Contents and Abstracts of BBB

(Vol.67 No.9 2003)

Suppression of Ethanol and Lipopolysaccharide-induced Liver Injury by Extracts of Hydrangeae Dulcis Folium in Rats
Erika HASHIZUME, Ryusuke NAKAGIRI, Akio SHIRAI, Shun KAYAHASHI, Sakai YASUSHI, and Toshikazu KAMIYA p.1857

Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process
Xu FANG,1 Yoshiyuki WATANABE,1 Shuji ADACHI,1, Yasuki MATSUMURA,2 Tomohiko MORI,2 Hirokazu MAEDA,3 Akihiro NAKAMURA,3 and Ryuichi MATSUNO1 p.1864

Oxidative Reaction of Oxindole-3-acetic Acids
Toshio NIWA,1,† Sayuri ISHII,1 Atsushi HIRAMATSU,1 and Toshihiko OSAWA2 p.1870

Characterization and Kinetics of 45 kDa Chitosanase from Bacillus sp. P16
You-Young JO,1, Kyu-Jong JO,1 Yu-Lan JIN,1 Kil-Yong KIM,1,2 Jae-Han SHIM,1,2 Yong-Woong KIM,1 and Ro-Dong PARK1,2, p.1875

Apoptosis Induction in HL-60 Cells and Inhibition of Topoisomerase II by Triterpene Celastrol
Masahiro NAGASE,1, Jinsei OTO,1 Sin SUGIYAMA,1 Kouichi YUBE,2 Yoshihisa TAKAISHI,3 and Nobuo SAKATO4 p.1883

Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) from Chlorella vulgaris C-27
Ken-ichi HONJOH,1, Ayano MIMURA,2 Eiko KUROIWA,2 Takahiro HAGISAKO,2 Koushirou SUGA,2,3 Hideyuki SHIMIZU,2 Rama Shanker DUBEY,4 Takahisa MIYAMOTO,1 Shoji HATANO,5 and Masayoshi IIO1 p.1888

Improved Functional Properties of the Ovoinhibitor by Conjugating with Galactomannan
Shamima BEGUM, Akira SAITO, Xianhua XU, and Akio KATO p.1897

Metabolism of Deuterium-labeled Jasmonic Acid and OPC 8:0 in the Potato Plant (Solanum tuberosum L.)
Hideyuki MATSUURA and Teruhiko YOSHIHARA p.1903

Malformation of Immature Starfish Oocytes by Theonellapeptolide Ie, a Tridecapeptide Lactone from a Marine Sponge Petrosia Species, through Disturbance of Cortical F-Actin Distribution
Emi OHTA,1,2 Hironobu OKADA,1 Shinji OHTA,2 Motomasa KOBAYASHI,3 Isao KITAGAWA,3 Shintaro HORIIKE,4 Tadao TAKAHASHI,4 Hiroshi HOSOYA,4 Kenya YAMAMOTO,5 and Susumu IKEGAMI1,2,　p.1908

Suppressive Effects of Genistein on Oxidative Stress and NFκB Activation in RAW 264.7 Macrophages
Chunyeon CHOI,1 Hyeyeon CHO,1 Jiyoung PARK,1 Chungwon CHO,2 and Youngsun SONG1,
p.1916

Production of D-Arabitol by Metschnikowia reukaufii AJ14787
Hiroyuki NOZAKI, Shun-ichi SUZUKI, Naoko TSUYOSHI, and Kenzo YOKOZEKI p.1923

Reduction of Noise-stress-induced Physiological Damage by Radices of Astragali and Rhodiolae: Glycogen, Lactic Acid and Cholesterol Contents in Liver of the Rat
Bei-Wei ZHU,1 Yu-Mei SUN,1 Xia YUN,1 Song HAN,2 Mei-Lan PIAO,2 Yoshiyuki MURATA,3 and Mikiro TADA2,† p.1930

Effects of Highly Purified Structured Lipids Containing Medium-chain Fatty Acids and Linoleic Acid on Lipid Profiles in Rats
Jun-ichi NAGATA,1,† Michio KASAI,2 Souichiro WATANABE,2 Ikuo IKEDA,3 and Morio SAITO1 p.1937

Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs
Hyen Joo PARK, Hyun-Jung LEE, Eun-Jin LEE, Hye Jin HWANG, Sang-Hee SHIN, Myung-Eun SUH, Choonmi KIM, Hwa Jung KIM, Eun-Kyung SEO, and Sang Kook LEE†, p.1944

Properties of a Novel Extracellular Cell-free Ice Nuclei from Ice-nucleating Pseudomonas antarctica IN-74
Naomi MURYOI, Hidehisa KAWAHARA, and Hitoshi OBATA p.1950

New Method for Synthesizing the Intermediates to 5-HETE from Yeast-mediated Reduction Products by Employing Baeyer-Villiger Oxidation with Complete Retention of Enantiomeric Excess
Satoshi YAMAUCHI, Yoshihiro KINOSHITA, and Yoshiro KINOSHITA p.1959

The Dual Functions of Biphenyl-degrading Ability of Pseudomonas sp. KKS102: Energy Acquisition and Substrate Detoxification
Mina DELAWARY,1,2 Yoshiyuki OHTSUBO,3 and Akinori OHTA1,† p.1970

Occurrence of a Specific Protein in Basidiomycete-lytic Enzyme Preparation Produced by Bacillus circulans KA-304 Inductively with a Cell-wall Preparation of Schizophyllum commune
Shigekazu YANO, Sachiko YAMAMOTO, Toshihiko TOGE, Mamoru WAKAYAMA, and Takashi TACHIKI p.1976

Note
Reconstitution and Characterization of NtrC Protein in a Deep-sea Piezophilic Bacterium, Shewanella violacea Strain DSS12
Hiroaki KAWANO,1,2 Akihiko IKEGAMI,3 Kaoru NAKASONE,4, Chiaki KATO,5 Ron USAMI,1 and Koki HORIKOSHI1,2 p.1983

Note
Glycerolipid Acyl Hydrolase Activity in the Brown Alga Cladosiphon okamuranus TOKIDA
Masaru TERASAKI and Yutaka ITABASHI p.1986

Note
A Study of Tryptophan Fluorescence Quenching of Bifunctional Alginate Lyase from a Marine Bacterium Pseudoalteromonas sp. Strain No. 272 by Acrylamide
Yoshiko IWAMOTO, Hidetoshi HIDAKA, Tatsuya ODA, and Tsuyoshi MURAMATSU p.1990

Note
Subsite Structure of Maltogenic Amylase from Thermomonospora viridis TF-35
Kosei TAKAHASHI and Teruo NAKAKUKI p.1993

Note
Generation of Reactive Oxygen Species from Hinokitiol under Near-UV Irradiation
Hitoshi SHIBATA, Takayuki NAGAMINE, Yong WANG, Takahiro ISHIKAWA, and Yoshihiro SAWA
p.1996

Note
(−)-Hydroxycitrate Ingestion Increases Fat Oxidation during Moderate Intensity Exercise in Untrained Men
Kyoko TOMITA,1, Yasuhide OKUHARA,1 Norihiro SHIGEMATSU,1 Heajung SUH,2 and Kiwon LIM3
p.1999

Note
Nucleotide Sequences of Genes Encoding Allosamidin-sensitive and -insensitive Chitinases Produced by Allosamidin-producing Streptomyces
Hiroshi MATSUURA,1 Susumu OKAMOTO,1 Sarintip ANAMNART,2 Qiuqi WANG,2 Ze-Yang ZHOU,2 Takuya NIHIRA,2 Yasuhiro YAMADA,2 Tomohisa KUZUYAMA,3 Haruo SETO,3 Jiro NAKAYAMA,1 Akinori SUZUKI,1 Hiromichi NAGASAWA,1 and Shohei SAKUDA1,† p.2002

Note
Construction of a Homologous Selectable Marker Gene for Lentinula edodes Transformation
Toshikazu IRIE,1 Toshitsugu SATO,1,† Kumiko SAITO,1 Yoichi HONDA,2 Takashi WATANABE,2 Masaaki KUWAHARA,3 and Hitoshi ENEI1 p.2006

Note
Cloning and Sequence Analysis of Endoglucanase Genes from an Industrial Fungus, Aspergillus kawachii
Yukari HARA,2 Yumi HINOKI,2 Hitoshi SHIMOI,1,2 and Kiyoshi ITO1,2, p.2010

Note
Structural Effects of Phenolic Acids on the Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers
Yutaka KONISHI,1,† Kazuo KUBO,2 and Makoto SHIMIZU3 p.2014

Note
Proteases of Maitake (Grifola frondosa) Responsible for Breakdown of Wheat Flour Dough and Their Reaction with Gluten Proteins
Makoto ABE1, and Masaharu SEGUCHI2 p.2018

Note
Root Growth-promoting Activity of Unsaturated Oligomeric Uronates from Alginate on Carrot and Rice Plants
Xu XU,1 Yoshiko IWAMOTO,1 Yoshie KITAMURA,2 Tatsuya ODA,1 and Tsuyoshi MURAMATSU1,† p.2022

Note
Isolation and Characterization of Phenol-catabolizing Bacteria from a Coking Plant
Wael S. El-SAYED,1 Mohamed K. IBRAHIM,1 Mohamed Abu-SHADY,1 Fawkia El-BEIH,1 Naoya OHMURA,2,† Hiroshi SAIKI,2 and Akikazu ANDO3 p.2026

Note
Fosmidomycin Resistance in Adenylate Cyclase Deficient (cya) Mutants of Escherichia coli
Yoshiko SAKAMOTO, Soichi FURUKAWA, Hirokazu OGIHARA, and Makari YAMASAKI p.2030

Note
A Simple and Efficient Method for High Fidelity PCR Cloning Using Antibody-neutralizing Technology
Masao KITABAYASHI,1,2 Yoshiaki NISHIYA,3 and Muneharu ESAKA1, p.2034

Note
Myrsinoic Acid E, an Anti-inflammatory Compound from Myrsine seguinii
Hidefumi MAKABE,1,† Shintaro MIYAZAKI,2 Tsunashi KAMO,2 and Mitsuru HIROTA2
p.2038

Note
Activin A Induces Phosphorylation of Smad2 but Not Complex Formation of Smad2 with Smad4 in Human Colon Cancer Cell Line HT-29
Pimara PHOLNUKULKIT, Kei SONOYAMA, and Jun KAWABATA p.2042

Note
Ethyl 4-[2-(6-Methyl-3-pyridyloxy)butyloxy]benzoate, a Novel Anti-juvenile Hormone Agent
Hanae ISHIGURO,1 Norihiro FUJITA,1 In-Hae KIM,1 Takahiro SHIOTSUKI,2 and Eiichi KUWANO1, p.2045

Note
A Long Acidic Domain Affects the Chromatographic Behaviour of a Neuronal Adaptor Protein on DEAE-Sepharose
Olimpia LONGO,1 Annalisa LAMBERTI,1 Nicola ZAMBRANO,1,2 and Paolo ARCARI1,2,†
p.2048

Note
Isolation of Lectins by Affinity Chromatography with Porcine Plasma Proteins Immobilized on Agarose
Akane KAJIYA,1 Yu KOYAMA,1 Takashi MITA,1 Tomohiro MEGA,2 Sumihiro HASE,2 Toshioki KAWAKAMI,3 Eiko HONDA,4 Hiroshi MUNAKATA,5 and Mamoru ISEMURA1,† p.2051

Communication
350-kDa Royal Jelly Glycoprotein (Apisin), Which Stimulates Proliferation of Human Monocytes, Bears the β1--3Galactosylated N-Glycan: Analysis of the N-Glycosylation Site
Mariko KIMURA,1 Yoshinobu KIMURA,2,† Kazunori TSUMURA,2 Kiyoshi OKIHARA,3 Hiroyuki SUGIMOTO,3 Hideo YAMADA,3 and Masami YONEKURA4 p.2055

-1-
Suppression of Ethanol and Lipopolysaccharide-induced Liver Injury by Extracts of Hydrangeae Dulcis Folium in Rats

Erika HASHIZUME, Ryusuke NAKAGIRI, Akio SHIRAI, Shun KAYAHASHI, Sakai YASUSHI, and Toshikazu KAMIYA

Kyowa Hakko Kogyo Co., LTD., Tsukuba Research Laboratories, 2 Miyukigaoka, Tsukuba-City, Ibaraki Prefecture 305-0841, Japan

Received November 1, 2002; Accepted May 26, 2003
In female SD rats that were injected with 4 g/kg BW ethanol p.o. followed by a 5 mg/kg BW lipopolysaccharide (LPS) i.v. injection, serum glutamic pyruvic transaminases (GPT) activity increased to about eight times that of normal rats. In this model, rats that had been fed a diet containing 1％ Hydrangeae Dulcis Folium (HDF) extracts for fifteen days showed significantly lower serum GPT activity (380.0±58.2 IU/l) than the control group (3527.0±774.1 IU/l). HDF′s efficacy was far superior to milk thistle in this model (2950.0±915.9 IU/l). When mouse macrophages were treated with HDF extracts at 50 μg/ml, TNF-α production induced by LPS was suppressed to about 10％ of the control. Rat serum TNF-α levels induced by LPS was decreased to 58.7％ of the control by administering 1000 mg/kg BW HDF extract p.o. These results indicate that HDF prevents alcohol-induced liver injury through the inhibition of TNF-α production.
Key words: Hydrangeae Dulcis Folium; lipopolysaccharide; ethanol; hepatitis; TNF-α

-2-
Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process

Xu FANG,1 Yoshiyuki WATANABE,1 Shuji ADACHI,1, Yasuki MATSUMURA,2 Tomohiko MORI,2 Hirokazu MAEDA,3 Akihiro NAKAMURA,3 and Ryuichi MATSUNO1

1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 2Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan 3New Ingredients Research Institute, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun, Ibaraki 300-2497, Japan

Received November 19, 2002; Accepted May 12, 2003
Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.
Key words: linoleic acid; microencapsulation; oxidation; soluble soybean polysaccharide

-3-
Oxidative Reaction of Oxindole-3-acetic Acids

Toshio NIWA,1,† Sayuri ISHII,1 Atsushi HIRAMATSU,1 and Toshihiko OSAWA2

1Department of Merchandise Development, San-ei Sucrochemical Co., Ltd., 24-5 Kitahama-machi, Chita 478-8503, Japan 2Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Furocho, Chikusa, Nagoya 464-8601, Japan

Received December 3, 2002; Accepted May 26, 2003
The oxindole-3-acetic acids, oxidative metabolites of indole-3-acetic acid, were isolated from a byproduct of a corn starch manufacturing plant, and were further converted to the 3-hydroxyl derivatives in the presence of metal ion. The mechanical study was followed by a chemical analysis including other byproducts, and suggested the presence of an intermediate that had a radical at the C-3 position of oxindole-3-acetic acids.
Key words: dimerization; oxindole-3-acetic acid; hydroxyl radical; radical reaction

-4-
Characterization and Kinetics of 45 kDa Chitosanase from Bacillus sp. P16

You-Young JO,1, Kyu-Jong JO,1 Yu-Lan JIN,1 Kil-Yong KIM,1,2 Jae-Han SHIM,1,2 Yong-Woong KIM,1 and Ro-Dong PARK1,2,

1Department of Agricultural Chemistry, Chonnam National University, Gwangju, 500-757, Korea 2Institute of Agricultural Science and Technology, Chonnam National University, Gwangju, 500-757, Korea

Received January 21, 2003; Accepted April 17, 2003
An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5--10.0 and under 50°C. The Km and Vmax were measured with a chitosan of a D.A. of 20.2％ as 0.52 mg/ml and 7.71×10−6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15--30％ and lesser activity for 0--15％ acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)7 in an endo-splitting manner producing a mixture of (GlcN)2--5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)7 to (GlcN)6 to (GlcN)5, as indicated by the apparent first order rate constants, k1 values, of 4.98×10−4, 2.3×10−4, and 9.3×10−6 sec−1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.
Key words: Bacillus sp. P16; chitosanase; chitooligosaccharides; chitosan

-5-
Apoptosis Induction in HL-60 Cells and Inhibition of Topoisomerase II by Triterpene Celastrol

Masahiro NAGASE,1, Jinsei OTO,1 Sin SUGIYAMA,1 Kouichi YUBE,2 Yoshihisa TAKAISHI,3 and Nobuo SAKATO4

1Department of Life Sciences, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761-0795, Japan 2Research Equipment Center, Kagawa Medical University, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan 3Faculty of Pharmaceutical Sciences, University of Tokushima, 1-78-1 Shomachi, Tokushima, Tokushima 770-8505, Japan 4United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan

Received February 6, 2003; Accepted May 16, 2003
Celastrol, which is a triterpene purified from Celastraceae plants, has anticancer and anti-inflammatory activities. In this study we investigated to clarify whether celastrol can induce apoptosis in a human leukemia HL-60 model system. Celastrol was found to induce apoptosis, and the rank order of the potency of celastrol and its derivatives to induce internucleosomal DNA fragmentation was found to be celastrol＞cela-H≫the other derivatives＝vehicle control. Many anticancer agents are known to possess the ability to inhibit topoisomerase II, so the inhibitory activities of celastrol and its derivatives on topoisomerase II were also explored. The rank order of the inhibitory activity was found to be celastrol＞etoposide＞cela-H, indicating that the apoptosis-inducing activities of cela derivatives correspond to their inhibitory activities on topoisomerase II. These data suggested that celastrol may cause its effects such as anticancer activity by the mechanism of apoptosis along with topoisomerase II inhibition.
Key words: celastrol; derivative; apoptosis; topoisomerase II; HL-60 cells

-6-
Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) from Chlorella vulgaris C-27

Ken-ichi HONJOH,1, Ayano MIMURA,2 Eiko KUROIWA,2 Takahiro HAGISAKO,2 Koushirou SUGA,2,3 Hideyuki SHIMIZU,2 Rama Shanker DUBEY,4 Takahisa MIYAMOTO,1 Shoji HATANO,5 and Masayoshi IIO1

1Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan 2Department of Bioscience and Biotechnology, Graduate School of Bioresource and Environmental Sciences, Kyushu University, Fukuoka 812-8581, Japan 3Chlorella Industries Co., Ltd., 1343 Hisatomi, Chikugo, Fukuoka 833-0056, Japan 4Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India 5Graduate School of Health and Social Welfare Science, Nishikyushu University, 4490-9 Ooazaozaki, Kanzaki-machi, Kanzaki-gun, Saga 842-8585, Japan

Received February 24, 2003; Accepted June 18, 2003
Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2＋ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.
Key words: Chlorella vulgaris C-27; freezing tolerance; glucose 6-phosphate dehydrogenase (G6PDH); purification

-7-
Improved Functional Properties of the Ovoinhibitor by Conjugating with Galactomannan

Shamima BEGUM, Akira SAITO, Xianhua XU, and Akio KATO

Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan

Received March 3, 2003; Accepted May 23, 2003
The chicken egg white ovoinhibitor, a multi-type proteinase inhibitor, was conjugated with galactomannan through the Maillard reaction in a controlled dry heating state at 60°C and 65％ relative humidity. The formation of an ovoinhibitor-galactomannan conjugate during dry heating was confirmed by SDS-PAGE. The resulting ovoinhibitor-galactomannan conjugate showed almost the same inhibitory activity toward trypsin, chymotrypsin and elastase as that of the untreated ovoinhibitor, while the conjugate showed stronger heat stability and better emulsifying properties than the untreated ovoinhibitor. These results suggest that the ovoinhibitor-galactomannan conjugate can be used as a protease inhibitor having heat stability and outstanding emulsifying properties for industrial application.
Key words: ovoinhibitor; galactomannan; protein-polysaccharide conjugate

-8-
Metabolism of Deuterium-labeled Jasmonic Acid and OPC 8:0 in the Potato Plant (Solanum tuberosum L.)

Hideyuki MATSUURA and Teruhiko YOSHIHARA

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received March 6, 2003; Accepted May 9, 2003
The metabolism of deuterium-labeled (±)-jasmonic
acid and 3-oxo-2-[(Z)pent-2′-enyl]-cyclopentan-1-
octanoic acid in potato (Solanum tuberosum L.) was examined by using cultures of potato single-node stems. Deuterium-labeled (±)-jasmonic acid and 3-oxo-2-[(Z) pent-2′-enyl]cyclopentan-1-octanoic acid, which had been prepared from commercially available methyl (±)-jasmonate, were fed to the cultures, and the metabolites were extracted from the plants and analyzed by a liquid chromatography-selected ion monitoring system. The metabolism of deuterium-labeled (±)-jasmonic acid and 3-oxo-2-[(Z)-2′-pentenyl]cyclopentan-1-octanoic acid to 5′ and 4′-O-glucopyranosyloxyjasmoic acids was strongly suggested.
Key words: Solanum tuberosum L.; jasmonate; tuberonic acid glucoside; tuberonic acid; jasmonic acid

-9-
Malformation of Immature Starfish Oocytes by Theonellapeptolide Ie, a Tridecapeptide Lactone from a Marine Sponge Petrosia Species, through Disturbance of Cortical F-Actin Distribution

Emi OHTA,1,2 Hironobu OKADA,1 Shinji OHTA,2 Motomasa KOBAYASHI,3 Isao KITAGAWA,3 Shintaro HORIIKE,4 Tadao TAKAHASHI,4 Hiroshi HOSOYA,4 Kenya YAMAMOTO,5 and Susumu IKEGAMI1,2,

1Laboratory of Molecular Cell Biology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan 2Instrument Center for Chemical Analysis, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan 3Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-Oka, Suita, Osaka 565-0871, Japan 4Department of Biological Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan 5Department of Biological Diversity and Resources, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received March 17, 2003; Accepted May 22, 2003
Theonellapeptolide Ie (Tp), an oligopeptide lactone isolated from a marine sponge, Petrosia sp., was shown
to induce an unprecedented morphological change in the
immature oocytes of the starfish Asterina pectinifera. The cortical F-actin was disturbed and assembled to form dots and rings, as evidenced by staining with rhodamine-conjugated phalloidin. The oocyte eventually became malformed. When Tp was added to an immature oocyte which had been pretreated with cytochalasin B or D, inhibitors of actin polymerization, no malformation was observed. When Tp was added to an oocyte which had been induced to mature by 1-methyladenine (1-MeAde), a maturation-inducing substance in starfishes, no morphological changes were observed in the maturing oocytes which reached the first meiotic prometaphase 40 min after the start of 1-MeAde treatment. This is the first description of a chemical that induces aberrant redistribution of F-actin-based cytoskeleton in an animal oocyte which is arrested at the first meiotic prophase.
Key words: actin; oocyte; starfish; theonellapeptolide Ie

-10-
Suppressive Effects of Genistein on Oxidative Stress and NFκB Activation in RAW 264.7 Macrophages

Chunyeon CHOI,1 Hyeyeon CHO,1 Jiyoung PARK,1 Chungwon CHO,2 and Youngsun SONG1,

1School of Food and Life Science, Food Science Institute and Biohealth Product Research Center, Inje University, Obang-dong, 607, Kimhae, 621-749, Korea 2School of Biomedical and Biotechnology, Inje University, Obang-dong, 607, Kimhae, 621-749, Korea

Received March 17, 2003; Accepted June 11, 2003
This study was designed to investigate whether genistein may ameliorate oxidative stress and nuclear factor κB (NFκB) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with genistein significantly reduced lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in a dose-dependent manner with an IC<Oh>_<Wa>{50} of 69.4 μM. Genistein at 50 μM and 100 μM concentrations reduced thiobarbituric acid-reactive substances (TBARS) accumulation, increasing the GSH level and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase. The specific DNA-binding activities of nuclear factor κB (NFκB) on nuclear extracts from 50 μM and 100 μM genistein treatments were significanly suppressed. These results suggest that genistein has mild antioxidant activity to suppress intracellular oxidative stress and NFκB activation.
Key words: genistein; oxidative stress; nuclear factor κB (NFκB); antioxidant; macrophages

-11-
Production of D-Arabitol by Metschnikowia reukaufii AJ14787

Hiroyuki NOZAKI, Shun-ichi SUZUKI, Naoko TSUYOSHI, and Kenzo YOKOZEKI

Aminoscience Laboratories, Ajinomoto Co., Ltd., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan

Received March 18, 2003; Accepted June 11, 2003
A potent producer of D-arabitol was isolated by screening of natural sources and identified as Metschnikowia reukaufii AJ14787. Resting cells of this strain can efficiently produce D-arabitol from D-glucose with a weight yield of more than 60％, and can also produce D-arabitol from several other types of sugars such as polyols, ketoses, and aldoses. To improve productivity, various culture conditions such as temperature and the concentrations of D-glucose and nitrogen sources were examined. Under optimal conditions, 206 g/l of D-arabitol was produced from D-glucose with a weight yield of 52％ in 100 hours.
Key words: D-arabitol; polyhydroxy alcohol; osomophilic yeast; Metschnikowia reukaufii

-12-
Reduction of Noise-stress-induced Physiological Damage by Radices of Astragali and Rhodiolae: Glycogen, Lactic Acid and Cholesterol Contents in Liver of the Rat

Bei-Wei ZHU,1 Yu-Mei SUN,1 Xia YUN,1 Song HAN,2 Mei-Lan PIAO,2 Yoshiyuki MURATA,3 and Mikiro TADA2,†

1College of Bio ＆ Food Technology, Dalian Institute of Light Industry, Dalian 116034, China 2Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan 3Department of Agriculture, Okayama University, Okayama 700-8530, Japan

Received March 19, 2003; Accepted May 13, 2003
Noise is one of the factors that induces critical stress in animals. The contents of glycogen, lactic acid and cholesterol in the liver of noise-stressed rats were analyzed in order to investigate the alleviation of noise-stress-induced physiological damages by traditional medicine using Astragali and Rhodiolae radices. More than 95 dB noise ranging from 2 to 4 kHz reduced the contents of these compounds in the liver of rats not injected with the extract of Astragali or Rhodiolae, but did not change the contents in the liver of rats injected with the Astragali or Rhodiolae extract. These results show that noise induced stress in the rats via a decrease in contents of these compounds in the liver and that Astragali or Rhodiolae maintained the contents of these compounds in the liver of the noise-stressed rats. The results indicate that Astragali or Rhodiolae improved the ability for rats to resist noise stress.
Key words: noise; Chinese medicine; glycogen; lactic acid; cholesterol

-13-
Effects of Highly Purified Structured Lipids Containing Medium-chain Fatty Acids and Linoleic Acid on Lipid Profiles in Rats

Jun-ichi NAGATA,1,† Michio KASAI,2 Souichiro WATANABE,2 Ikuo IKEDA,3 and Morio SAITO1

1Division of Food Science, Incorporated Administrative Agency, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan 2The Nisshin Oil Mills, LTD., Tokyo 104-8285, Japan 3Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Fukuoka 812-8581, Japan

Received March 27, 2003; Accepted June 2, 2003
The purpose of this study is to examine the effects of highly purified structured lipids on serum and liver lipid profiles in rats. We also investigated in vitro hydrolysis of lipid emulsions by porcine pancreas. Hydrolysis rates of medium chain (M)-linoleic (L)-medium chain (M) types were 2 to 3 times higher than those of L-M-L types. The diet containing structured lipids or corn oil was administered to rats for 4 weeks. There were no significant differences in growth and food efficiency. Serum cholesterol levels were significantly lower (P＜0.05) in the 2-octanoyl-1,3-dilinoleoyl-glycerol, 2-linoleoyl-1,3-didecanoyl-glycerol, and 2-decanoyl-1,3-dilinoleoyl-glycerol groups than in the corn-oil group. Serum triglyceride levels were significantly lower (P＜0.05) in rats fed L-M-L types than those in the other groups. Serum non-esterified fatty acid (NEFA) and β-hydroxybutylate levels were significantly higher (P＜0.01) in rats fed M-L-M types than those of the other groups. These results indicate that the feeding of highly purified L-M-L types could effectively improve serum and liver lipid profiles and that M-L-M types may be a preferable substrate for the pancreas and contribute to energy supply in rats.
Key words: structured lipids; lipid profiles; medium-chain fatty acid; rats

-14-
Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs

Hyen Joo PARK, Hyun-Jung LEE, Eun-Jin LEE, Hye Jin HWANG, Sang-Hee SHIN, Myung-Eun SUH, Choonmi KIM, Hwa Jung KIM, Eun-Kyung SEO, and Sang Kook LEE†,

College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea

Received April 11, 2003; Accepted June 2, 2003
A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50＜20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50＝0.4 μg/ml)) compared to colon (IC50＞20.0 μg/ml) and stomach (IC50＞20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.
Key words: topoisomerase I and II; benz[f]indole-4,9-diones; cytotoxicity

-15-
Properties of a Novel Extracellular Cell-free Ice Nuclei from Ice-nucleating Pseudomonas antarctica IN-74

Naomi MURYOI, Hidehisa KAWAHARA, and Hitoshi OBATA

Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan

Received April 15, 2003; Accepted June 12, 2003
Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium. A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica. Cell-free ice nuclei from P. antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization. Cell-free ice nuclei were purified by centrifugation, filtration (0.45 μm), ultrafiltration, and gel filtration. In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei. Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH. It was demonstrated that the components of cell-free ice nuclei were protein (33％), saccharide (12％), and lipid (55％), indicating that cell-free ice nuclei were lipoglycoproteins. Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.
Key words: ice nucleation activity; Pseudomonas antarctica IN-74; cell-free ice nuclei

-16-
New Method for Synthesizing the Intermediates to 5-HETE from Yeast-mediated Reduction Products by Employing Baeyer-Villiger Oxidation with Complete Retention of Enantiomeric Excess

Satoshi YAMAUCHI, Yoshihiro KINOSHITA, and Yoshiro KINOSHITA

Faculty of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan

Received April 17, 2003; Accepted May 21, 2003
(R) and (S)-Aldehydes 2, which are intermediates for the synthesis of (5R) and (5S)-HETE, were respectively synthesized from the yeast-mediated reductive products, hydroxy ester 3 and cis-lactone 4, through Baeyer-Villiger oxidation with complete retention of enantiomeric excess.
Key words: 5-HETE; yeast reduction

-17-
The Dual Functions of Biphenyl-degrading Ability of Pseudomonas sp. KKS102: Energy Acquisition and Substrate Detoxification

Mina DELAWARY,1,2 Yoshiyuki OHTSUBO,3 and Akinori OHTA1,†

1Department of Biotechnology, University of Tokyo, Tokyo 113-8657, Japan 2Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan 3Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan

Received April 24, 2003; Accepted June 9, 2003
The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.
Key words: Pseudomonas sp. KKS102; biphenyl; growth inhibition; detoxification

-18-
Occurrence of a Specific Protein in Basidiomycete-lytic Enzyme Preparation Produced by Bacillus circulans KA-304 Inductively with a Cell-wall Preparation of Schizophyllum commune

Shigekazu YANO, Sachiko YAMAMOTO, Toshihiko TOGE, Mamoru WAKAYAMA, and Takashi TACHIKI

Department of Bioscience and Biotechnology, Faculty of Science and Technology, Ritsumeikan University, Shiga 525-8577, Japan

Received May 1, 2003; Accepted June 10, 2003
KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation (CWP) of Schizophyllum commune, has been reported to have an activity to form protoplasts from S. commune mycelia. The SDS-polyacrylamide gel electrophoreses described here demonstrated that a specific proteinous component (molecular weight: 150,000) occurred in KA-prep. The protein (P150T) was also formed in culture filtrates with CWP of several basidiomycetes, which could release the protoplasts, suggesting that the component was an indispensable factor for protoplast formation. P150T, isolated from an ammonium sulfate fraction of KA-prep (0--30％ saturation), did not have any protoplast-forming activity. Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and β-glucanase(s) in KA-prep. The N-terminal amino acid sequence indicated an analogy of P150T to mutanase (α-1,3-glucanase) from Bacillus sp. RM1, and actually P150T hydrolyzed mutan as well as S-(α-1,3) glucan from S. commune.
Key words: Bacillus circulans KA-304; Schizophyllum commune; protoplast formation; α-1,3-glucanase

-19-
Note
Reconstitution and Characterization of NtrC Protein in a Deep-sea Piezophilic Bacterium, Shewanella violacea Strain DSS12

Hiroaki KAWANO,1,2 Akihiko IKEGAMI,3 Kaoru NAKASONE,4, Chiaki KATO,5 Ron USAMI,1 and Koki HORIKOSHI1,2

1Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-0852, Japan 2The DEEP STAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan 3Department of Oral Biology, State University of New York, 304 Foster Hall, 3435 Main Street, Buffalo, New York 14214-3092, USA 4Department of Biotechnology and Chemistry, School of Engineering, Kinki University, 1 Umenobe Takaya, Higashihiroshima, Hiroshima 739-2116, Japan 5Marine ecosystems Research Department, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan

Received December 11, 2002; Accepted May 28, 2003
NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.
Key words: Shewanella violacea; NtrC; high pressure; electrophoretic mobility shift assay; Western blot analysis

-20-
Note
Glycerolipid Acyl Hydrolase Activity in the Brown Alga Cladosiphon okamuranus TOKIDA

Masaru TERASAKI and Yutaka ITABASHI

Laboratory of Bioresources Chemistry, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan

Received December 18, 2002; Accepted May 21, 2003
The brown alga, Cladosiphon okamuranus TOKIDA, was found to contain a large amount of free fatty acid (45％ of the total lipids). A crude enzyme preparation from the alga showed activity for hydrolyzing the acyl groups of various glycerolipids. The results suggest that the free fatty acid in C. okamuranus was released mainly from glycoglycerolipids, which were the major lipid components in the alga, by such glycerolipid acyl hydrolases as galactolipase.
Key words: Cladosiphon okamuranus; brown alga; free fatty acid; galactolipase; glycoglycerolipid

-21-
Note
A Study of Tryptophan Fluorescence Quenching of Bifunctional Alginate Lyase from a Marine Bacterium Pseudoalteromonas sp. Strain No. 272 by Acrylamide

Yoshiko IWAMOTO, Hidetoshi HIDAKA, Tatsuya ODA, and Tsuyoshi MURAMATSU

Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan

Received January 14, 2003; Accepted May 23, 2003
A fluorescence quenching study of a sole tryptophan residue of a bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 was done in the presence and absence of substrates, oligomeric guluronic and its C5 isomer mannuronic acid, by a Stern-Volmer plot with a quencher, acrylamide. N-Acetyltryptophanamide and reduced and carboxymethylated alginate lyase showed large quenching constants, on the other hand, the native enzyme had small constants regardless of the presence or absence of the substrates. The result suggests that the tryptophan residue is located in a buried region of the enzyme molecule, but is barely accessible to acrylamide, and that the residue is not masked by the substrates with various degrees of polymerization.
Key words: alginate lyase; Pseudoalteromonas sp.; tryptophan fluorescence; fluorescence quenching; alginate oligomers

-22-
Note
Subsite Structure of Maltogenic Amylase from Thermomonospora viridis TF-35

Kosei TAKAHASHI and Teruo NAKAKUKI

Nihon Shokuhin Kako Co., Ltd., 30 Tajima, Fuji-shi, Shizuoka 417-8530, Japan

Received February 10, 2003; Accepted May 16, 2003
We estimated the subsite structure of the maltogenic amylase from Thermomonospora viridis TF-35 (TVA). TVA has six subsites, and the catalytic site is between the 3rd and 4th subsite. The subsite affinities, A−3, A−2, (A−1＋A＋1), A＋2 and A＋3 were calculated to be 0.47, 2.31, 0.49, 2.45, and 0.17 kcal mol−1, respectively.
Key words: subsite structure; maltogenic amylase; Thermomonospora viridis

-23-
Note
Generation of Reactive Oxygen Species from Hinokitiol under Near-UV Irradiation

Hitoshi SHIBATA, Takayuki NAGAMINE, Yong WANG, Takahiro ISHIKAWA, and Yoshihiro SAWA

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan

Received February 21, 2003; Accepted May 12, 2003
Near-UV irradiation caused the decomposition of hinokitiol in an aqueous solution. During the photochemical reaction, the distinct electron spin resonance signal characteristic of the adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with the hydroxyl radical was accompanied by small signals corresponding to the adduct of DMPO with the superoxide anion radical. More than 95％ of Escherichia coli cells were killed by the incubation with hinokitiol under near-UV irradiation by BLB fluorescent lamps. These results indicated the generation of reactive oxygen species during photochemical reaction of hinokitiol under near-UV irradiation.
Key words: bactericidal activity; hinokitiol; near UV; reactive oxygen species

-24-
Note
(−)-Hydroxycitrate Ingestion Increases Fat Oxidation during Moderate Intensity Exercise in Untrained Men

Kyoko TOMITA,1, Yasuhide OKUHARA,1 Norihiro SHIGEMATSU,1 Heajung SUH,2 and Kiwon LIM3

1Central Research Laboratory, Fancl Co., Yokohama, Japan 2Institute of Elderly Health, Seoul, Korea 3Department of Physical Education, Konkuk University, Seoul, Korea

Received February 21, 2003; Accepted May 7, 2003
We examined the effects of (−)-Hydroxycitrate (HCA) ingestion on fat oxidation during moderate intensity exercise in untrained men. Six subjects ingested 500 mg of HCA or a placebo for 5 days and did endurance exercise. Blood FFA concentrations were significantly increased and respiratory exchange ratio (RER) decreased by HCA ingestion. These results suggested short-term HCA ingestion increases fat oxidation in untrained men.
Key words: (−)-hydroxycitrate (HCA); fat oxidation; untrained; respiratory exchange ratio (RER); free fatty acid (FFA)

-25-
Note
Nucleotide Sequences of Genes Encoding Allosamidin-sensitive and -insensitive Chitinases Produced by Allosamidin-producing Streptomyces

Hiroshi MATSUURA,1 Susumu OKAMOTO,1 Sarintip ANAMNART,2 Qiuqi WANG,2 Ze-Yang ZHOU,2 Takuya NIHIRA,2 Yasuhiro YAMADA,2 Tomohisa KUZUYAMA,3 Haruo SETO,3 Jiro NAKAYAMA,1 Akinori SUZUKI,1 Hiromichi NAGASAWA,1 and Shohei SAKUDA1,†

1Department of Applied biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565-0871, Japan 3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received February 26, 2003; Accepted May 18, 2003
Allosamidin is a strong inhibitor of family 18 chitinases. We previously reported the presence of allosamidin-sensitive and -insensitive chitinases (chitinase S and IS) in the culture filtrate of the allosamidin-producing strain, Streptomyces sp. AJ9463. In this study, we cloned and sequenced the genes encoding the two chitinases, which clarified that chitinase S and IS belong to the family 18 and 19 chitinase, respectively.
Key words: allosamidin; family 18 chitinase; family 19 chitinase; Streptomyces

-26-
Note
Construction of a Homologous Selectable Marker Gene for Lentinula edodes Transformation

Toshikazu IRIE,1 Toshitsugu SATO,1,† Kumiko SAITO,1 Yoichi HONDA,2 Takashi WATANABE,2 Masaaki KUWAHARA,3 and Hitoshi ENEI1

1Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan 2Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 3Institute of Wood Technology, Akita Prefectural University, Noshiro, Akita 016-0876, Japan

Received February 27, 2003; Accepted May 22, 2003
We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 μg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.
Key words: Lentinula edodes; Pleurotus ostreatus; homobasidiomycetes; iron-sulfur protein (Ip) subunit

-27-
Note
Cloning and Sequence Analysis of Endoglucanase Genes from an Industrial Fungus, Aspergillus kawachii

Yukari HARA,2 Yumi HINOKI,2 Hitoshi SHIMOI,1,2 and Kiyoshi ITO1,2,

1National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan 2Department of Molecular Biotechnology, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan

Received March 7, 2003; Accepted May 23, 2003
Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.
Key words: endoglucanase; cellulose-binding domain; Aspergillus kawachii

-28-
Note
Structural Effects of Phenolic Acids on the Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers

Yutaka KONISHI,1,† Kazuo KUBO,2 and Makoto SHIMIZU3

1Applied Bioresearch Center, Research ＆ Development Department, and 2Pharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd., 3 Miyaharacho, Takasaki-shi, Gunma 370-1295, Japan 3Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received March 12, 2003; Accepted May 13, 2003
The structural specificity of the monocarboxylic acid transporter (MCT) for the transport of phenolic acids was investigated by measuring the inhibitory effect on the fluorescein transport in Caco-2 cell monolayers. Although most of the monohydroxylated derivatives had an inhibitory effect, the di- and tri-hydroxylated ones did not. The methoxylated derivatives were more inhibitory than the hydroxylated ones in all the meta-substituted derivatives, suggesting that meta-hydroxylation of the substrate would decrease the affinity for MCT.
Key words: fluorescein; monocarboxylic acid transporter; phenolic acid; structural specificity; Caco-2 cell

-29-
Note
Proteases of Maitake (Grifola frondosa) Responsible for Breakdown of Wheat Flour Dough and Their Reaction with Gluten Proteins

Makoto ABE1, and Masaharu SEGUCHI2

1Department of Japanese Studies, Faculty of Intercultural Studies, Gakushuin Women′s College, 3-20-1 Toyama, Shinjuku-ku, Tokyo 162-8650, Japan 2Laboratory of Food Technology, Faculty of Home Economics, Kobe Women′s University, 2-1 Aoyama, Higashi-Suma, Suma-ku, Kobe 654-8585, Japan

Received April 7, 2003; Accepted May 15, 2003
Two proteases capable of decreasing dough strength when added to wheat flour were purified from Maitake and these were both thought to be peptidyl-Lys metalloendopeptidase. The major purified protease SP-3-A hydrolyzed high-molecular-weight glutenin subunits preferably to the other glutenin subunits. SP-3-A cleaved peptide bonds adjacent to the N-terminal of lysine in the high-molecular-weight glutenin subunit.
Key words: Maitake; protease; glutenin subunit; dough strength; endopeptidase

-30-
Note
Root Growth-promoting Activity of Unsaturated Oligomeric Uronates from Alginate on Carrot and Rice Plants

Xu XU,1 Yoshiko IWAMOTO,1 Yoshie KITAMURA,2 Tatsuya ODA,1 and Tsuyoshi MURAMATSU1,†

1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan 2Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan

Received April 7, 2003; Accepted June 11, 2003
The root elongation activity of unsaturated oligomeric uronates from alginate on carrot and rice plants was investigated. Unsaturated oligomeric uronates were prepared by digesting polymannuronate (PM) and polyguluronate (PG) with an alginate lyase purified from Pseudoalteromonas sp. strain No. 272. The root elongation activity was measured by elongation in length of carrot- and rice-excised root incubated in the B5-medium containing 0.8％ agar in the dark. PM and PG showed no activity, but the enzymatic digestion mixtures of PG had promoting activity on roots of both plants at a final concentration of 0.5 mg/ml. The maximum activity was obtained at 0.75 mg/ml. The dependence of activity on degree of polymerization of the uronates was tested and the pentamer was most active, but the mechanism of the action of unsaturated uronates on the cells remains to be solved.
Key words: root elongation; uronate oligomer; alginate; carrot; rice

-31-
Note
Isolation and Characterization of Phenol-catabolizing Bacteria from a Coking Plant

Wael S. El-SAYED,1 Mohamed K. IBRAHIM,1 Mohamed Abu-SHADY,1 Fawkia El-BEIH,1 Naoya OHMURA,2,† Hiroshi SAIKI,2 and Akikazu ANDO3

1Microbiology Department, Faculty of Science, Ain Shams University, Cairo, Egypt 2Department of Bio-Science, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-city, Chiba 270-1194, Japan 3Department of Science and Technology, Chiba University, 648 Matsudo, Chiba 271-8510, Japan

Received April 7, 2003; Accepted May 1, 2003
New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a Vmax of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.
Key words: phenol; degradation; tolerance; meta-pathway

-32-
Note
Fosmidomycin Resistance in Adenylate Cyclase Deficient (cya) Mutants of Escherichia coli

Yoshiko SAKAMOTO, Soichi FURUKAWA, Hirokazu OGIHARA, and Makari YAMASAKI

Department of Food Science and Technology, College of Bioresouce Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-8510, Japan

Received April 10, 2003; Accepted June 16, 2003
Adenylate cyclase deficient (cya) mutants of E. coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin. E. coli glpT mutants were resistant to fosfomycin and also to fosmidomycin. This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT. DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP.
Key words: fosmidomycin; cAMP; Escherichia coli; DNA micro-array

-33-
Note
A Simple and Efficient Method for High Fidelity PCR Cloning Using Antibody-neutralizing Technology

Masao KITABAYASHI,1,2 Yoshiaki NISHIYA,3 and Muneharu ESAKA1,

1Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8528, Japan 2Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan 3Bio-division, Toyobo Co., Ltd., 2-8 Dojima Hama 2-chome, Kita-ku, Osaka 530-8230, Japan

Received April 11, 2003; Accepted June 13, 2003
We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.
Key words: high-fidelity PCR product; family B DNA polymerase; KOD DNA polymerase; TA cloning method; antibody

-34-
Note
Myrsinoic Acid E, an Anti-inflammatory Compound from Myrsine seguinii

Hidefumi MAKABE,1,† Shintaro MIYAZAKI,2 Tsunashi KAMO,2 and Mitsuru HIROTA2

1Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Minami-minowa, Kami-ina, Nagano 399-4598, Japan 2Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Minami-minowa, Kami-ina, Nagano 399-4598, Japan

Received April 17, 2003; Accepted May 22, 2003
The methanolic extract of Myrsine seguinii yielded the novel anti-inflammatory compound, myrsinoic acid E (1), whose structure was elucidated to be 3,5-digeranyl-4-hydroxy benzoic acid. We synthesized 1- and its 3,5-diprenyl (2) and 3,5-difarnesyl analogues (3). Compounds 1--3 suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation of mouse ears by 59％, 14％, and 69％ at a dose of 1.4 μmol.
Key words: anti-inflammatory; Myrsine seguinii;
12 - O - tetradecanoylphorbol - 13 - acetate
(TPA)-induced edema; Pd-catalyzed carbonylation

-35-
Note
Activin A Induces Phosphorylation of Smad2 but Not Complex Formation of Smad2 with Smad4 in Human Colon Cancer Cell Line HT-29

Pimara PHOLNUKULKIT, Kei SONOYAMA, and Jun KAWABATA

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

Received April 23, 2003; Accepted June 10, 2003
Western blotting coupled with immunoprecipitation showed that activin A treatment induced phosphorylation of Smad2 but not complex formation of Smad2/4 in human colon cancer-derived HT-29 cells. Because HT-29 cells expressed neither Smad4 mRNA nor Smad4 protein, it is suggested that deletion of Smad4 leads to a defect of formation of Smad2/4 complex upon activin A stimulation in HT-29 cells.
Key words: activin A; Smad2; Smad4; colon cancer; HT-29

-36-
Note
Ethyl 4-[2-(6-Methyl-3-pyridyloxy)butyloxy]benzoate, a Novel Anti-juvenile Hormone Agent

Hanae ISHIGURO,1 Norihiro FUJITA,1 In-Hae KIM,1 Takahiro SHIOTSUKI,2 and Eiichi KUWANO1,

1Laboratory of Pesticide Chemistry, Department of Applied Genetics and Pest Management, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan 2Laboratory of Insect Growth Regulation, Developmental Biology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan

Received April 23, 2003; Accepted May 23, 2003
Ethyl 4-[2-(6-methyl-3-pyridyloxy)butyloxy]benzoate (2) was prepared as a novel anti-juvenile hormone (anti-JH) agent. Compound 2 induced precocious metamorphosis in larvae of the silkworm and black pigmentation of the larval cuticle, which are clearly recognized as JH-deficiency symptoms. The 4-ethoxycarbonyl group on the benzene ring was indispensable for activity. The activity of compound 2 could be fully counteracted by methoprene, a JH agonist, but not by the dietary administration of 20-hydroxyecdysone.
Key words: anti-juvenile hormone; 6-methyl-3-pyridyl ethers; precocious metamorphosis; silkworm

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Note
A Long Acidic Domain Affects the Chromatographic Behaviour of a Neuronal Adaptor Protein on DEAE-Sepharose

Olimpia LONGO,1 Annalisa LAMBERTI,1 Nicola ZAMBRANO,1,2 and Paolo ARCARI1,2,†

1Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Via S. Pansini 5, I-80131, Naples, Italy 2CEINGE, Center of Genetic Engineering and Advanced Biotechnologies, Via S. Pansini 5, I-80131 Naples, Italy

Received April 23, 2003; Accepted May 22, 2003
The stepwise chromatographic behaviour on DEAE-Sepharose of rat Fe65, a neuronal protein, was tested, using as eluants KCl, CaCl2, and MgCl2. Assays by western blot showed that Fe65 was eluted by CaCl2, at a ionic strength 20％ lower than that of MgCl2 or KCl. Interestingly, in the case of a truncated Fe65, lacking a glutamic acid rich region at the N-terminus, the ionic strengths of the various eluants were almost identical. These results suggested a possible inhibitory role of calcium ions in the binding of the protein to DEAE and a specific affinity of these ions for long acidic stretches.
Key words: rat brain Fe65; deletion mutant; DEAE-chromatography; ionic strength; calcium and magnesium

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Note
Isolation of Lectins by Affinity Chromatography with Porcine Plasma Proteins Immobilized on Agarose

Akane KAJIYA,1 Yu KOYAMA,1 Takashi MITA,1 Tomohiro MEGA,2 Sumihiro HASE,2 Toshioki KAWAKAMI,3 Eiko HONDA,4 Hiroshi MUNAKATA,5 and Mamoru ISEMURA1,†

1School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan 2Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan 3Sanpo Co., Ltd., Atsuta-ku, Nagoya, Aichi 456-0023, Japan 4Life Science Institute, Kinki University, Osaka-Sayama, Osaka 589-8511, Japan 5Department of Biochemistry, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

Received May 19, 2003; Accepted June 23, 2003
To develop a convenient method to isolate lectins, we prepared an affinity gel by coupling plasma proteins with agarose beads under conditions where the pH did not exceed 7.5. The validity of the use of this affinity gel in combination with elution using a hapten saccharide was confirmed by isolation of concanavalin A from Jack bean meal. Successful application of the method was demonstrated by isolation of two novel vegetable lectins from udo (Aralia cordate) and wasabi (Wasabia japonica). The method would be useful to isolate new lectins from various sources including plant and animal tissues.
Key words: Aralia cordate; udo; Wasabia japonica; wasabi; lectin

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Communication
350-kDa Royal Jelly Glycoprotein (Apisin), Which Stimulates Proliferation of Human Monocytes, Bears the β1--3Galactosylated N-Glycan: Analysis of the N-Glycosylation Site

Mariko KIMURA,1 Yoshinobu KIMURA,2,† Kazunori TSUMURA,2 Kiyoshi OKIHARA,3 Hiroyuki SUGIMOTO,3 Hideo YAMADA,3 and Masami YONEKURA4

1Faculty of Food Culture, Department of Food System, Kurashiki Sakuyo University, Nagao-Tamashima, Kurashiki 710-0292, Japan 2Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1, Okayama 700-8530, Japan 3Yamada Apiculture Center Inc., Ichiba 194, Kagamino-cho, Tomada-gun, Okayama 708-0393, Japan 4Department of Bioresource Science, School of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan

Received June 6, 2003; Accepted June 28, 2003
While doing a structural analysis of minor component N-glycans linked to 350-kDa royal jelly glycoprotein (RJGP), which stimulates the proliferation of human monocytes, we found that a Galβ1--3GlcNAcβ1--4Man unit occurs on the insect glycoprotein. The structure of the fluorescence-labeled N-glycan was analyzed by sugar component analysis, IS-MS, and 1H-NMR. The structural analysis showed that the 350-kDa RJGP bears Galβ1--3GlcNAcβ1--4(GlcNAcβ1--2)Manα1--3 (Manα1--3Manα1--6)Manβ1--4GlcNAcβ1--4GlcNAc, suggesting this insect glycoprotein is one of the substrates for both β1--3 galactosyl and β1--4 N-acetylglucosamininyl transferases. To our knowledge, this is the first report that succeeded in identifing an insect glycoprotein bearing the β1--3 galactosylated N-glycan.
Key words: royal jelly; insect glycoprotein; N-glycosylation; site analysis; Apis mellifera