Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.10 2002)

Review
Molecular Bases of Aerobic Bacterial Degradation of Dioxins: Involvement of Angular Dioxygenation
Hideaki NOJIRI and Toshio OMORI p.2001

Review
Recent Trends in Functional Food Science and the Industry in Japan
Soichi ARAI,1 Yasushi MORINAGA,2 Toshikazu YOSHIKAWA,3 Eiichiro ICHIISHI,3 Yoshinobu KISO,4 Masatoshi YAMAZAKI,5 Masami MOROTOMI,6 Makoto SHIMIZU,7 Tamotsu KUWATA,8 and Shuichi KAMINOGAWA7, p.2017

Ferrous-iron-dependent Uptake of L-Glutamate by a Mesophilic, Mixotrophic Iron-oxidizing Bacterium Strain OKM-9
Takao INOUE, Kazuo KAMIMURA, and Tsuyoshi SUGIO p.2030

A Steroidal Glycoside from Polygonatum odoratum (Mill.) Druce. Improves Insulin Resistance but does not Alter Insulin Secretion in 90％ Pancreatectomized Rats
Soo Bong CHOI1 and Sunmin PARK2, p.2036

Appearance of Nitrite Reducing Activity of Cytochrome c upon Heat Denaturation
Seiji YAMADA, Kohei SURUGA, Masahiro OGAWA, Toshiyuki HAMA, Tadashi SATOH, Ryu KAWACHI, Toshiyuki NISHIO, and Tadatake OKU p.2044

Stable Ammonia-specific NAD Synthetase from Bacillus stearothermophilus: Purification, Characterization, Gene Cloning, and Applications
Fumihiko YAMAGUCHI,1, Shinji KOGA,2 Issei YOSHIOKA,2 Mamoru TAKAHASHI,2 Haruhiko SAKURABA,3 and Toshihisa OHSHIMA3 p.2052

Identification of Essential Ionizable Groups and Evaluation of Subsite Affinities in the Active Site of β-D-Glucosidase F1 from a Streptomyces sp.
Kenji FUKUDA,1 Haruhide MORI,1 Masayuki OKUYAMA,1 Atsuo KIMURA,1 Hachiro OZAKI,2 Michio YONEYAMA,3 and Seiya CHIBA1, p.2060

Purification of a Phi-type Glutathione S-Transferase from Pumpkin Flowers, and Molecular Cloning of Its cDNA
Mohammad Zakir HOSSAIN and Masayuki FUJITA p.2068

Effects of L-Ascorbic Acid on Lysyl Oxidase in the Formation of Collagen Cross-links
Miwa KUROYANAGI, Eriko SHIMAMURA, Mihyan KIM, Nobuhiko ARAKAWA, Yoko FUJIWARA, and Megumi OTSUKA p.2077

Two Genes Encoding Fruit Body Lectins of Pleurotus cornucopiae: Sequence Similarity with the Lectin of a Nematode-Trapping Fungus
Naoto IIJIMA, Hiroto YOSHINO, Lim Chee TEN, Akikazu ANDO, Katsunori WATANABE, and Yoshiho NAGATA p.2083

Biosynthesis of Bisorbicillinoid in Trichoderma sp. USF-2690; Evidence for the Biosynthetic Pathway, via Sorbicillinol, of Sorbicillin, Bisorbicillinol, Bisorbibutenolide, and Bisorbicillinolide
Naoki ABE,, Tadaharu ARAKAWA, Kazunobu YAMAMOTO, and Akira HIROTA p.2090

Glucose Metabolism of Lactic Acid Bacteria Changed by Quinone-mediated Extracellular Electron Transfer
Shin-ichi YAMAZAKI,1 Tsutomu KANEKO,2 Naoki TAKETOMO,2 Kenji KANO,2 and Tokuji IKEDA1
p.2100

Post-illumination Reduction of the Plastoquinone Pool in Chloroplast Transformants in which Chloroplastic NAD(P)H Dehydrogenase was Inactivated
Atsushi TAKABAYASHI,1 Tsuyoshi ENDO,1, Toshiharu SHIKANAI,2 and Fumihiko SATO1
p.2107

Novel Trimeric Adenylate Kinase from an Extremely Thermoacidophilic Archaeon, Sulfolobus solfataricus: Molecular Cloning, Nucleotide Sequencing, Expression in Escherichia coli, and Characterization of the Recombinant Enzyme
Toshihide OKAJIMA,1, Daisuke KITAGUCHI,1 Kumiko FUJII,2 Hidetada MATSUOKA,2, Sachio GOTO,2 Susumu UCHIYAMA,3 Yuji KOBAYASHI,4 and Katsuyuki TANIZAWA1 p.2112

Characterization of Subrepeat Regions within rDNA Intergenic Spacers of the Edible Basidiomycete Lentinula edodes
Takeshi SAITO,1,2, Norio TANAKA,2 and Takao SHINOZAWA1, p.2125

Molecular Cloning of Mouse Collectin Liver 1
Takao KAWAI,1, Yasuhiko SUZUKI,2, Souji EDA,3 Tetsuo KASE,4 Katsuki OHTANI,5 Yoshinori SAKAI,3 Hiroyuki KESHI,3 Atsushi FUKUOH,5 Takashi SAKAMOTO,3 Masami NOZAKI,6 Neal G. COPELAND,7 Nancy A. JENKINS,7 and Nobutaka WAKAMIYA5 p.2134

Gene Family of Oleosin Isoforms and Their Structural Stabilization in Sesame Seed Oil Bodies
Sorgan S. K. TAI, Miles C. M. CHEN, Chi-Chung PENG, and Jason T. C. TZEN p.2146

A Lipase-inhibiting Protein from Lipoxygenase-deficient Soybean Seeds
Kiyoshi SATOUCHI,1, Yoshifumi KODAMA,1 Kaoru MURAKAMI,1 Tamotsu TANAKA,1 Hiroyuki IWAMOTO,1 and Masao ISHIMOTO2 p.2154

Increased Staphylococcus-killing Activity of an Antimicrobial Peptide, Lactoferricin B, with Minocycline and Monoacylglycerol
Hiroyuki WAKABAYASHI, Susumu TERAGUCHI, and Yoshitaka TAMURA p.2161

Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae
Sunhwa KIM,1 Ichiro MATSUO,2, Katsumi AJISAKA,2 Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1, p.2168

Jasmonate-induced Changes in Flavonoid Metabolism in Barley (Hordeum vulgare) Leaves
Atsushi ISHIHARA,1,2, Yuki OGURA,1,2 Shin-ichi TEBAYASHI,2, and Hajime IWAMURA3
p.2176

Cloning and Expression of Cytochrome P450 Genes, Belonging to a New P450 Family, of the Basidiomycete Lentinula edodes
Ryoko AKIYAMA, Yoshihiro SATO, Susumu KAJIWARA, and Kazuo SHISHIDO p.2183

ESR Characterization of a Novel Spin-trapping Agent, 15N-Labeled N-tert-Butyl-α-phenylnitrone, as a Nitric Oxide Donor
Kieko SAITO and Hisashi YOSHIOKA p.2189

Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen from Thermococcus kodakaraensis KOD1
Masao KITABAYASHI,1,2, Yoshiaki NISHIYA,1 Muneharu ESAKA,2 Mitsuo ITAKURA,3 and Tadayuki IMANAKA4 p.2094

Identification of Non-Pseudomonad Bacteria from Fruit Bodies of Wild Agaricales Fungi That Detoxify Tolaasin Produced by Pseudomonas tolaasii
Takanori TSUKAMOTO,1, Hitoshi MURATA,2, and Akira SHIRATA1, p.2201

Effects of Iprodione and Fludioxonil on Glycerol Synthesis and Hyphal Development in Candida albicans
Noriyuki OCHIAI,1 Makoto FUJIMURA,1, Michiyo OSHIMA,1 Takayuki MOTOYAMA,2 Akihiko ICHIISHI,1 Hisafumi YAMADA-OKABE,3 and Isamu YAMAGUCHI2 p.2209

RNase G-Dependent Degradation of the eno mRNA Encoding a Glycolysis Enzyme Enolase in Escherichia coli
Naoko KAGA,1 Genryou UMITSUKI,2 Kazuo NAGAI,3 and Masaaki WACHI1, p.2216

Note
Inhibition by Ajoene of Skin-tumor Promotion in Mice
Tomoaki NISHIKAWA, Norihiko YAMADA, Atsuhiko HATTORI, Hiroyuki FUKUDA, and Tsuchiyoshi FUJINO p.2221

Note
Involvement of a CCAAT-binding Complex in the Expression of a Nitrogen-Starvation-Specific Gene, isp6＋, in Schizosaccharomyces pombe
Akio NAKASHIMA,1 Masaru UENO,1 Takashi USHIMARU,2 and Masahiro URITANI1, p.2224

Note
Apoptosis Induction by Wheat-flour Sphingoid Bases in DLD-1 Human Colon Cancer Cells
Tatsuya SUGAWARA,1 Mikio KINOSHITA,2 Masao OHNISHI,2 and Teruo MIYAZAWA1, p.2228

Note
Differential Effects of Dietary Casein and Soybean Protein Isolate on Lipopolysaccharide-induced Hepatitis in D-Galactosamine-sensitized Rats
Kimio SUGIYAMA, Yasuhiko SHIMADA, Kasumi IWAI, and Tatsuya MORITA p.2232

Note
Synthesis and Biological Activity of Isoamoenylin, a Metabolite of Dendrobium amoenum
Somepalli VENKATESWARLU, Mudunuri S. S. RAJU, and Gottumukkala V. SUBBARAJU p.2236

Note
Tryptophans 286 and 288 in the C-terminal Region of Protein B23.1 are Important for Its Nucleolar Localization
Yuki NISHIMURA, Takeshi OHKUBO, Yukio FURUICHI, and Hayato UMEKAWA p.2239

Note
Synthesis and Fungicidal Activity of 1-(α-tert-Butylcinnamoyl)imidazoles
Akio MANABE,1, Hirotaka TAKANO,1 Kunihiko FURUZAWA,1 Kazunori YANAGI,2 Yoshio HISADA,1 and Shizuya TANAKA1 p.2243

Note
PCR Detection of DNAs of Animal Origin in Feed by Primers Based on Sequences of Short and Long Interspersed Repetitive Elements
Kiyoshi TAJIMA,1, Osamu ENISHI,1 Masahiro AMARI,1 Makoto MITSUMORI,1 Hiroshi KAJIKAWA,1 Mitsunori KURIHARA,1 Satoshi YANAI,2 Hiroki MATSUI,2 Hiroshi YASUE,3 Tadayoshi MITSUHASHI,3 Tomoyuki KAWASHIMA,1 and Mitsuto MATSUMOTO1 p.2247

Note
Volatile Compounds of Headspace Gas in the Japanese Fish Sauce Ishiru
Toshihide MICHIHATA,1 Toshihiro YANO,2 and Toshiki ENOMOTO2, p.2251

Note
Inhibition of Proteasome Activity by Tyropeptin A in PC12 Cells
Isao MOMOSE, Ryuichi SEKIZAWA, Hironobu IINUMA, and Tomio TAKEUCHI p.2256

Note
Soft Gel Medium Solidified with Gellan Gum for Preliminary Screening for Root-associating, Free-living Nitrogen-fixing Bacteria Inhabiting the Rhizoplane of Plants
Yasuyuki HASHIDOKO,1, Motohiko TADA,1 Mitsuru OSAKI,2 and Satoshi TAHARA1 p.2259

Note
Specific Binding of Silkworm Bombyx mori 30-kDa Lipoproteins to Carbohydrates Containing Glucose
Minoru UJITA,1,2, Akihiro KIMURA,1 Daisuke NISHINO,1 Eiji YOKOYAMA,2 Yutaka BANNO,3 Hiroshi FUJII,3 and Akira HARA1,2 p.2264

Note
Access to Enantiomerically Pure Intermediates for (−)-Geosmin Synthesis Starting from (4aS,5S)-4,4a,5,6,7,8-Hexahydro-5-hydroxy-4a-methyl- naphthalen-2(3H)-one
Ken-ichi FUHSHUKU and Takeshi SUGAI p.2267

Synthesis of Novel α-C-Glycosylamino Acids and Reverse Regioselectivity in Larock′s Heteroannulation for the Synthesis of the Indole Nucleus
Toshio NISHIKAWA, Kyoko WADA, and Minoru ISOBE p.2273

Note
Molecular Cloning of a Pore-Forming Subunit (Kir6.2 Gene) of the ATP-Sensitive Potassium Channel in the Bullfrog, Rana catesbeiana Shaw
Pyo-Jam PARK,1 Joon-Yong CHUNG,2 Hee-Guk BYUN,1 Dae-Hyun SEOG,3 and Se-Kwon KIM1,
p.2279

Note
Ricinoleic Acid and Castor Oil as Substrates for Conjugated Linoleic Acid Production by Washed Cells of Lactobacillus plantarum
Shigenobu KISHINO, Jun OGAWA, Akinori ANDO, Yoriko OMURA, and Sakayu SHIMIZU p.2283

Note
Molecular Cloning and Functional Expression of cDNA Encoding a Cysteine Proteinase Inhibitor, Cystatin, from Job′s Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)
Koh-Ichi YOZA, Sumiko NAKAMURA, Miki YAGUCHI, Kazutomo HARAGUCHI, and Ken-Ichi OHTSUBO p.2287

Preliminary Communication
Isolation and Identification of Novel ADP-Ribosylated Proteins from Streptomyces coelicolor A3(2)
Kiyoshi SUGAWARA,1 Naoshi DOHMAE,2 Koji KASAI,1 Hisako SAIDO-SAKANAKA,1 Susumu OKAMOTO,1 Koji TAKIO,2 and Kozo OCHI1, p.2292

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Review
Molecular Bases of Aerobic Bacterial Degradation of Dioxins: Involvement of Angular Dioxygenation

Hideaki NOJIRI and Toshio OMORI

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

In the last decade, extensive investigation has been done on the bacterial degradation of dioxins and its related compounds, because this class of chemicals is highly toxic and has been widely distributed in the environment. These studies have revealed the primary importance of a novel dioxygenation reaction, called angular dioxygenation, in the aerobic bacterial degradation pathway of dioxin. Accompanied by the electron transport proteins, Rieske nonheme iron oxygenase catalyzes the incorporation of oxygen atoms to the ether bond-carrying carbon (the angular position) and an adjacent carbon, resulting in the irreversible cleavage of the recalcitrant aryl ether bond. The 2,2′,3-trihydroxybiphenyl or 2,2′,3-trihydroxydiphenyl ether derivatives formed are degraded through meta cleavage. In addition to the degradation system of dibenzofuran and dibenzo-p-dioxin (the nonchlorinated model compounds of dioxin), those of fluorene and carbazole were shown to function in dioxin degradation. Some dioxin degradation pathways have been studied biochemically and genetically. In addition, feasibility studies have shown that some dioxin-degrading strains can function in actual dioxin-contaminated soil. These studies provide useful information for the establishment of a bioremediation method for dioxin contamination. This review summarizes recent progress on molecular and biochemical bases of the bacterial aerobic degradation of dioxin and related compounds.
Key words: angular dioxygenation; bacterial degradation; carbazole; dioxin; fluorene

-2-
Review
Recent Trends in Functional Food Science and the Industry in Japan

Soichi ARAI,1 Yasushi MORINAGA,2 Toshikazu YOSHIKAWA,3 Eiichiro ICHIISHI,3 Yoshinobu KISO,4 Masatoshi YAMAZAKI,5 Masami MOROTOMI,6 Makoto SHIMIZU,7 Tamotsu KUWATA,8 and Shuichi KAMINOGAWA7

1Faculty of Applied Biological Sciences, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan 2Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki-shi, Kanagawa 210-8681, Japan 3First Department of Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto-city, Kyoto 602-8566, Japan 4Institute for Health Care Science, Suntory Ltd., Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan 5Faculty of Pharmaceutical Science, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan 6Yakult Central Institute for Microbiological Research, Kunitachi-shi, Tokyo 186-8650, Japan 7Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 8Meiji Dairies Corporation, Koto-ku, Tokyo 136-8908, Japan

International recognition of functional foods has resulted in the recent global development of this field, which originated in Japan. The national policy on functional foods, in terms of <Oh><0192><0192><Wa>foods for specified health use′′, also has been developing and has motivated the food industry to produce a variety of new food items. In Japan as well as in many other countries, academic and industrial scientists have been working in collaboration for the analysis and practical applications of functional food science. Emphasis has been placed on the study of antioxidant and anticarcinogenic food factors as well as pre- and probiotics. This review pinpoints recent trends in the science and industry in this field.
Key words: functional foods; oxidative stress; anticarcinogens; prebiotics; probiotics

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Ferrous-iron-dependent Uptake of L-Glutamate by a Mesophilic, Mixotrophic Iron-oxidizing Bacterium Strain OKM-9

Takao INOUE, Kazuo KAMIMURA, and Tsuyoshi SUGIO

Division of Science and Technology for Energy Conversion, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama 700-8530, Japan

Received November 26, 2001; Accepted March 28, 2002
Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2＋-dependent uptake activity of L-glutamate was 3.5--4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol・min−1・mg−1, respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 μM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2＋-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40％ of the iron-oxidizing activity of the resting cells and approximately 85％ of the Fe2＋-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1％ n-octyl-β-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2＋-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2＋ oxidation when using amino acids as carbon sources.
Key words: iron-oxidizing bacterium; mixotroph; L-glutamate uptake system; iron oxidase; L-glutamate transporter

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A Steroidal Glycoside from Polygonatum odoratum (Mill.) Druce. Improves Insulin Resistance but does not Alter Insulin Secretion in 90％ Pancreatectomized Rats

Soo Bong CHOI1 and Sunmin PARK2

1Department of Internal Medicine, Konkuk University, Kyohyun-Dong, Chungjoo-Si, Chungbuk-Do, Korea 2Department of Food and Nutrition, Hoseo University, Sechul-Ri, Asan-Si, Chungnam-Do 336-795, Korea

Received January 21, 2002; Accepted April 5, 2002
The aim of this study was to investigate the effects of three steroidal glycosides (SG-100, SG-280, and SG-460) obtained from Polygonatum odoratum (Mill.) Druce. on insulin secretion, insulin action, and relative glucose uptake in various tissues of 90％ pancreatectomized male Sprague-Dawley rats. One of the compounds (30 mg/kg body weight daily) with a 40％-fat diet was orally administered to a group of such rats for 13 weeks. On the day after a hyperglycemic clamp, euglycemic hyperinsulinemic clamp with 1 μCi of [1-14C]2-deoxyglucose per 100 g body weight was used. Serum glucose levels were lowest in the rats receiving SG-100. Insulin secretion from pancreatic β-cells did not change with SG administration. Whole-body glucose disposal rates increased with SG-100 administration by 39％. SG-100 increased the glycogen contents and glycogen synthase activity in the soleus muscle of pancreatectomized rats. Uptake of [1-14C]2-deoxyglucose into the soleus muscle was higher in such rats receiving SG-100 than in rats receiving other compounds. In conclusion, SG-100 has an antihyperglycemic effect by promoting peripheral insulin sensitivity without changing insulin secretion.
Key words: Polygonatum odoratum (Mill.) Druce.; hyperglycemic clamp; euglycemic hyperinsulinemic clamp; glucose uptake; glycogen synthase

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Appearance of Nitrite Reducing Activity of Cytochrome c upon Heat Denaturation

Seiji YAMADA, Kohei SURUGA, Masahiro OGAWA, Toshiyuki HAMA, Tadashi SATOH, Ryu KAWACHI, Toshiyuki NISHIO, and Tadatake OKU

Department of Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan

Received February 4, 2002; Accepted May 22, 2002
The appearance of NO−2 reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100°C for 30 min, had NO−2 reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO−2 reduction to NH＋4 via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.
Key words: cytochrome c; heat denaturation; immobilized protein; nitrite reduction

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Stable Ammonia-specific NAD Synthetase from Bacillus stearothermophilus: Purification, Characterization, Gene Cloning, and Applications

Fumihiko YAMAGUCHI,1, Shinji KOGA,2 Issei YOSHIOKA,2 Mamoru TAKAHASHI,2 Haruhiko SAKURABA,3 and Toshihisa OHSHIMA3

1Planova Technology Development, Asahi-Kasei Corporation, 6-4158 Asahimachi, Nobeoka, Miyazaki-ken 882-0847, Japan 2Diagnostics Research and Development Department, Asahi-Kasei Corporation, 632-1 Mifuku, Ohito-cho, Tagata-gun, Shizuoka-ken 410-2321, Japan 3Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minami-josanjima, Tokushima 770-8506, Japan

Received February 12, 2002; Accepted June 7, 2002
Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2＋ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60°C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60°C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32％ homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 μM) with this stable NAD synthetase was possible.
Key words: ammonia-specific NAD synthetase; NAD biosynthesis; gene cloning; Bacillus stearothermophilus

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Identification of Essential Ionizable Groups and Evaluation of Subsite Affinities in the Active Site of β-D-Glucosidase F1 from a Streptomyces sp.

Kenji FUKUDA,1 Haruhide MORI,1 Masayuki OKUYAMA,1 Atsuo KIMURA,1 Hachiro OZAKI,2 Michio YONEYAMA,3 and Seiya CHIBA1

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan 2Faculty of Education, Yamaguchi University, Yamaguchi 753-8511, Japan 3International Student Center, Hokkaido University, Sappro 060-0808, Japan

Received February 12, 2002; Accepted May 11, 2002
Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of β-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of β-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and Km values on pH, when the substrate concentration was sufficiently lower than Km, gave the values of 4.1 and 7.2 for the ionization constants, pKe1 and pKe2 of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10％ methanol, the pKe1 and pKe2, values shifted to higher, to ＋0.60 and ＋0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (--COO−, the group 1) and a carboxyl group (--COOH, the group 2). The subsite affinities Ai′s in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive Ai values (A1 was 1.10 kcal/mol and A2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative Ai values (A3 was −0.21 kcal/mol and A4 was −2.8 kcal/mol).
Key words: Streptomyces β-glucosidase; essential ionizable group; subsite affinity; laminarioligosaccharide hydrolase; β-fucosidase

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Purification of a Phi-type Glutathione S-Transferase from Pumpkin Flowers, and Molecular Cloning of Its cDNA

Mohammad Zakir HOSSAIN and Masayuki FUJITA

Department of Plant Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Received February 25, 2002; Accepted May 20, 2002
A major species of glutathione S-transferase (GST), Pugf, was highly purified from pumpkin flowers. Two-dimensional electrophoresis of the purified enzyme gave two adjacent protein spots. The specific activity of the purified enzyme was 2.4 μmol min−1 mg−1 protein for 1-chloro-2,4-dinitrobenzene. This value is one to two orders of magnitude lower than that of pumpkin tau-type GSTs. The expression pattern of Pugf in healthy pumpkin plants and responses to various stresses were examined by western blotting. Pugf was found in high concentrations in petioles, stems, and roots as well as flowers, and was more abundant in expanding young organs than in fully expanded mature organs. Dehydration caused a slight increase in its concentration, but high and low temperatures, salty stress, and 2,4-dichlorophenoxyacetic acid seemed to have no effects. A cDNA encoding Pugf was cloned and sequenced. Sequence comparison with other plant GSTs suggested that it should be classified as a phi-type GST.
Key words: cDNA cloning; Cucurbita maxima; glutathione S-transferase; pumpkin; enzyme purification

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Effects of L-Ascorbic Acid on Lysyl Oxidase in the Formation of Collagen Cross-links

Miwa KUROYANAGI, Eriko SHIMAMURA, Mihyan KIM, Nobuhiko ARAKAWA, Yoko FUJIWARA, and Megumi OTSUKA

Department of Nutrition and Food Science, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan

Received February 25, 2002; Accepted May 22, 2002
To clarify the role of L-ascorbic acid (AsA) in the formation of pyridinoline, we examined the effects of AsA in vitro using soluble collagen and partially purified lysyl oxidase from bovine aorta. The concentration of dehydrodihydroxylysinonorleucine decreased when AsA was added in the early stage of pyridinoline formation. However, when AsA was added in a later stage of pyridinoline formation, the concentration of pyridinoline was not affected. These findings indicated that AsA was involved in the initial enzymatic reaction in pyridinoline synthesis. We purified lysyl oxidase to confirm its association of AsA. AsA inhibited the enzyme activity. Erythorbic acid and 3,4-dihydroxybenzoate suppressed the enzyme activity as well as AsA did. The inhibition by AsA of the lysyl oxidase activity arose from characteristics of AsA structure. AsA might be important in the regulation of the oxidative reaction of lysine.
Key words: L-ascorbic acid; lysyl oxidase; pyridinoline; dehydrodihydroxylysinonorleucine; collagen cross-links

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Two Genes Encoding Fruit Body Lectins of Pleurotus cornucopiae: Sequence Similarity with the Lectin of a Nematode-Trapping Fungus

Naoto IIJIMA, Hiroto YOSHINO, Lim Chee TEN, Akikazu ANDO, Katsunori WATANABE, and Yoshiho NAGATA

Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan

Received March 7, 2002; Accepted May 19, 2002
Our previous studies on the fruit body lectin of Pleurotus cornucopiae revealed the existence of three isolectins, composed of two homodimers and one heterodimer of 16- and 15-kDa subunits. In this study, two genes encoding the lectins were cloned and characterized. Both genes encoded 144 amino acids and only 5 amino acids were different within the coding region, but the nucleotide sequences of the 5′-upstream and 3′-downstream regions differed extensively. Southern hybridization with gene-specific probes showed that one gene encoded the 16-kDa and the other encoded the 15-kDa subunit. Functional lectins were synthesized in Escherichia coli under the direction of these genes. On SDS-PAGE, the recombinant lectins showed the same banding patterns as the native lectins. In amino acid sequence, these lectins showed extensive similarity with the lectin from a nematode-trapping ascomycete fungus, Arthrobotrys oligospora, suggesting that the lectins might also function in capturing nematodes.
Key words: fruit body lectins of Pleurotus cornucopiae; lectin genes of P. cornucopiae; expression of the lectin genes in E. coli; sequence similarity to nematophagous fungus lectin

-11-
Biosynthesis of Bisorbicillinoid in Trichoderma sp. USF-2690; Evidence for the Biosynthetic Pathway, via Sorbicillinol, of Sorbicillin, Bisorbicillinol, Bisorbibutenolide, and Bisorbicillinolide

Naoki ABE,, Tadaharu ARAKAWA, Kazunobu YAMAMOTO, and Akira HIROTA

School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Received March 13, 2002; Accepted May 31, 2002
An incorporation study of [1-13C] and [1,2-13C2] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using 13C-labeled 1 prepared from [1-13C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated 13C-labeled 1 into their carbon skeletons. 13C-labeled bisorbicillinols 2 derived from [1-13C]- and [2-13C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.
Key words: sorbicillinol; bisorbicillinoids; sorbicillin; biosynthesis; Trichoderma sp. USF-2690

-12-
Glucose Metabolism of Lactic Acid Bacteria Changed by Quinone-mediated Extracellular Electron Transfer

Shin-ichi YAMAZAKI,1 Tsutomu KANEKO,2 Naoki TAKETOMO,2 Kenji KANO,2 and Tokuji IKEDA1

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan 2Food Functionality Research Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo 189-8530, Japan

Received March 15, 2002; Accepted June 3, 2002
It can be expected that extracellular electron transfer to regenerate NAD＋ changes the glucose metabolism of the homofermentative lactic acid bacteria. In this work, the glucose metabolism of Lactobacillus plantarum and Lactococcus lactis was examined in resting cells with 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as the electron transfer mediator and ferricyanide (Fe(CN)3−6) as the extracellular electron acceptor. NADH in the cells was oxidized by ACNQ with the aid of diaphorase, and the reduced ACNQ was reoxidized with Fe(CN)3−6. The extracellular electron transfer system promoted the generation of pyruvate, acetate, and acetoin from glucose, and restricted lactate production. Diaphorase activity increased when cultivation was aerobic, and this increased the concentrations of pyruvate, acetate, and acetoin relative to the concentration of lactate to increase in the presence of ACNQ and Fe(CN)3−6.
Key words: 2-amino-3-carboxy-1,4-naphthoquinone; Lactobacillus plantarum; Lactococcus lactis; diaphorase; lactate dehydrogenase

-13-
Post-illumination Reduction of the Plastoquinone Pool in Chloroplast Transformants in which Chloroplastic NAD(P)H Dehydrogenase was Inactivated

Atsushi TAKABAYASHI,1 Tsuyoshi ENDO,1, Toshiharu SHIKANAI,2 and Fumihiko SATO1

1Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 2Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan

Received March 18, 2002; Accepted May 3, 2002
We reported previously that an ndhB gene disruptant, ΔndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as ΔndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.
Key words: chloroplast transformation; cyclic electron transport; NAD(P)H dehydrogenase; Nicotiana tabacum

-14-
Novel Trimeric Adenylate Kinase from an Extremely Thermoacidophilic Archaeon, Sulfolobus solfataricus: Molecular Cloning, Nucleotide Sequencing, Expression in Escherichia coli, and Characterization of the Recombinant Enzyme

Toshihide OKAJIMA,1, Daisuke KITAGUCHI,1 Kumiko FUJII,2 Hidetada MATSUOKA,2, Sachio GOTO,2 Susumu UCHIYAMA,3 Yuji KOBAYASHI,4 and Katsuyuki TANIZAWA1

1Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan 2Faculty of Agriculture, Kinki University, Nara, Nara 631-8505, Japan 3RRF Research Inc., Tsukuba, Ibaraki 305-0856, Japan 4Graduate School of Pharmaceutical Science, Osaka University, Suita, Osaka 565-0871, Japan

Received March 20, 2002; Accepted May 13, 2002
A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.
Key words: adenylate kinase; archaea; Sulfolobus; thermoacidophile

-15-
Characterization of Subrepeat Regions within rDNA Intergenic Spacers of the Edible Basidiomycete Lentinula edodes

Takeshi SAITO,1,2, Norio TANAKA,2 and Takao SHINOZAWA1

1Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan 2The Mushroom Research Institute of Japan, Kiryu, Gunma 376-0051, Japan

Received March 20, 2002; Accepted June 15, 2002
For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.
Key words: rDNA; intergenic spacers; subrepeat regions; Lentinula edodes; DNA fingerprints

-16-
Molecular Cloning of Mouse Collectin Liver 1

Takao KAWAI,1, Yasuhiko SUZUKI,2, Souji EDA,3 Tetsuo KASE,4 Katsuki OHTANI,5 Yoshinori SAKAI,3 Hiroyuki KESHI,3 Atsushi FUKUOH,5 Takashi SAKAMOTO,3 Masami NOZAKI,6 Neal G. COPELAND,7 Nancy A. JENKINS,7 and Nobutaka WAKAMIYA5

Divisions of 1Food Microbiology, 2Pathology, and 4Virology, Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan 3Department of Biological Science Research, Research ＆ Development Center, Fuso Pharmaceutical Industries, Ltd., 2-3-30 Morinomiya, Joto-ku, Osaka 536-0025, Japan 5Department of Microbiology, Asahikawa Medical College, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan 6Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan 7Mouse Cancer Genetics Program, National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA

Received March 22, 2002; Accepted June 19, 2002
Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88％ identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.
Key words: collectin liver 1; collectin; mouse; cDNA; chromosome

-17-
Gene Family of Oleosin Isoforms and Their Structural Stabilization in Sesame Seed Oil Bodies

Sorgan S. K. TAI, Miles C. M. CHEN, Chi-Chung PENG, and Jason T. C. TZEN

Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan 40227, ROC

Received March 22, 2002; Accepted June 11, 2002
Oleosins are structural proteins sheltering the oil bodies of plant seeds. Two isoform classes termed H- and L-oleosin are present in diverse angiosperms. Two H-oleosins and one L-oleosin were identified in sesame oil bodies from the protein sequences deduced from their corresponding cDNA clones. Sequence analysis showed that the main difference between the H- and L-isoforms is an insertion of 18 residues in the C-terminal domain of H-oleosins. H-oleosin, presumably derived from L-oleosin, was duplicated independently in several species. All known oleosins can be classified as one of these two isoforms. Single copy or a low copy number was detected by Southern hybridization for each of the three oleosin genes in the sesame genome. Northern hybridization showed that the three oleosin genes were transcribed in maturing seeds where oil bodies are being assembled. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and sesame oleosin isoforms. The results indicated that reconstituted oil bodies could be stabilized by both isoforms, but L-oleosin gave slightly more structural stability than H-oleosin.
Key words: oil body; oleosin isoform; seed; sesame; structural stability

-18-
A Lipase-inhibiting Protein from Lipoxygenase-deficient Soybean Seeds

Kiyoshi SATOUCHI,1, Yoshifumi KODAMA,1 Kaoru MURAKAMI,1 Tamotsu TANAKA,1 Hiroyuki IWAMOTO,1 and Masao ISHIMOTO2

1Department of Applied Biological Science, Fukuyama University, Fukuyama 729-0292, Japan 2Biological Laboratory of Plant Biotechnology, National Agricultural Research Center for Western Region, Fukuyama 721-8514, Japan

Received April 1, 2002; Accepted June 26, 2002
A lipase-inhibiting protein was isolated from lipoxygenase (LOX)-deficient soybean seeds. The molecular mass of the protein was 56.0-kDa and the N-terminal amino acid was blocked. The protein was identified by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The masses of the lysyl endopeptidase-digested peptides of the 56.0-kDa inhibiting protein were almost identical to the calculated masses of the theoretically predicted lysyl endopeptidase-treated peptides of β-amylase from soybean seed. In a previous paper (Biosci. Biotechnol. Biochem., 62, 1498--1503, 1998), we reported that LOX-1, an isozyme of soybean seed LOX, inhibited hydrolysis of soybean oil by pancreatic lipase. Purified β-amylase also inhibited lipase activity, although the magnitude of inhibition was weaker than that by LOX-1. Thus, there are at least two lipase-inhibiting proteins, one is a LOX and the other is a β-amylase, in soybean seed.
Key words: lipase-inhibiting protein; lipoxygenase; soybean seed; β-amylase; peptide mass fingerprinting

-19-
Increased Staphylococcus-killing Activity of an Antimicrobial Peptide, Lactoferricin B, with Minocycline and Monoacylglycerol

Hiroyuki WAKABAYASHI, Susumu TERAGUCHI, and Yoshitaka TAMURA

Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228-8583, Japan

Received April 1, 2002; Accepted May 1, 2002
This study aimed to find antibiotics or other compounds that could increase the antimicrobial activity of an antimicrobial peptide, lactoferricin B (LFcin B), against Staphylococcus aureus, including antibiotic-resistant strains. Among conventional antibiotics, minocycline increased the bactericidal activity of LFcin B against S. aureus, but methicillin, ceftizoxime, and sulfamethoxazole-trimethoprim did not have such an effect. The combination of minocycline and LFcin B had synergistic effects against three antibiotic-resistant strains of S. aureus, according to result of checkerboard analysis. Screening of 33 compounds, including acids and salts, alcohols, amino acids, proteins and peptides, sugar, and lipids, showed that medium-chain monoacylglycerols increased the bactericidal activity of LFcin B against three S. aureus strains. The short-term killing test in water and the killing curve test in growing cultures showed that a combination of LFcin B and monolaurin (a monoacylglycerol with a 12-carbon acyl chain) killed S. aureus more rapidly than either agent alone. These findings may be helpful in the application of antimicrobial peptides in medical or other situations.
Key words: lactoferricin B; minocycline; monoacylglycerol; Staphylococcus aureus; bactericidal

-20-
Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

Sunhwa KIM,1 Ichiro MATSUO,2, Katsumi AJISAKA,2 Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1

1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Meiji Institute of Health Science, Naruda 540, Odawara 250-0862, Japan

Received April 2, 2002; Accepted June 14, 2002
We isolated a β-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed β-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of β-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture.
Key words: β-N-acetylglucosaminidase; nagA; Aspergillus nidulans

-21-
Jasmonate-induced Changes in Flavonoid Metabolism in Barley (Hordeum vulgare) Leaves

Atsushi ISHIHARA,1,2, Yuki OGURA,1,2 Shin-ichi TEBAYASHI,2, and Hajime IWAMURA3

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 2CREST, Japan Science and Technology Corporation (JST), Japan 3Department of Bio-technology, School of Biology-oriented Science and Technology, Kinki University, Uchita-cho, Naga-gun, Wakayama 649-6493, Japan

Received April 8, 2002; Accepted May 30, 2002
The effects of jasmonic acid (JA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. A reversed-phase HPLC analysis revealed that the amount of a particular compound increased in excised barley leaf segments that had been treated with JA. This compound was purified and identified as 6′<Oh>!<Wa>-feruloylsaponarin (1) by spectroscopic analyses and alkaline hydrolysis. A related compound, 6′<Oh>!<Wa>-sinapoylsaponarin (2), was also found to accumulate in excised leaves independently of the JA treatment. The accumulation of these compounds was accompanied by a decrease in the saponarin (3) content. [8,9-13C]p-Coumaric acid and [2,3,4,5,6-2H]L-phenylalanine were effectively incorporated into the hydroxycinnamoyl moieties in 1 and 2, while the degree of incorporation of the labeled precursors into the saponarin part was small. These findings indicate that the hydroxycinnamoyl moieties of 1 and 2 are synthesized de novo from phenylalanine via the phenylpropanoid pathway, and that the saponarin part is mainly provided by the constitutive pool of 3.
Key words: Holdeum vulgare; Gramineae; barley; jasmonic acid; hydroxycinnamoylsaponarin

-22-
Cloning and Expression of Cytochrome P450 Genes, Belonging to a New P450 Family, of the Basidiomycete Lentinula edodes

Ryoko AKIYAMA, Yoshihiro SATO, Susumu KAJIWARA, and Kazuo SHISHIDO

Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

Received April 8, 2002; Accepted June 8, 2002
Three cytochrome P450 genes, named Le. cyp1, Le. cyp2, and Le. cyp3, were isolated from the basidiomycete Lentinula edodes. Le. cyp1 and Le. cyp2 contained coding regions of 1500 bp and 1497 bp, respectively, but Le. cyp3 was found to be a defective gene. The deduced amino acid sequences of Le. CYP1 (CYP510A1, 500 amino acids) and Le. CYP2 (CYP510A3, 499 amino acids) were highly similar to each other (87％ identical) and they had 32--33％ identities to that of Coprinus cinereus P450 (CYP502) and 27--28％ identities to those of two Aspergillus P450s (CYP64 family). Quantitative RT-PCR analysis of the transcripts of Le. cyp1 and Le. cyp2 genes in the course of fruiting-body development of L. edodes showed that the primordium seems to contain larger amounts of these transcripts. The transcript levels of both of these genes in the stipe of the premature fruiting body were higher than those in the whole pileus and gill tissue.
Key words: cytochrome P450; gene clustering; transcriptional expression; fruiting-body formation; Lentinula edodes

-23-
ESR Characterization of a Novel Spin-trapping Agent, 15N-Labeled N-tert-Butyl-α-phenylnitrone, as a Nitric Oxide Donor

Kieko SAITO and Hisashi YOSHIOKA

Institute for Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan

Received April 8, 2002; Accepted May 30, 2002
We previously found that one of the pharmacological effects of N-tert-butyl-α-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15N to identify its corresponding 15NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15N-PBN, the starting material, ammonium-15N chloride, was converted to 2-amino-15N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-15N, and finally reacted with benzaldehyde to give 15N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)2, as a trapping agent to measure the NO levels of 15N-PBN or 14N-PBN in vitro, the peak intensity of 15NO[Fe(MGD)2] was over 50％ stronger than that of 14NO[Fe(MGD)2], and that 15NO and 14NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15NO derived from 15N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15NO from PBN and 14NO generated from cells. These results suggested that 15N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.
Key words: nitric oxide; N-tert-butyl-α-phenylnitrone (PBN); ESR; oxidative stress; spin trap

-24-
Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen from Thermococcus kodakaraensis KOD1

Masao KITABAYASHI,1,2, Yoshiaki NISHIYA,1 Muneharu ESAKA,2 Mitsuo ITAKURA,3 and Tadayuki IMANAKA4

1Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan 2Faculty of Applied Biological Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8528, Japan 3Institute for Genome Research, University of Tokushima, 3-8-15 Kuramoto-cho, Tokushima 770-8503, Japan 4Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, Japan

Received April 15, 2002; Accepted June 19, 2002
The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3％ identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 <Oh><0192><Wa>KOD DNA polymerase′. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.
Key words: PCNA; sliding clamp; DNA polymerase; Thermococcus kodakaraensis KOD1; PCR

-25-
Identification of Non-Pseudomonad Bacteria from Fruit Bodies of Wild Agaricales Fungi That Detoxify Tolaasin Produced by Pseudomonas tolaasii

Takanori TSUKAMOTO,1, Hitoshi MURATA,2, and Akira SHIRATA1

1Department of Sericulture, National Institute of Sericultural and Entomological Sciences, Ohwashi 1-2, Tsukuba-City 305-8634, Japan 2Division of Bio-Resource Development, Forestry ＆ Forest Products Research Institute, Kukizaki, Ibaraki 305-8687, Japan

Received April 17, 2002; Accepted June 24, 2002
Bacterial isolates from wild Agaricales fungi detoxified tolaasin, the inducer of brown blotch disease of cultivated mushrooms produced by Pseudomonas tolaasii. Mycetocola tolaasinivorans and Mycetocola lacteus were associated with fruit bodies of wild Pleurotus ostreatus and wild Lepista nuda, respectively. Tolaasin-detoxifying bacteria belonging to other genera were found in various wild mushrooms. An Acinetobacter sp. was isolated from fruit bodies of Tricholoma matsutake, Bacillus pumilus was isolated from Coprinus disseminatus, and Sphingobacterium multivorum was isolated from Clitocybe clavipes. A Pedobacter sp., which seemed not be identifiable as any known bacterial species, was isolated from a Clitocybe sp. Tolaasin-detoxifying bacteria identified thus far were attached to the surface of mycelia rather than residing within the fungal cells. M. tolaasinivorans, M. lacteus, B. pumilus, the Pedobacter sp., and S. multivorum efficiently detoxified tolaasin and strongly suppressed brown blotch development in cultivated P. ostreatus and Agaricus bisporus in vitro, but the Acinetobacter sp. did so less efficiently. These bacteria may be useful for the elucidation of mechanisms involved in tolaasin-detoxification, and may become biological control agents of mushroom disease.
Key words: Acinetobacter sp.; Bacillus pumilus; Mycetocola spp.; Pedobacter sp.; Sphingobacterium multivorum

-26-
Effects of Iprodione and Fludioxonil on Glycerol Synthesis and Hyphal Development in Candida albicans

Noriyuki OCHIAI,1 Makoto FUJIMURA,1, Michiyo OSHIMA,1 Takayuki MOTOYAMA,2 Akihiko ICHIISHI,1 Hisafumi YAMADA-OKABE,3 and Isamu YAMAGUCHI2

1Faculty of Life Science, University of Toyo, Itakura, Oura-gun 374-0193, Japan 2Plant Science Center, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan 3Department of Oncology, Nippon Roche Research Center, 200 Kajiwara, Kamakura 247-8530, Japan

Received May 7, 2002; Accepted June 6, 2002
We investigated the effects of iprodione and fludioxonil on the pathogenic yeast Candida albicans. Growth of the wild-type IFO1385 strain of C. albicans was inhibited by both fungicides, while Saccharomyces cerevisiae was basically unaffected by them even at a concentration of 25 μg/ml. Both fungicides stimulated glycerol synthesis in C. albicans but not in S. cerevisiae. The antioxidant α-tocopherol acetate and the cytochrome P-450 inhibitor piperonyl butoxide antagonized the fungitoxicity of iprodione and fludioxonil in C. albicans. It is known that mutations within the histidine kinase NIK1/OS-1 gene confer resistance to iprodione and fludioxonil in Neurospora crassa, while the fungicide-insensitive S. cerevisiae has only one histidine kinase SLN1 gene in its genome. In contrast, C. albicans has three histidine kinase genes, namely CaSLN1, CaNIK1/COS1, and CaHK1, the null mutants of which were found to impair the hyphal formation. Iprodione and fludioxonil were found to suppress filamentation when the IFO1385 strain was incubated on a solid medium containing fetal bovine serum. These observations suggest that iprodione and fludioxonil interfere with the CaNIK1/COS1 signal transduction pathway, resulting in glycerol synthesis stimulation and the inhibition of hyphal formation.
Key words: iprodione; fludioxonil; osmotic stress; two-component signal transduction; histidine kinase

-27-
RNase G-Dependent Degradation of the eno mRNA Encoding a Glycolysis Enzyme Enolase in Escherichia coli

Naoko KAGA,1 Genryou UMITSUKI,2 Kazuo NAGAI,3 and Masaaki WACHI1

1Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan 2Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba 278-0037, Japan 3Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai 487-8501, Japan

Received May 22, 2002; Accepted June 6, 2002
Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng＋ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng＋ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA.
Key words: RNase G; rng; eno mRNA; enolase

-28-
Note
Inhibition by Ajoene of Skin-tumor Promotion in Mice

Tomoaki NISHIKAWA, Norihiko YAMADA, Atsuhiko HATTORI, Hiroyuki FUKUDA, and Tsuchiyoshi FUJINO

Biodevelopment Division, Central Institute, Nagoya Seiraku Co., Ltd., 310 Nakasuna-cho, Tempaku-ku, Nagoya, Aichi 468-0065, Japan

Received February 14, 2002; Accepted May 30, 2002
Ajoene, a major compound containing sulfur in oil-macerated garlic products, inhibited in a two-stage carcinogenesis test on mouse skin. Treatment with ajoene suppressed skin tumor formation, depending on the amount. In particular, the group treated with 250 μg of ajoene had only 4.9％ the number of tumors per mouse compared with the control group at 18 weeks.
Key words: antitumor effect; cancer chemoprevention; ajoene; garlic; Allium sativum

-29-
Note
Involvement of a CCAAT-binding Complex in the Expression of a Nitrogen-Starvation-Specific Gene, isp6＋, in Schizosaccharomyces pombe

Akio NAKASHIMA,1 Masaru UENO,1 Takashi USHIMARU,2 and Masahiro URITANI1

1Department of Chemistry and 2Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan

Received February 26, 2002; Accepted May 16, 2002
The fission yeast gene isp6＋ is needed in nitrogen-starvation response but its transcriptional regulation has been unclear. isp6＋ was repressed under nutrient conditions, in which cAMP-dependent protein kinase A, the stress-activated protein kinase cascade, and the CCAAT-binding complex were concerned. The CCAAT-binding complex also was involved in the induction of isp6＋ during nitrogen starvation.
Key words: CCAAT-binding complex; cAMP-dependent protein kinase A; fission yeast; nitrogen starvation

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Note
Apoptosis Induction by Wheat-flour Sphingoid Bases in DLD-1 Human Colon Cancer Cells

Tatsuya SUGAWARA,1 Mikio KINOSHITA,2 Masao OHNISHI,2 and Teruo MIYAZAWA1

1Laboratory of Molecular Biodynamics, Graduate School of Life Science and Agriculture, Tohoku University, Sendai 981-8555, Japan 2Department of Bioresourse Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan

Received March 4, 2002; Accepted May 7, 2002
The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.
Key words: apoptosis; colon cancer; sphingoid bases; wheat flour

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Note
Differential Effects of Dietary Casein and Soybean Protein Isolate on Lipopolysaccharide-induced Hepatitis in D-Galactosamine-sensitized Rats

Kimio SUGIYAMA, Yasuhiko SHIMADA, Kasumi IWAI, and Tatsuya MORITA

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

Received March 11, 2002; Accepted May 25, 2002
Effects of dietary protein type on lipopolysaccharide (LPS)-induced hepatitis, as assessed by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, were investigated in D-galactosamine (GalN)-sensitized rats. The plasma ALT and AST activities in rats fed on 25％ soybean protein isolate (SPI) diet were significantly suppressed to about 1/4 and 1/5 of the values in rats fed on 25％ casein diet, respectively, 8 h after the injection of LPS＋GalN. Although hepatic ALT and AST activities of normal rats were also lower in the SPI group than in the casein group, this could not explain the differences in plasma enzyme activities between the two groups. The hepatic glutathione concentration of normal rats was lower in the SPI group than in the casein group, but it was reversed in rats injected with drugs. The results suggest that SPI can protect animals from LPS＋GalN-induced hepatitis, and that the hepatic glutathione level may participate in the effects of SPI.
Key words: casein; soybean protein isolate; lipopolysaccharide; hepatitis; glutathione

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Note
Synthesis and Biological Activity of Isoamoenylin, a Metabolite of Dendrobium amoenum

Somepalli VENKATESWARLU, Mudunuri S. S. RAJU, and Gottumukkala V. SUBBARAJU

Laila Impex R ＆ D Centre, Unit I, Phase III, Jawahar Autonagar, Vijayawada 520 007, India

Received March 22, 2002; Accepted May 20, 2002
Isoamoenylin (6), a dihydrostilbene from Dendrobium amoenum, was synthesised from 3,4,5-trimethoxybenzaldehyde (1) in four steps with an overall yield of 60％. The spectral data for synthetic 6 are in good agreement with those of the natural product. Isoamoenylin showed moderate antioxidative and weak antibacterial activities.
Key words: isoamoenylin; 3-[2-(3,4,5-trimethoxyphenyl)ethyl]phenol; Dendrobium amoenum; antioxidative activity

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Note
Tryptophans 286 and 288 in the C-terminal Region of Protein B23.1 are Important for Its Nucleolar Localization

Yuki NISHIMURA, Takeshi OHKUBO, Yukio FURUICHI, and Hayato UMEKAWA

Department of Sustainable Resource Science, Faculty of Bioresources, and Center for Molecular Biology and Genetics, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan

Received April 1, 2002; Accepted May 14, 2002
Nucleolar protein B23 can shuttle between the nucleolus and cytoplasm. However, the mechanism involved in the protein moving and staying in the nucleolus is not fully understood. To identify the nucleolar localization signal sequence of protein B23, we examined the subnuclear location of B23.1 mutant proteins fused with green fluorescent protein in HeLa cells. The results suggested that the two C-terminal tryptophan residues (Trp-286 and Trp-288) of protein B23.1 were important in this phenomenon.

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Note
Synthesis and Fungicidal Activity of 1-(α-tert-Butylcinnamoyl)imidazoles

Akio MANABE,1, Hirotaka TAKANO,1 Kunihiko FURUZAWA,1 Kazunori YANAGI,2 Yoshio HISADA,1 and Shizuya TANAKA1

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

Received April 1, 2002; Accepted May 30, 2002
Several 1-(α-tert-butylcinnamoyl)imidazoles were prepared to examine their fungicidal activity. The (Z)-4-chlorocinnamoyl derivative was prepared from (anti)-2-tert-butyl-3-(4-chlorophenyl)-3-hydroxypropanoic acid by treating with 1,1′-carbonyldiimidazole and a subsequent β-elimination reaction at an elevated temperature. The (Z)-isomer of the 4-chlorocinnamoyl derivative showed good fungicidal activity against Erysiphe graminis and Botrytis cinerea in pot tests, whereas the corresponding (E)-isomer derived from the (Z)-isomer through photoisomerization was much less active.
Key words: 1-cinnamoylimidazole; β-elimination reaction; geometrical isomer; azole fungicide; fungicidal activity

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Note
PCR Detection of DNAs of Animal Origin in Feed by Primers Based on Sequences of Short and Long Interspersed Repetitive Elements

Kiyoshi TAJIMA,1, Osamu ENISHI,1 Masahiro AMARI,1 Makoto MITSUMORI,1 Hiroshi KAJIKAWA,1 Mitsunori KURIHARA,1 Satoshi YANAI,2 Hiroki MATSUI,2 Hiroshi YASUE,3 Tadayoshi MITSUHASHI,3 Tomoyuki KAWASHIMA,1 and Mitsuto MATSUMOTO1

1National Institute of Livestock and Grassland Science, P.O. Box 5, Tsukuba, Norindanchi, Ibaraki 305-0901, Japan 2STAFF Institute, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan 3National Institute of Agrobiological Science, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-0806, Japan

Received April 2, 2002; Accepted May 23, 2002
PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01％ MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.
Key words: PCR detection; meat and bone meal; interspersed repetitive elements

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Note
Volatile Compounds of Headspace Gas in the Japanese Fish Sauce Ishiru

Toshihide MICHIHATA,1 Toshihiro YANO,2 and Toshiki ENOMOTO2

1Food Processing Laboratory, Industrial Research Institute of Ishikawa, Kanazawa, Ishikawa 920-0223, Japan 2Department of Food Science, Ishikawa Agricultural College, Nonoichi, Ishikawa 921-8836, Japan

Received April 2, 2002; Accepted May 29, 2002
Volatile compounds from the headspace gas of ten brands of the Japanese fish sauce ishiru were analyzed by GC-MS with a thermal-desorption cold-trap system. Many volatile peaks were detected and 51 compounds were identified. The major volatile compounds in ishiru included aldehydes (such as 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, and benzaldehyde), nitrogen-containing compounds (such as pyrazine derivatives and trimethylamine), sulfur-containing compounds (such as dimethyl disulfide), and ketones (such as 2-butanone and 3-methyl-2-butanone). On the other hand, volatile fatty acids were nearly absent in the headspace gas of ishiru.
Key words: ishiru; fish sauce; headspace gas; volatile compounds; GC-MS with thermal-desorption cold-trap system

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Note
Inhibition of Proteasome Activity by Tyropeptin A in PC12 Cells

Isao MOMOSE, Ryuichi SEKIZAWA, Hironobu IINUMA, and Tomio TAKEUCHI

Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

Received April 3, 2002; Accepted May 10, 2002
Tyropeptin A, a potent proteasome inhibitor not reported before, was produced by Kitasatospora sp. MK993-dF2. In this study, we investigated the effects of tyropeptin A on proteasome activity in PC12 cells. Tyropeptin A inhibited the intracellular proteasome activity in a dose-dependent way and seemed to cause neurite outgrowth. As expected, ubiquitinated proteins that should be substrates for the proteasome accumulated in cells treated with tyropeptin A. Hence, it appears that tyropeptin A can permeate into cells and there inhibit the intracellular proteasome activity.
Key words: tyropeptin A; proteasome inhibitor; neurite outgrowth

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Note
Soft Gel Medium Solidified with Gellan Gum for Preliminary Screening for Root-associating, Free-living Nitrogen-fixing Bacteria Inhabiting the Rhizoplane of Plants

Yasuyuki HASHIDOKO,1, Motohiko TADA,1 Mitsuru OSAKI,2 and Satoshi TAHARA1

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan 2Division of Bioresources and Product Science, Graduate School of Agriculture, Hokkaido, Kita-ku, Sapporo 060-8589, Japan

Received April 4, 2002; Accepted June 18, 2002
For preliminary screening for and characterization of free-living nitrogen-fixing bacteria from rhizoplane microflora, we used Winogradsky′s mineral mixture-based nitrogen-free medium solidified with 0.3％ gellan gum. The soft gel medium enabled some reference and wild free-living nitrogen-fixing bacteria to grow in characteristic colonies, including their reaction to oxygen and their motility change. Gellan gum is thus likely to be a better gel matrix than agarose for the investigation of root-associating, free-living nitrogen-fixing bacteria to identify their characteristic behaviors.
Key words: free-living nitrogen-fixing bacterium; gellan gum; Plantago lanceolata; rhizoplane microflora

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Note
Specific Binding of Silkworm Bombyx mori 30-kDa Lipoproteins to Carbohydrates Containing Glucose

Minoru UJITA,1,2, Akihiro KIMURA,1 Daisuke NISHINO,1 Eiji YOKOYAMA,2 Yutaka BANNO,3 Hiroshi FUJII,3 and Akira HARA1,2

1Laboratory of Biological Chemistry, Department of Applied Biological Chemistry, Faculty of Agriculture and 2Agricultural High-Tech Research Center, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan 3Institute of Genetic Resources, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan

Received April 5, 2002; Accepted June 5, 2002
Insect lectins are important as part of nonspecific self-defense, but their antifungal mechanisms remain to be elucidated. Fungi contain glucans on the cell surface and insect glucan-binding proteins are considered to be essential for antifungal mechanisms. We purified glucose-binding proteins from hemolymph of pupae of the silkworm Bombyx mori, and the amino acid sequence analysis showed that their two proteins are 30-kDa lipoproteins, major components of B. mori hemolymph. These lipoproteins specifically bound to glucose and glucans, suggesting that they are involved in insect self-defense systems.
Key words: Bombyx mori; hemolymph; fungi; glucan; lipoprotein

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Note
Access to Enantiomerically Pure Intermediates for (−)-Geosmin Synthesis Starting from (4aS,5S)-4,4a,5,6,7,8-Hexahydro-5-hydroxy-4a-methyl- naphthalen-2(3H)-one

Ken-ichi FUHSHUKU and Takeshi SUGAI

Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan

Received April 23, 2002; Accepted May 27, 2002
Enantiomerically pure key intermediates for the synthesis of the natural enantiomer of geosmin were synthesized from (4aS,5S)-4,4a,5,6,7,8-hexahydro-5-hydroxy-4a-methylnaphthalen-2(3H)-one.
Key words: geosmin; Wieland-Miescher ketone; cyclopropanation; hydrogenation

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Synthesis of Novel α-C-Glycosylamino Acids and Reverse Regioselectivity in Larock′s Heteroannulation for the Synthesis of the Indole Nucleus

Toshio NISHIKAWA, Kyoko WADA, and Minoru ISOBE

Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Received April 24, 2002; Accepted May 31, 2002
An α-C-iodoethynylglucose derivative was coupled with an L-serine-derived zinc-copper reagent to give α-C-glucosylpropargyl glycine, which underwent palladium catalyzed-heteroannulation with o-iodoaniline to give not α-C-glucosyl-tryptophan but α-C-glucosyl-iso-tryptophan. This is the first observation of complete reverse regioselectivity to Larock′s proposal.
Key words: C-glycosylamino acid; propargyl glycine; indole; iso-tryptophan; palladium catalyst

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Note
Molecular Cloning of a Pore-Forming Subunit (Kir6.2 Gene) of the ATP-Sensitive Potassium Channel in the Bullfrog, Rana catesbeiana Shaw

Pyo-Jam PARK,1 Joon-Yong CHUNG,2 Hee-Guk BYUN,1 Dae-Hyun SEOG,3 and Se-Kwon KIM1

1Department of Chemistry, Pukyong National University, Pusan 608-737, Korea Department of 2Parasitology and 3Microbiology, College of Medicine, Inje University, Pusan 614-735, Korea

Received April 25, 2002; Accepted May 28, 2002
A cDNA sequence encoding a pore-forming subunit of ATP-sensitive potassium channel (Kir6.2 gene) of the bullfrog, Rana catesbeiana Shaw, termed RcKir6.2, was isolated from a liver cDNA library. The cDNA contained a single open reading frame of 1,173 bp encoding 391 amino acids with a calculated molecular mass of 42.9 kDa, which has a structural motif (a GFG motif) of the putative pore-forming loop of Kir6.2. Analysis of its phlyogenetic position revealed that the RcKir6.2 is close to Kir6.2 of rabbits. The predicted amino acid sequence shared sequence identity with Kir6.2 of Homo sapiens, Cavia porcellus, Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus by 95.9, 95.6, 96.7, 96.7 and 99.7％, respectively. Expression of RcKir6.2 was detected in various tissues, including heart, kidney, liver, lung, spleen, and stomach of the bullfrog.
Key words: cloning; Kir6.2 gene; bullfrog; ATP-sensitive potassium channel

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Note
Ricinoleic Acid and Castor Oil as Substrates for Conjugated Linoleic Acid Production by Washed Cells of Lactobacillus plantarum

Shigenobu KISHINO, Jun OGAWA, Akinori ANDO, Yoriko OMURA, and Sakayu SHIMIZU

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received May 2, 2002; Accepted June 3, 2002
Ricinoleic acid (12-hydroxy-cis-9-octadecaenoic acid) was an effective substrate for conjugated linoleic acid (CLA) production by washed cells of Lactobacillus plantarum AKU 1009a. The CLA produced was a mixture of cis-9,trans-11- and trans-9,trans-11-octadecadienoic acids. Addition of α-linolenic acid to the culture medium increased the CLA productivity of the washed cells. In the presence of lipase, castor oil, in which the main fatty acid component is ricinoleic acid, also was a substrate for CLA.
Key words: conjugated linoleic acid; ricinoleic acid; castor oil; lactic acid bacteria; Lactobacillus plantarum

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Note
Molecular Cloning and Functional Expression of cDNA Encoding a Cysteine Proteinase Inhibitor, Cystatin, from Job′s Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

Koh-Ichi YOZA, Sumiko NAKAMURA, Miki YAGUCHI, Kazutomo HARAGUCHI, and Ken-Ichi OHTSUBO

National Food Research Institute, 2-1-12 Kannondai, Tsukuba City, Ibaraki 305-8642, Japan

Received May 14, 2002; Accepted June 11, 2002
A λZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job′s tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job′s-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.
Key words: Job′s tears; cystatin; cysteine proteinase inhibitor

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Preliminary Communication
Isolation and Identification of Novel ADP-Ribosylated Proteins from Streptomyces coelicolor A3(2)

Kiyoshi SUGAWARA,1 Naoshi DOHMAE,2 Koji KASAI,1 Hisako SAIDO-SAKANAKA,1 Susumu OKAMOTO,1 Koji TAKIO,2 and Kozo OCHI1

1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 2Biomolecular Characterization Division, RIKEN (Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Received June 17, 2002; Accepted July 15, 2002
An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785--3790; 178, 4935--4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.
Key words: protein ADP-ribosylation; membrane proteins; Streptomyces coelicolor A3(2)