Contents and Abstracts of BBB

(Vol.67 No.10 2003)

Characteristics of an Anaerobic, Syntrophic, Butyrate-degrading Bacterium in Paddy Field Soil
Bi-Zhen ZOU, Kiyoshi TAKEDA, Akio TONOUCHI, Shinji AKADA, and Takashi FUJITA p.2059

Isolation of GnafC, a Polysaccharide Constituent of Gnaphalium affine, and Synergistic Effects of GnafC and Ascorbate on the Phenotypic Expression of Osteoblastic MC3T3-E1 Cells
Yukihiro AOSHIMA,1 Yoshinori HASEGAWA,1 Sho HASEGAWA,1 Atsushi NAGASAKA,1 Tomofumi KIMURA,1 Satofumi HASHIMOTO,1 Yasuyoshi TORII2 and Norihiro TSUKAGOSHI3, p.2068

Transformation of the White-rot Basidiomycete Coriolus hirsutus Using the Ornithine Carbamoyltransferase Gene
Akira TSUKAMOTO, Yasushi KOJIMA, Yukio KITA, and Jun SUGIURA p.2075

Attenuating Effect of Chlorella Supplementation on Oxidative Stress and NFκB Activation in Peritoneal Macrophages and Liver of C57BL/6 Mice Fed on an Atherogenic Diet
Hyo-seon LEE,1 Chun-yeon CHOI,1 Chungwon CHO,2 and Youngsun SONG1, p.2083

Antimutagenic Activity of Flavonoids from Chrysanthemum morifolium
Mitsuo MIYAZAWA and Masayoshi HISAMA p.2091

Blood Coagulation and Fibrinolysis of Rats Fed Fish Oil: Reduced Coagulation Factors Especially Involved in Intrinsic Pathway and Increased Activity of Plasminogen Activator Inhibitor
Yoko SANO, Kaori SATO, Motoharu UCHIDA, and Masakazu MURATA p.2100

Biosynthesis of Branched-chain Fatty Acid in Bacilli: FabD (malonyl-CoA:ACP transacylase) Is Not Essential for In Vitro Biosynthesis of Branched-chain Fatty Acids
Hirosuke OKU, Naoya FUTAMORI, Kenichi MASUDA, Yumiko SHIMABUKURO, Tomoyo OMINE, and Hironori IWASAKI p.2106

Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase
Osao ADACHI,1,† Nozomi YOSHIHARA,1 Somboon TANASUPAWAT,2 Hirohide TOYAMA,1 and Kazunobu MATSUSHITA1 p.2115

3-Dehydroquinate Production by Oxidative Fermentation and Further Conversion of 3-Dehydroquinate to the Intermediates in the Shikimate Pathway
Osao ADACHI,1,† Somboon TANASUPAWAT,2 Nozomi YOSHIHARA,1 Hirohide TOYAMA,1 and Kazunobu MATSUSHITA1 p.2124

Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22
Soo Jeong CHO,2,, Woo Jin LIM,1, Su Young HONG,1 Sang Ryeol PARK,3 and Han Dae YUN1,2,† p.2132

Purification of Turkey Pancreatic Phospholipase A2
Riadh BEN SALAH,1 Nacim ZOUARI,1 Joseph REINBOLT,2 and Hafedh MEJDOUB3, p.2139

Purification and Characterization of an α-Haloketone-resistant Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA, and Cloning of the Gene
Hirokazu NANBA, Yasuko TAKAOKA, and Junzo HASEGAWA p.2145

Purification and Identification of Antimicrobial Sesquiterpene Lactones from Yacon (Smallanthus sonchifolius) Leaves
Fengqiu LIN, Morifumi HASEGAWA, and Osamu KODAMA p.2154

Cloning, Sequencing, and Expression of the Gene Encoding the Clostridium stercorarium α-Galactosidase Aga36A in Escherichia coli
SURYANI, Tetsuya KIMURA, Kazuo SAKKA, and Kunio OHMIYA p.2160

A Thermostable Laccase from Streptomyces lavendulae REN-7: Purification, Characterization, Nucleotide Sequence, and Expression
Takashi SUZUKI,1 Kohki ENDO,1 Masaaki ITO,1,† Hiroshi TSUJIBO,2 Katsushiro MIYAMOTO,2 and Yoshihiko INAMORI2 p.2167

Inhibition of Immunoglobulin E Production in Allergic Model Mice by Supplementation with Vitamin E and β-Carotene
Noriko BANDO, Rintaro YAMANISHI, and Junji TERAO p.2176

Systematic Strategy for the Synthesis of Cyanobacterin and Its Stereoisomers. 1. Asymmetric Total Synthesis of Dechloro-cyanobacterin and Its Enantiomer
Yasushi HAGA, Momotoshi OKAZAKI, and Yoshihiro SHUTO p.2183

Mode of α-Amylase Production by the Shochu Koji Mold Aspergillus kawachii
Kazuki NAGAMINE,1 Kenji MURASHIMA,2 Taku KATO,2 Hitoshi SHIMOI,1,2 and Kiyoshi ITO1,2,† p.2194

Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci
Hisashi KIMOTO1,† and Akira TAKETO2 p.2203

Synthesis of the Racemate and Both Enantiomers of Massoilactone
Masashi ISHIKAWA,1 Masayasu AMAIKE,1 Masamichi ITOH,1 Yasuhiro WARITA,1,† and Takeshi KITAHARA2 p.2210

Systematic Strategy for the Synthesis of Cyanobacterin and Its Stereoisomers. 2. Asymmetric Total Synthesis of the 2- and 3-Epimers of Dechloro-cyanobacterin
Yasushi HAGA, Momotoshi OKAZAKI, and Yoshihiro SHUTO p.2215

Synthesis of the Enantiomers of 21-Methyl-7-hentriacontanone and a Stereoisomeric Mixture of 5-Acetoxy-19-methylnonacosane, Candidates for the Female Sex Pheromone of the Screwworm Fly, Cochliomyia hominivorax
Kenji MORI p.2224

Structural Features of N-Glycans Linked to Glycoproteins from Oil Palm Pollen, an Allergenic Pollen
Yoshinobu KIMURA,1,2,† Takeo YOSHIIE,1 Woo Kwan KIT,2 Megumi MAEDA,2 Mariko KIMURA,3 and Siang Hee TAN4 p.2232

Synthesis and Absolute Configuration of (−)-3-Butyl-7-hydroxyphthalide, a Cytotoxic Metabolite of Penicillium vulpinum
Tomoya OHZEKI1 and Kenji MORI2,† p.2240

Expression Profiling of Translation-associated Genes in Sporulating Bacillus subtilis and Consequence of Sporulation by Gene Inactivation
Yoshiaki OHASHI,1,, Takashi INAOKA,1, Koji KASAI,1 Yasuhiro ITO,1 Susumu OKAMOTO,1 Hideo SATSU,1 Yuzuru TOZAWA,1 Fujio KAWAMURA,2 and Kozo OCHI1,† p.2245

Expression and Characterization of Bovine Lactoperoxidase by Recombinant Baculovirus
Tetsuya TANAKA, Sanae SATO, Haruto KUMURA, and Kei-ichi SHIMAZAKI p.2254

Note
Differential Expression of the Nonmuscle-type Cofilin Gene between Subcutaneous and Visceral Adipose Tissue
Ki-Choon CHOI,1 Sang-Gun ROH,1,† Daisuke HISHIKAWA,1 Hisae MIYAHARA,1 Masaaki KUNO,1 Hiroaki TSUZUKI,1 Ai TOMIMATSU,1 Yeon-Hee HONG,1 Kwang-Keun CHO,2 Kyung-Ho HAN,3 and Shin-ichi SASAKI1 p.2262

Note
Effects of the Marine Unicellular Alga Nannochloropsis sp. to Reduce the Plasma and Liver Cholesterol Levels in Male Rats Fed on Diets with Cholesterol
Moshe J. WERMAN,1 Assaf SUKENIK,2 and Shoshana MOKADY1,† p.2266

Note
Effect of α,α-Dialkyl Amino Acids on the Protease Resistance of Peptides
Hiroshi YAMAGUCHI,1 Hiroaki KODAMA,1,† Satoshi OSADA,1 Fumio KATO,2 Masood JELOKHANI-NIARAKI,3 and Michio KONDO1 p.2269

Note
PEG-Inhibitor Conjugates: Application of Diphenyl α-Aminoalkylphosphonate to Removal of Chymotrypsin
Makiko UMEZAKI,1 Takayoshi FUJII,1 Toshiaki YOSHIMURA,1 Isao YAMAZAKI,2 and Shin ONO1,† p.2273

Note
Production of Functional Lectin in Pichia pastoris Directed by Cloned cDNA from Aleuria aurantia
Koh AMANO, Mitsuyo TAKASE, Akikazu ANDO, and Yoshiho NAGATA p.2277

Note
Linoleic Acid 10-Hydroperoxide as an Intermediate during Formation of 1-Octen-3-ol from Linoleic Acid in Lentinus decadetes
Kenji MATSUI, Satomi SASAHARA, Yoshihiko AKAKABE, and Tadahiko KAJIWARA p.2280

Note
Acivicin Induce Filamentous Growth of Saccharomyces cerevisiae
Tsuyoshi MIYAKE,1,† Hiroyuki SAMMOTO,1 and Bun-ichiro ONO2 p.2283

Note
Isolation and Characterization of a Stilbene-degrading Strain of Pseudomonas fluorescens, and Production of Antioxidant Compounds by Stilbene Metabolism
Joseph G. LEAHY,1,† Patricia J. BATCHELOR,1 Mary C. SETZER,2 and William N. SETZER2
p.2286

Note
Efficient Production of Recombinant Human Pleiotrophin in Yeast, Pichia pastoris
Akira MURASUGI,1,† Isao KIDO,2 Hideshi KUMAI,2 and Yukio ASAMI1 p.2288

Note
Addition of a Peptide Tag at the C Terminus of AtHKT1 Inhibits Its Na＋ Transport
Yasuhiro KATO,1 Akihiro HAZAMA,2 Mutsumi YAMAGAMI,3 and Nobuyuki UOZUMI1,4,†
p.2291

Note
The Protein Component of Bacterial Ribonuclease P Flickers the Metal Ion Response to the Substrate Shape Preference of the Ribozyme
Tomoaki ANDO, Terumichi TANAKA, and Yo KIKUCHI p.2294

Note
Modulations of Food-derived Substances on Intestinal Permeability in Caco-2 Cell Monolayers
Yutaka KONISHI p.2297

Note
A Novel Gene Encoding an Enzyme That Degrades a Polysaccharide from the Sheath of Sphaerotilus natans
Minoru TAKEDA, Katsuya NISHINA, Yuji HANAOKA, Motoko HIGO, Mutsumi TERUI, Ichiro SUZUKI, and Jun-ichi KOIZUMI p.2300

Note
Increased Production of Antioxidative Sesaminol Glucosides from Sesame Oil Cake through Fermentation by Bacillus circulans Strain YUS-2
Takashi OHTSUKI,1 Junko AKIYAMA,1 Takao SHIMOYAMA,1 Shin-ichi YAZAKI,1 Sadaharu UI,1 Yuko HIROSE,2 and Akio MIMURA1,† p.2304

Communication
A New Type of RNase T2 Ribonuclease in Two Basidiomycetes Fungi, Lentinus edodes and Irpex lacteus
Hiroko KOBAYASHI,1 Tadashi ITAGAKI,1 Norio INOKUCHI,1 Kazuko OHGI,2,† Tatsuya WADA,2 Masanori IWAMA,2 and Masachika IRIE2 p.2307

-1-
Characteristics of an Anaerobic, Syntrophic, Butyrate-degrading Bacterium in Paddy Field Soil

Bi-Zhen ZOU, Kiyoshi TAKEDA, Akio TONOUCHI, Shinji AKADA, and Takashi FUJITA

Faculty of Agriculture and Life Science, Hirosaki University, Bunkyou-cho 3, Hirosaki 036-8561, Japan

Received January 10, 2003; Acceped June 27, 2003
The number of syntrophic butyrate-degrading bacte-
ria in a flooded paddy field soil was 1.7×103 MPN/g dry
soil. Butyrate was degraded to acetate and methane when paddy soils were incubated anaerobically with the addition of butyrate. However, butyrate degradation was completely suppressed by the addition of the specific inhibitor of methanogenesis, 2-bromoethanesulfonate (BES) to the soil. A hydrogen-using methanogen, strain TM-8, was isolated from flooded paddy field soil. Strain TM-8 was identified as Methanobacterium formicicum based on its physiology and phylogeny. Syntrophic butyrate-degrading bacteria were enumerated and isolated using strain TM-8. A syntrophic butyrate-degrading bacterium, strain TB-6, was isolated in coculture with strain TM-8 from paddy soil. The strain was Gram-negative, had curved rods, and grew on crotonate. Sulfate was not used as an electron acceptor. Strain TB-6 was closely related to S. wolfei subsp. wolfei. The relation between strain TB-6 and the members of Syntrophomonas are discussed.
Key words: butyrate-degrading bacteria; syntrophic association; β-oxidation; paddy field soil

-2-
Isolation of GnafC, a Polysaccharide Constituent of Gnaphalium affine, and Synergistic Effects of GnafC and Ascorbate on the Phenotypic Expression of Osteoblastic MC3T3-E1 Cells

Yukihiro AOSHIMA,1 Yoshinori HASEGAWA,1 Sho HASEGAWA,1 Atsushi NAGASAKA,1 Tomofumi KIMURA,1 Satofumi HASHIMOTO,1 Yasuyoshi TORII2 and Norihiro TSUKAGOSHI3,

1Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan 2Department of Food Science and Technology, Collage of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan 3Aichi Study Center, The University of the Air, 101-2 Yagoto-honnmachi, Showa, Nagoya 466-0825, Japan

Received January 17, 2003; Accepted June 12, 2003
After screening extensively factors in plant extracts that increase alkaline phosphatase activity, an osteoblastic differentiation marker protein in mouse calvarial osteoblast MC3T3-E1 cells, GnafC derived from Gnaphalium affine, was found to significantly enhance the alkaline phosphatase (ALPase) activity in a synergistic manner with ascorbate. GnafC was a polysaccharaide with an approximate molecular mass of 10,000 and comprised mannose, xylose, arabinose, galactose and glucose in a molar ratio of 1:2:4.3:2.5:2.7. Expression of the osteoblastic differentiation marker genes was examined by semiquantitative RT-PCR with RNAs prepared from cells at different developmental stages. With ascorbate in the culture, GnafC enhanced the expression of the ALPase and MMP13 genes from the early stage of differentiation, leading to maturation of the collagenous extracellular matrix (ECM), a prerequisite for mineralization.
Key words: GnafC; polysaccharide of Gnaphalium affine; ascorbate; MC3T3-E1; osteoblastic differentiation

-3-
Transformation of the White-rot Basidiomycete Coriolus hirsutus Using the Ornithine Carbamoyltransferase Gene

Akira TSUKAMOTO, Yasushi KOJIMA, Yukio KITA, and Jun SUGIURA

Advanced Technology Research Laboratory, Oji Paper Co., Ltd., 10-6 Shinonome 1-chome, Koto-ku, Tokyo 135-8558, Japan

Received February 14, 2003; Accepted July 10, 2003
An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg＋ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 103--104 transformants per μg DNA per 106--107 oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg＋ phenotype of the transformants was stably inherited through mitosis.
Key words: Coriolus hirsutus; basidiomycete; transformation; ornithine carbamoyltransferase

-4-
Attenuating Effect of Chlorella Supplementation on Oxidative Stress and NFκB Activation in Peritoneal Macrophages and Liver of C57BL/6 Mice Fed on an Atherogenic Diet

Hyo-seon LEE,1 Chun-yeon CHOI,1 Chungwon CHO,2 and Youngsun SONG1,

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

Received February 17, 2003; Accepted June 10, 2003
This study was designed to investigate whether chlorella supplementation may ameliorate oxidative stress and nuclear factor kappa B (NFκB) activation in peritoneal macrophages and liver of C57BL/6 mice fed on an atherogenic diet. The animals were maintained on an atherogenic diet (control), or an atherogenic diet supplemented with 3％ (w/w) chlorella or 5％ (w/w) chlorella for 12 wks. The plasma and hepatic lipid levels were not affected by chlorella supplementation. Hepatic thiobarbituric acid-reactive substances and superoxide anion production in peritoneal macrophages were significantly lower in the 5％ chlorella group (p＜0.05), but the glutathione level was not altered by chlorella supplementation. The hepatic antioxidative enzyme activities of Cu, Zn-superoxide dismutase and catalase were higher in the mice fed on the 5％ chlorella diet (p＜0.05). The plasma aspartate aminotransferase activity was lower in the mice fed on the chlorella-containing diets (p＜0.05), whereas the alanine aminotransferase activity was not affected by chlorella supplementation. The NFκB nuclear binding activities of peritoneal macrophages and liver were significantly lower in the 5％ chlorella groups (p＜0.05). These results suggest that chlorella supplementation may attenuate oxidative stress by reducing reactive oxygen production and increasing antioxidative processes, thus suppressing inflammatory mediator activation in peritoneal macrophages and liver.
Key words: chlorella; oxidative stress; nuclear factor kappa B (NFκB); C57BL/6 mice; inflammation

-5-
Antimutagenic Activity of Flavonoids from Chrysanthemum morifolium

Mitsuo MIYAZAWA and Masayoshi HISAMA

Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan

Received March 3, 2003; Accepted July 7, 2003
A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and 1H and 13C NMR spectroscopy. Compounds 1--4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1--4 suppressed 60.2, 75.7, 90.0, and 66.6％ of the SOS-inducing activity at a concentration of 0.70 μmol/ml. The ID50 (50％ inhibitory dose) values of 1--4 were 0.62, 0.55, 0.44, and 0.59 μmol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.
Key words: Chrysanthemum morifolium; flavonoids; antimutagenic activity; umu test; Ames test

-6-
Blood Coagulation and Fibrinolysis of Rats Fed Fish Oil: Reduced Coagulation Factors Especially Involved in Intrinsic Pathway and Increased Activity of Plasminogen Activator Inhibitor

Yoko SANO, Kaori SATO, Motoharu UCHIDA, and Masakazu MURATA

Laboratory of Applied Microorganisms, Marine Biochemistry Division, National Research Institute of Fisheries Science, Fisheries Research Agency, Kanazawa-ku, Yokohama 236-8648, Japan

Received March 4, 2003; Accepted June 23, 2003
Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.
Key words: fish oil; coagulation; fibrinolysis; intrinsic pathway; plasminogen activator inhibitor

-7-
Biosynthesis of Branched-chain Fatty Acid in Bacilli: FabD (malonyl-CoA:ACP transacylase) Is Not Essential for In Vitro Biosynthesis of Branched-chain Fatty Acids

Hirosuke OKU, Naoya FUTAMORI, Kenichi MASUDA, Yumiko SHIMABUKURO, Tomoyo OMINE, and Hironori IWASAKI

Division of Molecular Biotechnology, Center of Molecular Bioscience, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan

Received March 26, 2003; Accepted July 7, 2003
It was found that the partially purified β-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50--60％ of the control level for acyl-CoA primer and to 80％ for α-keto-β-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.
Key words: branched-chain fatty acid; biosynthesis; Bacillus; malonyl-CoA; ACP

-8-
Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase

Osao ADACHI,1,† Nozomi YOSHIHARA,1 Somboon TANASUPAWAT,2 Hirohide TOYAMA,1 and Kazunobu MATSUSHITA1

1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan 2Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand

Received April 7, 2003; Accepted July 17, 2003
Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6--7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.
Key words: acetic acid bacteria; Acinetobacter calcoaceticus; pyrroloquinoline quinone (PQQ); quinoprotein; quinate dehydrogenase

-9-
3-Dehydroquinate Production by Oxidative Fermentation and Further Conversion of 3-Dehydroquinate to the Intermediates in the Shikimate Pathway

Osao ADACHI,1,† Somboon TANASUPAWAT,2 Nozomi YOSHIHARA,1 Hirohide TOYAMA,1 and Kazunobu MATSUSHITA1

1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan 2Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand

Received April 7, 2003; Accepted July 24, 2003
3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100％ in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.
Key words: acetic acid bacteria; 3-dehydroquinate; 3-dehydroshikimate; quinate oxidation; shikimate pathway

-10-
Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22

Soo Jeong CHO,2,, Woo Jin LIM,1, Su Young HONG,1 Sang Ryeol PARK,3 and Han Dae YUN1,2,†

1Division of Applied Life Science and 2Research Institute of Life Science, Gyeongsang National University, Chinju 660-701, Korea 3Division of Molecular Physiology, National Institute of Agricultural Biotechnology, Suwon 441-707, Korea

Received April 8, 2003; Accepted June 30, 2003
Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270--1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 107 cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.
Key words: endophytic Bacillus sp. CY22; antibiotic iturin A; biological control

-11-
Purification of Turkey Pancreatic Phospholipase A2

Riadh BEN SALAH,1 Nacim ZOUARI,1 Joseph REINBOLT,2 and Hafedh MEJDOUB3,

1Laboratory of Biochemistry, ENIS Soukra Road, 3038 Sfax, Tunisia 2IBMC, UPR 9002 of CNRS, 15, Ren<0225>e Descartes Street, 67084 Strasbourg, France 3Laboratory of Biochemistry, Faculty of Sciences of Sfax, Soukra Road, 3038 Sfax, Tunisia

Received April 9, 2003; Accepted June 3, 2003
Turkey pancreatic phospholipase (TPP) has been purified from delipidated pancreases. The purification included ammonium sulfate fractionation, acidic (pH 5) treatment, followed by sequencial column chromatographies on DEAE-cellulose, Sephadex G-75, and reverse phase high pressure liquid chromatography. The purified enzyme was found to be a monomeric protein with molecular mass of 14 kDa. The optimal activity was measured at pH 8 and 37°C using egg yolk emulsion as substrate. Our results show that the enzyme (TPP) was not stable for 1 h at 60°C, and that bile salt and Ca2＋ were required for the expression of the purified enzyme. The sequence of the N-terminal amino acids of the purified enzyme shows a very close similarity between TPP and all other known pancreatic phospholipases.
Key words: pancreatic phospholipase; amino acids sequence; bile salt; Ca2＋; egg yolk

-12-
Purification and Characterization of an α-Haloketone-resistant Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA, and Cloning of the Gene

Hirokazu NANBA, Yasuko TAKAOKA, and Junzo HASEGAWA

Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan

Received April 10, 2003; Accepted July 7, 2003
Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to α-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with α-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD＋ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.
Key words: NAD-dependent formate dehydrogenase; Thiobacillus; α-haloketone-resistance

-13-
Purification and Identification of Antimicrobial Sesquiterpene Lactones from Yacon (Smallanthus sonchifolius) Leaves

Fengqiu LIN, Morifumi HASEGAWA, and Osamu KODAMA

Laboratory of Phytochemical Ecology, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan

Received April 14, 2003; Accepted June 11, 2003
The extraction of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson; Asteraceae] leaves and chromatographic separation yielded two new antibacterial melampolide-type sesquiterpene lactones, 8β-tigloyloxymelampolid-14-oic acid methyl ester and 8β-methacryloyloxymelampolid-14-oic acid methyl ester, as well as the four known melampolides, sonchifolin, uvedalin, enhydrin and fluctuanin. The newly identified compound, 8β-methacryloyloxymelampolid-14-oic acid methyl ester, exhibited potent antimicrobial activity against Bacillus subtilis and Pyricularia oryzae, while 8β-tigloyloxymelampolid-14-oic acid methyl ester showed lower activity. Fluctuanin exhibited the strongest antibacterial activity against B. subtilis among these six sesquiterpene lactones.
Key words: Smallanthus sonchifolius; yacon; antibacterial compound; sesquiterpene lactone; melampolide

-14-
Cloning, Sequencing, and Expression of the Gene Encoding the Clostridium stercorarium α-Galactosidase Aga36A in Escherichia coli

SURYANI, Tetsuya KIMURA, Kazuo SAKKA, and Kunio OHMIYA

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

Received April 14; 2003; Accepted June 30, 2003
The α-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786. Aga36A is an enzyme classified in family 36 of the glycoside hydrolases and showed sequence similarity with some enzymes of family 36 such as Geobacillus (formerly Bacillus) stearothermophilus GalA (57％) and AgaN (52％). The enzyme purified from a recombinant E. coli is optimally active at 70°C and pH 6.0. The enzyme hydrolyzed raffinose and guar gum with specific activities of 3.0 U/mg and 0.46 U/mg for the respective substrates.
Key words: Clostridium stercorarium; α-galactosidase

-15-
A Thermostable Laccase from Streptomyces lavendulae REN-7: Purification, Characterization, Nucleotide Sequence, and Expression

Takashi SUZUKI,1 Kohki ENDO,1 Masaaki ITO,1,† Hiroshi TSUJIBO,2 Katsushiro MIYAMOTO,2 and Yoshihiko INAMORI2

1Central Laboratory, Rengo Co., Ltd., 4-1-186 Ohhiraki, Fukushima-ku, Osaka 553-0007, Japan 2Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Received April 21, 2003; Accepted July 7, 2003
We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0％. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70°C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.
Key words: polyphenoloxidase; laccase; Streptomyces; thermostable laccase

-16-
Inhibition of Immunoglobulin E Production in Allergic Model Mice by Supplementation with Vitamin E and β-Carotene

Noriko BANDO, Rintaro YAMANISHI, and Junji TERAO

Department of Nutrition, School of Medicine, The University of Tokushima, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan

Received April 24, 2003; Accepted June 23, 2003
A diet containing different amounts of vitamin E (α-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of β-carotene was supplemented to the basal diet containing vitamin E as α-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with β-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus β-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and β-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and β-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.
Key words: IgE antibody; vitamin E; β-carotene; allergy

-17-
Systematic Strategy for the Synthesis of Cyanobacterin and Its Stereoisomers. 1. Asymmetric Total Synthesis of Dechloro-cyanobacterin and Its Enantiomer

Yasushi HAGA, Momotoshi OKAZAKI, and Yoshihiro SHUTO

The United Graduate School of Agricultural Science, Ehime University, Tarumi 3-5-7, Matsuyama 790-8566, Japan

Received May 6, 2003; Accepted June 16, 2003
The stereocontrolled total synthesis of the non-chlorinated analog of cyanobacterin, a potent photosynthesis inhibitor, was achieved by 12 steps in a 10.0％ overall yield. Its enantiomer was also synthesized from the same starting material. This synthetic strategy is expected to be applicable to prepare cyanobacterin and all its stereoisomers, together with other similar bioactive compounds.
Key words: cyanobacterin; photosynthesis inhibitor; β-
hydroxy-γ-ylidene-γ-butyrolactone framework; enantiodivergent synthesis; Evans and non-Evans aldol reaction

-18-
Mode of α-Amylase Production by the Shochu Koji Mold Aspergillus kawachii

Kazuki NAGAMINE,1 Kenji MURASHIMA,2 Taku KATO,2 Hitoshi SHIMOI,1,2 and Kiyoshi ITO1,2,†

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

Received May 16, 2003; Accepted July 15, 2003
Aspergillus kawachii produces two kinds of α-amylase, one is an acid-unstable α-amylase and the other is an acid-stable α-amylase. Because the quality of the shochu depends strongly on the activities of the α-amylases, the culture conditions under which these α-amylases are produced were examined. In liquid culture, acid-unstable α-amylase was produced abundantly, but, acid-stable α-amylase was not produced. The acid-unstable α-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no α-amylase activity was recognized in the medium. Instead, the α-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable α-amylase. Usually, acid-unstable α-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of α-amylase were produced. The production of acid-stable α-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.
Key words: Aspergillus kawachii; α-amylase; solid-state culture-specific

-19-
Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci

Hisashi KIMOTO1,† and Akira TAKETO2

1Department of Biochemistry I, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan 2Department of Environmental and Biotechnological Frontier Engineering, Fukui University of Technology, 3-6-1 Gakuen, Fukui 910-8505, Japan

Received May 20, 2003; Accepted July 10, 2003
Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1×107 transformants per μg of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo− or sagB− mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.
Key words: electroporation; transformation; streptococci; gene targeting; streptolysin

-20-
Synthesis of the Racemate and Both Enantiomers of Massoilactone

Masashi ISHIKAWA,1 Masayasu AMAIKE,1 Masamichi ITOH,1 Yasuhiro WARITA,1,† and Takeshi KITAHARA2

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

Received May 22, 2003; Accepted June 30, 2003
A simple and efficient synthesis of (±)-massoilactone (1) as a key substance for the butter and milk flavor was accomplished from n-hexanal in only a few steps. Application of its racemic synthesis enabled natural (R)-(−)- and unnatural (S)-(＋)-massoilactone (1a, 1b) to be synthesized by starting from commercially available (R)-(＋)-1,2-epoxyheptane (5).
Key words: massoilactone; (R)-(＋)-1,2-epoxyheptane; Mitsunobu inversion; flavor

-21-
Systematic Strategy for the Synthesis of Cyanobacterin and Its Stereoisomers. 2. Asymmetric Total Synthesis of the 2- and 3-Epimers of Dechloro-cyanobacterin

Yasushi HAGA, Momotoshi OKAZAKI, and Yoshihiro SHUTO

The United Graduate School of Agricultural Science, Ehime University, Tarumi 3-5-7, Matsuyama 790-8566, Japan

Received May 22, 2003; Accepted June 27, 2003
Stereocontrolled total syntheses of the (2S,3R)- and (2R,3S)-isomers of the non-chlorinated analog of cyanobacterin, a potent photosynthesis inhibitor, were achieved. Since both the (2R,3R)- and (2S,3S)-isomers of this compound had been previously synthesized from the same starting material, a systematic strategy for the synthesis of all stereoisomers could be established.
Key words: cyanobacterin; natural photosynthesis inhibitor; synthesis of stereoisomers; Evans and non-Evans aldol condensation; diastereoselective alkynylation

-22-
Synthesis of the Enantiomers of 21-Methyl-7-hentriacontanone and a Stereoisomeric Mixture of 5-Acetoxy-19-methylnonacosane, Candidates for the Female Sex Pheromone of the Screwworm Fly, Cochliomyia hominivorax

Kenji MORI

Insect Pheromone and Traps Division, Fuji Flavor Co., Ltd., Midorigaoka 3-5-8, Hamura-City, Tokyo 205-8503, Japan

Received May 28, 2003; Accepted June 23, 2003
The enantiomers of 21-methyl-7-hentriacontanone (1), which might show weak bioactivity as the female sex pheromone of the screwworm fly (Cochliomyia hominivorax), were synthesized by starting from the enantiomers of citronellal. (±)-Citronellol was converted to a racemic and diastereomeric mixture of 5-acetoxy-19-methylnonacosane (2), which was considered to be a candidate for the female sex pheromone of C. hominivorax. Synthetic 2 exhibited no pheromone activity against male C. hominivorax.
Key words: Cochliomyia hominivorax; methyl-branched ketone; methyl-branched secondary acetate; pheromone; screwworm fly

-23-
Structural Features of N-Glycans Linked to Glycoproteins from Oil Palm Pollen, an Allergenic Pollen

Yoshinobu KIMURA,1,2,† Takeo YOSHIIE,1 Woo Kwan KIT,2 Megumi MAEDA,2 Mariko KIMURA,3 and Siang Hee TAN4

1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan 2Division of Biomolecular Science, The Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan 3Department of Food System, Faculty of Food Culture, Kurashiki Sakuyo University, Kurashiki, Okayama 710-0292, Japan 4Genome Centre, Institute Bioscience, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia

Received June 4, 2003; Accepted July 15, 2003
The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820--827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing α1--3 fucose and/or β1--2 xylose residues accounts for 36.9％ of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6％), GlcNAc2Man3Xyl1GlcNAc2 (4.4％), Man3Xyl1Fuc1-
GlcNAc2 (1.1％), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6％), and GlcNAc1Man3Xyl1GlcNAc2 (1.2％). The remaining 63.1％ of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8％), Man8GlcNAc2 (32.1％), Man7GlcNAc2 (19.9％), Man6GlcNAc2 (5.3％).
Key words: plant glycoprotein; antigenic N-glycan; pollen allergy; oil palm pollen; Elaeis guineensis Jacq

-24-
Synthesis and Absolute Configuration of (−)-3-Butyl-7-hydroxyphthalide, a Cytotoxic Metabolite of Penicillium vulpinum

Tomoya OHZEKI1 and Kenji MORI2,†

1Department of Chemistry, Graduate School of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan 2Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology c/o Seikagaku Corporation, Tateno 3-1253, Higashiyamato City, Tokyo 207-0021, Japan

Received June 11, 2003; Accepted July 7, 2003
Both the enantiomers as well as the racemate of 3-butyl-7-hydroxyphthalide (1) were synthesized, and the absolute configuration of the naturally occurring (−)-1 (a weakly cytotoxic metabolite of Penicillium vulpinum) was identified as S.
Key words: asymmetric dihydroxylation; cytotoxic compound; Penicillium vulpinum; phthalide

-25-
Expression Profiling of Translation-associated Genes in Sporulating Bacillus subtilis and Consequence of Sporulation by Gene Inactivation

Yoshiaki OHASHI,1,, Takashi INAOKA,1, Koji KASAI,1 Yasuhiro ITO,1 Susumu OKAMOTO,1 Hideo SATSU,1 Yuzuru TOZAWA,1 Fujio KAWAMURA,2 and Kozo OCHI1,†

1National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan 2Laboratory of Molecular Genetics, College of Science, Rikkyo (St. Paul′s) University, Toshima-ku, Tokyo 171-8501, Japan

Received June 13, 2003; Accepted July 24, 2003
A DNA microarray technique was used to demonstrate global changes in the transcription pattern of translation-associated genes that encode fifty-four ribosomal proteins including a putative ribosomal gene, and eleven translation factors in sporulating B. subtilis. We found that the mRNA levels of nine genes involved in the translation system, which include the genes for three ribosomal proteins (rpmA, rpmGB, and ctc) and two translation factors (efp, and frr), were maintained at a high level at the onset of sporulation. The ypfD gene, which encodes the ribosomal protein S1 homologue, was also found to be expressed significantly during the early sporulation stage. In order to demonstrate the significance of these genes for sporulation, mutants were constructed using the pMutinT3 disruption vector. We detected an impaired sporulation in the mutants of rpmA (gene for the ribosomal protein L27), efp (elongation factor P), frr (ribosome recycling factor), and ypfD. The effect was especially pronounced in the efp mutant, sporulation of which was entirely abolished without affecting growth. The reduced expression of rpmGB (ribosomal protein L33) resulted in an impaired sporulation only at a high temperature (47°C). Only the rplI mutant, which encodes the ribosomal protein L9, could not be obtained, implying that its function is essential for viability. Thus, we successfully demonstrated the significance of several translation-associated genes in sporulation by using the results of the gene expression profiling.
Key words: DNA microarray; ribosome; sporulation; translation; gene expression

-26-
Expression and Characterization of Bovine Lactoperoxidase by Recombinant Baculovirus

Tetsuya TANAKA, Sanae SATO, Haruto KUMURA, and Kei-ichi SHIMAZAKI

Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan

Received June 25, 2003; Accepted July 22, 2003
Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of δ-aminolevulinic acid, which is a heme precursor. Differences in the δ-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.
Key words: lactoperoxidase; baculovirus; glycosylation; heme; peroxidase activity

-27-
Note
Differential Expression of the Nonmuscle-type Cofilin Gene between Subcutaneous and Visceral Adipose Tissue

Ki-Choon CHOI,1 Sang-Gun ROH,1,† Daisuke HISHIKAWA,1 Hisae MIYAHARA,1 Masaaki KUNO,1 Hiroaki TSUZUKI,1 Ai TOMIMATSU,1 Yeon-Hee HONG,1 Kwang-Keun CHO,2 Kyung-Ho HAN,3 and Shin-ichi SASAKI1

1Department of Food Production Science, Faculty of Agriculture, Shinshu University, Nagano-ken 399-4598, Japan 2Department of International Livestock Industry, Chinju National University, Chinju 660-758, Korea 3Department of Animal Science ＆ Technology, School of Agricultural Biotechnology, Seoul National University, Suweon 441-744, Korea

Received January 20, 2003; Accepted July 9, 2003
Visceral adipocytes differ in various biochemical properties from adipocytes of subcutaneous origin. However, information on differences in gene expression between visceral and subcutaneous fat depots is limited. Expression of the genes for the nonmuscle and muscle isoforms of the actin-binding protein cofilin was examined in subcutaneous and visceral fat depots of mice, pigs, and cattle by semiquantative reverse transcription and polymerase chain reaction analysis. The abundance of nonmuscle-type cofilin mRNA was markedly higher in visceral adipose tissue than in subcutaneous adipose tissue of mouse and pig. This difference was more pronounced in mice fed a high-fat diet than in those fed a standard diet. In cattle, however, the amount of nonmuscle-type cofilin mRNA was greater in subcutaneous fat than in visceral fat. Muscle-type cofilin mRNA was not detected in either adipose tissue of any of the three species. These results suggest that the nonmuscle isoform of cofilin, and therefore the cytoskeleton, may play a role in lipid accumulation in visceral adipose tissue.
Key words: nonmuscle-type cofilin gene; visceral adipocyte; subcutaneous adipocyte

-28-
Note
Effects of the Marine Unicellular Alga Nannochloropsis sp. to Reduce the Plasma and Liver Cholesterol Levels in Male Rats Fed on Diets with Cholesterol

Moshe J. WERMAN,1 Assaf SUKENIK,2 and Shoshana MOKADY1,†

1Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel 2The National Institute of Oceanography, Israel Oceanographic and Limnological Research, Haifa, Israel

Received February 12, 2003; Accepted May 7, 2003
The effects of Nannochloropsis were studied on rats consuming hypercholesterolemic diets. The whole biomass and the hexane/ethanol extract increased the plasma and hepatic eicosapentaenoic and docosahexaenoic acids levels, and reduced the cholesterol levels. We also observed a higher level of propionate, and a lower ratio between acetate and propionate. These data suggest the efficacy of Nannochloropsis in reducing cholesterol levels.
Key words: Nannochloropsis sp.; cholesterol; plasma; liver; rat

-29-
Note
Effect of α,α-Dialkyl Amino Acids on the Protease Resistance of Peptides

Hiroshi YAMAGUCHI,1 Hiroaki KODAMA,1,† Satoshi OSADA,1 Fumio KATO,2 Masood JELOKHANI-NIARAKI,3 and Michio KONDO1

1Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840-8502, Japan 2Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan 3Department of Chemistry, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada

Received March 14, 2003; Accepted June 19, 2003
A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of α,α-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1′ positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1′ and P2 positions resulted in complete resistance.
Key words: 2-aminoisobutyric acid; α,α-dialkyl amino acid; peptide antibiotic; protease resistance; trypsin

-30-
Note
PEG-Inhibitor Conjugates: Application of Diphenyl α-Aminoalkylphosphonate to Removal of Chymotrypsin

Makiko UMEZAKI,1 Takayoshi FUJII,1 Toshiaki YOSHIMURA,1 Isao YAMAZAKI,2 and Shin ONO1,†

1Department of Material Systems Engineering and Life Science, Faculty of Engineering, Toyama University, Gofuku, Toyama 930-8555, Japan 2Yayoi Kagaku Kogyo Co., Ltd., Fukuoka-machi, Nishitonami-gun, Toyama 939-0135, Japan

Received April 21, 2003; Accepted June 21, 2003
Diphenyl 1-amino-2-phenylethylphosphonate was introduced to poly(ethylene glycol)s (PEGs) with average molecular masses of 300, 400, and 600 to prepare water-insoluble PEG-inhibitor conjugates. Interestingly, only the conjugate from PEG with an average molecular weight of 600 formed a precipitate with chymotrypsin but not with trypsin. The results demonstrated that the PEG-inhibitor conjugate is useful for separation of chymotrypsin.
Key words: affinity separation; diphenyl α-aminoalkylphosphonate; irreversible inhibitor; poly(ethylene glycol); serine proteases

-31-
Note
Production of Functional Lectin in Pichia pastoris Directed by Cloned cDNA from Aleuria aurantia

Koh AMANO, Mitsuyo TAKASE, Akikazu ANDO, and Yoshiho NAGATA

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

Received April 21, 2003; Accepted June 22, 2003
A plasmid bearing a nucleotide sequence of fucose-specific lectin of Aleuria aurantia was constructed and expressed in a methylotrophic yeast, Pichia pastoris. The product showed almost the same hemagglutinating activity as the lectin produced in Escherichia coli, the properties of which were quite similar to the native one. Because of glycosylation of the product, the molecular mass was larger than that of the native one, and it acquired higher thermostability.
Key words: Aleuria aurantia lectin; heterogenous gene expression; glycosylation of lectin; improvement of thermostability

-32-
Note
Linoleic Acid 10-Hydroperoxide as an Intermediate during Formation of 1-Octen-3-ol from Linoleic Acid in Lentinus decadetes

Kenji MATSUI, Satomi SASAHARA, Yoshihiko AKAKABE, and Tadahiko KAJIWARA

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

Received April 22, 2003; Accepted June 13, 2003
In order to confirm the biosynthetic pathway to 1-octen-3-ol from linoleic acid, a crude enzyme solution was prepared from the edible mushroom, Lentinus decadetes. When the reaction was performed in the presence of glutathione peroxidase, which can reduce organic hydroperoxide to the corresponding hydroxide, the amount of 1-octen-3-ol formed from linoleic acid was decreased. At the same time, an accumulation of linoleic acid 10-hydroxide could be detected. The 10-hydroperoxide therefore seems to be an intermediate on the biosynthetic pathway.
Key words: Lentinus decadetes; flavor compound; 1-octen-3-ol, linoleic acid 10-hydroperoxide

-33-
Note
Acivicin Induce Filamentous Growth of Saccharomyces cerevisiae

Tsuyoshi MIYAKE,1,† Hiroyuki SAMMOTO,1 and Bun-ichiro ONO2

1Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama 701-1296, Japan 2Department of Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan

Received May 1, 2003; Accepted June 23, 2003
The antibiotic acivicin is a known inhibitor of γ-glutamyl transpeptidase (γGTP). We found that acivicin can induce filamentous growth in both diploid and haploid cells of Saccharomyces cerevisiae. This phenomenon is not related to the inhibition of γGTP or interference in glutathione metabolism. Interestingly, yeasts used in the brewing industry are more sensitive to acivicin, suggesting that this dimorphological differentiation may be related to some characteristics of these particular strains.
Key words: yeast; acivicin; filamentous growth; industrial strain

-34-
Note
Isolation and Characterization of a Stilbene-degrading Strain of Pseudomonas fluorescens, and Production of Antioxidant Compounds by Stilbene Metabolism

Joseph G. LEAHY,1,† Patricia J. BATCHELOR,1 Mary C. SETZER,2 and William N. SETZER2

1Department of Biological Sciences and 2Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, U.S.A.

Received May 20, 2003; Accepted June 17, 2003
In this study, we consider the use of hydrocarbon-degrading bacteria that degrade trans-stilbene as a novel approach for synthesizing potentially bioactive hydroxylated stilbenes. A trans-stilbene-degrading bacterium, MN2, was isolated from activated sludge through enrichment culture, and identified as Pseudomonas fluorescens using conventional techniques. Degradation of trans-stilbene by this strain yielded two metabolites that had significant antioxidant activity.
Key words: antioxidant; Pseudomonas fluorescens; stilbene

-35-
Note
Efficient Production of Recombinant Human Pleiotrophin in Yeast, Pichia pastoris

Akira MURASUGI,1,† Isao KIDO,2 Hideshi KUMAI,2 and Yukio ASAMI1

1Meiji Institute of Health Science, and 2Meiji Cell Technology Center, Meiji Milk Products Co., Ltd., 540 Naruda, Odawara, Kanagawa 250-0862, Japan

Received May 27, 2003; Accepted June 30, 2003
Approximately 260 mg/l of authentic recombinant human pleiotrophin (rhPTN) was expressed into the medium of high-cell density fermentation using a Pichia pastoris protein expression system. The prepro-sequence of yeast α-mating factor was used successfully. The recombinant hPTN was efficiently recovered from the medium by expanded bed adsorption, and purified using successive column chromatography steps. In the purified rhPTN preparation, modified rhPTN were scarcely detected. Circular dichroism measurement of the purified PTN showed the presence of the characteristic β-structures in the protein.
Key words: human pleiotrophin; fermentor culture; α-factor signal; Pichia pastoris; circular dichroism

-36-
Note
Addition of a Peptide Tag at the C Terminus of AtHKT1 Inhibits Its Na＋ Transport

Yasuhiro KATO,1 Akihiro HAZAMA,2 Mutsumi YAMAGAMI,3 and Nobuyuki UOZUMI1,4,†

1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan 2Fukushima Medical University, Fukushima 960-1295, Japan 3Institute of Environmental Sciences, Aomori 039-3212, Japan 4Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan

Received May 27, 2003; Accepted June 27, 2003
Eight transmembrane segments in the Arabidopsis Na＋ transporter, AtHKT1, have been proposed to be arranged around the axis of the pore. We report here that the addition of a GFP or a FLAG tag to the C terminus of AtHKT1 impairs the Na＋ transport in Xenopus ooytes as detected by electrophysiological measurements.
Key words: AtHKT1; transporter; sodium; membrane protein; Xenopus laevis oocyte

-37-
Note
The Protein Component of Bacterial Ribonuclease P Flickers the Metal Ion Response to the Substrate Shape Preference of the Ribozyme

Tomoaki ANDO, Terumichi TANAKA, and Yo KIKUCHI

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan

Received June 3, 2003; Accepted July 23, 2003
The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion. At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA). The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion. Considering that the homologous E. coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell.
Key words: specificity; ribonuclease P; Escherichia coli; ribozyme; holo enzyme

-38-
Note
Modulations of Food-derived Substances on Intestinal Permeability in Caco-2 Cell Monolayers

Yutaka KONISHI

Applied Bioresearch Center, Research ＆ Development, Department, Kirin Brewery Co., Ltd., 3 Miyaharacho, Takasaki-shi, Gunma 370-1295, Japan

Received June 5, 2003; Accepted July 1, 2003
The effects of more than 300 kinds of food extracts on intestinal permeability were investigated in Caco-2 cells with the use of model compounds: Lucifer Yellow (LY) for the paracellular pathway, Fluorescein (FC) for the monocarboxylic acid transporter-mediated pathway, and Rhodamine 123 (RH) for the p-glycoprotein-mediated efflux pathway. With several extracts of increasing or decreasing LY permeation, increasing FC or RH permeation was also observed, indicating modulation by dietary substances in several pathways for intestinal absorption.
Key words: fluorescein; monocarboxylic acid transporter; intestinal absorption; Caco-2 cells

-39-
Note
A Novel Gene Encoding an Enzyme That Degrades a Polysaccharide from the Sheath of Sphaerotilus natans

Minoru TAKEDA, Katsuya NISHINA, Yuji HANAOKA, Motoko HIGO, Mutsumi TERUI, Ichiro SUZUKI, and Jun-ichi KOIZUMI

Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, Tokiwadai 79-5, Hodogaya, Yokohama 240-8501, Japan

Received June 11, 2003; Accepted June 30, 2003
A gene encoding an enzyme that is able to depolymerize the basic polysaccharide prepared from the sheath of Sphaerotilus natans was identified in a sheath-degrading bacterium, Paenibacillus koleovorans. The gene was constructed from 2217 bp coding for 738 amino acids, including the signal sequence of 34 amino acids. No closely related protein or gene was indicated by a homology search. The gene was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The fusion protein depolymerized the sheath polysaccharide into an oligosaccharide, introducing an unsaturated sugar residue, suggesting that the gene codes for a polysaccharide lyase acting on a basic polysaccharide.
Key words: expression; Sphaerotilus natans; sheath polysaccharide; degradation; Paenibacillus koleovorans

-40-
Note
Increased Production of Antioxidative Sesaminol Glucosides from Sesame Oil Cake through Fermentation by Bacillus circulans Strain YUS-2

Takashi OHTSUKI,1 Junko AKIYAMA,1 Takao SHIMOYAMA,1 Shin-ichi YAZAKI,1 Sadaharu UI,1 Yuko HIROSE,2 and Akio MIMURA1,†

1Department of Applied Chemistry and Biotechnology, Faculty of Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan 2Department of Chemistry, Faculty of Education and Human Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan

Received June 23, 2003; Accepted July 28, 2003
Bacillus circulans strain YUS-2 was isolated as the strongest antioxidant-producer in fermentation of sesame oil cake (SOC, defatted residue yielded from sesame seed oil production). Two major strong antioxidants from fermented SOC were purified and identified as known sesaminol triglucoside and sesaminol diglucoside, however, our results demonstrated that the fermentation process with B. circulans YUS-2 was highly effective to gain the extraction efficiency of the sesaminol glucosides.
Key words: antioxidative activity; Bacillus circulans; fermentation; sesaminol glucosides; sesame oil cake

-41-
Communication
A New Type of RNase T2 Ribonuclease in Two Basidiomycetes Fungi, Lentinus edodes and Irpex lacteus

Hiroko KOBAYASHI,1 Tadashi ITAGAKI,1 Norio INOKUCHI,1 Kazuko OHGI,2,† Tatsuya WADA,2 Masanori IWAMA,2 and Masachika IRIE2

1Department of Microbiology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan 2Department of Applied Microbiology, Faculty of Pharmaceutical Science, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan

Received June 17, 2003; Accepted July 30, 2003
Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.
Key words: RNase T2 ribonucleases; Basidiomycetes; excretion; Irpex lacteus; Lentinus edodes