Contents and Abstracts of BBB

(Vol.67 No.6 2003)

Autonomic Nervous Responses According to Preference for the Odor of Jasmine Tea
Naohiko INOUE,1 Kyoko KURODA,1 Akio SUGIMOTO,2 Takami KAKUDA,2 and Tohru FUSHIKI1,† p.1206

Pro-oxidative Properties of Flavonoids in Human Lymphocytes
Gow-Chin YEN,1, Pin-Der DUH,2 Hui-Ling TSAI,1 and Shih-Li HUANG3 p.1215

Antigen Feeding Increases Frequency and Antigen-specific Proliferation Ability of Intraepithelial CD4＋ T Cells in αb T Cell Receptor Transgenic Mice
Masao GOTO,1, Satoshi HACHIMURA,1, Akio AMETANI,1, Takehito SATO,2 Yoshihiro KUMAGAI,3 Sonoko HABU,2 Mamoru TOTSUKA,1 Hiromichi ISHIKAWA,4 and Shuichi KAMINOGAWA1 p.1223

Synthesis and Insecticidal Activity of Novel N-Oxydihydropyrrole Derivatives with a Substituted Spirocyclohexyl Group
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.1230

Production and Characterization of Biosurfactants from Bacillus licheniformis F2.2
Jiraporn THANIYAVARN,1,2,3, Niran ROONGSAWANG,2 Takayuki KAMEYAMA,2 Mitsuru HARUKI,2 Tadayuki IMANAKA,3 Masaaki MORIKAWA,2 and Shigenori KANAYA2 p.1239

Accumulation of Hydroxycinnamic Acid Amides in Winter Wheat under Snow
Shigeki JIN,1,† Midori YOSHIDA,1 Takashi NAKAJIMA,2 and Akio MURAI3 p.1245

Polyphenols from Some Foodstuffs as Inhibitors of Ovalbumin Permeation through Caco-2 Cell Monolayers
Shoko KOBAYASHI,1, Jun WATANABE,2 Eri FUKUSHI,3 Jun KAWABATA,3 Mitsutoshi NAKAJIMA,4 and Michiko WATANABE1 p.1250

Effects of Dietary Eritadenine on the Liver Microsomal Δ6-Desaturase Activity and Its mRNA in Rats
Yasuhiko SHIMADA, Akihiro YAMAKAWA, Tatsuya MORITA, and Kimio SUGIYAMA p.1258

Biogenesis of Volatile Methyl Esters in Snake Fruit (Salacca edulis, Reinw) cv. Pondoh
SUPRIYADI,1 Masayuki SUZUKI,1 Shuiqin WU,3 Naomi TOMITA,2 Akira FUJITA,2 and Naoharu WATANABE3,† p.1267

Insulin Stimulates Expression of the Pyruvate Kinase M Gene in 3T3-L1 Adipocytes
Yuuki ASAI,1 Kazuya YAMADA,2 Toyoaki WATANABE,1 Vincent W. KENG,1 and Tamio NOGUCHI1,
p.1272

Angiotensin I-Converting Enzyme Inhibitory Peptides Isolated from Tofuyo Fermented Soybean Food
Megumi KUBA,1 Kumi TANAKA,1 Shinkichi TAWATA,1 Yasuhito TAKEDA,2 and Masaaki YASUDA1,
p.1278

Effects of Oleamide on Choline Acetyltransferase and Cognitive Activities
Ho-Jin HEO,1 Young-June PARK,1 Young-Min SUH,1 Soo-Jung CHOI,1 Mi-Jeong KIM,1 Hong-Yon CHO,1 Yun-Jeong CHANG,1 Bumshik HONG,1 Hye-Kyung KIM,2 Eunki KIM,3 Chang-Ju KIM,4 Byung-Gee KIM,5 and Dong-Hoon SHIN1, p.1284

Hypolipidemic Effect of an Exo-biopolymer Produced from a Submerged Mycelial Culture of Hericium erinaceus
Byung-Keun YANG,1 Jun-Bo PARK,2 and Chi-Hyun SONG1,† p.1292

Identification of Suberization-associated Anionic Peroxidase as a Possible Allergenic Protein from Tomato
Thanakorn WEANGSRIPANAVAL,1 Noriko NOMURA,1 Tatsuya MORIYAMA,1 Nobuo OHTA,2 and Tadashi OGAWA1, p.1299

Antihypertensive Effects of Onion on NO Synthase Inhibitor-induced Hypertensive Rats and Spontaneously Hypertensive Rats
Yoko SAKAI,1 Tetsuo MURAKAMI,2 and Yukiko YAMAMOTO1, p.1305

Effect of Neuronal PC12 Cells on the Functional Properties of Intestinal Epithelial Caco-2 Cells
Hideo SATSU, Tatsuya YOKOYAMA, Nobumasa OGAWA, Yoko FUJIWARA-HATANO, and Makoto SHIMIZU p.1312

Cloning, Sequencing, and Heterologous Expression of a Cellobiohydrolase cDNA from the Basidiomycete Corticium rolfsii
Daisuke YASOKAWA, Takeshi SHIMIZU, Ryoji NAKAGAWA, Takayuki IKEDA, and Koji NAGASHIMA
p.1319

A Novel Enzyme of Bacillus sp. 217C-11 That Produces Inulin from Sucrose
Tadashi WADA, Masao OHGUCHI, and Yoshio IWAI p.1327

Identification by the Phage-display Technique of Peptides That Bind to H7 Flagellin of Escherichia coli
Teruhiko IDE,1, Sang-Ho BAIK,1 Takao MATSUBA,2 and Shigeaki HARAYAMA1 p.1335

Simultaneous Measurement of Fluoroquinolones in Eggs by a Combination of Supercritical Fluid Extraction and High Pressure Liquid Chromatography
Jae Han SHIM,1,† Mi Hyun LEE,1 Mi Ra KIM,1 Chang Joo LEE,2 and In Seon KIM3 p.1342

Purification and Characterization of Laminaran Hydrolases from Trichoderma viride
Rika NOBE,1 Yoichi SAKAKIBARA,1 Nobuhiro FUKUDA,1 Naoto YOSHIDA,1 Kihachiro OGAWA,2 and Masahito SUIKO1, p.1349

Molecular and Immunochemical Characteristics of Monoclonal and Recombinant Antibodies Specific to Bisphenol A
Kosuke NISHI,1 Mikio TAKAI,1 Kosuke MORIMUNE,2 and Hideo OHKAWA1,† p.1358

Preparation and Characterization of Recombinant Murine p65/L-Plastin Expressed in Escherichia coli and High-titer Antibodies against the Protein
Hiroto SHINOMIYA,1,† Kozo NAGAI,1 Hajime HIRATA,4 Naoto KOBAYASHI,2 Hitoshi HASEGAWA,3 Fengzhi LIU,1 Kohsuke SUMITA,1 and Yoshihiro ASANO1 p.1368

Preparation of a Water-in-oil-in-water (W/O/W) Type Microcapsules by a Single-droplet-drying Method and Change in Encapsulation Efficiency of a Hydrophilic Substance during Storage
Shuji ADACHI, Hanaho IMAOKA, Yuri HASEGAWA, and Ryuichi MATSUNO p.1376

Light-responsive psbA Transcription Requires the −35 Hexamer in the Promoter and Its Proximal Upstream Element, UPE, in Cyanobacteria
Yoko ITO, Munehiko ASAYAMA, and Makoto SHIRAI p.1382

Note
Inhibition of Linoleic Acid Hydroperoxide-induced Toxicity in Cultured Human Fibroblasts by Anthocyanidins
Takao KANEKO,1,† Shoichi TAHARA,1 and Naomichi BABA2 p.1391

Note
A Common Structure of Substrate Shared by Lignostilbenedioxygenase Isozymes from Sphingomonas paucimobilis TMY1009
Shigehiro KAMODA,2,† Tamami TERADA,1 and Yoshimasa SABURI1 p.1394

Note
Molecular Cloning of Quinohemoprotein Alcohol Dehydrogenase, ADH IIB, from Pseudomonas putida HK5
Hirohide TOYAMA, Takaaki FUJII, Nahoko AOKI, Kazunobu MATSUSHITA, and Osao ADACHI
p.1397

Note
Cytotoxic Screening of Medicinal and Edible Plants in Okinawa, Japan, and Identification of the Main Toxic Constituent of Rhodea japonica (Omoto)
Toshiya MASUDA,1,3, Yasuo OYAMA,1 Natsuko YAMAMOTO,1 Chisato UMEBAYASHI,1 Hiromi NAKAO,1 Yukiko TOI,1 Yoshio TAKEDA,1 Katsuo NAKAMOTO,2 Hideki KUNINAGA,2 Yukari NISHIZATO,2 and Akira NONAKA2 p.1401

Note
A T42M Substitution in Bacterial 5-Enolpyruvylshikimate-3-phosphate Synthase (EPSPS) Generates Enzymes with Increased Resistance to Glyphosate
Ming HE, Yan-Fang NIE, and Peilin XU p.1405

Note
Three New Cytotoxic Acylspermidines from the Soft Coral, Sinularia sp.
Makoto OJIKA,1,† Mohammad Kamrul ISLAM,2 Tomoaki SHINTANI,2 Yang ZHANG,2 Tetsuji OKAMOTO,2 and Youji SAKAGAMI1 p.1410

Note
βg-Dehydrocurvularin and Related Compounds as Nematicides of Pratylenchus penetrans from the Fungus Aspergillus sp.
Miyako KUSANO,1 Kazumi NAKAGAMI,2 Shozo FUJIOKA,3 Tsuyoshi KAWANO,2 Atsumi SHIMADA,4 and Yasuo KIMURA2, p.1413

Note
Purification and Properties of a Carbonyl Reductase Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate from Cylindrocarpon sclerotigenum IFO 31855
Yuri SARATANI,1 Eiji UHEDA,1 Hiroaki YAMAMOTO,2 Atsuo NISHIMURA,1 and Fumiki YOSHIZAKO1,†, p.1417

Note
Antidiabetic Effect of Lactobacillus GG in Streptozotocin-induced Diabetic Rats
Mihoko TABUCHI,1, Miyo OZAKI,1 Asako TAMURA,1 Noriko YAMADA,1 Tetsuo ISHIDA,1 Masataka HOSODA,2 and Akiyoshi HOSONO3 p.1421

Note
Effects of Carrageenans on the Binding, Phagocytotic, and Killing Abilities of Macrophages to Salmonella
Yoshiko SUGITA-KONISHI,1 Shusaku YAMASHITA,2 Fumio AMANO,3, and Makoto SHIMIZU2,† p.1425

Note
Purification of Conjugated Linoleic Acid Isomers through a Process Including Lipase-catalyzed Selective Esterification
Toshihiro NAGAO,1 Yoshie YAMAUCHI-SATO,2 Akio SUGIHARA,3 Toshio IWATA,2 Koji NAGAO,4 Teruyoshi YANAGITA,4 Shuji ADACHI,5 and Yuji SHIMADA1,† p.1429

Note
Cloning and Overexpression of the avi2 Gene Encoding a Major Cellulase Produced by Humicola insolens FERM BP-5977
Tatsuki MORIYA, Manabu WATANABE, Naomi SUMIDA, Kaoru OKAKURA, and Takeshi MURAKAMI
p.1434

Note
Structural Revision of Epoxyrollins A and B, Biosynthetic Precursors of Annonaceous Acetogenins
Hiroyuki KONNO1, and Hidefumi MAKABE2 p.1438

-1-
Promoting Effect of Kaempferol on the Differentiation and Mineralization of Murine Pre-osteoblastic Cell Line MC3T3-E1

Masaki MIYAKE, Norie ARAI, Shimpei USHIO, Kanso IWAKI, Masao IKEDA, and Masashi KURIMOTO

Fujisaki Institute, Hayashibara Biochemical Laboratories Inc., 675-1 Fujisaki, Okayama 702-8006, Japan

Received August 12, 2002; Accepted January 16, 2003
A number of agents have been reported to influence osteoblastic differentiation and to prevent and treat bone loss. We found that kaempferol, a flavonoid identified in extracts of the medicinal plant, Polygonum tinctorium. Lour, had stimulatory effects on the differentiation and mineralization of the murine pre-osteoblastic cell line, MC3T3-E1. After enhancing the alkaline phosphatase activity, significant augmentation of calcification by kaempferol was observed between concentrations of 10 and 20 μM, without any marked effect on cell proliferation. When kaempferol was combined with ipriflavone, which is clinically applied to treat bone loss, calcification was synergistically augmented, suggesting that these two flavonoids may have different mechanisms of action. These results suggest that kaempferol may be a promising agent for the prevention or treatment of bone loss, especially when combined with ipriflavone.
Key words: kaempferol; osteoblasts; alkaline phosphatase; mineralization; differentiation

-2-
Autonomic Nervous Responses According to Preference for the Odor of Jasmine Tea

Naohiko INOUE,1 Kyoko KURODA,1 Akio SUGIMOTO,2 Takami KAKUDA,2 and Tohru FUSHIKI1,†

1Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Kyoto 606-8502, Japan 2Central Research Institute, Itoen Ltd., 21 Mekami, Sagara-cho, Haibara-gun, Shizuoka 421-0516, Japan
Received September 27, 2002; Accepted January 14, 2003

The effect of jasmine tea odor on the autonomic nervous system was investigated by a power spectral analysis of the heart rate variability. We assigned eight volunteers to two groups with either a predilection for or antipathy toward the jasmine tea odor. We tested both high- and low-intensity jasmine tea odors. The low-intensity odor was produced by diluting 20-fold the jasmine tea used for the high-intensity odor test. The low-intensity odor produced an increase in parasympathetic nervous activity in both the predilection and antipathy groups. The high-intensity odor produced an increase in parasympathetic nervous activity in the predilection group, but an increase in sympathetic nervous activity in the antipathy group. The odor of Chinese green tea, a basic ingredient of jasmine tea, produced no effects similar to those of the jasmine tea odor. These results suggest that the jasmine tea odor activated the parasympathetic nerve, whereas the higher-intensity odor activated the sympathetic nerve in those subjects who disliked the odor.
Key words: jasmine tea; odor; autonomic nervous system; power spectral analysis; preference

-3-
Pro-oxidative Properties of Flavonoids in Human Lymphocytes

Gow-Chin YEN,1, Pin-Der DUH,2 Hui-Ling TSAI,1 and Shih-Li HUANG3

1Department of Food Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan 2Department of Food Health, Chia Nan University of Pharmacy and Science, 60 Erh-jen Road, Section 1, Pao-An, Jen-te Hsiang, Tainan Hsien, Taiwan 3Department of Baking Management, National Kaoshiung Hospitality College, Kaoshiung, Taiwan

Received October 2, 2002; Accepted January 6, 2003
The pro-oxidative properties of the four flavonoids, quercetin, morin, naringenin and hesperetin, in human lymphocyte system were investigated. Naringenin and hesperetin accelerated the oxidation of deoxyribose induced by Fe3＋/H2O2 in a concentration range of 0--200 μM, but quercetin and morin decreased it when the concentration was greater than 100 μM. The generation of hydrogen peroxide and the superoxide anion and the production of TBARS in lymphocytes were increased with increasing concentration of a flavonoid. Cell membrane protein thiols of the lymphocytes decreased when treated with the four flavonoids. Quercetin and hesperetin had no significant effect (p＞0.05) on the activity of glutathione reductase, but morin and naringenin could inhibit the activity of the enzyme at a concentration of 200 μM, when compared to the control group. The glutathione S-transferase activity was slightly decreased by treatment with each of the four flavonoids only at a concentration of 200 μM. Therefore, the DNA damage in lymphocytes induced by the flavonoids in the model system might have been due to their stimulation of oxidative stress in the lymphocytes, which resulted in the decrease of cell membrane protein thiols, increase of lipid peroxidation in cell membrane and in the influence of the antioxidative enzyme activities.
Key words: flavonoid; pro-oxidant; DNA damage; lymphocyte

-4-
Antigen Feeding Increases Frequency and Antigen-specific Proliferation Ability of Intraepithelial CD4＋ T Cells in αb T Cell Receptor Transgenic Mice

Masao GOTO,1, Satoshi HACHIMURA,1, Akio AMETANI,1, Takehito SATO,2 Yoshihiro KUMAGAI,3 Sonoko HABU,2 Mamoru TOTSUKA,1 Hiromichi ISHIKAWA,4 and Shuichi KAMINOGAWA1

1Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 2Division of Host Defense Mechanisms, Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan 3Department of Microbiology and Immunology, Nippon Medical School, Tokyo 113-8602, Japan 4Department of Microbiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan

Received October 8, 2002; Accepted February 22, 2003
To study how intestinal intraepithelial lymphocytes (IEL) are affected by orally ingested antigen, the phenotypes and responses of the IEL in mice expressing a transgenic T cell receptor αb (TCR αb) specific for ovalbumin (OVA) were analyzed after feeding OVA. In the OVA-fed mice, the abundance of αb-IEL as a proportion of the total IEL population increased and the frequency of CD4＋ cells increased within the TCR αb＋ IEL population. CD4＋ IEL from OVA-fed transgenic mice proliferated in vitro more markedly in response to antigen stimulation than IEL from mice fed the control diet. These results indicate that antigen-specific proliferation of CD4＋ IEL was amplified as a result of oral administration of antigen.
Key words: intestinal intraepithelial lymphocytes (IEL); transgenic mice; ovalbumin; food antigen

-5-
Synthesis and Insecticidal Activity of Novel N-Oxydihydropyrrole Derivatives with a Substituted Spirocyclohexyl Group

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, Sankyo Agro Co., Ltd., 1041 Yasu, Yasu-cho, Yasu-gun, Shiga 520-2342, Japan 2Product Development Department, Sankyo Agro Co., Ltd., 4-23-14 Hongo, Bunkyo-ku, Tokyo 113-0033, 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 October 15, 2002; Accepted January 21, 2003
A series of 5-spirocyclohexyl-3-(2,6-dimethylphenyl)-1,5-dihydro-2H-pyrrol-2-one derivatives (3) with various substituents on the spirocyclohexyl ring was synthesized and evaluated for its insecticidal activity against the aphid, Myzus persicae. Substituents at the 1- and 4-positions of the dihydropyrrole ring were also varied to optimize the activity. An investigation of the structure-activity relationship revealed that methoxy, alkoxyalkoxy, ethylenedioxy and methoxyimino groups were favorable as substituents at the 4-position of the spirocyclohexyl ring. The activity was optimized by the respective substitution of a methoxy or methoxymethoxy moiety and cyclopropylcarbonyloxy group at the 1- and 4-positions of the dihydropyrrole ring.
Key words: N-oxydihydropyrrole derivative; spirocyclohexyl group; systemic insecticide; sucking pest; Myzus persicae

-6-
Production and Characterization of Biosurfactants from Bacillus licheniformis F2.2

Jiraporn THANIYAVARN,1,2,3, Niran ROONGSAWANG,2 Takayuki KAMEYAMA,2 Mitsuru HARUKI,2 Tadayuki IMANAKA,3 Masaaki MORIKAWA,2 and Shigenori KANAYA2

1Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 2Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan 3Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 686-8501, Japan

Received October 23, 2002; Accepted February 22, 2003
A biosurfactant-producing strain, Bacillus licheniformis F2.2, was isolated from a fermented food in Thailand. The strain was capable of producing a new biosurfactant, BL1193, as well as two kinds of popular lipopeptide biosurfactants, plipastatin and surfactin. Mass spectrometry and FT-IR analysis indicated that BL1193 had a molecular mass of 1,193 Da with no peptide portion in the molecule. While plipastatin and surfactin were abundantly produced in a nutrient YPD medium, BL1193 was produced only in a synthetic DF medium containing no amino acids. According to an oil displacement activity test, the specific activity of BL1193 (6.53 kBS units/mg) is equivalent to that of surfactin (5.78--6.83 kBS units/mg).
Key words: Bacillus licheniformis; biosurfactant(s); lipopeptide(s); plipastatin; surfactin

-7-
Accumulation of Hydroxycinnamic Acid Amides in Winter Wheat under Snow

Shigeki JIN,1,† Midori YOSHIDA,1 Takashi NAKAJIMA,2 and Akio MURAI3

1National Agricultural Research Center for Hokkaido Region, Toyohira-ku, Sapporo 062-8555, Japan 2National Agricultural Research Center for Kyushu Okinawa Region, Nishigoshi, Kumamoto 861-1192, Japan 3Division of Chemistry, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan

Received November 5, 2002; Accepted January 17, 2003
It was found that the content of antifungal compounds p-coumaroylagmatine [1-(trans-4′-hydroxycinnamoylamino)-4-guanidinobutane] and p-coumaroyl-3-hydroxyagmatine [1-(trans-4′-hydroxycinnamoylamino)-3-hydroxy-4-guanidinobutane] in the crown of winter wheat (Triticum aestivum L. cv Chihokukomugi) significantly increased under snow cover. This finding suggests that the accumulation of these hydroxycinnamic acid amides was caused by winter stress and related to protecting the plant against snow mold under snow cover.
Key words: Triticum aestivum; wheat; snow; p-coumaroylagmatine; p-coumaroyl-3-hydroxyagmatine

-8-
Polyphenols from Some Foodstuffs as Inhibitors of Ovalbumin Permeation through Caco-2 Cell Monolayers

Shoko KOBAYASHI,1, Jun WATANABE,2 Eri FUKUSHI,3 Jun KAWABATA,3 Mitsutoshi NAKAJIMA,4 and Michiko WATANABE1

1Department of Health and Nutrition, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan 2Food Function Division, National Food Research Institute, Tsukuba 305-8642, Japan 3Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan 4Food Engineering Division, National Food Research Institute, Tsukuba 305-8642, Japan

Received November 11, 2002; Accepted December 27, 2002
Some spices showed high inhibitory activity against ovalbumin permeation through Caco-2 cell monolayers. Pimentol from allspice, rosmarinic acid and luteolin-7-O-β-glucuronide from thyme, quercetin-3-O-β-glucuronide from coriander and rutin from tarragon were identified as the active principles. A structure-activity relationship study among the active isolates and their related compounds indicated that the presence of a catechol structure played an important role in the inhibitory activity of each compound.
Key words: pimentol; rosmarinic acid; luteolin-7-
O-β-glucuronide; quercetin-3-O-β-glucuronide; rutin

-9-
Effects of Dietary Eritadenine on the Liver Microsomal Δ6-Desaturase Activity and Its mRNA in Rats

Yasuhiko SHIMADA, Akihiro YAMAKAWA, Tatsuya MORITA, and Kimio SUGIYAMA

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan

Received November 11, 2002; Accepted January 10, 2003
Eritadenine, a hypocholesterolemic factor of Lentinus edodes mushroom, has a wide range of effects on lipid metabolism such as an increase in the liver microsomal phosphatidylethanolamine (PE) concentration, a decrease in the liver microsomal Δ6-desaturase activity, and an alteration of the fatty acid and molecular species profile of liver and plasma lipids. In this study, the time-dependent effects of dietary eritadenine on several variables concerning lipid metabolism were investigated in rats to clarify the sequence of metabolic changes caused by eritadenine, with special interest in the association of the liver microsomal phospholipid profile and the activity of Δ6-desaturase. The effect of dietary eritadenine on the abundance of mRNA for Δ6-desaturase was also investigated. When the time required for a half-change of variables was estimated during the first 5 days after the change from the control diet to the eritadenine-supplemented (50 mg/kg) diet, the change rates of the variables were fastest in the following order: alteration of the liver microsomal phospholipid profile＞decrease in liver microsomal Δ6-desaturase activity＞alteration of the fatty acid and molecular species profiles of microsomal and plasma phosphatidylcholine (PC)＞decrease in the plasma cholesterol concentration. There was a significant correlation between the Δ6-desaturase activity and liver microsomal PE concentration, but not PC concentration, or the proportion of PC and PE or the PC/PE ratio. The suppression of Δ6-desaturase activity by dietary eritadenine was accompanied by a significant reduction in the abundance of mRNA for the enzyme. These results suggest that dietary eritadenine might suppress the activity of liver microsomal Δ6-desaturase by altering the microsomal phospholipid profile, as represented by an increase in PE concentration, and that the effect of eritadenine is mediated by the regulation of gene expression.
Key words: eritadenine; Δ6-desaturase; mRNA; phosphatidylethanolamine; linoleic acid metabolism

-10-
Biogenesis of Volatile Methyl Esters in Snake Fruit (Salacca edulis, Reinw) cv. Pondoh

SUPRIYADI,1 Masayuki SUZUKI,1 Shuiqin WU,3 Naomi TOMITA,2 Akira FUJITA,2 and Naoharu WATANABE3,†

1The United Graduate School of Agriculture Sciences, Gifu University (Shizuoka University), 836 Ohya, Shizuoka 422-8529, Japan 2Technical Research Center, T. Hasegawa Co., Ltd., 335 Kariyado, Nakahara-ku, Kawasaki-shi 211-0022, Japan 3Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan

Received November 19, 2002; Accepted February 14, 2003
The methyl esters of carboxylic acids are characteristic olfactory volatile compounds for the sweet aroma of snake fruit, (Salacca edulis, Reinw) cv. Pondoh. Although methanol was not detected as a volatile constituent, the crude enzymes showed activity to synthesize the methyl esters in the presence of acyl-CoA and methanol. Therefore, the biosynthetic origin of methanol was investigated, resulting in the detection of pectin methyl transferase activity in the flesh. This pectin methyl transferase activity increased during fruit maturation, in parallel with the level of methanol originating from hand-squeezed juice and with the methyl esters extracted from flesh of the fruit. Based on these results, the origin of methanol was confirmed to be the methyl esters of pectins. The crude enzyme also catalyzed the formation of methyl hexanoate, one of the esters of the fruit, in the presence of methyl pectins and hexanoyl-CoA that were used as precursors for a model reaction.
Key words: snake fruit; Salacca edulis, Reinw, cultivar Pondoh; pectin methyl-esterase; methyl ester biogenesis

-11-
Insulin Stimulates Expression of the Pyruvate Kinase M Gene in 3T3-L1 Adipocytes

Yuuki ASAI,1 Kazuya YAMADA,2 Toyoaki WATANABE,1 Vincent W. KENG,1 and Tamio NOGUCHI1,

1Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Chikusa-ku, Nagoya 464-8601, Japan 2Department of Biochemistry, Fukui Medical University and CREST, Japan Science and Technology, Matsuoka, Fukui 910-1193, Japan

Received November 29, 2002; Accepted February 22, 2003
M2-type pyruvate kinase (M2-PK) mRNA is produced from the PKM gene by an alternative RNA splicing in adipocytes. We found that insulin increased the level of M2-PK mRNA in 3T3-L1 adipocytes in both time- and dose-dependent manners. This induction did not require the presence of glucose or glucosamine in the medium. The insulin effect was blocked by pharmacological inhibitors of insulin signaling pathways such as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase. A stable reporter expression assay showed that the promoter activity of an about 2.2-kb 5′-flanking region of the rat PKM gene was stimulated by insulin, but the extents of these stimulations were lower than those of the mRNA stimulation. Thus, we suggest that insulin increases the level of M2-PK mRNA in adipocytes by acting at transcriptional and post-transcriptional levels through signaling pathways involving both PI3K and MAPK kinase.
Key words: insulin; pyruvate kinase M gene; 3T3-L1 adipocytes; phosphatidylinositol 3-kinase; mitogen-activated protein kinase kinase

-12-
Angiotensin I-Converting Enzyme Inhibitory Peptides Isolated from Tofuyo Fermented Soybean Food

Megumi KUBA,1 Kumi TANAKA,1 Shinkichi TAWATA,1 Yasuhito TAKEDA,2 and Masaaki YASUDA1,

1Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan 2Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan

Received December 2, 2002; Accepted January 20, 2003
Angiotensin I-converting enzyme (ACE) inhibitory activity was observed in a tofuyo (fermented soybean food) extract with an IC50 value of 1.77 mg/ml. Two ACE inhibitors were isolated to homogeneity from the extract by adsorption and gel filtration column chromatography, and by reverse-phase high-performance liquid chromatography (HPLC). The purified substances reacted with 2,4,6-trinitrobenzensulfonic acid sodium salt. The amino acid sequences of these inhibitors determined by Edman degradation were Ile-Phe-Leu (IC50, 44.8 μM) and Trp-Leu (IC50, 29.9 μM). The Ile-Phe-Leu sequence is found in the α- and β-subunits of β-conglycinin, while the Trp-Leu sequence is in the B-, B1A- and BX-subunits of glycinin from soybean. Both of the peptides are non-competitive inhibitors. The inhibitory activity of Trp-Leu was completely preserved after a treatment with pepsin, chymotrypsin or trypsin. Even after successive digestion by these gastrointestinal proteases, the activity remained at 29％ of the original value.
Key words: angiotensin I-converting enzyme inhibitor; bioactive peptide; fermented soybean food; tofuyo

-13-
Effects of Oleamide on Choline Acetyltransferase and Cognitive Activities

Ho-Jin HEO,1 Young-June PARK,1 Young-Min SUH,1 Soo-Jung CHOI,1 Mi-Jeong KIM,1 Hong-Yon CHO,1 Yun-Jeong CHANG,1 Bumshik HONG,1 Hye-Kyung KIM,2 Eunki KIM,3 Chang-Ju KIM,4 Byung-Gee KIM,5 and Dong-Hoon SHIN1,

1Graduate School of Biotechnology, Korea University Seoul 136-701, Korea 2Department of Food and Biotechnology, Hanseo University Seosan 356-820, Korea 3Department of Biological Engineering, Inha University Inchon 402-751, Korea 4Department of Physiology, Kyung Hee University Seoul 130-701, Korea 5School of Chemical Engineering, and Institute for Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea

Received December 2, 2002; Accepted February 24, 2003
We screened 50 Korean traditional natural plants to measure the activation effect on choline acetyltransferase and attenuation of scopolamine-induced amnesia. The methanolic extracts from Zizyphus jujuba among the tested 50 plants, showed the highest activatory effect (34.1％) on choline acetyltransferase in vitro. By sequential fractionation of Zizyphus jujuba, the active component was finally identified as cis-9-octadecenoamide (oleamide). After isolation, oleamide showed a 65％ activation effect. Administration of oleamide (0.32％) to mice significantly reversed the scopolamine-induced memory and/or cognitive impairment in the passive avoidance test and Y-maze test. Injection of scopolamine to mice impaired performance on the passive avoidance test (31％ decrease in step-through latency), and on the Y-maze test (16％ decrease in alternation behavior). In contrast, mice treated with oleamide before scopolamine injection were protected from these changes (12--25％ decrease in step-through latency; 1--10％ decrease in alternation behavior). These results suggest that oleamide should be a useful chemo-preventive agent against Alzheimer′s disease.
Key words: choline acetyltransferase; Zizyphus jujuba; oleamide; Alzheimer′s disease; cognitive impairment

-14-
Hypolipidemic Effect of an Exo-biopolymer Produced from a Submerged Mycelial Culture of Hericium erinaceus

Byung-Keun YANG,1 Jun-Bo PARK,2 and Chi-Hyun SONG1,†

1Department of Biotechnology, Research Center for Processing ＆ Application of Agricultural Products, Daegu University, Kyungsan, Kyungbuk 712-714, South Korea 2Mega Biotech Co., Bonli-dong 1217-4, Dalseo-gu, Daegu 704-120, South Korea

Received December 16, 2002; Accepted February 21, 2003
The hypolipidemic effect of an exo-biopolymer produced from a submerged mycelial culture of Hericium erinaceus was investigated in dietary-induced hyperlipidemic rats. Hypolipidemic effects were proportionally increased with the increasing concentration of the exo-biopolymer for oral administration. The exo-biopolymer, at the dose of 200 mg/kg body weight, substantially reduced the plasma total cholesterol (32.9％), LDL cholesterol (45.4％), triglyceride (34.3％), phospholipid (18.9％), atherogenic index (58.7％), and hepatic HMG-CoA reductase activity (20.2％). It increased the plasma HDL cholesterol level (31.1％) as compared to the control group. The molecular mass of this exo-biopolymer measured by HPLC was under 40 kDa. Total sugar and protein contents were 91.2 and 8.8％, respectively. The sugar and amino acid compositions of the exo-biopolymer were analyzed in detail.
Key words: exo-biopolymer; HMG-CoA reductase; hypolipidemic effect; Hericium erinaceus; submerged mycelial culture

-15-
Identification of Suberization-associated Anionic Peroxidase as a Possible Allergenic Protein from Tomato

Thanakorn WEANGSRIPANAVAL,1 Noriko NOMURA,1 Tatsuya MORIYAMA,1 Nobuo OHTA,2 and Tadashi OGAWA1,

1Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 2Ohta Pediatric and Allergy Clinic, Kasuga-Cho, Takamatsu 761-0101, Japan

Received December 19, 2002; Accepted March 4, 2003
A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.
Key words: allergen; tomato; IgE; suberization-associated anionic peroxidase 1; pathogenesis-related protein

-16-
Antihypertensive Effects of Onion on NO Synthase Inhibitor-induced Hypertensive Rats and Spontaneously Hypertensive Rats

Yoko SAKAI,1 Tetsuo MURAKAMI,2 and Yukiko YAMAMOTO1,

1Graduate School of Human Life Science, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan 2Faculty of Agriculture, Kinki University, Nakamachi 3327-204, Nara 631-8505, Japan

Received December 19, 2002; Accepted February 26, 2003
This study was designed to show the effects of onion on blood pressure in NG-nitro-L-arginine methyl ester (L-NAME) induced-hypertensive rats and stroke prone spontaneously hypertensive rats (SHRSP) using dried onion at 5％ in their diets. For the experiment with L-NAME induced-hypertensive rats, male 6-weeks-old Sprague-Dawley rats were given tap water containing L-NAME to deliver 50 mg/kg BW/day. In this experiment, we found distinct antihypertensive effects of onion on the L-NAME induced-hypertensive rats and the SHRSP. Dietary onion decreased the thiobarbituric acid reactive substances (TBARS) in plasma in these hypertensive rats. Also, onion increased the nitrate/nitrite (products of nitric oxide (NO)) excreted in urine and the NO synthase (NOS) activity in the kidneys in SHRSP. These results suggested that the increased NO caused by the greater NOS activity, and additionally by the increased saving of NO by the antioxidative activity of onion, was one of the cause of the antihypertensive effect of onion in SHRSP. In the L-NAME induced hypertensive rats, onion did not significantly block the inhibition of NOS activity by L-NAME, and decreased nitrate/nitrite excretion in urine was not restored. The mechanism of the antihypertensive effect of onion probably involves increased saving of NO by antioxidative activity of onion in L-NAME induced-hypertensive rats.
Key words: hypertension; onion; NO synthase inhibitor; SHRSP; antioxidant

-17-
Effect of Neuronal PC12 Cells on the Functional Properties of Intestinal Epithelial Caco-2 Cells

Hideo SATSU, Tatsuya YOKOYAMA, Nobumasa OGAWA, Yoko FUJIWARA-HATANO, and Makoto SHIMIZU

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received December 20, 2002; Accepted January 25, 2003
The effect of neuronal cells on the functional properties of intestinal epithelial cells was examined by using an in vitro coculture system. Two cell lines, Caco-2 and PC12, were respectively used as intestinal epithelial and enteric neuronal cell models. Coculture of differentiated Caco-2 cells with PC12 caused a significant decrease in the transepithelial electrical resistance (TER) value of the Caco-2 monolayer. The permeability to lucifer yellow (LY) was also significantly increased, suggesting that the tight junction (TJ) of the Caco-2 monolayers was modulated by coculturing with PC12. To identify the TJ-modulating factor presumably secreted from PC12, the effects of the major neurotransmitters on the TER value and LY transport were examined, but no influence was apparent. The TJ-modulating effect of PC12 was prevented by exposing PC12 to cycloheximide, suggesting that new protein synthesis in PC12 was necessary for this regulation.
Key words: intestinal epithelial cell; Caco-2; PC12; coculture; tight junction

-18-
Cloning, Sequencing, and Heterologous Expression of a Cellobiohydrolase cDNA from the Basidiomycete Corticium rolfsii

Daisuke YASOKAWA, Takeshi SHIMIZU, Ryoji NAKAGAWA, Takayuki IKEDA, and Koji NAGASHIMA

Section of Biotechnology, Department of Applied Technology, Hokkaido Food Processing Research Center, 589-4 Bunkyodai Midorimachi, Ebetsu, Hokkaido 069-0836, Japan

Received December 24, 2002; Accepted March 12, 2003
From a Corticium rolfsii cDNA library, a clone homologous to other fungal cellobiohydrolase (CBH1) genes was isolated using the polymerase chain reaction. In the nucleotide sequence, one 1.6 kb long open reading frame coding for a polypeptide of 530 amino acid residues was detected which showed 64％ identity with CBH1 of Phanerochaete chrysosporium. With expression of the 1.8 kb cDNA using the Aspergillus oryzae expression system, we detected microcrystalline cellulose (Avicel) hydrolyzing activity in the culture supernatant. The secreted protein, accompanied by the activity, was 89 kDa by SDS-polyacrylamide gel electrophoresis.
Key words: cDNA; sequencing; expression; cellobiohydrolase

-19-
A Novel Enzyme of Bacillus sp. 217C-11 That Produces Inulin from Sucrose

Tadashi WADA, Masao OHGUCHI, and Yoshio IWAI

Fuji Nihon Seito Corporation, 1-4-10 Seikai, Shimizu City, Shizuoka 424-8737, Japan

Received December 24, 2002; Accepted February 25, 2003
We found a bacterium that converts sucrose to a useful material, using about 6,000 samples of bacteria isolated from soil. This bacterium, Bacillus sp. 217C-11, was identified according to Bergey′s manual, and produced a highly efficient enzyme that converted sucrose into inulin. So, the enzyme was purified to homogeneity through five chromatographic steps, to identify its enzymatic properties. The molecular mass of the enzyme was estimated to be 45,000, and this enzyme was a monomer protein (by SDS-PAGE). The optimum pH and temperature of this enzyme were 7--8 and 45--50°C, respectively. The enzyme reacted only with sucrose, but did not with other disaccharides, fructooligosaccharides and inulin. This paper will show that our enzyme is a novel one, which is different from the other well-known enzymes concerned in inulin production.
Key words: inulin; sucrose; fructosyltransferase; inulin-producing enzyme (IPE); Bacillus sp. 217-11

-20-
Identification by the Phage-display Technique of Peptides That Bind to H7 Flagellin of Escherichia coli

Teruhiko IDE,1, Sang-Ho BAIK,1 Takao MATSUBA,2 and Shigeaki HARAYAMA1

1Marine Biotechnology Institute, Heita, Kamaishi, Iwate 026-0001, Japan 2Tosoh Corporation, Tokyo Laboratories, Hayakawa, Ayaseshi, Kanagawa 252-1123, Japan

Received January 6, 2003; Accepted February 24, 2003
The four peptides interacting with H7 flagellin of Escherichia coli were selected from a phage display library. The library was selected four times, and the interacting phage peptides were competitively eluted with H7 flagellin. An enzyme-linked immunosorbent assay (ELISA) showed that these peptides were reactive with the H7 flagellin in a dose-dependent manner. Among them, a D1 phage clone showed the highest binding affinity to the H7 flagellin. We synthesized the D1 peptide (LHIHRPTLSIQG) corresponding to the peptide-encoding region of the D1 phage clone. The synthetic peptide showed micro-molar affinity (EC50 value＝1.9 μM) for the H7 flagellin. Furthermore, this D1 peptide interacted more specifically with the H7 flagellin than with the other flagellins (H1, H5, H12, or H23) of E. coli. In situ hybridization clearly showed that the peptide only detected those cells harboring the H7 flagellin gene (fliC). The peptide may specifically bind to the H7 flagellin on the cell surface. These results suggest that the phage-display technique could be used as a tool for identifying peptides as an alternative to using a ligand as a diagnostic reagent in food products or in clinical testing.
Key words: H7 flagellin; flagella; antigen; phage display

-21-
Simultaneous Measurement of Fluoroquinolones in Eggs by a Combination of Supercritical Fluid Extraction and High Pressure Liquid Chromatography

Jae Han SHIM,1,† Mi Hyun LEE,1 Mi Ra KIM,1 Chang Joo LEE,2 and In Seon KIM3

1Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, College of Agriculture and Life Science, Chonnam National University, Gwangju 500-757, South Korea 2Department of Civil and Environmental Engineering, Kwangju University, Gwangju 503-703, South Korea 3Department of Environmental Science and Engineering, Kwangju Institute of Science and Technology (K-JIST), Gwangju 500-712, South Korea

Received January 7, 2003; Accepted February 17, 2003
Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO2 alone, and then the fluoroquinolones were extracted by SFE with supercritical CO2 containing 20％ (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96％ in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r2 value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.
Key words: antibiotic; fluoroquinolones; high pressure liquid chromatography; supercritical fluid extraction

-22-
Purification and Characterization of Laminaran Hydrolases from Trichoderma viride

Rika NOBE,1 Yoichi SAKAKIBARA,1 Nobuhiro FUKUDA,1 Naoto YOSHIDA,1 Kihachiro OGAWA,2 and Masahito SUIKO1,

1Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan 2Department of Food Technology, Faculty of Horticulture, Minami Kyushu University, 11609 Minamitakanabe, Takanabe 884-0003, Japan

Received January 16, 2003; Accepted March 15, 2003
At least three extracellular laminaran hydrolases which hydrolyzed laminaran (β-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a β-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for β-1,3-linkages, but lam B was specific for β-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.
Key words: Trichoderma viride; β-1,3-glucanase; β-1,6-glucanase; extracellular enzyme; Eisenia bicyclis

-23-
Molecular and Immunochemical Characteristics of Monoclonal and Recombinant Antibodies Specific to Bisphenol A

Kosuke NISHI,1 Mikio TAKAI,1 Kosuke MORIMUNE,2 and Hideo OHKAWA1,†

1Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan 2Naruto Research Center, Otsuka Chemical Co., Ltd., 615 Hanamen, Satoura-cho, Naruto, Tokushima 772-0021, Japan

Received January 16, 2003; Accepted February 25, 2003
Four anti-bisphenol A monoclonal antibodies (mabs) were obtained and each characterized by an enzyme-linked immunosorbent assay (ELISA). Among these mabs, BBA-2187 was the most reactive towards bisphenol A. The quantitation limit of the ELISA assay for bisphenol A was 0.13 ng/ml, which is more sensitive than the other immunoassays reported. Then, the cDNA clones encoding variable heavy and variable light chains of these four mabs were isolated, and used for construction of four single-chain Fv (scFv) antibody genes, which were expressed in Escherichia coli cells. The reactivity of four scFv antibodies towards bisphenol A in ELISA was comparable to those of the parent mabs. The most sensitive assay was achieved with BBA-2187scFv. Its cross-reactivity to the related compounds was similar to that of the parent mab. Based on the reactivity of heterologous combinations of VH and VL fragments, it was found that the unique structure of the framework region 2 in the VL of BBA-2187 appeared to be important for specific assembly together with the VH.
Key words: bisphenol A; monoclonal antibody; single-chain Fv (scFv); enzyme-linked immunosorbent assay (ELISA); immunoassay

-24-
Preparation and Characterization of Recombinant Murine p65/L-Plastin Expressed in Escherichia coli and High-titer Antibodies against the Protein

Hiroto SHINOMIYA,1,† Kozo NAGAI,1 Hajime HIRATA,4 Naoto KOBAYASHI,2 Hitoshi HASEGAWA,3 Fengzhi LIU,1 Kohsuke SUMITA,1 and Yoshihiro ASANO1

Departments of 1Immunology and Host Defenses, 2Anatomy and Embryology, and 3Internal Medicine I, Ehime University School of Medicine, Ehime 791-0295, Japan 4Department of Life Science, Himeji Institute of Technology, Hyogo 678-1297, Japan

Received January 20, 2003; Accepted March 7, 2003
We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca2＋-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to β-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.
Key words: p65/L-plastin; actin-binding protein; macrophage; phosphorylation; cancer

-25-
Preparation of a Water-in-oil-in-water (W/O/W) Type Microcapsules by a Single-droplet-drying Method and Change in Encapsulation Efficiency of a Hydrophilic Substance during Storage

Shuji ADACHI, Hanaho IMAOKA, Yuri HASEGAWA, and Ryuichi MATSUNO

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received January 24, 2003; Accepted March 6, 2003
Microcapsules of a water-in-oil-in-water (W/O/W) emulsion, which contained a hydrophilic substance, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTSA), in its inner aqueous phase, was prepared by hot-air-drying or freeze-drying the emulsion using a single-droplet-drying method. Pullulan, maltodextrin, or gum arabic was used as a wall material, and the oily phase was tricaprylin, oleic acid, olive oil, or a mixture of tricaprylin and olive oil. An encapsulation efficiency higher than 0.95 was reached except for the microcapsules prepared using gum arabic and oleic acid. The hot-air-dried microcapsules were generally more stable than the freeze-dried microcapsules at 37°C and various relative humidities. The stability was higher for the microcapsules with tricaprylin as the oily phase than for the microcapsules with oleic acid. The higher stability of the microcapsules with tricaprylin would be ascribed to the lower partition coefficient of PTSA to the oily phase. There was a tendency for the stability to be higher at lower relative humidity for both the hot-air- and freeze-dried microcapsules. The volumetric fraction of olive oil in its mixture with tricaprylin did not significantly affect either the encapsulation efficiency or the stability of the hot-air-dried microcapsules.
Key words: microcapsule; microencapsulation; water-in-oil-in-water (W/O/W) emulsion; encapsulation efficiency

-26-
Light-responsive psbA Transcription Requires the −35 Hexamer in the Promoter and Its Proximal Upstream Element, UPE, in Cyanobacteria

Yoko ITO, Munehiko ASAYAMA, and Makoto SHIRAI

Laboratory of Molecular Genetics, School of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki 300-0393, Japan

Received February 17, 2003; Accepted March 17, 2003
We characterized the function of the −35 hexamer in the promoter and an element just upstream, UPE, in the expression in a unicellular cyanobacterium, Microcystis aeruginosa K-81, of the light-responsive gene psbA2, which encodes a reaction center key protein for photosynthesis. A series of mutants with mutations at the −35 hexamer (−35 to −30) and a novel conserved upstream element (UPE: −45 to −36, ＋1 referring to the transcription start point) were constructed. Expression of the mutants was examined in vivo and in vitro by analyses using a β-galactosidase assay, primer extension, and a DNA-mobility shift assay with RNA polymerases. Results indicated that the −35 hexamer and its proximal UPE act as effective cis-elements for the light-responsive and/or basal transcription, respectively. A model of the 5′-upstream region with cis- and possible trans-acting factors is presented for the psbA regulatory system.
Key words: transcription; RNA polymerase; sigma factor; curved DNA; photosynthesis

-27-
Note
Inhibition of Linoleic Acid Hydroperoxide-induced Toxicity in Cultured Human Fibroblasts by Anthocyanidins

Takao KANEKO,1,† Shoichi TAHARA,1 and Naomichi BABA2

1Redox Regulation Research Group, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan 2Faculty of Agriculture, Okayama University, 1-1-1 Tsushimanaka, Okayamashi, Okayama 700-0082, Japan

Received July 24, 2002; Accepted February 22, 2003
The protective effect of anthocyanidins against the toxicity induced by linoleic acid hydroperoxide (LOOH) was examined in cultured human fetal lung fibroblasts, TIG-7. Cyanidin was more effective than pelargonidin or delphinidin in inhibiting LOOH-induced cytotoxicity. The presence of a catechol moiety in the B ring is shown to be important for the protective activities against the cytotoxicity of LOOH.
Key words: anthocyanidin; cytotoxicity; linoleic acid hydroperoxide; lipid peroxide; human fibroblasts

-28-
Note
A Common Structure of Substrate Shared by Lignostilbenedioxygenase Isozymes from Sphingomonas paucimobilis TMY1009

Shigehiro KAMODA,2,† Tamami TERADA,1 and Yoshimasa SABURI1

1Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan 2The University Forest in Hokkaido, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yamabe, Furano-shi, Hokkaido 079-1561, Japan

Received August 7, 2002; Accepted March 15, 2003
A common structure of substrates of lignostilbenedioxygenases was investigated using synthesized stilbenes. Cell-free extracts of Sphingomonas paucimobilis TMY1009 degraded only trans-4-hydroxystilbene and trans-4-hydroxy-3-methoxystilbene. Other stilbenes that had no 4-hydroxyl group and had a cis structure were not substrates for lignostilbenedioxygenases. These results indicate that a 4-hydroxyl group and trans-structure is necessary for the common structure for substrates of lignostilbenedioxygenases.
Key words: lignostilbenedioxygenase; lignin; stilbene; Sphingomonas; Pseudomonas

-29-
Note
Molecular Cloning of Quinohemoprotein Alcohol Dehydrogenase, ADH IIB, from Pseudomonas putida HK5

Hirohide TOYAMA, Takaaki FUJII, Nahoko AOKI, Kazunobu MATSUSHITA, and Osao ADACHI

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

Received October 30, 2002; Accepted February 25, 2003
Molecular cloning of the gene of quinohemoprotein alcohol dehydrogenase (ADH IIB) from Pseudomonas putida HK5 was done. The gene (qbdA) was 690 amino acids in length, containing a 22-amino acid signal sequence. Another gene (qbdB) upstream of qbdA, probably in the same transcriptional unit, was found. Further upstream, a gene divergently transcribed against qbdBA had identity with NAD-dependent aldehyde dehydrogenase.
Key words: quinoprotein; quinohemoprotein; pyrroloquinoline quinone; PQQ; alcohol dehydrogenase

-30-
Note
Cytotoxic Screening of Medicinal and Edible Plants in Okinawa, Japan, and Identification of the Main Toxic Constituent of Rhodea japonica (Omoto)

Toshiya MASUDA,1,3, Yasuo OYAMA,1 Natsuko YAMAMOTO,1 Chisato UMEBAYASHI,1 Hiromi NAKAO,1 Yukiko TOI,1 Yoshio TAKEDA,1 Katsuo NAKAMOTO,2 Hideki KUNINAGA,2 Yukari NISHIZATO,2 and Akira NONAKA2

1Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan 2Nakazen Inc., Chinen, Okinawa 901-1513, Japan 3Tropical Biosphere Research Center, University of Ryukyus, Taketomi, Okinawa 907-1441, Japan

Received November 25, 2002; Accepted January 20, 2003
The cytotoxic activity of ethanol extracts from 53 parts of 36 species of medicinal and edible plants cultivated in Okinawa was measured by using K562 human leukemia cells by a flow cytometric method. Two extracts from Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 μg/ml. The main cytotoxic constituent of Rhodea japonica was isolated and identified to be rhodexin A, which has been isolated as a cardetonic agent of the plant. The IC50 value for rhodexin A against the growth of K562 cells was 19 nM, this activity being much stronger than that of ouabain (IC50, 60 nM).
Key words: cytotoxic activity; edible and medicinal plant; Okinawa; Rhodea japonica; rhodexin A

-31-
Note
A T42M Substitution in Bacterial 5-Enolpyruvylshikimate-3-phosphate Synthase (EPSPS) Generates Enzymes with Increased Resistance to Glyphosate

Ming HE, Yan-Fang NIE, and Peilin XU

Biotechnology Research Center, The Key Laboratory of Gene Engineering of Education Ministry, Zhongshan University, Guangzhou, 510275, The People′s Republic of China

Received November 26, 2002; Accepted February 12, 2003
Mutants of class I enolpyruvylshikimate 3-phosphate synthase (EPSPS) with resistance to glyphosate were produced in a previous study using the staggered extension process with aroA genes from S. typhimurium and E. coli. Two of these mutants shared a common amino acid substitution, T42M, near the hinge region between the large globular domains of EPSPS. Using site-directed mutagenisis, we produced the T42M mutants without the other amino acid changes of the original mutants. The T42M substitution alone produced enzymes with a 9- to 25-fold decreased Km[PEP] and a 21- to 26-fold increased Ki[glyphosate] compared to the wild-type enzymes. These results provide more testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design.
Key words: bacterial aroA; glyphosate; mutagenesis; herbicide; 5-enolpyruvylshkimate-3-phosphate synthase (EPSPS)

-32-
Note
Three New Cytotoxic Acylspermidines from the Soft Coral, Sinularia sp.

Makoto OJIKA,1,† Mohammad Kamrul ISLAM,2 Tomoaki SHINTANI,2 Yang ZHANG,2 Tetsuji OKAMOTO,2 and Youji SAKAGAMI1

1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan 2Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan

Received November 28, 2002; Accepted January 11, 2003
Three new acylated spermidines and two known related compounds were isolated from the Okinawan soft coral, Sinularia sp. These compounds were N′,N″,N″-trimethylspermidines that are acylated by a methyl-branched unsaturated fatty acid. These acylspermidines showed potent cytotoxicity against human tumor cell lines at an IC50 value of 17 ng/ml and the induction of apoptotic phenomena.
Key words: spermidine; Sinularia; marine natural products; cytotoxicity; apoptosis

-33-
Note
βg-Dehydrocurvularin and Related Compounds as Nematicides of Pratylenchus penetrans from the Fungus Aspergillus sp.

Miyako KUSANO,1 Kazumi NAKAGAMI,2 Shozo FUJIOKA,3 Tsuyoshi KAWANO,2 Atsumi SHIMADA,4 and Yasuo KIMURA2,

1Umea Plant Science Centre, Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, SE-901 83 Umea, Sweden 2Department of Biological and Environmental Chemistry, Faculty of Agriculture, Tottori University, Tottori-shi 680-8553, Japan 3The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan 4Department of Environmental Chemistry, Faculty of Engineering, Kyushu Kyoritsu University, Jiyugaoka, Yahatanishi-ku, Kitakyushu-shi 807-8585, Japan

Received November 28, 2002; Accepted January 18, 2003
The new nematicidal compound, βg-dehydrocurvularin (1), together with three known compounds, αb-dehydrocurvularin (2), 8-β-hydroxy-7-oxocurvularin (3) and 7-oxocurvularin (4), were isolated from the culture filtrate and mycelial mats of Aspergillus sp. The structures of 1--4 were established by spectroscopic methods including 2D NMR. The biological activities of 1--4 were examined by bioassays with root-lesion nematodes, and lettuce and rice seedlings.
Key words: βγ-dehydrocurvularin; nematicide; root-lesion nematode; Pratylenchus penetrans

-34-
Note
Purification and Properties of a Carbonyl Reductase Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate from Cylindrocarpon sclerotigenum IFO 31855

Yuri SARATANI,1 Eiji UHEDA,1 Hiroaki YAMAMOTO,2 Atsuo NISHIMURA,1 and Fumiki YOSHIZAKO1,†,

1Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan 2Tsukuba Research Center, Daicel Chemical Industries Ltd., 27 Miyukigaoka, Tsukuba-City, Ibaraki 305-0841, Japan

Received November 29, 2002; Accepted February 24, 2003
A NADPH-dependent carbonyl reductase (CSCR1) was purified to homogeneity from Cylindrocarpon sclerotigenum IFO 31855. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a ＞99％ enantiomer excess. The relative molecular mass of the enzyme was estimated to be 68,000 by gel filtration chromatography and 24,800 on SDS polyacrylamide gel electrophoresis. The enzyme had an extremely narrow substrate specificity and it highly reduced conjugated diketone, 2,3-butanedion, in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by HgCl2 (100％), 5,5′-dithiobis(2-nitrobenzoic acid) (56％), dicoumarol (42％), and CuSO4 (46％). The N-terminal amino acid sequence of the enzyme (P-Q-G-I-P-T-A-S-R-L) showed no apparent similarity with those of other oxidoreductases.
Key words: carbonyl reductase; Cylindrocarpon sclerotigenum IFO 31855; (S)-ethyl 4-chloro-3-hydroxybutanoate

-35-
Note
Antidiabetic Effect of Lactobacillus GG in Streptozotocin-induced Diabetic Rats

Mihoko TABUCHI,1, Miyo OZAKI,1 Asako TAMURA,1 Noriko YAMADA,1 Tetsuo ISHIDA,1 Masataka HOSODA,2 and Akiyoshi HOSONO3

1Yonezawa Women′s College of Yamagata Prefecture, Yonezawa 992-0025, Japan 2Technical Research Laboratory, Takanashi Milk Products Co., Ltd., Yokohama 241-0023, Japan 3Graduate School of Agriculture, Shinshu University, Nagano-Minamiminowa 399-4598, Japan

Received December 9, 2002; Accepted March 3, 2003
Neonatally streptozotocin-induced diabetic (n-STZ) rats were given food containing Lactobacillus GG cells (GG) or a control diet (control), from 9 to 18 weeks of age. The GG cells significantly lowered the blood hemoglobin A1C (HbA1C) level and improved glucose tolerance in n-STZ rats (p＜0.05). In the GG group, the serum insulin level at 30 min after glucose loading was significantly higher than in the control group (p＜0.05).
Key words: Lactobacillus GG; antidiabetic effect; glucose tolerance; HbA1C; n-STZ rats

-36-
Note
Effects of Carrageenans on the Binding, Phagocytotic, and Killing Abilities of Macrophages to Salmonella

Yoshiko SUGITA-KONISHI,1 Shusaku YAMASHITA,2 Fumio AMANO,3, and Makoto SHIMIZU2,†

1Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 2Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 3Department of Biochemistry and Cell Biochemistry, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

Received December 9, 2002; Accepted March 4, 2003
The effects of carrageenans (CGNs) on the host defense mechanisms of macrophages against Salmonella infection were examined in vitro by using macrophage-like J774.1 cells. Iota-CGN reduced the Salmonella-binding and phagocytotic activities of J774.1 cells, but it increased the killing activity of the cells. Kappa-CGN increased the binding activity, but reduced the killing ability. CGNs would affect the host defense mechanisms by modulating the macrophage functions.
Key words: carrageenan; Salmonella infection; host defense; macrophage, J774.1 cells

-37-
Note
Purification of Conjugated Linoleic Acid Isomers through a Process Including Lipase-catalyzed Selective Esterification

Toshihiro NAGAO,1 Yoshie YAMAUCHI-SATO,2 Akio SUGIHARA,3 Toshio IWATA,2 Koji NAGAO,4 Teruyoshi YANAGITA,4 Shuji ADACHI,5 and Yuji SHIMADA1,†

1Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan 2Rinoru Oil Mills Co., Ltd., 2-13-12 Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan 3Faculty of Engineering, Tokushima Bunri University, 1314-1 Sido, Sanuki, Kagawa 769-2101, Japan 4Department of Applied Biological Sciences, Saga University, 1 Honjo-machi, Saga 840-8502, Japan 5Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received December 16, 2002; Accepted February 24, 2003
A mixture of conjugated linoleic acids (CLAs) was prepared by alkali conjugation of high purity linoleic acid. The preparation contained 45.1 wt％ cis-9, trans-11 (c9,t11)-CLA, 46.8 wt％ trans-10, cis-12 (t10,c12)-CLA, and 5.3 wt％ other CLAs. A process comprising Candida rugosa lipase-catalyzed selective esterification with lauryl alcohol, molecular distillation, and urea adduct fractionation under strict conditions in ethanol was very effective for purification of c9,t11- and t10,c12-CLAs. In particular, the urea adduct fractionation efficiently eliminated CLAs except c9,t11- and t10,c12-isomers. Purification of c9,t11- and t10,c12-CLAs from 1.0 kg of the CLA mixture increased the c9,t11-CLA purity to 93.1％ with 34％ recovery of the initial content, and increased the t10,c12-CLA purity to 95.3％ with 31％ recovery.
Key words: conjugated linoleic acid; Candida rugosa lipase; esterification; urea adduct fractionation; molecular distillation

-38-
Note
Cloning and Overexpression of the avi2 Gene Encoding a Major Cellulase Produced by Humicola insolens FERM BP-5977

Tatsuki MORIYA, Manabu WATANABE, Naomi SUMIDA, Kaoru OKAKURA, and Takeshi MURAKAMI

Microbiological Resources and Technology Laboratories, Meiji Seika Kaisya, Ltd., 788 Kayama, Odawara-shi 250-0852, Japan

Received December 20, 2002; Accepted March 15, 2003
The avi2 gene encoding Avi2, which is a major cellulase produced by Humicola insolens FERM BP-5977, was cloned and sequenced. Avi2 showed high homology with other family 6 cellulases. The expression vector pNCE4 containing the avi2 gene was constructed, and this strain was transformed using a protoplast method. As a result, the pNCE4 transformant secreted 8-fold more Avi2 than the recipient strain.
Key words: cellulase; Humicola insolens; avi2; protoplast

-39-
Note
Structural Revision of Epoxyrollins A and B, Biosynthetic Precursors of Annonaceous Acetogenins

Hiroyuki KONNO1, and Hidefumi MAKABE2

1Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan 2Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan

Received December 26, 2002; Accepted January 30, 2003
Chemical structural elucidation of epoxyrollins A and B, representatives of biosynthetic precursors of tetrahydrofuran and tetrahydropyran annonaceous acetogenins, is described. These chemical structures were diepoxyreticanin 1 (4) and diepoxymuricanin A (6) by a 13C-NMR and mass spectral study of the natural and synthetic sample data.
Key words: natural product; mass spectrum; annonaceous acetogenin