Contents and Abstracts of Latest Issue of BBB

(Vol.67 No.1 2003)

Production and Characterization of Recombinant Phanerochaete chrysosporium β-Glucosidase in the Methylotrophic Yeast Pichia pastoris
Rie KAWAI, Makoto YOSHIDA, Tomomi TANI, Kiyohiko IGARASHI, Tsuyoshi OHIRA, Hiromichi NAGASAWA, and Masahiro SAMEJIMA p.1

Extracellular Acidic Polysaccharide Production by a Two-membered Bacterial Coculture
Shinya KURATA,1,2 Kazutaka YAMADA,1 Kyoko TAKATSU,1 Satoshi HANADA,3 Osamu KOYAMA,1 Toyokazu YOKOMAKU,1 Yoichi KAMAGATA,3 Takahiro KANAGAWA,2,3, and Ryuichiro KURANE3
p.8

Effect of Phytate in Soy Protein on the Serum and Liver Cholesterol Levels and Liver Fatty Acid Profile in Rats
Kazunori KOBA,1, Jim-Wen LIU,2 Emil BOBIK, Jr.,2 David E. MILLS,3 Michihiro SUGANO,4 and Yung-Sheng HUANG2 p.15

Regulation of the Peripheral Body Temperature by Foods: A Temperature Decrease Induced by the Japanese Persimmon (kaki, Diospyros kaki)
Gaku HIBINO,1, Tomonori NADAMOTO,2, Fumiko FUJISAWA,2 and Tohru FUSHIKI1 p.23

Purification and Characterization of a New Type of α-Glucosidase from Paecilomyces lilacinus That Has Transglucosylation Activity to Produce α-1,3- and α-1,2-Linked Oligosaccharides
Isao KOBAYASHI,1, Masahiro TOKUDA,1 Hiroyuki HASHIMOTO,2 Takashi KONDA,3 Hirofumi NAKANO,4 and Sumio KITAHATA4 p.29

Purification and Characterization of meta-Cleavage Compound Hydrolase from a Carbazole Degrader Pseudomonas resinovorans Strain CA10
Hideaki NOJIRI,1 Hiroko TAIRA,1 Kenichi IWATA,1 Kenichi MORII,1 Jeong-Won NAM,1 Takako YOSHIDA,1 Hiroshi HABE,1 Shugo NAKAMURA,2 Kentaro SHIMIZU,2 Hisakazu YAMANE,1 and Toshio OMORI1, p.36

Effect of Medium-chain Triglycerides on the Postprandial Triglyceride Concentration in Healthy Men
Michio KASAI,1, Hideaki MAKI,1 Naohisa NOSAKA,1 Toshiaki AOYAMA,1 Katsuhiko OOYAMA,1 Harumi UTO,2 Mitsuko OKAZAKI,3 Osamu IGARASHI,4 and Kazuo KONDO2 p.46

Antioxidative Activity of the Blue Pigment Formed in a D-Xylose-Glycine Reaction System
Satomi SHIZUUCHI and Fumitaka HAYASE p.54

Structural Characterization of Triacylglycerol in Several Oils Containing Gamma-linolenic Acid
Satoru SHIMIZU1,2 and Masuo NAKANO3, p.60

β-Galactosidase and Its Significance in Ripening of <Oh><0192><0192><Wa>Saijyo′′ Japanese Persimmon Fruit
Akihiro NAKAMURA,1, Hirokazu MAEDA,1 Misae MIZUNO,2 Yoko KOSHI,2 and Yasunori NAGAMATSU2 p.68

Identification and Characterization of Scp15, a Protein from Streptomyces coelicolor A3(2) Inducing Neurites in PC12 Cells
Satoru NAKASHIMA, Shuji WAKATSUKI, Tatsuya YOKOYAMA, Manabu ARIOKA, and Katsuhiko KITAMOTO p.77

Novel Aminopeptidase Specific for Glycine from Actinomucor elegans
Kiyoshi ITO,1 Xiaohang MA,1, Nik AZMI,1 Hua-Shan HUANG,1 Mikio FUJII,2 and Tadashi YOSHIMOTO1, p.83

Effect of Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Susceptibility to Listeria Infection
Yoshiko SUGITA-KONISHI,1, Kazuo KOBAYASHI,1 Hiroyuki NAITO,2 Katuhiro MIURA,3 and Yoshihiko SUZUKI2 p.89

Binding Properties of Bacillus thuringiensis Cry1C δ-Endotoxin to the Midgut Epithelial Membranes of Culex pipiens
Shinya KAMAUCHI,1 Masashi YAMAGIWA,1 Motoyuki ESAKI,2 Kanao OTAKE,2 and Hiroshi SAKAI1, p.94

Interactions of Heterologous Mycelia Colonized in the Substrate Govern Fruit Body Production in the Cultivated Homobasidiomycete Pholiota nameko
Katsuhiko BABASAKI,1 Kazuhiko MASUNO,2 and Hitoshi MURATA1, p.100

Specific RNA Interference in psbP Genes Encoded by a Multigene Family in Nicotiana tabacum with a Short 3′-Untranslated Sequence
Kentaro IFUKU, Yumiko YAMAMOTO, and Fumihiko SATO p.107

Purification and Characterization of NADP-Linked Isocitrate Dehydrogenase from the Copper-tolerant Wood-rotting Basidiomycete Fomitopsis palustris
Jeong-Jun YOON, Takefumi HATTORI, and Mikio SHIMADA p.114

Need for Cytochrome bc1 Complex for Dissimilatory Nitrite Reduction of Pseudomonas aeruginosa
Norio HASEGAWA, Hiroyuki ARAI, and Yasuo IGARASHI p.121

Characterization of the Biosynthetic Gene Cluster of Rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243
Hiroyasu ONAKA, Shin-ichi TANIGUCHI, Yasuhiro IGARASHI, and Tamotsu FURUMAI p.127

Mepanipyrim, a Novel Inhibitor of Pharmacologically Induced Golgi Dispersion
Michiko NAKAMURA,1,2 Yoshiki KONO,2 and Akira TAKATSUKI1, p.139

Nordihydroguaiaretic Acid, of a New Family of Microtubule-stabilizing Agents, Shows Effects Differentiated from Paclitaxel
Machiko NAKAMURA,1,3 Junko NAKAZAWA,2 Takeo USUI,2 Hiroyuki OSADA,2 Yoshiki KONO,3 and Akira TAKATSUKI1, p.151

Note
Nuclear Localization of Senescence Marker Protein-30, SMP30, in Cultured Mouse Hepatocytes and Its Similarity to RNA Polymerase
Akihito ISHIGAMI,2 Setsuko HANDA,2 Naoki MARUYAMA,2 and Prakash C. SUPAKAR1, p.158

Note
Selective Accumulation of Delphinidin Derivatives in Tobacco Using a Putative Flavonoid 3′,5′-Hydroxylase cDNA from Campanula medium
Yasushi OKINAKA,, Yukihisa SHIMADA, Ritsuko NAKANO-SHIMADA, Masaya OHBAYASHI, Shigeto KIYOKAWA, and Yasuhiro KIKUCHI p.161

Note
Adjuvant Activity of Alum in Inducing Antigen Specific IgE Antibodies in BALB/c Mice: a Reevaluation
Rintaro YAMANISHI, Ikuko YUSA, Noriko BANDO, and Junji TERAO p.166

Note
Stimulation by Caffeic Acid, Coumalic Acid, and Corilagin of the Germination of Resting Spores of the Clubroot Pathogen Plasmodiophora brassicae
Michio OHI,1, Takashi KITAMURA,1 and Shuichi HATA2 p.170

Note
Primary Structure of Cytochrome c Gene from the White Root Rot Fungus Rosellinia necatrix
Tadanori AIMI, Hiroyuki TAGUCHI, and Tsutomu MORINAGA p.174

Note
Structural Analysis of Conjugated Linoleic Acid Produced by Lactobacillus plantarum, and Factors Affecting Isomer Production
Shigenobu KISHINO,1 Jun OGAWA,1 Akinori ANDO,1 Takashi IWASHITA,2 Tsuyoshi FUJITA,2 Hiroshi KAWASHIMA,3 and Sakayu SHIMIZU1, p.179

Note
A Novel Cellulolytic, Anaerobic, and Thermophilic Bacterium, Moorella sp. Strain F21
Shuichi KARITA,1, Kengo NAKAYAMA,1 Masakazu GOTO,1 Kazuo SAKKA,1 Wan-Jae KIM,1 and Satoru OGAWA2 p.183

Note
Putative Homologs of SSK22 MAPKK Kinase and PBS2 MAPK Kinase of Saccharomyces cerevisiae Encoded by os-4 and os-5 Genes for Osmotic Sensitivity and Fungicide Resistance in Neurospora crassa
Makoto FUJIMURA,1, Noriyuki OCHIAI,2 Michiyo OSHIMA,1 Takayuki MOTOYAMA,3 Akihiko ICHIISHI,1 Ron USAMI,2 Koki HORIKOSHI,1 and Isamu YAMAGUCHI3 p.186

Note
Biocatalytic Deprotection of a Cetraxate Ester by Microbacterium sp. Strain 7-1W Cells
Kohsuke HONDA, Keiji SAKAMOTO, Shinji KITA, Michihiko KATAOKA, and Sakayu SHIMIZU
p.192

Note
Antioxidative Effect of Citrus Essential Oil Components on Human Low-density Lipoprotein In Vitro
Yasuo TAKAHASHI, Nobuya INABA, Shigeru KUWAHARA, and Wataru KUKI p.195

Note
Efficient Release of Overproduced Gene Products from Escherichia coli BL21(DE3) by Lytic Infection with Newly Isolated Bacteriophages
Yuichiro IIDA,1 Yoshinori MATSUDA,1, Ryuichiro SAITO,1 Masanori NAKASATO,1 Teruo NONOMURA,1 Koji KAKUTANI,2 Yukio TOSA,3 Shigeyuki MAYAMA,3 and Hideyoshi TOYODA1
p.198

Note
Catalytic Properties of Lipases Immobilized on Various Mesoporous Silicates
Katsuya KATO,1, Roxana IRIMESCU,1 Takao SAITO,1 Yoshiyuki YOKOGAWA,1 and Haruo TAKAHASHI2 p.203

Note
Differential Scanning Calorimetry of the Effects of Ca2＋ on the Thermal Unfolding of Pseudomonas cepacia Lipase
Akiyoshi TANAKA,1, Hayuki SUGIMOTO,1 Yuko MUTA,2 Takafumi MIZUNO,1 Keishi SENOO,3 Hitoshi OBATA,1 and Kuniyo INOUYE2 p.207

Note
Construction of a Vector Plasmid for Use in Gluconobacter oxydans
Naoto TONOUCHI, Masakazu SUGIYAMA, and Kenzo YOKOZEKI p.211

Note
Functions of Malonate Decarboxylase Subunits from Pseudomonas putida
Shigeru CHOHNAN, Kayo AKAGI, and Yoshichika TAKAMURA p.214

Preliminary Communication
Biodegradation of Bisphenol A by Cultured Cells of Caragana chamlagu
Wen CHAI,1 Hiroshi SAKAMAKI,2 Susumu KITANAKA,3 Michihiko SAITO,4 and C. Akira HORIUCHI1, p.218

Preliminary Communication
Plant Chitinase as a Possible Biocontrol Agent for Use Instead of Chemical Fungicides
Shuji KARASUDA,1 Shuhei TANAKA,1 Hiroshi KAJIHARA,2 Yuji YAMAMOTO,2 and Daizo KOGA1,
p.221

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Production and Characterization of Recombinant Phanerochaete chrysosporium β-Glucosidase in the Methylotrophic Yeast Pichia pastoris

Rie KAWAI, Makoto YOSHIDA, Tomomi TANI, Kiyohiko IGARASHI, Tsuyoshi OHIRA, Hiromichi NAGASAWA, and Masahiro SAMEJIMA

Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

Received March 14, 2002; Accepted August 16, 2002
The extracellular β-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days′ cultivation in an induction medium containing 1％ (v/v) methanol, the expression level of the recombinant enzyme was 28,500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-β-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-β-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.
Key words: β-glucosidase; glycoside hydrolase; Pichia pastoris; Phanerochaete chrysosporium; heterologous expression

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Extracellular Acidic Polysaccharide Production by a Two-membered Bacterial Coculture

Shinya KURATA,1,2 Kazutaka YAMADA,1 Kyoko TAKATSU,1 Satoshi HANADA,3 Osamu KOYAMA,1 Toyokazu YOKOMAKU,1 Yoichi KAMAGATA,3 Takahiro KANAGAWA,2,3, and Ryuichiro KURANE3

1Kankyo Engineering Corporation, 1 Kimitsu, Kimitsu-shi, Chiba 299-1141, Japan 2Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan 3Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

Received March 22, 2002; Accepted September 20, 2002
A two-membered coculture of strains KYM-7 and KYM-8, identified as Cellulomonas cellulans and Agrobacterium tumefaciens, respectively, produced a large amount of an extracellular polysaccharide, designated APK-78, from starch. Each strain in pure culture produced only very little amount of polysaccharide from starch; the coexistence of the two strains from the early stage of cultivation was indispensable for a large amount of polysaccharide to be produced. The polysaccharide APK-78 was acidic and composed of glucose, galactose, succinic acid, and pyruvic acid with a molar ratio of 8.1:1.0:1.7:1.0, indicating that it is a succinoglycan type of polysaccharide.
Key words: coculture; extracellular polysaccharide; succinoglycan; Cellulomonas; Agrobacterium

-3-
Effect of Phytate in Soy Protein on the Serum and Liver Cholesterol Levels and Liver Fatty Acid Profile in Rats

Kazunori KOBA,1, Jim-Wen LIU,2 Emil BOBIK, Jr.,2 David E. MILLS,3 Michihiro SUGANO,4 and Yung-Sheng HUANG2

1Siebold University of Nagasaki, 1-1-1 Manabino, Nagayo, Nagasaki 851-2195, Japan 2Ross Products Division, Abbott Laboratories, 625 Cleveland Av., Columbus, OH 43215-1724, USA 3Scientific Laboratory Division, Department of Health of the State of New Mexico, Albuquerque, NM 87196-4700, USA 4Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan

Received April 15, 2002; Accepted September 4, 2002
Dietary soy protein, in comparison with casein, generally lowers the serum cholesterol concentration in rats fed on a cholesterol-enriched diet, while mixed results were observed in rats fed on a diet free of cholesterol. Soy protein also suppresses the conversion of linoleic acid to arachidonic acid in the rat liver. The present study examines whether phytate, a minor component of a soy protein isolate, is responsible for these beneficial effects of soy protein. Weanling male rats were fed for 4 weeks on a purified diet containing a 20％ level of protein (either casein (CAS), soy protein (SOY), phytate-depleted SOY (PDSOY) or phytate-replenished PDSOY (PRSOY)) and cholesterol (0 or 0.5％). The dietary protein source and phytate level only affected the serum and liver cholesterol concentrations when the animals were fed on the cholesterol-enriched diet, being significantly lower in those rats fed on the SOY and PRSOY diets than in those fed on the CAS diet, while the concentrations in the rats fed on the PDSOY diet were intermediate. When the animals were fed on the cholesterol-free diet, the ratio of (20:3n-6＋20:4n-6)/18:2n-6 in liver phosphatidylcholine, a Δ6 desaturation index, was significantly lower in the SOY diet group than in the CAS, PDSOY and PRSOY diet groups. Dietary cholesterol significantly depressed the ratio, but neither depletion nor replenishment of phytate affected the ratio. These results suggest that phytate in soy protein played a limited role in the cholesterol-lowering effect of soy protein and was not involved in the metabolism of linoleic acid.
Key words: phytate; soy protein; serum cholesterol; linoleic acid metabolism; rat

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Regulation of the Peripheral Body Temperature by Foods: A Temperature Decrease Induced by the Japanese Persimmon (kaki, Diospyros kaki)

Gaku HIBINO,1, Tomonori NADAMOTO,2, Fumiko FUJISAWA,2 and Tohru FUSHIKI1

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 2School of Human Cultures, The University of Shiga Prefecture, Shiga 522-8533, Japan

Received April 23, 2002; Accepted August 24, 2002
We investigated whether the ingestion of the Japanese persimmon (kaki, Diospyros kaki) could lower the human peripheral body temperature. It was found that the temperatures recorded at the foot and wrist were depressed after kaki consumption compared to after the same amount of water consumption. The effects of ingesting freeze-dried kaki and eating a cookie (as its nutritional counterpart) containing the same amount of carbohydrate, protein, fat, and water were compared. A similar temperature-reducing effect of kaki was observed. The recovery of finger temperature after soaking the finger in ice-cooled water was also studied. The temperature recovery was delayed after kaki consumption. It was thus quantitatively demonstrated that ingesting kaki indeed had the effect of lowering (or repressing the rise) of the peripheral human body temperature, as has been traditionally believed in China for many hundreds of years.
Key words: persimmon; peripheral body temperature; autonomic nerve; Chinese traditional medicine; human

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Purification and Characterization of a New Type of α-Glucosidase from Paecilomyces lilacinus That Has Transglucosylation Activity to Produce α-1,3- and α-1,2-Linked Oligosaccharides

Isao KOBAYASHI,1, Masahiro TOKUDA,1 Hiroyuki HASHIMOTO,2 Takashi KONDA,3 Hirofumi NAKANO,4 and Sumio KITAHATA4

1Honen Corporation, 1-2-3 Ohtemachi, Chiyoda-ku, Tokyo 100-8150, Japan 2Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan 3Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan 4Osaka Municipal Technical Research Institute, 6-50 Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan

Received April 26, 2002; Accepted August 26, 2002
A fungus producing an α-glucosidase that synthesizes α-1,3- and α-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65°C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitrophenyl α-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing α-1,3- and α-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-α-nigerosyl-D-glucose, and 4-O-α-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.
Key words: α-glucosidase; Paecilomyces lilacinus; kojibiose; nigerose; transglucosylation

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Purification and Characterization of meta-Cleavage Compound Hydrolase from a Carbazole Degrader Pseudomonas resinovorans Strain CA10

Hideaki NOJIRI,1 Hiroko TAIRA,1 Kenichi IWATA,1 Kenichi MORII,1 Jeong-Won NAM,1 Takako YOSHIDA,1 Hiroshi HABE,1 Shugo NAKAMURA,2 Kentaro SHIMIZU,2 Hisakazu YAMANE,1 and Toshio OMORI1,

1Biotechnology Research Center and 2Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received May 14, 2002; Accepted September 24, 2002
2-Hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-
dienoic acid [6-(2′-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0--7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 μM and kcat of 2.14 (s−1) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25°C). The effect of the presence of an amino group or hydroxyl group at the 2′-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2′-aminophenyl)-HODA (2.44 U/mg)＞6-phenyl-HODA (1.99 U/mg)＞2-hydroxy-6-oxo-6-(2′-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2′-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.
Key words: meta-cleavage compound hydrolase; carbazole; dibenzofuran; Pseudomonas resinovorans strain CA10

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Effect of Medium-chain Triglycerides on the Postprandial Triglyceride Concentration in Healthy Men

Michio KASAI,1, Hideaki MAKI,1 Naohisa NOSAKA,1 Toshiaki AOYAMA,1 Katsuhiko OOYAMA,1 Harumi UTO,2 Mitsuko OKAZAKI,3 Osamu IGARASHI,4 and Kazuo KONDO2

1Division of Healthcare Science Research Laboratory, Nisshin Oil Mills LTD., 1 Shinmei-cho, Yokosuka, Kanagawa 239-0832, Japan 2Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan 3Kagawa Nutrition University, 3-9-21 Chiyoda, Sakado, Saitama 350-0288, Japan 4Ibaraki Christian University, 6-11-1 Oomika-cho, Hitachi, Ibaraki 319-1295, Japan

Received June 3, 2002; Accepted September 9, 2002
This study compared the serum lipid concentrations after a single dose of medium-chain triglycerides (MCT) or long-chain triglycerides (LCT) between individuals grouped according to the body mass index (BMI). Twenty-five males participated as volunteers, the test diet containing 10 g of MCT or LCT. Blood samples were collected up to 6 h after the intake of a test diets. The LCT diet resulted in significantly greater increases in areas under the curves (AUCs) for serum and chylomicron triglyceride in the BMI<10-38>23 kg/m2 group than those in the BMI＜23 kg/m2 group. The magnitude of response after intake of the MCT diet by the BMI<10-38>23 kg/m2 group was significantly lower than that after the LCT diet. These results suggest that, in subjects with BMI<10-38>23 kg/m2, the intake of MCT is preferable to that of LCT for maintaining postprandial triglyceride at a low concentration.
Key words: medium-chain triglycerides; long-chain triglycerides; body mass index; triglyceride; chylomicron

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Antioxidative Activity of the Blue Pigment Formed in a D-Xylose-Glycine Reaction System

Satomi SHIZUUCHI and Fumitaka HAYASE

Department of Agricultural Chemistry, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan

Received June 4, 2002; Accepted August 23, 2002
A blue compound was prepared from 1 M D-xylose and 0.1 M glycine, and designated Blue-M1, an intermediate color product of melanoidins. As melanoidins are well known to have antioxidative activity as well as high scavenging activity against active oxygen species, the antioxidative activity of Blue-M1 against the peroxidation of linoleic acid was investigated, in addition to the scavenging activity of Blue-M1 toward hydroxyl and DPPH radicals. Blue-M1 suppressed the peroxidation of linoleic acid as effectively as melanoidins did. The scavenging activity of Blue-M1 toward hydroxyl and DPPH radicals was also as strong as that of melanoidins. Blue-M1 showed higher activity with increasing concentration. The pyrrolopyrrole ring and a methine bridge between two pyrrolopyrrole rings in Blue-M1 could be related to the ability for radical scavenging activity, but not four carboxyl groups.
Key words: melanoidin; Maillard reaction; blue pigment; antioxidative activity; scavenging activity of hydroxyl and DPPH radicals

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Structural Characterization of Triacylglycerol in Several Oils Containing Gamma-linolenic Acid

Satoru SHIMIZU1,2 and Masuo NAKANO3,

1The United Graduate School of Agriculture Sciences, Iwate University, Morioka, Iwate 020-8550, Japan 2Total Science Institute, Zukohsha Co. Ltd., Obihiro, Hokkaido 080-0048, Japan 3Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

Received June 17, 2002; Accepted September 10, 2002
The differences are reported in the triacylglycerol (TG) structures of oils containing gamma-linolenic acid (GLA) from Oenothera biennis Linn seed oil (OBLO) from the wild plant, evening primrose seed oil (EPO) from a cultured plant, and bio-GLA oil (BIO) from a mold, the physiological functions of which were ascertained by animal testing. Reverse-phase high-performance liquid chromatographic separation detected 12 TG peaks each for OBLO and EPO, and 28 TG peaks for BIO. TG-containing GLA were composed of five molecular species each in OBLO and EPO, and ten molecular species in BIO. The totals of the molecular species containing GLA were 29.8％ in OBLO, 23.8％ in EPO, and 56.6％ in BIO. In OBLO, the GLA level at the sn-2 position of the major TG species was higher than that in EPO. In BIO, the GLA level at the sn-2 position of the major TG species was lower than those in OBLO and EPO.
Key words: gamma-linolenic acid; Oenothera biennis Linn; triacylglycerol; molecular species

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β-Galactosidase and Its Significance in Ripening of <Oh><0192><0192><Wa>Saijyo′′ Japanese Persimmon Fruit

Akihiro NAKAMURA,1, Hirokazu MAEDA,1 Misae MIZUNO,2 Yoko KOSHI,2 and Yasunori NAGAMATSU2

1Tsukuba R＆D Center, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun, Ibaraki 300-2497, Japan 2Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan

Received June 19, 2002; Accepted September 14, 2002
The fruit extracts of ripening cv. Japanese Persimmon, ''Saijyo′′, contained a number of glycosidases and glycanases. Among them, β-galactosidase appeared to be the most significant, and the activity increased in parallel with tissue ripening. Persimmon β-galactosidase was presented in at least three isoforms, β-galactosidase-I (pI＝4.88), β-galactosidase-II (pI＝6.76), and β-galactosidase-III (pI＝7.05). β-Galactosidase-III had exo-type galactanase activity, while the others did not. The activity of endo-type glycanases was a maximum in immature green or yellow fruits. The firmness of the pulp tissue decreased dramatically, and the amount of water-soluble polysaccharide (WSS) increased. The enzyme activities of exo-type glycosidases, especially β-galactosidase, appeared maximal in mature red fruits. The amount of extractable pectin remained unchanged, although the galactose content of the high-molecular-weight fraction in WSS decreased dramatically. These results suggest that the ripening of persimmon was caused by the solubilization of pectic polysaccharide by endo-type glycanases and digestion by exo-type glycosidases. β-Galactosidase, in particular, seemed to play a major role in ripening the fruit.
Key words: β-galactosidase; isozyme; persimmon; pectin; ripening

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Identification and Characterization of Scp15, a Protein from Streptomyces coelicolor A3(2) Inducing Neurites in PC12 Cells

Satoru NAKASHIMA, Shuji WAKATSUKI, Tatsuya YOKOYAMA, Manabu ARIOKA, and Katsuhiko KITAMOTO

Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received June 28, 2002; Accepted August 5, 2002
We previously showed that a fungal protein, p15, induces neurite outgrowth and differentiation of rat pheochromocytoma PC12 cells. We report here the identification and characterization of a protein similar to p15, found in Streptomyces coelicolor A3(2). This hypothetical protein, tentatively named Scp15, has significant similarity with p15, including conserved positions of four cysteine residues involved in the formation of essential disulfide bonds in p15. Hexahistidine-tagged recombinant Scp15 proteins were produced in Escherichia coli, purified, and analyzed for their neurite-inducing activity. Although they were less active than p15, they dose-dependently induced neurites and the expression of neurofilament M. Neurite outgrowth by Scp15 was inhibited by nicardipine, suggesting that Scp15 induces neurites via activation of a calcium signaling pathway.
Key words: PC12; neurite outgrowth; differentiation; L-type Ca2＋ channel; Streptomyces coelicolor A3(2)

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Novel Aminopeptidase Specific for Glycine from Actinomucor elegans

Kiyoshi ITO,1 Xiaohang MA,1, Nik AZMI,1 Hua-Shan HUANG,1 Mikio FUJII,2 and Tadashi YOSHIMOTO1,

1School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan 2Foods Technology Laboratory, Japan Tobacco Inc., 443-1 Shirayamado, Ohito-cho, Tagata-gun, Shizuoka 410-2318, Japan

Received July 2, 2002; Accepted August 31, 2002
Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3％ of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly≫Ala≫Met＞Arg＞Ser＞Leu. The Km value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40°C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.
Key words: glycine; aminopeptidase; glycyl aminopeptidase; Actinomuco; peptidase

-13-
Effect of Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Susceptibility to Listeria Infection

Yoshiko SUGITA-KONISHI,1, Kazuo KOBAYASHI,1 Hiroyuki NAITO,2 Katuhiro MIURA,3 and Yoshihiko SUZUKI2

1Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 2Department of Veterinary Science, Azabu University, 1-17-1 Fuchinobe, Sagamihara-shi, Kanagawa 229-0006, Japan 3Department of Safety Research, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba-shi, Ibaraki 305-0856, Japan

Received July 3, 2002; Accepted September 18, 2002
We studied the effect of lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the susceptibility to Listeria infection of offspring in C57BL/6NCji mice. The offspring were nursed by TCDD-treated dams and exposed to TCDD from birth to weaning via milk. The exposure had little effect on the weights of immune organs and the spleen or the thymus cell population in the dams and offspring, but it enhanced the production of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in the serum after Listeria infection. The clearance of Listeria monocytogenes from the spleen was impaired in the offspring. These results suggest that the exposure to TCDD of the offspring via milk disrupted the host resistance of the offspring, even though the main immune parameters were unchanged.
Key words: TCDD; lactation; Listeria monocytogenes; drinking water; suckling mice

-14-
Binding Properties of Bacillus thuringiensis Cry1C δ-Endotoxin to the Midgut Epithelial Membranes of Culex pipiens

Shinya KAMAUCHI,1 Masashi YAMAGIWA,1 Motoyuki ESAKI,2 Kanao OTAKE,2 and Hiroshi SAKAI1,

1Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan 2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received July 4, 2002; Accepted October 4, 2002
The Cry1C δ-endotoxin from Bacillus thuringiensis is toxic to both lepidopteran and dipteran insect larvae. To analyze the dipteran-specific insecticidal mechanisms, we investigated the properties of Cry1C binding to the epithelial cell membrane of the larval midgut from the mosquito Culex pipiens in comparison with dipteran-specific Cry4A. Immunohistochemical staining of the larval midgut sections from Culex pipiens showed that Cry1C and Cry4A bound to the microvilli of the epithelial cells. The Cry1C binding to brush border membrane vesicles from the mosquito larvae was specific and irreversible, and did not compete with Cry4A. By ligand blotting analyses, we detected several Cry1C-binding proteins, the Cry1C binding to which did compete with excess unlabeled Cry4A. These results suggested that Cry1C and Cry4A recognized the same binding site(s) on the epithelial cell surface but that their interaction with the target membrane differed.
Key words: Bacillus thuringiensis; Cry toxin; Culex pipiens; midgut; membrane binding

-15-
Interactions of Heterologous Mycelia Colonized in the Substrate Govern Fruit Body Production in the Cultivated Homobasidiomycete Pholiota nameko

Katsuhiko BABASAKI,1 Kazuhiko MASUNO,2 and Hitoshi MURATA1,

1Department of Applied Microbiology and Mushroom Sciences, Forestry ＆ Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan 2Department of Mushroom Science, Nagano Forest Research Center, Shiojiri Kataoka, Nagano 399-0711, Japan

Received July 5, 2002; Accepted September 25, 2002
The spawn of cultivated mushrooms are generally produced, propagated, and distributed to growers as a mycelial culture without genetic purification, in which phenotypic variants frequently occur. We investigated how heterologous mycelia present in a spawn influence fruit body production in the cultivated basidiomycete Pholiota nameko. The <Oh><0192><Wa>di-mon′ dual cultivation of protoplast clones produced mosaic fruit bodies, which could result from the <Oh><0192><Wa>di-mon′ mating. In the <Oh><0192><Wa>di-di′ dual cultivation of heterologous strains with different fruiting times, authentic fruit bodies of each dikaryon and chimera showing a feature combining characteristics of the two dikaryons emerged simultaneously. Mycelia isolated from the chimera produced all three types of fruit bodies, indicating unlikeliness of the occurrence of anastomosis. These results suggest that mycelia colonized in the substrate interact with each other and coordinately promote fruit body production in P. nameko. This phenomenon masks a clonal variability that may be surfaced through multiplication and distribution of the spawn, occasionally bringing about abnormal fruiting.
Key words: basidiomycetes; cell-to-cell communication; fruit body production; Pholiota nameko

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Specific RNA Interference in psbP Genes Encoded by a Multigene Family in Nicotiana tabacum with a Short 3′-Untranslated Sequence

Kentaro IFUKU, Yumiko YAMAMOTO, and Fumihiko SATO

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received July 12, 2002; Accepted September 10, 2002
RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3′-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3′-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3′-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3′-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.
Key words: gene family; Nicotiana tabacum; psbP; RNA interference; small interfering RNA

-17-
Purification and Characterization of NADP-Linked Isocitrate Dehydrogenase from the Copper-tolerant Wood-rotting Basidiomycete Fomitopsis palustris

Jeong-Jun YOON, Takefumi HATTORI, and Mikio SHIMADA

Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan

Received July 18, 2002; Accepted September 3, 2002
NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), a key enzyme of the tricarboxylic acid cycle, was purified 672-fold as a nearly homogeneous protein from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris. The purified enzyme, with a molecular mass of 115 kDa, consisted of two 55-kDa subunits, and had the Km of 12.7, 2.9, and 23.9 μM for isocitrate, NADP, and Mg2＋, respectively, at the optimal pH of 9.0. The enzyme had maximum activity in the presence of Mg2＋, which also helped to prevent enzyme inactivation during the purification procedures and storage. The enzyme activity was competitively inhibited by 2-oxoglutarate (Ki, 127.0 μM). Although adenine nucleotides and other compounds, including some of the metabolic intermediates of glyoxylate and tricarboxylic acid cycles, had no or only slight inhibition, a mixture of oxaloacetate and glyoxylate potently inhibited the enzyme activity and the inhibition pattern was a mixed type.
Key words: NADP-linked isocitrate dehydrogenase; enzyme purification; enzyme inhibition; tricarboxylic acid cycle; wood-rotting basidiomycete Fomitopsis palustris

-18-
Need for Cytochrome bc1 Complex for Dissimilatory Nitrite Reduction of Pseudomonas aeruginosa

Norio HASEGAWA, Hiroyuki ARAI, and Yasuo IGARASHI

Department of Biotechnology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

Received July 19, 2002; Accepted September 19, 2002
Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.
Key words: Pseudomonas aeruginosa; electron transport; cytochrome bc1; cytochrome c551; azurin

-19-
Characterization of the Biosynthetic Gene Cluster of Rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243

Hiroyasu ONAKA, Shin-ichi TANIGUCHI, Yasuhiro IGARASHI, and Tamotsu FURUMAI

Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan

Received July 29, 2002; Accepted August 19, 2002
The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11′-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11′-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.
Key words: rebeccamycin; indolocarbazole; biosynthetic pathway; Lechevarielia aerocolonigenes ATCC 39243; chromopyrrolic acid

-20-
Mepanipyrim, a Novel Inhibitor of Pharmacologically Induced Golgi Dispersion

Michiko NAKAMURA,1,2 Yoshiki KONO,2 and Akira TAKATSUKI1,

1Animal and Cellular Systems Laboratory, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan 2School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0332, Japan

Received August 7, 2002; Accepted September 10, 2002
Mepanipyrim inhibited retrograde Golgi-to-ER trafficking induced by brefeldin A (BFA), nordihydroguaiaretic acid, clofibrate, and arachidonyltrifluoromethyl ketone in NRK and other types of cells, but did not inhibit anterograde trafficking of Golgi-resident proteins translocated to ER by BFA and newly synthesized VSV-G. However, mepanipyrim did not block the TGN38 dispersion induced by any of these compounds. Mepanipyrim acted on the Golgi, and swollen vesicular Golgi structures were formed and similar structures accumulated during rebuilding of the Golgi after BFA removal. These actions of mepanipyrim were readily reversed after its removal. Mepanipyrim did not stabilize microtubules, but prevented nocodazole-induced fragmentation and dispersion of the Golgi. These results suggest that the mepanipyrim-sensitive molecules participated in stabilizing the Golgi and its anchoring in the perinuclear region, and equally importantly, that the novel action of mepanipyrim may be used as a pharmacological tool for investigating membrane transport, Golgi membrane dynamics, and differentiation of the Golgi from TGN.
Key words: mepanipyrim; Golgi membrane dynamics; retrograde trafficking; microtubule; brefeldin A

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Nordihydroguaiaretic Acid, of a New Family of Microtubule-stabilizing Agents, Shows Effects Differentiated from Paclitaxel

Machiko NAKAMURA,1,3 Junko NAKAZAWA,2 Takeo USUI,2 Hiroyuki OSADA,2 Yoshiki KONO,3 and Akira TAKATSUKI1,

1Animal and Cellular Systems Laboratory and 2Antibiotic Laboratory, RIKEN (Institute of Physical and Chemical Research), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan 3School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0332, Japan

Received August 27, 2002; Accepted October 7, 2002
Nordihydroguaiaretic acid (NDGA) protected microtubules in NRK cells from depolymerization caused by structurally and functionally diverse drugs such as nocodazole, colchicine, vinblastine, and ilimaquinone. Hitherto reported drugs, although structurally unrelated to paclitaxel, stabilize microtubules in a way similar to that of paclitaxel and compete for paclitaxel binding to tubulin. However, NDGA had activity toward microtubules different from the effects of paclitaxel. In NRK cells, paclitaxel caused microtubule bundle formation in the presence and absence of microtubule-depolymerizing drugs. However, microtubule bundle did not form, and microtubules radiated from the microtubule-organizing center, in cells treated with NDGA. Acceleration of tubulin polymerization in vitro by paclitaxel was strong but that by NDGA was weak. Microtubules polymerized in vitro in the presence of paclitaxel, but not those polymerized in the presence of NDGA, resisted the effects of cold. NDGA seemed to bind to tubulin, but did not compete for [3H]paclitaxel binding to tubulin. These observations indicate that NDGA belongs to a novel family of microtubule-stabilizing drugs.
Key words: nordihydroguaiaretic acid; microtubule stabilization; paclitaxel; microtubule-organizing center; microtubule depolymerization

-22-
Note
Nuclear Localization of Senescence Marker Protein-30, SMP30, in Cultured Mouse Hepatocytes and Its Similarity to RNA Polymerase

Akihito ISHIGAMI,2 Setsuko HANDA,2 Naoki MARUYAMA,2 and Prakash C. SUPAKAR1,

1Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar-751 023, India 2Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan

Received April 1, 2002; Accepted August 16, 2002
Senescence marker protein-30 (SMP30), expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity. By immunohistochemistry and western blotting, we found that SMP30 was in both the nuclei and cytoplasm of cultured mouse hepatocytes. By a homology search, we found that a domain of the SMP30 sequence 51 amino acid residues long was 60--66％ similar to bacterial and yeast RNA polymerases.
Key words: SMP30; nuclear localization

-23-
Note
Selective Accumulation of Delphinidin Derivatives in Tobacco Using a Putative Flavonoid 3′,5′-Hydroxylase cDNA from Campanula medium

Yasushi OKINAKA,, Yukihisa SHIMADA, Ritsuko NAKANO-SHIMADA, Masaya OHBAYASHI, Shigeto KIYOKAWA, and Yasuhiro KIKUCHI

Tsukuba Research Laboratories, Kyowa Hakko Co., Ltd., Tsukuba 305, Japan

Received May 10, 2002; Accepted August 20, 2002
Blue flowers generally contain 3′,5′-hydroxylated anthocyanins (delphinidin derivatives) as pigments, which are formed only in the presence of flavonoid 3′,5′-hydroxylases (F3′5′H). Heterologous expression of a F3′5′H gene therefore provides an opportunity to produce novel blue flowers for a number of ornamental plants missing blue flowering varieties. However, our previous study indicated difficulties in obtaining good accumulation of delphinidin derivatives in plants expressing F3′5′H. Here we report the isolation of a putative F3′5′H cDNA (Ka1) from canterbury bells (Campanula medium) and its expression in tobacco. Surprisingly, compared with other F3′5′H cDNAs, Ka1 encoded a protein with a unique primary structure that conferred high competence in the accumulation of delphinidin derivatives (up to 99％ of total anthocyanins) and produced novel purple flowers. These results suggest that, among F3′5′H cDNAs, Ka1 is the best genetic resource for the creation of fine blue flowers by genetic engineering.
Key words: anthocyanin biosynthesis; flavonoid 3′,5′-hydroxylases; cytochrome P450; flower color; plant breeding

-24-
Note
Adjuvant Activity of Alum in Inducing Antigen Specific IgE Antibodies in BALB/c Mice: a Reevaluation

Rintaro YAMANISHI, Ikuko YUSA, Noriko BANDO, and Junji TERAO

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

Received May 13, 2002; Accepted September 9, 2002
The IgE production was compared in the presence and absence of aluminum hydroxide gel (alum). Without alum, the IgE production was induced within a suitable range of the antigen dosage; however, alum enhanced it. Alum did not affect the minimum requirement for the antigen dosage, indicating that alum may not take part in the efficiency of antigen presentation.
Key words: adjuvant; alum; antigen dosage; IgE

-25-
Note
Stimulation by Caffeic Acid, Coumalic Acid, and Corilagin of the Germination of Resting Spores of the Clubroot Pathogen Plasmodiophora brassicae

Michio OHI,1, Takashi KITAMURA,1 and Shuichi HATA2

1Faculty of Agriculture, Shinshu University, Minamiminowa-mura 8304, Nagano Prefecture 399-4598, Japan 2Tama Biochemical Co., Ltd., Gohdo 450, Isehara, Kanagawa Prefecture 259-1138, Japan

Received June 13, 2002; Accepted August 30, 2002
Some chemicals were examined for their effects on the germination of resting spores of the clubroot pathogen Plasmodiophora brassicae, and on the control of clubroots in Chinese cabbage. Caffeic acid, coumalic acid, and corilagin stimulated the germination of Plasmodiophora spores and prevented the formation of clubroots in Chinese cabbage. Clubroot might be controlled by agents with germination-stimulating effects.
Key words: germination; Plasmodiophora brassicae; caffeic acid; coumalic acid; corilagin

-26-
Note
Primary Structure of Cytochrome c Gene from the White Root Rot Fungus Rosellinia necatrix

Tadanori AIMI, Hiroyuki TAGUCHI, and Tsutomu MORINAGA

Department of Bioresource Development, Hiroshima Prefectural University, Nanatsuka-cho 562, Shobara-shi, Hiroshima 727-0023, Japan

Received June 14, 2002; Accepted August 9, 2002
The nucleotide sequence of the cytochrome c (CytC) gene of the white root rot fungus Rosellinia necatrix was analyzed. The structure of this gene, which had three introns in the coding region, was similar to that of Aspergillus nidulans. The second intron of the R. necatrix CytC gene was not present in Neurospora crassa or Fusarium oxysporum. However, the amino acid sequence of R. necatrix was most similar to that of Neurospora crassa. Thus, it seemed that the second intron of the R. necatrix CytC gene was inserted into its present position after R. necatrix and its closest relatives diverged evolutionarily.
Key words: mitochondria; Rosellinia; intron; PCR; cytochrome c

-27-
Note
Structural Analysis of Conjugated Linoleic Acid Produced by Lactobacillus plantarum, and Factors Affecting Isomer Production

Shigenobu KISHINO,1 Jun OGAWA,1 Akinori ANDO,1 Takashi IWASHITA,2 Tsuyoshi FUJITA,2 Hiroshi KAWASHIMA,3 and Sakayu SHIMIZU1,

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 2Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan 3Institute for Fundamental Research, Suntory Ltd., Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan

Received June 18, 2002; Accepted September 26, 2002
An isomer of the conjugated linoleic acid (CLA) produced from linoleic acid by Lactobacillus plantarum was identified as cis-9,trans-11-octadecadienoic acid by proton nuclear magnetic resonance spectroscopy. Together with earlier results, we concluded that the bacterium produces two CLA isomers, cis-9,trans-11- and trans-9,trans-11-octadecadienoic acid from linoleic acid. The addition of L-serine, glucose, AgNO3, or NaCl to the reaction mixture reduced production of the latter.

-28-
Note
A Novel Cellulolytic, Anaerobic, and Thermophilic Bacterium, Moorella sp. Strain F21

Shuichi KARITA,1, Kengo NAKAYAMA,1 Masakazu GOTO,1 Kazuo SAKKA,1 Wan-Jae KIM,1 and Satoru OGAWA2

1Department of Sustainable Resource Science, Faculty of Bioresources, and 2Laboratory of Electron Microscope, School of Medicine, Mie University, Tsu 514-8507, Japan

Received June 24, 2002; Accepted September 9, 2002
A cellulolytic and thermophilic anaerobe was isolated from soil. This bacterium made a halo on a roll-tube culture containing Avicel. Analysis of the PCR-based 16S rRNA gene sequence showed that the bacterium was closely related to Moorella thermoacetica. Scanning electron microscopy showed the bacterium is a rod and has no protuberant structure on the surface of cells growing on cellulose, suggesting that this strain is a non-cellulosomal cellulolytic bacterium. Carboxymethyl cellulase and xylanase activities were detected in the culture broth. A major fermentation product from ball-milled cellulose was acetate. This strain has a potential to convert cellulosic biomass to acetate, directly.
Key words: cellulase; xylanase; Moorella sp.; anaerobic thermophile

-29-
Note
Putative Homologs of SSK22 MAPKK Kinase and PBS2 MAPK Kinase of Saccharomyces cerevisiae Encoded by os-4 and os-5 Genes for Osmotic Sensitivity and Fungicide Resistance in Neurospora crassa

Makoto FUJIMURA,1, Noriyuki OCHIAI,2 Michiyo OSHIMA,1 Takayuki MOTOYAMA,3 Akihiko ICHIISHI,1 Ron USAMI,2 Koki HORIKOSHI,1 and Isamu YAMAGUCHI3

1Faculty of Life Science, University of Toyo, Itakura, Oura-gun 374-0193, Japan 2Department of Engineering, University of Toyo, Kujirai, Kawagoe 350-8585, Japan 3Plant Science Center, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan

Received June 27, 2002; Accepted August 8, 2002
We cloned and characterized Neurospora NcSSK22 and NcPBS2 genes, similar to yeast SSK22 mitogen-activated protein (MAP) kinase kinase kinase and the PBS2 MAP kinase kinase genes, respectively. Disruptants of the NcSSK22 gene were sensitive to osmotic stress and resistant to iprodione and fludioxonil. Their phenotypes were similar to those of osmotic-sensitive (os) mutants os-1, os-2, os-4, and os-5. The os-4 mutant strain transformed with the wild-type NcSSK22 gene grew on a medium containing 4％ NaCl and was sensitive to iprodione and fludioxonil. In contrast, the NcPBS2 gene complemented the osmotic sensitivity and fungicide resistance of the os-5 mutant strain. We sequenced the NcPBS2 gene of the os-5 mutant strain (NM216o) and found five nucleotides deleted within the kinase domain. This result suggests that the gene products of os-4 and os-5 are components of the MAP kinase cascade, which is probably regulated upstream by two-component histidine kinase encoded by the os-1/nik1 gene.
Key words: fungicide; osmotic stress; two-component signal transduction; histidine kinase; mitogen-activated protein kinases

-30-
Note
Biocatalytic Deprotection of a Cetraxate Ester by Microbacterium sp. Strain 7-1W Cells

Kohsuke HONDA, Keiji SAKAMOTO, Shinji KITA, Michihiko KATAOKA, and Sakayu SHIMIZU

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

Received July 2, 2002; Accepted September 9, 2002
Enzymatic deprotection of the terminal ester bond of a cetraxate methyl ester was done with resting cells of Microbacterium sp. strain 7-1W, which produces an esterase catalyzing a regioselective hydrolysis reaction, as the catalyst. When 20 g of cetraxate methyl ester in 50 ml of a reaction mixture was incubated with 5 g of wet cells for 17 h, 96％ of the substrate was converted to the desired product, cetraxate, quantitatively.
Key words: esterase; cetraxate; regioselective hydrolysis; Microbacterium

-31-
Note
Antioxidative Effect of Citrus Essential Oil Components on Human Low-density Lipoprotein In Vitro

Yasuo TAKAHASHI, Nobuya INABA, Shigeru KUWAHARA, and Wataru KUKI

Wakayama Agricultural Processing Research Corporation, 398 Tsukatsuki, Momoyama-cho, Naga-gun, Wakayama 649-6112, Japan

Received July 3, 2002; Accepted September 7, 2002
We studied the antioxidative action to evaluate the effect of citrus essential oil components on human LDL in vitro. Among the authentic volatile compounds tested, γ-terpinene showed the strongest antioxidative effect, and inhibited both the Cu2＋-induced and AAPH-induced oxidation of LDL. γ-Terpinene added after 30 min (mid-lag phase) and 60 min (propagation phase) of incubation of LDL with Cu2＋ inhibited LDL oxidation.
Key words: antioxidative effect; low-density lipoprotein; γ-terpinene

-32-
Note
Efficient Release of Overproduced Gene Products from Escherichia coli BL21(DE3) by Lytic Infection with Newly Isolated Bacteriophages

Yuichiro IIDA,1 Yoshinori MATSUDA,1, Ryuichiro SAITO,1 Masanori NAKASATO,1 Teruo NONOMURA,1 Koji KAKUTANI,2 Yukio TOSA,3 Shigeyuki MAYAMA,3 and Hideyoshi TOYODA1

1Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University, Nara 631-8505, Japan 2Pharmaceutical Research and Technology Institute, Kinki University, Higashi-Osaka 577-8502, Japan 3Laboratory of Plant Pathology, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan

Received July 8, 2002; Accepted September 20, 2002
Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with β-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.
Key words: β-glucosidase; chitinase; chitosanase; chiSH1; csnSM1

-33-
Note
Catalytic Properties of Lipases Immobilized on Various Mesoporous Silicates

Katsuya KATO,1, Roxana IRIMESCU,1 Takao SAITO,1 Yoshiyuki YOKOGAWA,1 and Haruo TAKAHASHI2

1Bio-functional Ceramics Group, Ceramics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan 2Toyota Central R ＆ D Lab. Inc., Nagakute, Aichi 480-1192, Japan

Received July 30, 2002; Accepted September 18, 2002
Lipases SP525, AK, LIP, and PS were immobilized on three kinds of mesoporous silicates (FMS, PESO, and SBA) with diameters of 27 to 92 <Oh><0197><Wa>. The amount of lipase activity adsorbed on these supports was related to the pore size of the silicate. Enantioselectivities of immobilized lipases were similar to those of free lipases, and recycling could be done in both aqueous and organic solvents.
Key words: mesoporous silica; immobilization; lipase; enantioselective acetylation; secondary alcohol

-34-
Note
Differential Scanning Calorimetry of the Effects of Ca2＋ on the Thermal Unfolding of Pseudomonas cepacia Lipase

Akiyoshi TANAKA,1, Hayuki SUGIMOTO,1 Yuko MUTA,2 Takafumi MIZUNO,1 Keishi SENOO,3 Hitoshi OBATA,1 and Kuniyo INOUYE2

1Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan 2Graduate School of Agriculture, Kyoto University, Kyoto, Kyoto 606-8502, Japan 3Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan

Received August 9, 2002; Accepted September 27, 2002
Thermal unfolding of P. cepacia lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme. The temperature of unfolding was higher at higher concentrations of Ca2＋. From the Ca2＋ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one. These results confirmed the validity of the unfolding mechanism proposed previously: NCa2＋<Oh><0248><Wa>D＋Ca2＋, where N and D represent the native and denatured states, respectively, of the enzyme.
Key words: calcium ion; differential scanning calorimetry; lipase; thermal unfolding

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Note
Construction of a Vector Plasmid for Use in Gluconobacter oxydans

Naoto TONOUCHI, Masakazu SUGIYAMA, and Kenzo YOKOZEKI

AminoScience Laboratories, Ajinomoto. Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan

Received August 26, 2002; Accepted September 20, 2002
A host vector system in Gluconobacter oxydans was constructed. An Acetobacter-Escherichia coli shuttle vector was introduced with the efficiency of 104 transformants/μg of DNA. Next, aiming for a self-cloning vector, we found a cryptic plasmid (which we named pAG5) of 5648 bp in G. oxydans strain IFO 3171, and sequenced the nucleotides. The plasmid seemed to have only one open reading flame (ORF) for a possible replication protein. Shuttle vectors of Gluconobacter-E. coli were constructed with the plasmid pAG5 and an E. coli vector, pUC18.
Key words: Gluconobacter; host-vector system; cryptic plasmid

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Note
Functions of Malonate Decarboxylase Subunits from Pseudomonas putida

Shigeru CHOHNAN, Kayo AKAGI, and Yoshichika TAKAMURA

Department of Bioresource Science, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan

Received August 29, 2002; Accepted October 2, 2002
Malonate decarboxylase from Pseudomonas putida is composed of five subunits, α, β, γ, δ, and ε. Two subunits, δ and ε, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coli formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the α subunit was an acetyl-S-ACP:malonate ACP transferase and that the βg-subunit complex was a malonyl-S-ACP decarboxylase.
Key words: malonate decarboxylase; Pseudomonas putida; subunit function; expression in Escherichia coli; GroEL/ES

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Preliminary Communication
Biodegradation of Bisphenol A by Cultured Cells of Caragana chamlagu

Wen CHAI,1 Hiroshi SAKAMAKI,2 Susumu KITANAKA,3 Michihiko SAITO,4 and C. Akira HORIUCHI1,

1Department of Chemistry, Rikkyo (St. Paul′s) University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan 2College of Science and Technology, Nihon University, 7-24-1 Funabashi-shi, Chiba 274-8501, Japan 3College of Pharmacy, Nihon University, 7-7-1 Funabashi-shi, Chiba 274-8501, Japan 4Food Packaging Lab., National Food Research Institute, 2-1-12 Kannondai, Tsukuba-shi, Ibaraki 305-8642, Japan

Received July 29, 2002; Accepted October 24, 2002
The biological degradation of 2,2-bis(4-hydroxyphenol)propane (1; bisphenol A, BPA), a representative endocrine disruptor, was studied with plant-cultured cells of Caragana chamlagu. An initial BPA concentration of 425 μM in an aqueous solution was degraded by C. chamlagu at 25°C for 2 days in the dark, and two intermediates were then completely dissipated after 10 days.
Key words: bisphenol A; plant-cultured cells; Caragana chamlagu; biological degradation; endocrine disrupter

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Preliminary Communication
Plant Chitinase as a Possible Biocontrol Agent for Use Instead of Chemical Fungicides

Shuji KARASUDA,1 Shuhei TANAKA,1 Hiroshi KAJIHARA,2 Yuji YAMAMOTO,2 and Daizo KOGA1,

1Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan 2Yamaguchi Agricultural Experimental Station, Yamaguchi 753-0214, Japan

Received September 2, 2002; Accepted September 27, 2002
We investigated whether a plant chitinase can be used as a biocontrol agent instead of chemical fungicides by spraying chitinase E (family 19; class IV) from a yam (Dioscorea opposita Thunb) alone or together with β-1,3-glucanase directly onto the surface of a powdery mildew infecting strawberry berries and leaves. Results were observed by eye and with a scanning electron microscope. The powdery mildew infecting the strawberries was degraded, mainly by the chitinase, and the disease did not appear again for more than 2 weeks. These results indicated that this kind of plant chitinase might be safe and biodegradable biocontrol agent for use instead of conventional fungicides.
Key words: biocontrol agent; class IV chitinase; family 19 chitinase; powdery mildew; yam