Muroran Institute of Technology, 27-1 Mizumoto-cho, Muroran-shi, Hokkaido 050-8585, Japan

A and B subunits of the V-type Na+-ATPase from Enterococcus hirae were suggested to possess nucleotide binding sites (Murata, T. et al., J. Biochem., 132, 789-794 (2002)), although the B subunit did not have the consensus sequence for nucleotide binding. To further characterize the binding sites in the V-ATPase, we did the photoaffinity labeling study using 8-azido-[ α -32P]ATP. A and B subunits were labeled with 8-azido-[ α -32P]ATP when analysed with SDS polyacrylamide gel electrophoresis. The peptide fragment of A subunit obtained by lysyl endopeptidase digestion after labeling showed a molecular size of 9 kDa and its amino acid sequencing revealed that it corresponded to residues Arg423-Lys494. The peptide fragment from B subunit after photoaffinity labeling and lysyl endopeptidase digestion showed the size of 5 kDa and corresponded to residues Phe404-Lys443. In our structure model, these peptides were close to the adenine ring of ATP. We suggest that non-catalytic B subunit of E. hirae V-ATPase has a nucleotide binding site, similarly to eukaryotic V-ATPases and F-ATPases.

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A Novel Method for Producing a Foodstuff from Defatted Black Sesame Seed That Inhibits Allergen Absorption

Shoko Kobayashi,1,† Jun Watanabe,2 Jun Kawabata,3 Eri Fukushi,3 and Hiroshi Shinmoto2

1Department of Food and Life-science, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan
2Food Function Division, National Food Research Institute, Tsukuba 305-8642, Japan
3Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

A method is proposed to produce a foodstuff that inhibits allergen absorption through the intestinal tract. Defatted black sesame (Sesamum indicum) seeds as a starting material were hydrolyzed with a crude preparation of trypsin at 40°C and pH 8 for 3 hrs while gently stirring to generate an active peptide. The resulting hydrolysate was heated to inactivate the trypsin and make the active components soluble. An extract was obtained by centrifugation and then freeze-dried. Ser-Asn-Ala-Leu-Val-Ser-Pro-Asp-Trp-Ser-Met-Thr-Gly-His (compound 1) as an active peptide, and sesamino1 2’-O- β -glucopyranosyl-(1→2)-O-[ β -glucopyranosyl(1→6)]-O- β -glucopyranoside (compound 2) and sesamino1 2’-O- β -glucopyranosyl (1 → 2)-O- β -glucopyranoside (compound 3) were identified as active lignan glycosides in an in vitro model by using Caco-2 cells. Compound 1 was active at 10-7 M and compounds 2 and 3 at 10-5 M.

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Characterization of Two Genes Encoding Putative Cysteine Synthase Required for Cysteine Biosynthesis in Schizosaccharomyces pombe

Yasuko Fujita1,2 and Kaoru Takegawa1,†

1Department of Life Sciences, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan
2Research Center, Asahi Glass Co., Ltd., Kanagawa, Yokohama 221-8755, Japan

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the S. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04 ), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.

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MS Fragment Isotope Ratio Analysis for Evaluation of Citrus Essential Oils by HRGC-MS

Atsushi Satake,1,2,† Kiyoshi Furukawa,2 Takao Ueno,1 Hiroyuki Ukeda,1 and Masayoshi Sawamura1

1Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan
2Nagaoka Perfumery Co., Ltd., 3-30, 1-chome, Itsukaichi, Ibaraki-City, Osaka 567-0005, Japan

To evaluate the origin of citrus essential oils, the isotope ratio of fragment peaks on HRGC-MS of the volatile compounds from various citrus oils was measured. The MS fragment ratio was found by the ratio of fragment peak intensity, m + 1/m (m/z). This ratio reflects the isotope effect of volatile compounds, that is, it provides information about locality, quality, and species for essential oils. Multivariate analysis based on the MS fragment ratio of monoterpene hydrocarbons clearly distinguished three citrus species, yuzu, lemon, and lime. The carbonyl fractions were also extracted from citrus essential oils by the sodium hydrogensulfite method. The isotope ratio of MS fragments of octanal, nonanal, and decanal was also examined. The results suggest that there was no significant difference in the individual fragment isotope ratios of the three aldehydes.

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Pseudomonas putida NCTC 10936 Balances Membrane Fluidity in Response to Physical and Chemical Stress by Changing the Saturation Degree and the Trans/cis Ratio of Fatty Acids

Norbert Loffhagen,† Claus Hartig, and Wolfgang Babel

UFZ Centre for Environmental Research Leipzig-Halle Ltd., Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany

This study explored the capability of Pseudomonas putida NCTC 10936 to maintain homeoviscosity after changing the growth temperature, incubating resting cells at different temperatures or at a constant temperature in the presence of 4-chlorophenol (4-CP). After raising the growth temperature from 20 to either 30 or 35°C, the degree of saturation of the organism’s fatty acids increased and the ratio of trans to cis unsaturated fatty acids decreased somewhat. In contrast, after the incubation temperature of resting cells was raised (grown at 30°C) from 20 to 30 or 35°C the degree of saturation of the fatty acids remained nearly constant, while the ratio of trans to cis unsaturated fatty acids increased. Incubating resting cells (grown at 30°C) at 20°C in the presence of 4-CP again caused no major changes in the degree of saturation, but cis to trans conversion of unsaturated fatty acids was induced, with a corresponding increase in the trans/cis ratios. Increases in both the saturation degree of the fatty acids and the trans/cis ratio of the unsaturated fatty acids correlated with increases in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene intercalated in the bilayers of liposomes prepared from the cells of P. putida NCTC 10936.
Electron transport phosphorylation (ETP) could be stabilized by adaptive adjustments in the fluidity of the cytoplasmic membrane mediated by changes in fatty acid composition such as those observed. Whether changes in the degree of saturation or in the trans/cis ratio are more effective can be decided by studying P. putida NCTC 10936.

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Molecular Cloning, Functional Expression, and Mutagenesis of cDNA Encoding Class I Chitinase from Rye (Secale cereale) Seeds

Takayuki Ohnuma,1 Toki Taira,2 Takeshi Yamagami,1 Yoichi Aso,1 and Masatsune Ishiguro1,†

1Laboratory of Protein Chemistry and Engineering, Graduate School of Bioresource and Environmental Science, Kyushu University, Fukuoka 812-8581, Japan
2Department of Bioscience and Biotechnology, Faculty of Agriculture, Ryukyu University, Okinawa 903-0213, Japan

A cDNA encoding rye seed chitinase-a (RSC-a) was cloned by rapid amplification of cDNA ends and PCR procedures. It consists of 1,191 nucleotides and encodes an open reading frame of 321 amino acid residues. Recombinant RSC-a (rRSC-a) was produced in the oxidative cytoplasm of Escherichia coli Origami(DE3) in a soluble form by inducing bacteria at a low temperature (20°C). Purified rRSC-a showed properties similar to the original enzyme from rye seeds in terms of chitinase activity toward a soluble substrate, glycolchitin, and an insoluble substrate, chitin beads, in chitin-binding ability to chitin, and in antifungal activity against Trichoderma sp. in vitro. rRSC-a mutants were subsequently produced and purified by the same procedures as those for rRSC-a. Mutation of Trp23 to Ala decreased the chitinase activity toward both substrates and impaired the chitin-binding ability. Furthermore, the antifungal activity of this mutant was weakened with increasing of the NaCl concentration in the culture medium. Complete abolishment of both activities was observed upon the mutation of Glu126 to Gln. The roles of these residues in both activities are discussed.

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Cytotoxicity and Oxidative Stress Induced by the Glyceraldehyde-related Maillard Reaction Products for HL-60 Cells

Teruyuki Usui,1 Satomi Shizuuchi,1 Hirohito Watanabe,2 and Fumitaka Hayase1,†

1Department of Agricultural Chemistry, Faculty of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
2Department of Life Sciences, Faculty of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan

We demonstrated the cytotoxicity of glyceraldehyde-related Maillard reaction products for HL-60 cells. Glyceraldehyde-modified bovine serum albumin and glyceraldehyde-modified casein inhibited the proliferation of HL-60 cells. The reaction products formed from glyceraldehyde and Nα-acetyllysine had also a cytotoxic effect on HL-60 cells. The cytotoxic effect was prevented by N-acetylcysteine or pyrrolidinedithiocarbamate as the antioxidants. In addition, the reaction products depressed the intracellular glutathione level, and induced the reactive oxygen species (ROS) production. These results suggested that the glyceraldehyde-related advanced glycation end products (AGEs) induced the cytotoxicity and the oxidative stress.
We previously reported that the glyceraldehyde-related AGE was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named GLAP (glyceraldehyde-derived pyridinium compound), formed from glyceraldehyde and Nα-acetyllysine (Biosci. Biotechnol. Biochem., 67, 930-932 (2003)). In this study, GLAP inhibited the proliferation of HL-60 cells, and the inhibitory effect was prevented by the antioxidants. Furthermore, GLAP depressed the intracellular glutathione level, and induced the ROS production.
This work indicated the possibility that the cytotoxicity and the oxidative stress in the progression of diabetic complications and chronic renal disease might be induced by GLAP.

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Molecular Cloning and Expression of the Gene Encoding Family 19 Chitinase from Streptomyces sp. J-13-3*

Katsuichiro Okazaki,1,† Yousuke Yamashita,1 Minoru Noda,1 Noriyuki Sueyoshi,1 Isamu Kameshita,1 and Shigeru Hayakawa2

1Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan
2Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40°C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.

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Soybean β-Conglycinin Diet Suppresses Serum Triglyceride Levels in Normal and Genetically Obese Mice by Induction of β-Oxidation, Downregulation of Fatty Acid Synthase, and Inhibition of Triglyceride Absorption

Tatsuya Moriyama,† Keiko Kishimoto, Kiyoko Nagai, Reiko Urade, Tadashi Ogawa, Shigeru Utsumi, Nobuyuki Maruyama, and Motohiro Maebuchi

Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

The purpose of this study was to discover the effects of soybean β-conglycinin (7S-globulin) and glycinin (11S-globulin) on serum lipid levels and metabolism in the livers of normal and genetically obese mice. Male normal (ICR) and obese (KK-Ay) mice were fed ad libitum high fat diets for two weeks, followed by a 2-week restriction of diet (2 g diet/mouse/day) containing 20% casein, soybean β-conglycinin, or soybean glycinin, and then sacrificed immediately. Serum triglyceride (TG), glucose, and insulin levels of β-conglycinin-fed mice were lower than in casein- and glycinin-fed mice of both strains. In order to analyze the related events to these effects, enzyme activities and relative mRNA levels of lipid metabolism-related proteins were measured. The activities of two enzymes related to fatty acid β-oxidation were higher while that of fatty acid synthase was lower in livers of β-conglycinin-fed mice than of casein-fed both mice. Messenger RNA levels of acyl-CoA oxidase (fatty acid β-oxidation related enzyme) were significantly higher in livers of β-conglycinin-fed mice than of both casein-fed mice. On the contrary, mRNA levels of SREBP-1 and 2 tended to be lowered in livers of soy protein-fed mice than of both casein-fed mice. Fecal excretion of TG was higher in β-conglycinin-fed mice than in casein-fed mice. Our results demonstrated that the soy β-conglycinin diet reduced serum TG levels by acceleration of β-oxidation, suppression of fatty acid synthase and/or increased TG fecal excretion, and also diminished serum glucose and insulin levels. Some of these events might be caused at the transcriptional levels, judged from the result that relative messenger RNA levels of lipid metabolism-related proteins were altered. These results suggest that soy β-conglycinin could be a potentially useful dietary protein source for the prevention of hypertriglyceridemia, hyperinsulinemia, and hyperglycemia, which are recognized as risk factors for atherosclerosis.

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Identification of Single Base-pair Mutation on uidA Gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) Mediated PCR Clamping

Toshiyuki Takiya,1,4 Satoshi Futo,4 Mika Tsuna,4 Takanori Namimatsu,5 Tetsuya Sakano,5 Keiichi Kawai,1,2 and Tohru Suzuki1,3,†

1United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
2Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
3Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
4Nippon Flour Mills Co., Ltd., Atsugi, Kanagawa 243-0041, Japan
5Zen-noh Institute of Animal Health, Sakura, Chiba 285-0043, Japan

Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57°C and 45°C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results.

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Structure-activity Relationships for α-Glucosidase Inhibition of Baicalein, 5,6,7-Trihydroxyflavone: the Effect of A-Ring Substitution

Hong Gao, Tetsuo Nishioka, Jun Kawabata,† and Takanori Kasai

Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 060-8589, Japan

In order to estimate the effects of the A-ring hydroxyl group of baicalein (5,6,7-trihydroxyflavone, 1) on rat intestinal α-glucosidase inhibition, flavone, monohydroxyflavones, dihydroxyflavones, and methylated derivatives of 5,6,7-trihydroxyflavone were used for the structure-activity relationship (SAR) study. The importance of the 6-hydroxyl group of baicalein was validated for an exertion of the activity. And also, the tested flavones which lacked a hydroxyl substituent on any of positions 5, 6, or 7, showed no activity. Hence, the 5,6,7-trihydroxyflavone structure was concluded to be crucial for the potent inhibitory activity. In addition, an introduction of electron-withdrawing or electron-donating groups at position 8 of baicalein led to a dramatic decrease for activity, except for 8-fluoro-5,6,7-trihydroxyflavone, which carried a less bulky substituent on position 8. Hence, this result suggested that a sterically bulky substituent on C-8 of baicalein was detrimental for the activity regardless of its electronic nature. Through examining the inhibitory mechanism of baicalein against rat intestinal α-glucosidase, it was suggested to be a mixed type inhibition.

-15-
Structural and Functional Characterization of Ovotransferrin Produced by Pichia pastoris

Kimihiko Mizutani,1 Ikuko Okamoto,1 Kiyotaka Fujita,2 Kenji Yamamoto,2 and Masaaki Hirose1,†

1The Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan
2Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin. We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system. The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly. The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l. The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein. The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein. Endoglycosidases, such as endo-β-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF), and endo-β-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms. The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac and 10 mannoses.

-16-
Ingestion of Raffinose Promotes Calcium Absorption in the Large Intestine of Rats

Rieko Mitamura, Hiroshi Hara,† and Yoritaka Aoyama

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan

We examined the effects of feeding raffinose on intestinal calcium absorption in ovariectomized rats by two separate experiments. In experiment 1, female Sprague-Dawley rats (6 wk old) were divided into two groups: sham operation and ovariectomy, and fed diets with or without raffinose (30 g/kg diet) for 4 wk. In experiment 2, ovariectomized rats with cecocolonectomy or transsection and reanastomosis (sham) were divided into two groups as in experiment 1 and fed the same diets for 3 wk. In experiment 1, calcium absorption was lower in the ovariectomized rats than in the sham rats but calcium absorption in rats fed the raffinose diet was higher than that in rats fed the raffinose-free diet. In experiment 2, increased calcium absorption in the raffinose group was abolished by cecocolonectomy. The impaired absorption in ovariectomized rats was restored by feeding raffinose. The large intestine is involved in the beneficial effects of raffinose.

-17-
Biotransformation of Cystothiazole A, a Myxobacterial Antibiotic, into Novel Derivatives by the Mother Producer, Cystobacter fuscus

Yoshihiro Suzuki, Makoto Ojika,† and Youji Sakagami

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

The bioconversion of the myxobacterial antibiotic, cystothiazole A, by the antibiotic producer, Cystobacter fuscus, was investigated. In our previous study, an adsorbent resin was added to the fermentation mixture to achieve high productivity of cystothiazole A, the major and most active component. On the other hand, a relative increase in the metabolic derivatives of cystothiazole A was observed when cultured without the resin. Furthermore, when cystothiazole A was externally added to the culture of C. fuscus without the resin, cystothiazole A was rapidly metabolized by the culture to a number of polar metabolic derivatives, among them being novel ones. The identification and structural elucidation of the known and novel derivatives were performed by spectroscopic analyses. Based on the time-dependent production profile and chemical structures of these derivatives, pathways for the conversion of cystothiazole A to the more polar derivatives of this antibiotic by C. fuscus are proposed.

-18-
Ureases of Extreme Halophiles of the Genus Haloarcula with a Unique Structure of Gene Cluster

Toru Mizuki,1 Masahiro Kamekura,2,† Shiladitya DasSarma,3 Tadamasa Fukushima,1 Ron Usami,1 Yasuhiko Yoshida,1 and Koki Horikoshi1

1Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe 350-8585, Japan
2Noda Institute for Scientific Research, 399 Noda, Noda 278-0037, Japan
3Center for Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, U.S.A.

We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method. Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts. Only 4 strains of the genus Haloarcula, Har. aidinensis, Har. hispanica, Har. japonica, and Har. marismortui were finally shown as the urease producers. A partially purified urease from Har. hispanica was a typical halophilic enzyme in that it showed maximum activity at 18-23% NaCl and lost the activity irreversibly in the absence of NaCl. Partial genes (1596 bp) of the urease encoding from upstream of the β subunit down to the N-terminal 139 amino acids of the α subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains. Strains of other genera, which were urease-negative, did not yield PCR products. The deduced amino acid sequences of the β subunit and partial α subunit were similar to each other (92-100% similarities) and to those from other organisms. Analysis of the draft genome sequence of Har. marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was β-α-γ-UreG-UreD-UreE-UreF. This order is quite unique, since in other microorganisms the order is γ-β-α-UreE-UreF-UreG-UreD in most cases. No open reading frames were detected in the PCR-amplified upstream of the β subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.

-19-
Absolute Configuration of a Polyol Fragment of Blasticidin A, a Specific Inhibitor of Aflatoxin Production

Shohei Sakuda,† Hiroyuki Ikeda, Takefumi Nakamura, and Hiromichi Nagasawa

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Blasticidin A (1) and aflastatin A (2), Streptomyces metabolites with similar structures, are specific inhibitors of aflatoxin production by Aspergillus parasiticus. The stereochemistry of the polyol fragment of 1 (3a) containing ten chiral centers was elucidated by applying acetonide and MTPA methods to a variety of acetonide derivatives of 3a, which determined the absolute configuration of 3a. By using the similar methods, the absolute configuration of the polyol fragment of 2 (4a) was determined, which was the same as that elucidated by J-based and other chemical methods previously.

-20-
Purification, Characterization, and Subsite Affinities of Thermoactinomyces vulgaris R-47 Maltooligosaccharide-metabolizing Enzyme Homologous to Glucoamylases

Kazuhiro Ichikawa, Takashi Tonozuka, Rie Uotsu-Tomita,* Hiromi Akeboshi, Atsushi Nishikawa, and Yoshiyuki Sakano†

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

A maltooligosaccharide-metabolizing enzyme from Thermoactinomyces vulgaris R-47 (TGA) homologous to glucoamylases does not degrade starch efficiently unlike most glucoamylases such as fungal glucoamylases (Uotsu-Tomita et al., Appl. Microbiol. Biotechnol., 56, 465-473 (2001)). In this study, we purified and characterized TGA, and determined the subsite affinities of the enzyme. The optimal pH and temperature of the enzyme are 6.8 and 60°C, respectively. Activity assays with 0.4% substrate showed that TGA was most active against maltotriose, but did not prefer soluble starch. Kinetic analysis using maltooligosaccharides ranging from maltose to maltoheptaose revealed that TGA has high catalytic efficiency for maltotriose and maltose. Based on the kinetics, subsite affinities were determined. The A1+A2 value of this enzyme was highly positive whereas A4-A6 values were negative and little affinity was detected at subsites 3 and 7. Thus, the subsite structure of TGA is different from that of any other GA. The results indicate that TGA is a metabolizing enzyme specific for small maltooligosaccharides.

-21-
Note
Inhibition of the Bacterial Surface Protein Anchoring Transpeptidase Sortase by Isoquinoline Alkaloids

Soo-Hwan Kim,1 Dong-Sun Shin,1 Mi-Na Oh,2 Soon-Chun Chung,2 Jang-Suk Lee,2 and Ki-Bong Oh1,2,†

1Natural Products Research Institute, Seoul National University, Seoul 110-460, Korea
2Graduate School of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 151-742, Korea

The inhibitory activity of Coptis chinensis rhizome-derived material was evaluated against sortase, a bacterial surface protein anchoring transpeptidase, from Staphylococcus aureus ATCC 6538p and compared to that of four commercially available isoquinoline alkaloids. The biologically active constituent of C. chinensis extract was characterized as the isoquinoline alkaloid, berberine chloride, by spectral analysis. The isolate was a potent inhibitor of sortase, with an IC50 value of 8.7 μg/ml and had antibacterial activity against Gram-positive bacteria with a minimum inhibitory concentration (MIC) in the range of 50-400 μg/ml. Among the four isoquinoline alkaloids tested, berberine chloride had strong inhibitory activity. These results indicate that berberine is a possible candidate for the development of a bacterial sortase inhibitor.

-22-
Note
New Trifluoromethanesulfonanilide Compounds Having High Miticidal Activity against House Dust Mites

Tatsuya Mori,† Yoji Takada, Makoto Hatakoshi, and Noritada Matsuo

Agricultural Chemicals Research Laboratory, Sumitomo Chemical Co., Ltd., 4-2-1 Takatsukasa, Takarazuka, Hyogo 665-8555, Japan

Several 2-alkoxycarbonyltrifluoromethanesulfonanilides were prepared to examine their miticidal activity. 4-Halo-2-alkoxycarbonyltrifluoromethanesulfonanilides in particular showed good miticidal activity against Tyrophagus putrescentiae (Tp), Dermatophagoides farinae (Dp) and Chelacaropsis moorei (Cm). Amidoflumet (methyl 5-chloro-2-[(trifluoromethyl)sulfonyl]aminobenzoate) was selected and has been developed as a new miticide for house dust mites.

-23-
Note
Isoflavones Inhibit 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase in Vitro

Jang Hoon Sung,1 Sung-Joon Lee,2 Kwan Hwa Park,1 and Tae Wha Moon1,†

1Department of Food Science and Technology, School of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742, Korea
2Stanford School of Medicine, Palo Alto Medical Research Foundation, Palo Alto, CA 94301, USA

Isoflavones identified as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in soybean paste were assayed using the catalytic portion of Syrian hamster HMG-CoA reductase, and the kinetic values were measured using HMG-CoA and NADPH. The inhibition of HMG-CoA reductase by these inhibitors was competitive with HMG-CoA and noncompetitive with NADPH. Ki values for genistein, daidzein, and glycitein were 27.7, 49.5, and 94.7 μM, respectively.

-24-
Note
Effects of Ionized Cyclodextrin on Decomposition of Allyl Isothiocyanate in Alkaline Solutions

Yoshio Ohta,1,† Kenichi Takatani,1,* and Shunro Kawakishi2

1Hiroshima Prefectural Food Technology Research Center, Hijiyamahon-machi, Minami-ku, Hiroshima 732-0816, Japan
2Nagoya University, Furo-chou, Chikusa-ku, Nagoya 464-8601, Japan

The secondary hydroxyl ion (alkoxide) ion for cyclodextrin (CD) may affect the decomposition of allyl isothiocyanate (AITC) in alkaline solutions. The equilibrium and kinetic parameters involved in the reaction processes were measured. It was found that the interaction between the alkoxide ion for CD and the included AITC occurred more than pKa 12.7. From the identified kinetic parameters and discussion, it was suggested that the reaction site of AITC was located in the vicinity of one of the secondary hydroxyl groups of CD in the cavity.

-25-
Note
Suppressive Effects of Natural and Synthetic Agents on Dextran Sulfate Sodium-induced Interleukin-1β Release from Murine Peritoneal Macrophages

Ki Han Kwon, Akira Murakami, and Hajime Ohigashi†

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Interleukin (IL)-1β, an anti-apoptotic and pro-inflammatory cytokine, plays an important role in the onset of inflammation-associated disease. We examined the suppressive effects of a total of 39 synthetic or natural compounds on dextran sulfate sodium-induced IL-1β production in murine peritoneal macrophages. Several compounds, including α-tocopherol, gallic acid, (-)-catechin and rutin, were found to be highly effective for attenuating IL-1β production, suggesting that they would be useful for anti-inflammatory application.

-26-
Note
Specification of Amino Acid Residues Essential for the Catalytic Reaction of Cold-active Protein-tyrosine Phosphatase of a Psychrophile, Shewanella sp.

Hiroki Tsuruta,2,† Jun Tamura,1 Hiroshi Yamagata,1 and Yasuo Aizono1

1Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe 657-8501, Japan
2Center for Cooperative Research and Development, Kobe University, Nada-ku, Kobe 657-8501, Japan

Protein-tyrosine phosphatase [EC 3.1.3.48] from a psychrophile, Shewanella sp. shows high activity at low temperatures and has the conserved amino acid sequence of protein-Ser/Thr-phosphatases. Site-directed mutagenesis with the conserved amino acid residues indicated that His148 could be important as a general acid catalyst and Asp115 assists the protonation with His148 of the leaving group of a substrate, and that Asp76 and Asp112 were involved in binding to magnesium ions.

-27-
Note
The Effect of Pre-germinated Brown Rice Intake on Blood Glucose and PAI-1 Levels in Streptozotocin-induced Diabetic Rats

Hiromi Hagiwara, Taiichiro Seki,† and Toyohiko Ariga

Laboratory of Nutrition and Physiology, Department of Agricultural and Biological Chemistry, Nihon University College of Bioresource Sciences, Nihon University Graduate School of Bioresource Sciences, Kanagawa 252-8510, Japan

Effects of pre-germinated brown rice (PGBR) on streptozotocin-induced diabetic rats were studied. The feeding of a PGBR diet to diabetic rats ameliorated the elevation of blood glucose and PAI-1 concentrations significantly, and tended to decrease the plasma lipid peroxide concentrations in comparison with rats fed a white rice diet. These results suggest that intake of PGBR instead of white rice is effective for the prevention of diabetic vascular complications.

-28-
Note
Dehydrotrametenonic Acid and Dehydroeburiconic Acid from Poria cocos and Their Inhibitory Effects on Eukaryotic DNA Polymerase α and β

Toshihiro Akihisa,1,† Yoshiyuki Mizushina,2 Motohiko Ukiya,1 Manabu Oshikubo,1 Seizo Kondo,3 Yumiko Kimura,4 Takashi Suzuki,4 and Takaaki Tai5

1College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan
2Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
3Central Research Laboratory, Kotaro Pharmaceutical Co., Ltd., Suga-cho 47-3, Takatsuki 569-0022, Japan
4College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
5Morishita Jintan Co., Ltd., 1-30, Tamatsukuri 1-chome, Chuo-ku, Osaka 540-8566, Japan

A new lanostane-type triterpene acid, (20 ξ )-3-oxolanosta-7,9(11),24-trien-21-oic acid (1; dehydrotrametenonic acid), along with a known triterpene acid, dehydroeburiconic acid (2), were isolated from the epidermis of the sclerotia of Poria cocos. The structure of 1 was analyzed on the basis of spectroscopic methods. Compounds 1 and 2 inhibited calf DNA polymerase α and rat DNA polymerase β , with the 50% inhibition values of 45.5 μ M (1) and 40.8 μ M (2), and 86.5 μ M (1) and 30.0 μ M (2), respectively.

-29-
Note
Reduction in Photostability by the Esterification of β -Cryptoxanthin

Shin Arita,1 Kojiro Otsuki,2 Ken-ichi Osaki,1 Yoshiyuki Murata,2 Yasuaki Shimoishi,2 and Mikiro Tada1,†

1Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan
2Faculty of Agriculture, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan

Liposomes, in which β -carotene, β -cryptoxanthin, zeaxanthin, β -cryptoxanthin palmitate or β -cryptoxanthin acetate had been embedded, were irradiated by UVA, and the rate of degradation of each carotenoid was measured. There was no significant difference in the degradation rate between β -carotene, β -cryptoxanthin and zeaxanthin. The degradation rates of β -cryptoxanthin palmitate and β -cryptoxanthin acetate were faster than that of β -cryptoxanthin, and the degradation rate of β -cryptoxanthin palmitate was faster than that of β -cryptoxanthin acetate.

-30-
Note
“Aqua-space®”, a New Headspace Method for Isolation of Natural Floral Aromas Using Humidified Air as a Carrier Gas

Masashi Ishikawa,1,† Tsutomu Honda,1 Akira Fujita,1 Yoshiko Kurobayashi,1 and Takeshi Kitahara2

1Technical Research Center, T. Hasegawa Co., Ltd., 335 Kariyado, Nakahara-ku, Kawasaki-shi 211-0022, Japan
2Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

A new method called “Aqua-space®” was developed for the isolation of the natural fragrances of plants. Living flowers were enclosed in a space under simulated natural conditions, and humidified air was pumped into the space as a fragrance carrier. In a comparison among three isolation methods, i.e., Aqua-space®, headspace, and solvent extraction, the Aqua-space® method proved to be the most effective in retaining natural fragrances with abundant oxygenated components key to floral fragrances.

-31-
Note
Transcriptional Analysis of the ostA/imp Gene Involved in Organic Solvent Sensitivity in Escherichia coli

Iwao Ohtsu,1 Nobuto Kakuda,1 Norihiko Tsukagoshi,1 Noriyuki Dokyu,1 Hiroshi Takagi,2 Masaaki Wachi,3,† and Rikizo Aono1

1Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
2Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan
3Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

Transcriptional initiation sites of the ostA gene involved in organic solvent sensitivity in Escherichia coli were found by primer extension analysis. Two transcriptional initiation sites were newly identified at -133 and -48 nucleotides from the initiation codon of ostA, but the previously reported σ E-dependent one at -227 could not be detected. No heat-inducible expression of ostA was observed by Northern blotting analysis, indicating that the contribution of σ E-dependent transcription was very small if any. σ D-Dependent promoter-like sequences were found just upstream of the newly identified transcriptional initiation sites by computer-aided analysis. Deletion analysis of ostA-lacZ fusions demonstrated that these two promoters contributed almost equally to the constitutive expression of the ostA gene.

-32-
Note
Comparative Studies of the AHP Histidine-containing Phosphotransmitters Implicated in His-to-Asp Phosphorelay in Arabidopsis thaliana

Yasushi Tanaka, Tomomi Suzuki, Takafumi Yamashino, and Takeshi Mizuno†

Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku Nagoya, Nagoya 464-8601, Japan

The evolutionarily-conserved histidine to aspartate (His-to-Asp) phosphorelay signal transduction is common in both prokaryotes and eukaryotes. Such a phosphorelay system is generally made up of ‘a histidine (His)-kinase’, ‘a histidine-containing phosphotransmitter (HPt)’, and ‘a phospho-accepting response regulator (RR)’. In general, an HPt factor acts as an intermediate in a given multistep His-to-Asp phosphorelay. In Arabidopsis thaliana, this model higher plant has five genes (named AHP1 to AHP5), each of which seems to encode an HPt factor. Recent studies suggested that the His-to-Asp phosphorelay involving the AHP factors is at least partly implicated in signal transduction in response to cytokinin (a plant hormone). Nevertheless, the properties of AHPs have not yet been fully clarified. Here we did comparative studies of all the AHP factors, in terms of (i) expression profiles in plants, (ii) intracellular localization, (iii) ability to acquire a phosphoryl group in vitro, and (iv) ability to interact with the downstream components, ARRs (Arabidopsis response regulators). The results of this study provided us with a comprehensive view at the molecular level for understanding the functions of the AHP phosphotransmitters in the His-to-Asp phosphorelay.

-33-
Note
Hydrogen Gas Generation from Refuse-derived Fuel (RDF) under Wet Conditions

Makiko Sakka,† Tetsuya Kimura, Kazuo Sakka, and Kunio Ohmiya

Faculty of Bioresources, Mie University, 1515 Kamihamacho, Tsu 514-8507, Japan

An explosion has recently occurred at a silo containing refuse-derived fuels (RDF) in Japan. There is a possibility that microorganisms are involved in generation of combustible gas from RDF and this study was aimed at showing the presence of bacteria that can ferment RDF pellets. All RDF samples tested contained a relatively high number of viable bacterial cells, 1.4 × 105 to 3.2 × 106 viable cells/g. These bacteria in the RDF samples fermented them to generate heat and hydrogen gas.

-34-
Communication
Isolation of Genes Specifically Expressed in the Fruit Body of the Edible Basidiomycete Lentinula edodes

Tatsuya Hirano,1,† Toshitsugu Sato,2 and Hitoshi Enei2

1Faculty of Agriculture, Meijo University, Shiogamaguchi, Tenpaku, Nagoya 468-8502, Japan
2Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan

In order to isolate the genes expressed specifically and abundantly in the mature fruit body of Lentinula edodes, the cDNAs derived from the gill of the fruit body were compared with the cDNAs from the mycelia by differential screening. Consequently, six clones were identified as fruit-body-specific genes (fbg03, 08, 13, 14, 16, and 21). The deduced amino acid sequence of fbg14 (Le.cypfb) had significant homology with the cytochrome P450 protein. The transcriptional level of fbg16, which showed 29.9% identity with the riboflavin aldehyde-forming enzyme of Agaricus bisporus, was highest among all of the fbg clones. This result indicates that the promoter region of fbg16 may become a powerful candidate for the expression signal of the vector for the gene manipulation in the mature fruit body.

-35-
Communication
Nitrous Oxide-forming Codenitrification Catalyzed by Cytochrome P450nor

Fei Su,1 Naoki Takaya,1 and Hirofumi Shoun2,†

1Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
2Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-Ku, Tokyo 113-8657, Japan

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.