Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.11 2002)

Molecular Characterization of Sucrose:Sucrose 1-Fructosyltransferase and Sucrose:Fructan 6-Fructosyltransferase Associated with Fructan Accumulation in Winter Wheat during Cold Hardening
Akira KAWAKAMI and Midori YOSHIDA p.2297

Increasing Effect of Nori on the Fecal Excretion of Dioxin by Rats
Kunimasa MORITA and Kazuhiro TOBIISHI p.2306

Main Polyol Dehydrogenase of Gluconobacter suboxydans IFO 3255, Membrane-bound D-Sorbitol Dehydrogenase, That Needs Product of Upstream Gene, sldB, for Activity
Masako SHINJOH, Noribumi TOMIYAMA, Taro MIYAZAKI, and Tatsuo HOSHINO p.2314

Recombinant Agrobacterium AgaE-like Protein with Fructosyl Amino Acid Oxidase Activity
Kozo HIROKAWA and Naoki KAJIYAMA p.2323

Effects of External Factors on the Interaction of Tea Catechins with Lipid Bilayers
Katsuko KAJIYA, Shigenori KUMAZAWA, and Tsutomu NAKAYAMA p.2330

Antimutagenicity of Mono-, Di-, and Tricaffeoylquinic Acid Derivatives Isolated from Sweetpotato (Ipomoea batatas L.) Leaf
Makoto YOSHIMOTO,1, Shoji YAHARA,2 Shigenori OKUNO,1 Md. Shahidul ISLAM,1 Koji ISHIGURO,1 and Osamu YAMAKAWA1 p.2336

Milk Calcium Taken with Cheese Increases Bone Mineral Density and Bone Strength in Growing Rats
Ken KATO,1, Yukihiro TAKADA,2 Hiroaki MATSUYAMA,2 Yoshihiro KAWASAKI,2 Seiichiro AOE,2 Hideo YANO,3 and Yasuhiro TOBA2 p.2342

Purification and Characterization of a Novel Cholesterol Esterase from Pseudomonas aeruginosa, with Its Application to Cleaning Lipid-stained Contact Lenses
Akio SUGIHARA,1, Yuji SHIMADA,1 Atsuo NOMURA,2 Tadamasa TERAI,2 Masaki IMAYASU,3 Yusuke NAGAI,3 Toshihiro NAGAO,1 Yomi WATANABE,1 and Yoshio TOMINAGA1 p.2347

Fusicoccins P and Q, and 3-Epifusicoccins H and Q, New Polar Fusicoccins from Isolate Niigata 2-A of a Peach Fusicoccum Canker Fungus
Takeshi SASSA,1,2, Naoto TAJIMA,2 Mitsuyoshi SATO,1 Ai TAKAHASHI,1 and Nobuo KATO3
p.2356

Cloned Cytosine Deaminase Gene Expression of Bifidobacterium longum and Application to Enzyme/Pro-drug Therapy of Hypoxic Solid Tumors
Toshiyuki NAKAMURA,1, Takayuki SASAKI,1, Minoru FUJIMORI,1, Kazuyuki YAZAWA,1 Yasunobu KANO,2 Jun AMANO,1 and Shun′ichiro TANIGUCHI3 p.2362

Stable Form of Ascorbate Peroxidase from the Red Alga Galdieria partita Similar to Both Chloroplastic and Cytosolic Isoforms of Higher Plants
Sakihito KITAJIMA,1, Masami UEDA,1 Satoshi SANO,1, Chikahiro MIYAKE,2, Takayuki KOHCHI,2 Ken-ichi TOMIZAWA,1 Shigeru SHIGEOKA,3 and Akiho YOKOTA1, p.2367

Bilirubin Dehydrogenase, an Enzyme in Aspergillus ochraceus IB-3 Useful for Diagnostic Measurement of Bilirubin
Jun OGAWA,1 Woro Triarsi SULISTYANINGDYAH,1 Hiromi TANAKA,1 Kenji KANO,2 Tokuji IKEDA,2 and Sakayu SHIMIZU1, p.2376

Equilibrium Dialysis Measurements of the Ca2＋-Binding Properties of Recombinant Radish Vacuolar Ca2＋-Binding Protein Expressed in Escherichia coli
Koji YUASA and Masayoshi MAESHIMA p.2382

Novel Reversible Indole-3-carboxylate Decarboxylase Catalyzing Nonoxidative Decarboxylation
Toyokazu YOSHIDA, Kohei FUJITA, and Toru NAGASAWA p.2388

Identification of the Bile Acid-binding Region in the Soy Glycinin A1aB1b Subunit
Seon-Kang CHOI, Motoyasu ADACHI, and Shigeru UTSUMI p.2395

Epitope Analysis of Antibodies in Japanese to Human Cytomegalovirus Phosphoprotein 150 with Synthetic Peptides
Isao TAKAHASHI, Sayoko SUGIURA, Hirotoshi OHTA, Kazuo OZAWA, and Tadashi KAMIYA
p.2402

Synthesis and Insecticidal Activity of N-Oxydihydropyrroles: 4-Hydroxy-3-mesityl-5,5-dimethyl Derivatives with Various Substituents at the 1-Position
Mitsuru ITO,1, Hideshi OKUI,1 Harumi NAKAGAWA,1 Shigeru MIO,1 Ayako KINOSHITA,1 Takashi OBAYASHI,1 Takako MIURA,1 Junko NAGAI,1 Shinji YOKOI,1 Reiji ICHINOSE,2 Keiji TANAKA,1 Seiichiro KODAMA,3 Toshiaki IWASAKI,4 Takaaki MIYAKE,4 Miho TAKASHIO,4 and Jun IWABUCHI4 p.2406

Purification and Characterization of a Novel Fungal α-Glucosidase from Mortierella alliacea with High Starch-hydrolytic Activity
Yoshio TANAKA, Tsunehiro AKI, Y<Oh><0226><Wa>uki HIDAKA, Y<Oh><0226><Wa>uji FURUYA, Seiji KAWAMOTO, Seiko SHIGETA, Kazuhisa ONO, and Osamu SUZUKI p.2415

Repellents in the Japanese Cedar, Cryptomeria japonica, against the Pill-bug, Armadillidium vulgare
Jun MORISAWA, Chul-Sa KIM, Takehiro KASHIWAGI, Shin-ichi TEBAYASHI, and Michio HORIIKE p.2424

Compilation and Characterization of aNovel WNK Family of Protein Kinases in Arabiodpsis thaliana with Reference to Circadian Rhythms
Norihito NAKAMICHI, Masaya MURAKAMI-KOJIMA, Eriko SATO, Yasuko KISHI, Takafumi YAMASHINO, and Takeshi MIZUNO p.2429

Effects of Partial Suppression of Ribosomal Protein S6 on Organ Formation in Arabidopsis thaliana
Takashi MORIMOTO, Yoshihito SUZUKI, and Isomaro YAMAGUCHI p.2437

Paecilopeptin, a New Cathepsin S Inhibitor Produced by Paecilomyces carneus
Kazutoshi SHINDO,1, Hidefumi SUZUKI,2 and Toru OKUDA3 p.2444

Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers and Use of Such Transport in In Vitro Evaluation of Phenolic Acid Availability
Yutaka KONISHI,1, Keiko HAGIWARA,1 and Makoto SHIMIZU2 p.2449

Note
Biotransformation of (−)-Verbenone by Human Liver Microsomes
Mitsuo MIYAZAWA, Atsushi SUGIE, and Masaki SHINDO p.2458

Note
Identification of the Sex Pheromone Components Secreted by Female Moths of Peridroma saucia (Noctuidae: Noctuinae)
Shin-ichi INOMATA,1 Satoshi TSUCHIYA,1 Kazutaka IKEDA,1 Osamu SAITO,2 and Tetsu ANDO1, p.2461

Note
Influence of Dietary Methionine Level on the Liver Metallothionein mRNA Level in Rats
Komang Ayu NOCIANITRI,1 Shoji SAKAKIBARA,1 Takashi KANNO,2 Hiroto KIKUCHI,2 Masaaki KURASAKI,3 and Yoritaka AOYAMA1, p.2465

Note
Siderophore Production and Induction of Iron-regulated Proteins by a Microorganism from Rhizosphere of Barley
Hiroshi TERANO,1, Kyosuke NOMOTO,1 and Shigehiro TAKASE2 p.2471

Note
Characterization of Five Phyllosphere Bacteria Isolated from Rosa rugosa Leaves, and Their Phenotypic and Metabolic Properties
Yasuyuki HASHIDOKO, Eriko ITOH, Kentaro YOKOTA, Tadashi YOSHIDA, and Satoshi TAHARA
p.2474

Note
Carotenoid Pigments in GAC Fruit (Momordica cochinchinensis SPRENG)
Hiromitsu AOKI,1 Nguyen Thi Minh KIEU,2 Noriko KUZE,1 Kazue TOMISAKA,2 and Nguyen Van CHUYEN2, p.2479

Note
Anthocyanin Compositions in Sweetpotato (Ipomoea batatas L.) Leaves
Md. Shahidul ISLAM,1 Makoto YOSHIMOTO,1, Norihiko TERAHARA,2 and Osamu YAMAKAWA1
p.2483

Note
Identification of a Wheat Allergen, Tri a Bd 36K, as a Peroxidase
Hiromi YAMASHITA, Yoko NANBA, Miki ONISHI, Masumi KIMOTO, Miki HIEMORI, and Hideaki TSUJI p.2487

Note
Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells by a Coptidis Rhizoma Extract and Protoberberine Alkaloids
Koji SHIGETA,1 Keisuke OOTAKI,1 Hideki TATEMOTO,1, Tsutomu NAKANISHI,2 Akira INADA,2 and Norio MUTO1, p.2491

Note
Isolation and Characterization of cDNAs That Encode Homologs of a Pathogenesis-related Protein Allergen from Cryptomeria japonica
Norihiro FUTAMURA,1 Yuzuru MUKAI,2 Masahiro SAKAGUCHI,3 Hiroshi YASUEDA,4 Sakae INOUYE,3, Terumi MIDORO-HORIUTI,5 Randall M. GOLDBLUM,5 and Kenji SHINOHARA1,p.2495

Note
Synthesis of Both Enantiomers of Isorobinal, a Novel Cyclic Monoterpene Isolated from the Astigmatid Mite, Rhizoglyphus sp.
Ting LIANG and Shigefumi KUWAHARA p.2501

Note
Total Synthesis of (±)-syn-Copalol
Hiroaki TOSHIMA,1, Hideaki OIKAWA,2 Hiroshi YADA,3 Hiroshi ONO,3 Tomonobu TOYOMASU,4 and Takeshi SASSA4 p.2510

Note
Molecular Cloning and mRNA Expression of Geraniol-inducible Genes in Cultured Shoot Primordia of Matricaria chamomilla
Yoshiyuki ASHIDA, Masaki NISHIMOTO, Akihito MATSUSHIMA, Junko WATANABE, and Toshifumi HIRATA p.2511

Note
Purification and Characterization of β-1,6-Glucanase of Streptomyces rochei Application in the Study of Yeast Cell Wall Proteins
Hong WU, Hitoshi SHIMOI, and Kiyoshi ITO p.2515

Preliminary Communication
Mutual Binding Inhibition of Tetrodotoxin and Saxitoxin to Their Binding Protein from the Plasma of the Puffer Fish, Fugu pardalis
Mari YOTSU-YAMASHITA,1, Yuki SHOJI,1 Takahiro TERAKAWA,1 Shunichi YAMADA,1 Teruo
p.2520

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Molecular Characterization of Sucrose:Sucrose 1-Fructosyltransferase and Sucrose:Fructan 6-Fructosyltransferase Associated with Fructan Accumulation in Winter Wheat during Cold Hardening

Akira KAWAKAMI and Midori YOSHIDA

National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Sapporo 062-8555, Japan

Received March 7, 2002; Accepted May 22, 2002
We isolated two cDNAs of winter wheat (Triticum aestivum L.), designated wft1 and wft2, which encoded sucrose:fructan 6-fructosyltransferase (6-SFT) and sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), respectively, which are involved in the synthesis of fructan in wheat. wft1 and wft2 were cloned by screening of a cDNA library with probed-cDNA fragments corresponding to plant fructosyltransferase and invertase. The identity of the clones was verified by functional characterization of recombinant proteins expressed in methylotrophic yeast, Pichia pastoris. Northern blotting showed that the level of wft2 transcripts increased from autumn to early winter in the crown tissues of all field-grown wheat cultivars examined. Higher levels of wft1 and wft2 transcripts were found in leaf tissues of snow mold-resistant cultivars, which accumulated more fructan than other cultivars. Our results showed that Wft1 and Wft2 were important in fructan accumulation during cold hardening of winter wheat.
Key words: fructan; sucrose:fructan 6-fructosyltransferase; sucrose:sucrose 1-fructosyltransferase; hardening; Triticum aestivum L.

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Increasing Effect of Nori on the Fecal Excretion of Dioxin by Rats

Kunimasa MORITA and Kazuhiro TOBIISHI

Fukuoka Institute of Health and Environmental Sciences, Dazaifu City, Fukuoka 818-0135, Japan

Received March 11, 2002; Accepted May 27, 2002
The effects of nori (Porphyra yezoensis), a kind of red alga, on the gastrointestinal absorption and reabsorption of 17 types of dioxin were investigated in male Wistar rats. The rats were fed with 4 g of the control diet or 4 g of the nori diet containing a standard dioxin solution (233 ngTEQ/kg of body weight) for five consecutive days. In the group fed with the 10％ nori diet, the fecal excretion of dioxin from days 1 to 5 was higher (p＜0.01) than that of the control group by 5.5-fold for 2,3,7,8-TCDD, 6.6-fold for 1,2,3,7,8-pentaCDD, and 6.0-fold for 2,3,4,7,8-pentaCDF. In another experiment, the rats were fed with 4 g of the control diet containing a standard dioxin solution (2991 ngTEQ/kg of body weight) on the first day of the experiment and then given the control diet for 7 consecutive days, before being given either the control diet or the nori diet for 28 consecutive days more. In the group fed with the 10％ Nori diet, the fecal excretion of dioxin during the period from days 8 to 35 was higher (p＜0.01 or p＜0.05) than that of the control group by 2.4-fold for 2,3,7,8-TCDD, 2.3-fold for 1,2,3,7,8-pentaCDD, and 2.4-fold for 2,3,4,7,8-pentaCDF. These results suggest that the administration of nori prevented dioxin from being efficiently absorbed and reabsorbed from the gastrointestinal tract, and might be useful for protecting humans exposed to dioxin from ill effects.
Key words: dioxin; polychlorinated dibenzo-p-dioxin; polychlorinated dibenzofuran; nori; rats

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Main Polyol Dehydrogenase of Gluconobacter suboxydans IFO 3255, Membrane-bound D-Sorbitol Dehydrogenase, That Needs Product of Upstream Gene, sldB, for Activity

Masako SHINJOH, Noribumi TOMIYAMA, Taro MIYAZAKI, and Tatsuo HOSHINO

Department of Applied Microbiology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan

Received March 15, 2002; Accepted May 31, 2002
The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.
Key words: pyrroloquinoline-quinone-dependent D-sorbitol dehydrogenase; L-sorbose production by Gluconobacter cells

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Recombinant Agrobacterium AgaE-like Protein with Fructosyl Amino Acid Oxidase Activity

Kozo HIROKAWA and Naoki KAJIYAMA

Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278-0037, Japan

Received April 1, 2002; Accepted June 17, 2002
Agrobacterium tumefaciens AgaE-like protein had a similar sequence to that of a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1. To characterize the AgaE-like protein, we produced the enzyme in Escherichia coli, and purified it to homogeneity. The molecular mass of recombinant AgaE-like protein was 42 kDa on SDS-PAGE and 85 kDa on gel filtration. The protein acted on N-fructosyl valine and N-fructosyl glycine as substrates, but not on glycated protein or Nε-fructosyl lysine. Apparent Km for N-fructosyl valine and N-fructosyl glycine were 1.64 and 0.31 mM, respectively. The AgaE-like protein had maximum activity at pH 7.8 and 35°C in 0.1 M potassium phosphate, but more than 80％ of its activity was lost at 40°C or more. In contrast to eukaryotic fructosyl amino acid oxidases, the AgaE-like protein contained noncovalently bound FAD as a cofactor and was inactive against Nε-fructosyl Nα-Z(benzyloxycarbonyl)-lysine. These characteristics were similar to a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1, suggesting that these prokaryotic enzymes comprise a new family of fructosyl amino acid oxidases.
Key words: fructosyl amino acid oxidase; glycated protein; enzymatic measurement of HbA1C; mannityl opine; Agrobacterium tumefaciens C58

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Effects of External Factors on the Interaction of Tea Catechins with Lipid Bilayers

Katsuko KAJIYA, Shigenori KUMAZAWA, and Tsutomu NAKAYAMA

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

Received April 17, 2002; Accepted June 11, 2002
Green tea contains a high concentration of such catechins as (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECg), and (−)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.
Key words: chemical factor; lipid bilayer; liposome; tea catechin

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Antimutagenicity of Mono-, Di-, and Tricaffeoylquinic Acid Derivatives Isolated from Sweetpotato (Ipomoea batatas L.) Leaf

Makoto YOSHIMOTO,1, Shoji YAHARA,2 Shigenori OKUNO,1 Md. Shahidul ISLAM,1 Koji ISHIGURO,1 and Osamu YAMAKAWA1

1Department of Upland Farming Research, National Agricultural Research Center for Kyushu Okinawa Region, Miyakonojo, Miyazaki 885-0091, Japan 2Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan

Received April 22, 2002; Accepted June 4, 2002
The caffeoylquinic acid derivatives, 3-mono-O-caffeoylquinic acid (chlorogenic acid, ChA), 3,4-di-O-caffeoylquinic acid (3,4-diCQA), 3,5-di-O-caffeoylquinic acid (3,5-diCQA), 4,5-di-O-caffeoylquinic acid (4,5-diCQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-triCQA), and caffeic acid (CA) were isolated from the sweetpotato (Ipomoea batatas L.) leaf. We examined the antimutagenicity of these caffeoylquinic acid compounds to promote new uses of the sweetpotato leaf. These caffeoylquinic acid derivatives effectively inhibited the reverse mutation induced by Trp-P-1 on Salmonella typhimurium TA 98. The antimutagenicity of these derivatives was 3,4,5-triCQA＞3,4-diCQA＝3,5-diCQA＝4,5-diCQA＞ChA in this order. There was no difference in the antimutagenicity of all dicaffeoylquinic acid derivatives. A comparison of the activities and structures of these compounds suggested that the number of caffeoyl groups bound to quinic acid played a role in the antimutagenicity of the caffeoylquinic acid derivatives. The sweetpotato leaves contained distinctive polyphenolic components with a high content of mono-, di-, and tricaffeoylquinic acid derivatives and could be a source of physiological functions.
Key words: antimutagenicity; caffeoylquinic acid derivative; sweetpotato leaf; polyphenol

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Milk Calcium Taken with Cheese Increases Bone Mineral Density and Bone Strength in Growing Rats

Ken KATO,1, Yukihiro TAKADA,2 Hiroaki MATSUYAMA,2 Yoshihiro KAWASAKI,2 Seiichiro AOE,2 Hideo YANO,3 and Yasuhiro TOBA2

1Product Planning and Development Division, Snow Brand Frozen Foods Co., Ltd., Kawagoe, Saitama 350-1165, Japan 2Technology and Research Institute, Snow Brand Milk Products Co., Ltd., Kawagoe, Saitama 350-1165, Japan 3Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received May 2, 2002; Accepted July 5, 2002
We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6-week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times per day) for 2 weeks. The rats were then divided into three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca-Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were significantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.
Key words: cheese; milk calcium; bioavailability; bone; rats

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Purification and Characterization of a Novel Cholesterol Esterase from Pseudomonas aeruginosa, with Its Application to Cleaning Lipid-stained Contact Lenses

Akio SUGIHARA,1, Yuji SHIMADA,1 Atsuo NOMURA,2 Tadamasa TERAI,2 Masaki IMAYASU,3 Yusuke NAGAI,3 Toshihiro NAGAO,1 Yomi WATANABE,1 and Yoshio TOMINAGA1

1Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan 2Department of Chemistry, Osaka Institute of Technology, 5-16-1 Oomiya, Asahi-ku, Osaka 535-8585, Japan 3Central Research Laboratories, Menicon Co. Ltd., 5-1-10 Takamoridai, Kasugai 487-0032, Japan

Received May 2, 2002; Accepted July 18, 2002
With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53°C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25°C, and retained its activity up to 53°C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate＞
oleate＞stearate＞palmitate＞caprylate＞myristate＞
laurate, caprate＞caproate＞butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.
Key words: cholesterol esterase; cholesteryl ester; Pseudomonas aeruginosa; contact lens cleaner

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Fusicoccins P and Q, and 3-Epifusicoccins H and Q, New Polar Fusicoccins from Isolate Niigata 2-A of a Peach Fusicoccum Canker Fungus

Takeshi SASSA,1,2, Naoto TAJIMA,2 Mitsuyoshi SATO,1 Ai TAKAHASHI,1 and Nobuo KATO3

1Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan 2Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University) 3Institute of Advanced Material Study, Kyushu University, Kasuga 816-8580, Japan

Received May 2, 2002; Accepted June 14, 2002
Our search for new polar fusicoccins biosynthetically related to fusicoccin A from the culture filtrate of isolate Niigata 2-A of a peach Fusicoccum canker fungus resulted in the isolation of new fusicoccins named fusicoccins P and Q, and 3-epifusicoccins H and Q, together with 3′-deacetylfusicoccin A and 16-O-demethyl-3-epifusicoccin J. The structures of fusicoccins P and Q, and of 3-epifusicoccin Q were determined to be those of deisopentenylfusicoccin J, 12α-hydroxyfusicoccin H and 12α-hydroxy-3-epifusicoccin H, respectively, by NMR spectrometry and chemical derivation from known fusicoccins. 3-Epifusicoccin H was identified by comparing its 400 MHz NMR spectra with those of fusicoccin H. The lettuce seed germination-stimulating activity of these new fusicoccins was examined in the presence of ABA: fusicoccin P was highly active, while 3-epifusicoccins H and Q were slightly active, and fusicoccins H and Q were almost inactive. Possible biosynthetic pathways incorporating these new fusicoccins and 3-epifusicoccins from geranylgeranyl diphosphate to 3′-deacetlyfusicoccin A and 16-O-demethyl-3-epifusicoccin J are discussed.
Key words: fusicoccin P; fusicoccin Q; 3-epifusicoccin H; 3-epifusicoccin Q; Phomopsis amygdali

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Cloned Cytosine Deaminase Gene Expression of Bifidobacterium longum and Application to Enzyme/Pro-drug Therapy of Hypoxic Solid Tumors

Toshiyuki NAKAMURA,1, Takayuki SASAKI,1, Minoru FUJIMORI,1, Kazuyuki YAZAWA,1 Yasunobu KANO,2 Jun AMANO,1 and Shun′ichiro TANIGUCHI3

1Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan 2Department of Molecular Genetics, Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan 3Department of Molecular Oncology and Angiology, Angio-Aging Research Division, Research Center on Aging and Adaptation, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan

Received May 7, 2002; Accepted June 27, 2002
Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors.
Key words: Bifidobacterium longum; cytosine deaminase; enzyme/pro-drug therapy; pBLES100- S-eCD

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Stable Form of Ascorbate Peroxidase from the Red Alga Galdieria partita Similar to Both Chloroplastic and Cytosolic Isoforms of Higher Plants

Sakihito KITAJIMA,1, Masami UEDA,1 Satoshi SANO,1, Chikahiro MIYAKE,2, Takayuki KOHCHI,2 Ken-ichi TOMIZAWA,1 Shigeru SHIGEOKA,3 and Akiho YOKOTA1,

1Research Institute of Innovative Technology for the Earth (RITE), Soraku-gun, Kyoto, 619-0292, Japan 2Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Ikoma, Nara 630-01, Japan 3Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan

Received May 8, 2002; Accepted June 28, 2002
Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.
Key words: cDNA; inactivation; reactive oxygen species; recombinant protein

-12-
Bilirubin Dehydrogenase, an Enzyme in Aspergillus ochraceus IB-3 Useful for Diagnostic Measurement of Bilirubin

Jun OGAWA,1 Woro Triarsi SULISTYANINGDYAH,1 Hiromi TANAKA,1 Kenji KANO,2 Tokuji IKEDA,2 and Sakayu SHIMIZU1,

1Division of Applied Life Sciences, Laboratory of Fermentation Physiology and Applied Microbiology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 2Division of Applied Life Sciences, Laboratory of Bio-Analytical and Physical Chemistry, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received May 15, 2002; Accepted July 30, 2002
Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 μM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.
Key words: bilirubin; biliverdin; bilirubin dehydrogenase; bilirubin oxidase; ditaurobilirubin

-13-
Equilibrium Dialysis Measurements of the Ca2＋-Binding Properties of Recombinant Radish Vacuolar Ca2＋-Binding Protein Expressed in Escherichia coli

Koji YUASA and Masayoshi MAESHIMA

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

Received May 21, 2002; Accepted July 11, 2002
Vacuoles of radish (Raphanus sativus) contained a Ca2＋-binding protein (RVCaB) of 43 kDa. We investigated the Ca2＋-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2＋-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2＋ and small dialysis buttons. The protein was estimated to bind 19Ca2＋ ions per molecule with a Kd for Ca2＋ of 3.4 mM. Ca2＋ was bound to the protein even in the presence of high concentrations of Mg2＋ or K＋. The results suggested that the protein bound Ca2＋ with high ion selectivity, high capacity, and low affinity.
Key words: Ca2＋-binding protein; recombinant protein; plant vacuole; Raphanus sativus

-14-
Novel Reversible Indole-3-carboxylate Decarboxylase Catalyzing Nonoxidative Decarboxylation

Toyokazu YOSHIDA, Kohei FUJITA, and Toru NAGASAWA

Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan

Received May 21, 2002; Accepted July 15, 2002
After enrichment culture with indole-3-carboxylate in static culture, a novel reversible decarboxylase, indole-3-carboxylate decarboxylase, was found in Arthrobacter nicotianae FI1612 and several molds. The enzyme reaction was examined in resting-cell reactions with A. nicotianae FI1612. The enzyme activity was induced specifically by indole-3-carboxylate, but not by indole. The indole-3-carboxylate decarboxylase of A. nicotianae FI1612 catalyzed the nonoxidative decarboxylation of indole-3-carboxylate into indole, and efficiently carboxylated indole and 2-methylindole by the reverse reaction. In the presence of 1 mM dithiothreitol, 50 mM Na2S2O3, and 20％ (v/v) glycerol, indole-3-carboxylate decarboxylase was partially purified from A. nicotianae FI1612. The purified enzyme had a molecular mass of approximately 258 kDa. The enzyme did not need any cofactor for the decarboxylating and carboxylating reactions.
Key words: decarboxylase; nonoxidative decarboxylation; carboxylation; indole-3-carboxylate; indole

-15-
Identification of the Bile Acid-binding Region in the Soy Glycinin A1aB1b Subunit

Seon-Kang CHOI, Motoyasu ADACHI, and Shigeru UTSUMI

Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received May 22, 2002; Accepted June 28, 2002
Soy glycinin has five major subunits which are classified into two groups according to their homology in amino acid sequences (group I, A1aB1b, A1bB2 and A2B1a; group II, A3B4 and A5A4B3). It has been reported that the peptide fragments derived from the A1a and A2 chains of the A1aB1b and A2B1a subunits had bile acid-binding ability and that the region of 114--161 residues of the A1a chain was responsible for this bile acid-binding ability. In this study, we constructed A1a, A3 and 9 deletion mutants of A1a lacking various numbers of residues at the C-terminus, and evaluated their bile acid-binding ability by a cholic acid-conjugated column and fluorescence analysis. The bile acid-binding ability of A1a was higher than that of A3 and there was a remarkable decrease in the bile acid-binding ability between the Δ[138--291] and Δ[130--291] mutants. The 130--138 region is rich in hydrophobic residues. In this regard, when we constructed the Δ[129--134] mutant lacking six contiguous hydrophobic residues (VAWWMY) and evaluated its bile acid-binding ability, a similar remarkable decrease in the bile acid-binding ability was observed. These results indicate that the 129--134 residue region (VAWWMY) with high hydrophobicity was important for bile acid-binding of A1a.
Key words: soy glycinin; A1aB1b subunit; bile acid-binding

-16-
Epitope Analysis of Antibodies in Japanese to Human Cytomegalovirus Phosphoprotein 150 with Synthetic Peptides

Isao TAKAHASHI, Sayoko SUGIURA, Hirotoshi OHTA, Kazuo OZAWA, and Tadashi KAMIYA

Japanese Red Cross Aichi Blood Center, 539-3 Minami-yamaguchi, Seto, Aichi 489-8555, Japan

Received May 22, 2002; Accepted July 26, 2002
Serological detection of antibodies specific to human cytomegalovirus (HCMV) is not reliable because the assay uses the whole HCMV protein fraction. Antigenic materials composed of well-characterized viral proteins are being tried for serodiagnosis in Europe. Epitopes of antibodies to HCMV phosphoprotein 150 (pp150) encoded by UL32 in Japanese individuals were investigated for comparison with the results in Europe. The major epitopes on amino acid residues 496 to 652 of HCMV pp150 were identified and the detection of antibodies with an enzyme-linked immunosorbent assay (ELISA) of synthetic peptides against the main epitopes was established. Fifteen seropositive and five seronegative serum samples for the epitope mapping and 131 seropositive and 50 seronegative samples for ELISA were investigated. Overlapping 15-mer peptides moving by two amino acids through V496-H652 were synthesized. The main epitope regions were V508-D530, L526-Q544, S536-D554, T616-G634, S624-P642, and L632-H652. When each peptide was conjugated with bovine serum albumin for ELISA, 80.9％ of the seropositive samples were judged to be positive. The results of this study are the same as those for European sera, so the antigenic materials developed in Europe might be used to replace the whole HCMV protein fraction in Japanese.
Key words: epitope mapping; virus infection; human cytomegalovirus; synthetic peptide

-17-
Synthesis and Insecticidal Activity of N-Oxydihydropyrroles: 4-Hydroxy-3-mesityl-5,5-dimethyl Derivatives with Various Substituents at the 1-Position

Mitsuru ITO,1, Hideshi OKUI,1 Harumi NAKAGAWA,1 Shigeru MIO,1 Ayako KINOSHITA,1 Takashi OBAYASHI,1 Takako MIURA,1 Junko NAGAI,1 Shinji YOKOI,1 Reiji ICHINOSE,2 Keiji TANAKA,1 Seiichiro KODAMA,3 Toshiaki IWASAKI,4 Takaaki MIYAKE,4 Miho TAKASHIO,4 and Jun IWABUCHI4

1Agroscience Research Laboratories, Crop Protection Company, Sankyo Co. Ltd., 1041 Yasu, Yasu-cho, Yasu-gun, Shiga 520-2342, Japan 2Crop Protection Department, Crop Protection Company, Sankyo Co. Ltd., 7-12 Ginza 2-chome, Chuo-ku, Tokyo 104-8113, Japan 3Marketing Department, Agrochemicals Division, Agro ＆ Specialty Chemicals Group, Nippon Kayaku Co. Ltd., 11-2 Fujimi 1-chome, Chiyoda-ku, Tokyo 102-8172, Japan 4Research ＆ Development Laboratories, Agro ＆ Specialty Chemicals Group, Nippon Kayaku Co. Ltd., 225-1 Horigome, Koshikiya, Ageo-city, Saitama 362-0064, Japan

Received May 23, 2002; Accepted June 24, 2002
A new series of N-oxydihydropyrrole derivatives was synthesized and evaluated for insecticidal activity against Nilaparvata lugens and Myzus persicae. Various substituents were introduced to the 1-position of the dihydropyrrole ring, and the derivatives obtained exhibited systemic and/or contact insecticidal activity. The structure-activity relationship revealed that small alkyoxy and alkoxyalkoxy groups were more favorable than alkylcarbonyloxy, alkoxycarbonyloxy, or sulfonyloxy groups as substituents at the 1-position.
Key words: N-oxydihydropyrrole derivatives; 1,3-diketone moiety; systemic insecticide; sucking insects; phytotoxicity

-18-
Purification and Characterization of a Novel Fungal α-Glucosidase from Mortierella alliacea with High Starch-hydrolytic Activity

Yoshio TANAKA, Tsunehiro AKI, Y<Oh><0226><Wa>uki HIDAKA, Y<Oh><0226><Wa>uji FURUYA, Seiji KAWAMOTO, Seiko SHIGETA, Kazuhisa ONO, and Osamu SUZUKI

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan

Received May 27, 2002; Accepted July 18, 2002
The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable α-amylase activity. Here, a α-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal α-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55°C and in a pH range of 5.0--6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-α-D-glucoside was optimal at pH 5.0 and a temperature range of 45--50°C.
Key words: arachidonic acid; carbohydrate hydrolase; α-glucosidase; Mortierella alliacea; starch

-19-
Repellents in the Japanese Cedar, Cryptomeria japonica, against the Pill-bug, Armadillidium vulgare

Jun MORISAWA, Chul-Sa KIM, Takehiro KASHIWAGI, Shin-ichi TEBAYASHI, and Michio HORIIKE

Department of Bioresouces Science, Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku 783-8502, Japan

Received June 6, 2002; Accepted July 9, 2002
Sandaracopimarinol and (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one were isolated and identified from Cryptomeria japonica as repellents against Armadillidium vulgare which is well known as an unpleasant pest in the house and as vegetable pest in Japan. These compounds strongly repelled A. vulgare when they were combined, although each compound alone did not show any activity.
Key words: Armadillidium vulgare; Cryptomeria japonica; repellent; sandaracopimarinol; (1S,6R) - 2,7(14),10 - bisabolatrien - 1 - ol-4-one

-20-
Compilation and Characterization of aNovel WNK Family of Protein Kinases in Arabiodpsis thaliana with Reference to Circadian Rhythms

Norihito NAKAMICHI, Masaya MURAKAMI-KOJIMA, Eriko SATO, Yasuko KISHI, Takafumi YAMASHINO, and Takeshi MIZUNO

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

Received June 10, 2002; Accepted July 13, 2002
The complete genome sequence of Arabidopsis thaliana revealed that this higher plant has a tremendous number of protein kinases. We recently isolated a novel type of protein kinase, named AtWNK1, which shows an in vitro ability to phosphorylate the APRR3 member of the APRR1/TOC1 quintet that has been implicated in a mechanism underlying circadian rhythms in Arabidopsis. We here address two issues, one general and one specific, as to this novel protein kinase. We first asked the general question of how many WNK family members are present in this higher plant, then whether or not other members are also relevant to circadian rhythms. The results of our analyses showed that Arabidopsis has at least 9 members of the WNK1 family of protein kinases (designated here as WNK1 to WNK9), the structural design of which is clearly distinct from those of other known protein kinases, such as receptor-like kinases and mitogen-activated protein kinases. They were examined with special reference to the circadian-related APRR1/TOC1 quintet. It was found that not only the transcription of the WNK1 gene, but also those of three other members (WNK2, WNK4, and WNK6) are under the control of circadian rhythms. These results suggested that certain members of the WNK family of protein kinases might play roles in a mechanism that generates circadian rhythms in Arabidopsis.
Key words: Arabidopsis; protein kinase; circadian rhythm

-21-
Effects of Partial Suppression of Ribosomal Protein S6 on Organ Formation in Arabidopsis thaliana

Takashi MORIMOTO, Yoshihito SUZUKI, and Isomaro YAMAGUCHI

Department of Applied Biological Chemistry, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received June 11, 2002; Accepted July 15, 2002
An expression library of Arabidopsis thaliana cDNAs was randomly introduced into A. thaliana. The transformant pool was used to obtain a line, c105, with reduced apical dominance and irregular positioning of leaves and flowers. The inserted DNA was a 3′-fragment of the ribosomal protein S6 gene with antisense orientation. The transcriptional level of the ribosomal protein S6 was lower in c105 than in the wild-type plant. Introduction of the same fragment into the wild-type plant gave phenotypes similar to those of c105, so the phenotypes of c105 were due to the S6 antisense expression. The phenotypes suggest selectively reduced function of specific proteins rather than an overall decrease in protein function caused by defective ribosomal biogenesis.
Key words: ribosomal protein S6; Arabidopsis thaliana; organ formation; ribosomal biogenesis

-22-
Paecilopeptin, a New Cathepsin S Inhibitor Produced by Paecilomyces carneus

Kazutoshi SHINDO,1, Hidefumi SUZUKI,2 and Toru OKUDA3

1Department of Food and Nutrition, Japan Women′s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan 2Pharmaceutical Reseach Laboratories, Kirin Brewery Co. Ltd., 3 Miyahara-cho, Takasaki 370-1295, Japan 3Tamagawa University Research Institute, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan

Received June 13, 2002; Accepted June 28, 2002
Paecilopeptin, a novel cathepsin S inhibitor, was produced and isolated from the culture supernatant of the fungal strain, Paecilomyces carneus. A spectroscopic analysis revealed the planar structure of paecilopeptin to be acetyl-Leu-Val-CHO. The stereochemistry of the constituent amino acids was analysed by chiral HPLC after oxidation and 6N HCl hydrolysis of paecilopeptin. The total synthesis of paecilopeptin was completed in six steps. Paecilopeptin inhibited human cathepsin S with an IC50 value of 2.1 nM in vitro.
Key words: cathepsin S; enzyme inhibitor; Paecilomyces carneus; structural elucidation; peptide

-23-
Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers and Use of Such Transport in In Vitro Evaluation of Phenolic Acid Availability

Yutaka KONISHI,1, Keiko HAGIWARA,1 and Makoto SHIMIZU2

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

Received June 25, 2002; Accepted July 26, 2002
Fluorescein is a marker-dye customary applied to the evaluation of tight-junctional permeability of epithelial cell monolayers. However, the true mechanism for the permeation has not been elucidated. Transepithelial transport of fluorescein in Caco-2 cell monolayers was therefore examined. Fluorescein transport was dependent on pH, and in a vectorical way in the apical-basolateral direction, but it was independent of the tight-junctional permeability of monolayers of these human intestinal cells. The permeation of fluorescein was concentration-dependent and saturable; the Michaelis constant was 7.7 mM and the maximum velocity was 40.3 nmol min−1 (mg protein)−1. Benzoic acid competitively inhibited fluorescein transport, suggesting that fluorescein is transported by a monocarboxylic acid transporter (MCT). Antioxidative polyphenolic compounds such as ferulic acid from dietary sources, competitively inhibited the permeation of fluorescein. These compounds probably share a transport carrier with fluorescein. Measurement of the effects of phenolic acids on fluorescein transport across Caco-2 monolayers would be a useful way to evaluate the intestinal absorption or bioavailability of dietary phenolic acids.
Key words: fluorescein; monocarboxylic acid transporter; ferulic acid; availability; Caco-2 cells

-24-
Note
Biotransformation of (−)-Verbenone by Human Liver Microsomes

Mitsuo MIYAZAWA, Atsushi SUGIE, and Masaki SHINDO

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

Received March 11, 2002; Accepted June 19, 2002
The biotransformation of (−)-verbenone was investigated with human liver microsomes by using GC-MS. Regioselective biotransformation was observed when (−)-verbenone was incubated with the liver microsomes. (−)-10-Hydroxyverbenone was formed from (−)-verbenone of kinetic analysis showed that the Km and Vmax values for the hydroxylation of (−)-verbenone by liver microsomes from three human samples, HG-70, HG-56 and HG-23, were 1.1 mM and 4.8 nmol/min/nmol P450, 0.6 mM and 2.1 nmol/min/nmol P450, and 2.8 mM and 4.6 nmol/min/nmol P450, respectively.
Key words: biotransformation; (−)-verbenone; 10-hydroxylation; (−)-10-hydroxyverbenone; human liver microsomes

-25-
Note
Identification of the Sex Pheromone Components Secreted by Female Moths of Peridroma saucia (Noctuidae: Noctuinae)

Shin-ichi INOMATA,1 Satoshi TSUCHIYA,1 Kazutaka IKEDA,1 Osamu SAITO,2 and Tetsu ANDO1,

1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan 2National Agricultural Research Center for Western Region, Fukuyama, Hiroshima 721-8514, Japan

Received March 19, 2002; Accepted June 3, 2002
The variegated cutworm, Peridroma saucia H<Oh><0228><Wa>ubner, is a lepidopteran pest to a large number of crops in Canada, the United States, and Europe. It was probably naturalized in Japan in the 1970s. The pheromone glands of the female moth include two components with electroantennographic activity in a ratio of 3:1. GC-MS analyses of pheromone extracts untreated and treated with dimethyl disulfide revealed the major component to be (Z)-11-hexadecenyl acetate and the minor component to be (Z)-9-tetradecenyl acetate. The synthetic pheromone was used to attract a large number of males in a vegetable field in Tokyo, which suggests that this species has already become a harmful pest in Japan.
Key words: Lepidoptera; insect pheromone; attractant; monoenyl acetate; filed attraction

-26-
Note
Influence of Dietary Methionine Level on the Liver Metallothionein mRNA Level in Rats

Komang Ayu NOCIANITRI,1 Shoji SAKAKIBARA,1 Takashi KANNO,2 Hiroto KIKUCHI,2 Masaaki KURASAKI,3 and Yoritaka AOYAMA1,

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Nishi-9, Kita-9, Kita-ku, Sapporo 060-8589, Japan 2Nippon Beet Sugar Mfg. Co. Ltd., Nishi-13, Minami-9, Inada-cho, Obihiro 080-0831, Japan 3Graduate School of Environmental Earth Science, Hokkaido University, Nishi-6, Kita-10, Kita-ku, Sapporo 060-0810, Japan

Received March 20, 2002; Accepted June 28, 2002
The effects of some methyl-containing compounds added to a choline-deficient diet on the metallothionein mRNA level in the rat liver were studied. The addition of choline or carnitine to the choline-deficient diet did not induce a gain in body weight, while the addition of either betaine or methionine to the choline-deficient diet, or of methionine to the choline-deficient diet with choline significantly increased the body weight. The metallothionein mRNA level in the liver of rats fed on the choline-deficient diet was similar to that of rats fed on the choline-deficient diet with choline, betaine or carnitine. However, the addition of methionine to the choline-deficient diet with or without choline caused a marked suppression in the metallothionein mRNA level in the liver. It is thus surmised that the metallothionein mRNA level in the liver might be regulated by the dietary content of methionine.
Key words: choline deficiency; betaine; carnitine; methionine; liver metallothionein mRNA

-27-
Note
Siderophore Production and Induction of Iron-regulated Proteins by a Microorganism from Rhizosphere of Barley

Hiroshi TERANO,1, Kyosuke NOMOTO,1 and Shigehiro TAKASE2

1Faculty of Life Sciences, Toyo University, Itakura, Gunma 374-0193, Japan 2Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Tsukuba, Ibaraki 300-2698, Japan

Received April 1, 2002; Accepted July 12, 2002
This study provides new information on the Fe uptake system capable of supporting growth of the organism. Pseudomonas fluorescens isolated from the rhizosphere of barley, a gramineous plant, produced a siderophore under iron-limiting conditions. Its chemical structure was identified as pyochelin, on the basis of 1H and 13C NMR data of a stable methyl ester derivative. The same iron-limiting conditions induced a new set of outer membrane proteins (75 and 55 kDa), consistent with a siderophore-mediated iron-uptake system.
Key words: Pseudomonas fluorescens; siderophore production; pyochelin; outer membrane protein

-28-
Note
Characterization of Five Phyllosphere Bacteria Isolated from Rosa rugosa Leaves, and Their Phenotypic and Metabolic Properties

Yasuyuki HASHIDOKO, Eriko ITOH, Kentaro YOKOTA, Tadashi YOSHIDA, and Satoshi TAHARA

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

Received April 1, 2002; Accepted July 8, 2002
Five Gram-negative bacteria, all of which were Enterobacteriaceae, were isolated from the phyllosphere of green or senescing leaves of Rosa rugosa, and their phenotypic and physiological characteristics were examined. Partial 16S rDNA sequences led to identification of these isolates as Pantoea agglomerans, Klebsiella terrigena, Erwinia rhapontici, and two strains of Rahnella aquatilis. Interestingly, these phyllosphere bacteria had certain phenotypic and physiological convergences, while they showed their own metabolic properties toward phenolic compounds of plant origin. In particular, the two Ra. aquatilis isolates from the green leaves had a substrate-inducible gallate decarboxylase activity in the resting cells that had been cultured in 1 mM gallic acid- or protocatechuic acid-containing medium. The other three isolates from the senescing leaves did not have this enzyme activity. Simple phenolics that the Ra. aquatilis decarboxylatively produced from benzoic acid derivatives had better antimicrobial activities than those of the substrates.
Key words: leaf epiphytic bacteria; phyllosphere; gallate decarboxylase; Rahnella aquatilis; Rosa rugosa

-29-
Note
Carotenoid Pigments in GAC Fruit (Momordica cochinchinensis SPRENG)

Hiromitsu AOKI,1 Nguyen Thi Minh KIEU,2 Noriko KUZE,1 Kazue TOMISAKA,2 and Nguyen Van CHUYEN2,

1Food Color Laboratory Division, San-Ei Gen F.F.I. Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka 561-8588, Japan 2Department of Food and Nutrition, Japan Women′s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan

Received April 24, 2002; Accepted June 6, 2002
The carotenoids in Gac fruit (Momordica Cochinchinensis spreng) were analysed by high-performance liquid chromatography (HPLC), and the concentrations of β-carotene, lycopene, zeaxanthin and β-cryptoxanthin were measured. Lycopene was found to be predominantly present in the Gac seed membrane at a concentration of up to 380 μg/g of seed membrane. The concentration of lycopene in the Gac seed membrane was about ten-fold higher than that in known lycopene-rich fruit and vegetables, indicating that Gac fruit could be a new and potentially valuable source of lycopene.
Key words: carotenoid; momordica; β-carotene; lycopene; Gac

-30-
Note
Anthocyanin Compositions in Sweetpotato (Ipomoea batatas L.) Leaves

Md. Shahidul ISLAM,1 Makoto YOSHIMOTO,1, Norihiko TERAHARA,2 and Osamu YAMAKAWA1

1Department of Upland Farming Research, National Agricultural Research Center for the Kyushu-Okinawa Region, Yokoichi 6651-2, Miyakonojo, Miyazaki 885-0091, Japan 2Department of Food Science and Technology, College of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0003, Japan

Received April 26, 2002; Accepted July 11, 2002
The anthocyanin composition of three varieties, Simon No. 1, Kyushu No. 119, and Elegant Summer, in sweetpotato (Ipomoea batatas L.) leaves was examined for promoting new uses. Fifteen anthocyanin compounds were identified and measured. HPLC clearly showed quantitative differences, but not qualitative ones. The anthocyanins were acylated cyanidin and peonidin type. The result suggests that the major anthocyanin composition of sweetpotato leaves is cyanidin type.
Key words: anthocyanin; sweetpotato leaf; cyanidin; peonidin

-31-
Note
Identification of a Wheat Allergen, Tri a Bd 36K, as a Peroxidase

Hiromi YAMASHITA, Yoko NANBA, Miki ONISHI, Masumi KIMOTO, Miki HIEMORI, and Hideaki TSUJI

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja-shi, Okayama 719-1197, Japan

Received May 9, 2002; Accepted July 5, 2002
A 36-kDa allergen, Tri a Bd 36K, was purified from wheat albumin and characterized. The protein was similar to barley peroxidase BP-1 both in its amino acid sequence and peroxidase activity. The enzyme seemed to contain L-fucose and D-mannose and the glycan moiety reacted with IgE antibodies in a patient′s serum.
Key words: wheat allergen; Tri a Bd 36K; lectin; peroxidase

-32-
Note
Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells by a Coptidis Rhizoma Extract and Protoberberine Alkaloids

Koji SHIGETA,1 Keisuke OOTAKI,1 Hideki TATEMOTO,1, Tsutomu NAKANISHI,2 Akira INADA,2 and Norio MUTO1,

1School of Bioresources, Hiroshima Prefectural University, 562 Nanatsuka, Shobara 727-0023, Japan 2Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge, Hirakata 573-0101, Japan

Received May 13, 2002; Accepted June 19, 2002
A methanol extract of Coptidis Rhizoma effectively enhanced the outgrowth of neurite in PC12 cells induced by nerve growth factor (NGF). Following solvent partition and preparative HPLC, berberine was isolated as the major active compound. Berberine enhanced the proportion of neurite-bearing cells in a dose-dependent manner without cytotoxicity. Its structural relatives, palmatine and coptisine, showed a slightly weaker NGF-enhancing effect than berberine. These three alkaloids inhibited acetylcholinesterase activity at a level comparable to that of physostigmine, but this inhibition was not responsible for the potentiation of NGF-induced neurite outgrowth. It is demonstrated for the first time that protoberberine alkaloids potentiated the NGF-induced differentiation of neural cells.
Key words: neurite outgrowth; PC12 cell; nerve growth factor; protoberberine alkaloid; Coptidis Rhizoma

-33-
Note
Isolation and Characterization of cDNAs That Encode Homologs of a Pathogenesis-related Protein Allergen from Cryptomeria japonica

Norihiro FUTAMURA,1 Yuzuru MUKAI,2 Masahiro SAKAGUCHI,3 Hiroshi YASUEDA,4 Sakae INOUYE,3, Terumi MIDORO-HORIUTI,5 Randall M. GOLDBLUM,5 and Kenji SHINOHARA1,

1Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute, Ibaraki 305--8687, Japan 2Faculty of Agriculture, Shizuoka University, Shizuoka 422--8529, Japan 3Department of Immunology and Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo 162--8640, Japan 4Clinical Research Center for Allergy and Rheumatology, National Sagamihara Hospital, Kanagawa 228--8522, Japan 5Department of Child Health Research Center, University of Texas Medical Branch, Galveston, TX 77555, U.S.A.

Received May 13, 2002; Accepted July 5, 2002
Many plant pathogenesis-related (PR) proteins are allergenic. We isolated three cDNAs, Cry j 3.1, Cry j 3.2, and Cry j 3.3, that encoded homologs of Jun a 3, a PR protein allergen in Juniperus ashei, from a cDNA library derived from the pollen of Cryptomeria japonica. The predicted amino acid sequences encoded by the three cDNAs were more than 85％ identical to each other and about 57％ identical to the sequence of Jun a 3. The Cry j 3 genes seemed to form a small multigene family in the genome of C. japonica. Expression of Cry j 3 was strong in roots and in female and male strobili; expression was weaker in cotyledons, leaves, stems, and pollen grains.
Key words: cDNA cloning; Cryptomeria japonica; pathogenesis-related protein; pollen allergen

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Note
Synthesis of Both Enantiomers of Isorobinal, a Novel Cyclic Monoterpene Isolated from the Astigmatid Mite, Rhizoglyphus sp.

Ting LIANG and Shigefumi KUWAHARA

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received May 21, 2002; Accepted June 21, 2002
Both enantiomers of isorobinal, a cyclic monoterpene isolated from the astigmatid mite (Rhizoglyphus sp.), were synthesized from the enantiomers of perillaldehyde in four steps by using PCC-oxidation of a tertiary allylic alcohol intermediate as the key step.
Key words: isorobinal; Rhizoglyphus; terpenoid

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Note
Total Synthesis of (±)-syn-Copalol

Hiroaki TOSHIMA,1, Hideaki OIKAWA,2 Hiroshi YADA,3 Hiroshi ONO,3 Tomonobu TOYOMASU,4 and Takeshi SASSA4

1Department of Bioresource Science, College of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan 2Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan 3National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 4Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan

Received May 23, 2002; Accepted June 26, 2002
The labdane diterpene derivative, syn-copalol [(＋)-5] is the alcohol part of syn-copalyl diphosphate [(＋)-4]. In this paper, racemic (±)-5 was synthesized from a known racemic lactone in 8 steps. The current and our previous syntheses provide all four copalol derivatives [(＋)-3, (−)-3 and (±)-5] which are required for the biosynthetic study of polycyclic diterpenes.
Key words: labdane; diterpene; biosynthesis; syn-copalyl diphosphate; syn-copalol

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Note
Molecular Cloning and mRNA Expression of Geraniol-inducible Genes in Cultured Shoot Primordia of Matricaria chamomilla

Yoshiyuki ASHIDA, Masaki NISHIMOTO, Akihito MATSUSHIMA, Junko WATANABE, and Toshifumi HIRATA

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan

Received June 12, 2002; Accepted July 10, 2002
Genes for two geraniol-responsive factors, designated McEREBP1 and McWRKY1, from cultured shoot primordia of Matricaria chamomilla were cloned. The deduced amino acid sequences of these genes were highly similar to those of the family of ethylene-responsive element binding proteins and elicitor-induced DNA-binding proteins containing a WRKY domain, respectively. The levels of McEREBP1 and McWRKY1 mRNAs were maximum when measured 1 h after treatment of the cultured cells with geraniol.
Key words: Matricaria chamomilla; mRNA expression; geraniol; transcription factor

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Note
Purification and Characterization of β-1,6-Glucanase of Streptomyces rochei Application in the Study of Yeast Cell Wall Proteins

Hong WU, Hitoshi SHIMOI, and Kiyoshi ITO

National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima 739-0046, Japan

Received June 17, 2002; Accepted July 15, 2002
A β-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies. The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE. The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range. The enzyme was stable under 50°C but lost almost all activity at 60°C. The enzyme was specific to β-1,6-glucan and had little activity towards β-1, 3-glucan and β-1, 4-glucan. When the β-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20％ glucose, 36％ gentiobiose, and 44％ other oligosaccharides, suggesting that the enzyme is an endo-type glucanase. When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins.
Key words: yeast cell wall; β-1,6-glucanase; hydrolysis; cell-wall proteins

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Preliminary Communication
Mutual Binding Inhibition of Tetrodotoxin and Saxitoxin to Their Binding Protein from the Plasma of the Puffer Fish, Fugu pardalis

Mari YOTSU-YAMASHITA,1, Yuki SHOJI,1 Takahiro TERAKAWA,1 Shunichi YAMADA,1 Teruo MIYAZAWA,1 and Takeshi YASUMOTO2

1Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan 2Japan Food Research Laboratories, Tama Laboratory, 6-11-10 Nagayama, Tama-shi, Tokyo 206-0025, Japan

Received June 6, 2002; Accepted August 9, 2002
The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 μM) binding to this protein (1.2 μM) for saxitoxin, and of saxitoxin (0.47 μM) binding to that (0.30 μM) for tetrodotoxin were 0.35±0.057 μM and 81±16 μM (n＝2), respectively.
Key words: tetrodotoxin; saxitoxin; binding protein; puffer fish; mutual inhibition