Contents and Abstracts of BBB

(Vol.68 No.9 2004)

Prooxidative Activities of Tea Catechins in the Presence of Cu2+
Fumiko Hayakawa1, Yoko Ishizu1, Nobuo Hoshino2, Akira Yamaji2, Takashi Ando3, and Takahide Kimura3 p.1825

Influence of the Physical Form of Processed Rice Products on the Enzymatic Hydrolysis of Rice Starch in Vitro and on the Postprandial Glucose and Insulin Responses in Patients with Type 2 Diabetes Mellitus
Jae Cherl Kim1,2, Jung-In Kim1,3, Byoung-Wook Kong1, Min-Jung Kang1, Myo-Jeong Kim1,3, and In-June Cha4 p.1831

Umbelliferone Released from Hairy Root Cultures of Pharbitis nil Treated with Copper Sulfate and Its Subsequent Glucosylation
Sayaka Yaoya1, Hideki Kanho1, Youji Mikami1, Tomio Itani1, Kaoru Umehara2, and Masanori Kuroyanagi1 p.1837

Fragrances in Oolong Tea That Enhance the Response of GABAA Receptors
Sheikh Julfikar Hossain1, Hitoshi Aoshima1, Hirofumi Koda2, and Yoshinobu Kiso2
p.1842

Isolation of a Novel Promoter for Efficient Protein Production in Aspergillus oryzae
Hiroki Ishida1, Yoji Hata1, Akitsugu Kawato1, Yasuhisa Abe1, and Yutaka Kashiwagi2
p.1849

Importance of the B Ring and Its Substitution on the ±-Glucosidase Inhibitory Activity of Baicalein, 5,6,7-Trihydroxyflavone
Hong Gao and Jun Kawabata p.1858

Four Rice Genes Encoding NADP Malic Enzyme Exhibit Distinct Expression Profiles
Wei Chi, Jianghua Yang, Naihu Wu, and Fang Zhang p.1865

Thermal Behavior of Fowl Feather Keratin
Koji Takahashi1, Hirosaburo Yamamoto1, Yoshiko Yokote2, and Makoto Hattori1 p.1875

Comparative Effect of Repeated Ingestion of Difructose Anhydride III and Palatinose on the Induction of Gastrointestinal Symptoms in Humans
Akiko Tamura1,2, Takuya Shiomi1, Noriko Tamaki1, Norihiro Shigematsu1, Fusao Tomita2, and Hiroshi Hara2 p.1882

Purification and Characterization of Glutamine Synthetase of Pseudomonas taetrolens Y-30: An Enzyme Usable for Production of Theanine by Coupling with the Alcoholic Fermentation System of Bakers Yeast
Sachiko Yamamoto, Kousuke Uchimura, Mamoru Wakayama, and Takashi Tachiki p.1888

Target Genes of the Developmental Regulator PRIB of the Mushroom Lentinula edodes
Yasumasa Miyazaki, Yuta Sakuragi, Takashi Yamazaki, and Kazuo Shishido p.1898

Mode of AmyR Binding to the CGGN8AGG Sequence in the Aspergillus oryzae taaG2 Promoter
Tatsuo Ito, Shuji Tani, Tomoyuki Itoh, Norihiro Tsukagoshi, Masashi Kato, and Tetsuo Kobayashi p.1906

Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products
Kazumi Funane1, Tadashi Ishii2, Kazue Terasawa1, Tomoko Yamamoto1, and Mikihiko Kobayashi3 p.1912

Purification and Characterization of Aromatic Amine Dehydrogenase from Alcaligenes xylosoxidans
Tetsuya Kondo1, Emi Kondo2, Hitomi Maki2, Kyoden Yasumoto2, Kazuyoshi Takagi3, Kenji Kano4, and Tokuji Ikeda4 p.1921

A Soluble Flavonoid-glycoside, ±G-Rutin, Is Absorbed as Glycosides in the Isolated Gastric and Intestinal Mucosa
Megumi Matsumoto1,2, Noriko Matsukawa2, Hitoshi Mineo2, Hideyuki Chiji2, and Hiroshi Hara1 p.1929

Molecular Characterization of Two Highly Homologous Receptor-like Kinase Genes, RLK902 and RKL1, in Arabidopsis thaliana
Yoshiaki Tarutani1, Takashi Morimoto1, Akiko Sasaki1, Michiko Yasuda2, Hideo Nakashita2, Shigeo Yoshida2, Isomaro Yamaguchi1, and Yoshihito Suzuki1 p.1935

Functional Expression and Characterization of a Bacterial Light-harvesting Membrane Protein in Escherichia coli and Cell-free Synthesis Systems
Yuichiro Shimada, Zheng-Yu Wang, Yushi Mochizuki, Masayuki Kobayashi, and Tsunenori Nozawa p.1942

Characterization of Starch Synthase I and II Expressed in Early Developing Seeds of Kidney Bean (Phaseolus vulgaris L.)
Takeshi Senoura, Naoto Isono, Motoyo Yoshikawa, Ayako Asao, Shigeki Hamada, Kenji Watanabe, Hiroyuki Ito, and Hirokazu Matsui p.1949

First Synthesis of (±)-Robinlin
Hirosato Takikawa, Shintaro Hosoe, Keiko Ueda, and Mitsuru Sasaki p.1961

Rapid Response of Arabidopsis T87 Cultured Cells to Cytokinin through His-to-Asp Phosphorelay Signal Transduction
Hisami Yamada, Nobuya Koizumi, Norihito Nakamichi, Takatoshi Kiba, Takafumi Yamashino, and Takeshi Mizuno p.1966

Note
Effects of Green Tea Polyphenol on Cognitive and Acetylcholinesterase Activities
Hye Kyung Kim1, Mija Kim2, Sunki Kim3, Mujo Kim4, and Joo Ho Chung5 p.1977

Note
Improved Bile Acid-binding Ability of Soybean Glycinin A1a Polypeptide by the Introduction of a Bile Acid-binding Peptide (VAWWMY)
Seon-kang Choi, Motoyasu Adachi, and Shigeru Utsumi p.1980

Note
Tyrosinase Inhibitor Isolated from the Leaves of Zanthoxylum piperitum
Chang Ho Jeong and Ki Hwan Shim p.1984

Note
Biosynthetic Pathway for the C45 Polyprenol, Solanesol, in Tobacco
Ei-ichiro Fukusaki1, Shinya Takeno1, Takeshi Bamba1, Hiroshi Okumoto2, Hiroko Katto2, Shin-ichiro Kajiyama1, and Akio Kobayashi1 p.1988

Note
Soybean Glycinin A1aB1b Subunit Has a Molecular Chaperone-like Function to Assist Folding of the Other Subunit Having Low Folding Ability
Seon-kang Choi1, Motoyasu Adachi1, Masaaki Yoshikawa2, Nobuyuki Maruyama1, and Shigeru Utsumi1 p.1991

Note
Synthesis of Microfolicoumarin, a Constituent of Cedrelopsis microfoliata
Marellapudi S. Ramachandra, Somepalli Venkateswarlu, and Gottumukkala V. Subbaraju]
p.1995

Note
The Reversible Change in the Redox State of Type I Cu in Myrothecium verrucaria Bilirubin Oxidase Depending on pH
Giorgio Zoppellaro1, Nobuhiko Sakurai2, Kunishige Kataoka1, and Takeshi Sakurai1
p.1998

Communication
Diterpene Cyclases Responsible for the Biosynthesis of Phytoalexins, Momilactones A, B, and Oryzalexins AF in Rice
Kazuko Otomo1, Yuri Kanno1, Akihiro Motegi1, Hiromichi Kenmoku1, Hisakazu Yamane2, Wataru Mitsuhashi1, Hideaki Oikawa3, Hiroaki Toshima4, Hironori Itoh5, Makoto Matsuoka5, Takeshi Sassa1, and Tomonobu Toyomasu1 p.2001

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Prooxidative Activities of Tea Catechins in the Presence of Cu2+

Fumiko Hayakawa1, Yoko Ishizu1, Nobuo Hoshino2, Akira Yamaji2, Takashi Ando3, and Takahide Kimura3

1School of Human Cultures, The University of Shiga Prefecture, Hassaka-cho, Hikone, Shiga 522-8533, Japan
2Department of Pharmacy, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan
3Department of Chemistry, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan

The ability of various tea catechins to generate H2O2 and the hydroxyl radical in the presence of the Cu2+ ion was investigated and compared with the effect of iron ions. The presence of Cu2+ accelerated the generation of H2O2 by EGC, while EGCg with Cu2+ generated a little H2O2. The presence of iron ions inhibited the generation of H2O2 by EGC. EGC and EC with Cu2+ generated the hydroxyl radical, while EGCg and ECg with Cu2+ did not. The fact that EGCg showed less prooxidative activity than EGC can be explained by the chelating ability of catechin gallates to metal ions under the experimental conditions.

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Influence of the Physical Form of Processed Rice Products on the Enzymatic Hydrolysis of Rice Starch in Vitro and on the Postprandial Glucose and Insulin Responses in Patients with Type 2 Diabetes Mellitus

Jae Cherl Kim1,2, Jung-In Kim1,3, Byoung-Wook Kong1, Min-Jung Kang1, Myo-Jeong Kim1,3, and In-June Cha4

1Food Science Institute and School of Food and Life Science, Inje University, Gimhae 621-749, Republic of Korea
2Institute of Basic Sciences, Inje University, Gimhae 621-749, Republic of Korea
3Biohealth Products Research Center, Inje University, Gimhae 621-749, Republic of Korea
4Department of Pharmacology, Inje University, and College of Medicine and Clinical Pharmacology Center, Pusan Paik Hospital, Busan 614-165, Republic of Korea

The manufacturing processes used determined the physicochemical properties of the three kinds of rice food, garaeduk, bagsulgi, and cooked rice. The initial rate of hydrolysis by porcine pancreatic ±-amylase (PPA) was affected by the food form. The firmer structure of garaeduk was apparently responsible for the difficulty in maceration, resulting in less digestion than with easily digestible food for the same maceration time. The initial rate of hydrolysis of each rice product by PPA increased with increasing maceration time in a Waring Blender for all of the processed rice products. The postprandial glucose and insulin responses to the three processed rice products were also studied in ten patients with type 2 diabetes mellitus (4 men and 6 women aged 56.8 ± 2.3 yr; duration of diabetes, 3.6 ± 1.2 yr; body mass index (BMI), 23.7 ± 2.6 kg/m2; fasting serum glucose, 143.9 ± 5.1 mg/dl; serum insulin, 20.8 ± 2.2 ¼U/ml). Each subject ingested of the three rice foods after a 12-h overnight fast, and the serum glucose and insulin levels were measured over a 0240 min period. The postprandial serum glucose and insulin levels at 90 min after ingesting bagsulgi and cooked rice were less than those at 60 min, while the levels at 90 min after ingesting garaeduk were higher than those at 60 min. Garaeduk also significantly decreased the incremental responses of glucose and insulin when compared with bagsulgi and cooked rice. The results suggest that garaeduk would be the most unlikely to increase the postprandial serum glucose and insulin levels among the three rice foods. The food form, which eventually differentiated each food by its specific surface area with the same degree of maceration because of the characteristic physical strength, therefore affected the rate of rice starch hydrolysis both in vitro and in vivo.

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Umbelliferone Released from Hairy Root Cultures of Pharbitis nil Treated with Copper Sulfate and Its Subsequent Glucosylation

Sayaka Yaoya1, Hideki Kanho1, Youji Mikami1, Tomio Itani1, Kaoru Umehara2, and Masanori Kuroyanagi1

1School of Bioresources, Hiroshima Prefectural University, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan
2School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a ²-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin.

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Fragrances in Oolong Tea That Enhance the Response of GABAA Receptors

Sheikh Julfikar Hossain1, Hitoshi Aoshima1, Hirofumi Koda2, and Yoshinobu Kiso2

1Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, Yoshida, Yamaguchi 753-8512, Japan
2Institute for Health Care Science, Suntory Limited, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan

We electrophysiologically investigated the effect of some fragrant compounds in oolong tea on the response of ionotropic ³-aminobutyric acid (GABA) receptors (GABAA receptors) which were expressed in Xenopus oocytes. Of the tested fragrances in oolong tea, cis-jasmone, jasmine lactone, linalool oxide and methyl jasmonate significantly potentiated the response. Among these, cis-jasmone and methyl jasmonate potently potentiated the response, having a respective dissociation constant of the compound (Kp) and maximum potentiation (Vm) of 0.49 mM and 322% for cis-jasmone, and 0.84 mM and 450% for methyl jasmonate. Inhalation of 0.1% cis-jasmone or methyl jasmonate significantly increased the sleeping time of mice induced by pentobarbital, suggesting that these fragrant compounds were absorbed by the brain and thereby potentiated the GABAA receptor response. Both of these compounds may therefore have a tranquillizing effect on the brain.

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Isolation of a Novel Promoter for Efficient Protein Production in Aspergillus oryzae

Hiroki Ishida1, Yoji Hata1, Akitsugu Kawato1, Yasuhisa Abe1, and Yutaka Kashiwagi2

1Research Institute, Gekkeikan Sake Co., Ltd., 300 Katahara-cho, Fushimi-ku, Kyoto 612-8361, Japan
2National Food Research Institute, 2-1-12 Kanondai, Tsukuba, Ibaraki 305-8642, Japan

A novel protein overexpression system of Aspergillus oryzae was constructed. Five promoters which originate from A. oryzae expressed sequence tag (EST) clones in submerged culture were obtained by genome walking. These were subjected to ²-glucuronidase (GUS) reporter assays. The promoter of manganese superoxide dismutase-encoding gene (sodM) showed the most GUS production. The sodM gene was abundantly expressed in submerged culture but little expressed in solid-state culture. The sodM promoter was approximately 3-fold induced by the addition of 0.01% H2O2. Glucoamylase production in A. oryzae using the sodM promoter led to secretion of approximately 1 g/l-broth in Czapek-Dox medium for 3 d. Fucose lectin production in A. oryzae using the sodM promoter led to overexpression as a specific and abundant intracellular protein.

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Importance of the B Ring and Its Substitution on the ±-Glucosidase Inhibitory Activity of Baicalein, 5,6,7-Trihydroxyflavone

Hong Gao and Jun Kawabata

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

Hydroxychromones and B-ring-substituted 5,6,7-trihydroxyflavones were prepared to evaluate the contribution of the B ring of baicalein (5,6,7-trihydroxyflavone, 1) to its potent ±-glucosidase inhibitory activity. Hydroxychromones, which lack 6-hydroxyl substitution, did not show any inhibitory activity, while 5,6,7-trihydroxy-2-methylchromone (5) showed high activity. Among the tested B-ring-substituted 5,6,7-trihydroxyflavones, the 4-hydroxy-, 3,4-dihydroxy-, and 3,4,5-trihydroxy-substituted derivatives were found to give more activity than that of 1. The methoxy-substituted derivatives, however, showed less activity than 1. The results suggest that the B ring of 1 was not essential, although advantageous to the activity; hydroxyl substitution on the B ring of 5,6,7-trihydroxyflavones was favorable to the activity, whereas methoxyl substitution was unfavorable; at least 4-hydroxyl substitution of 5,6,7-trihydroxyflavones was required for enhanced activity, in which the number of hydroxyl groups did not take part.

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Four Rice Genes Encoding NADP Malic Enzyme Exhibit Distinct Expression Profiles

Wei Chi, Jianghua Yang, Naihu Wu, and Fang Zhang

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 3 Nanyitiao, Zhongguancun, Haidian District, Beijing 100080, Peoples Republic of China

In plants, the NADP malic enzymes (NADP-MEs) are encoded by small gene families. These NADP-ME gene families are relatively well described in C4 plants but not well studied in C3 plants. In this study, we investigated the NADP-ME gene family in a model C3 monocot plant (rice, Oryza sativa) based on its recently released genomic DNA sequence. We found that the rice NADP-ME family is composed of four members, one plastidic NADP-ME and three cytosolic versions. Although the rice NADP-ME genes identified share a high degree of similarity with one another, one cytosolic NADP-ME (OscytME3) contains several unique amino acid substitutions within highly conserved amino acid regions. Phylogenetic analysis showed that OscytME3 might be derived from a different evolutionary branch than the other three rice genes. Expression analysis of the four rice NADP-ME genes indicated that each had a different tissue-specific and developmental profile, although all four responded to stress stimuli.

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Thermal Behavior of Fowl Feather Keratin

Koji Takahashi1, Hirosaburo Yamamoto1, Yoshiko Yokote2, and Makoto Hattori1

1Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
2Department of Chemistry, Faculty of Science, Josai University, Saitama 350-0248, Japan

Differential scanning calorimetry (DSC) was applied to elucidate the thermal behavior of fowl feather keratins (barbs, rachis, and calamus) with different morphological features. The DSC curves exhibited a clear and relatively large endothermic peak at about 110160 °C in the wet condition. A considerable decrease in transition temperature with urea and its helical structure content estimated by Fourier transform infrared spectroscopy (FT-IR), and the disappearance of one of the diffraction peaks with heating at 160 °C for 30 min, indicated that DSC could be used to evaluate the thermal behavior of keratin. Barbs showed a lower denaturation temperature than rachis and calamus. The pulverized samples showed a slightly higher denaturation temperature than the native samples. In the dry condition, thermal transition occurred in a markedly higher temperature region close to 170200 °C. It is hence concluded that fowl feather keratins have very high thermal stability, and that the elimination of water brings about even greater thermal stability.

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Comparative Effect of Repeated Ingestion of Difructose Anhydride III and Palatinose on the Induction of Gastrointestinal Symptoms in Humans

Akiko Tamura1,2, Takuya Shiomi1, Noriko Tamaki1, Norihiro Shigematsu1, Fusao Tomita2, and Hiroshi Hara2

1Central Research Laboratory, FANCL Co., Ltd., 12-13 Kamishinano, Totsuka-ku, Yokohama 244-0806, Japan
2Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Nishi 9, Kita 9, Kita-ku, Sapporo 060-8589, Japan

We evaluated the safety and change in fermentability from repeated ingestion of difructose anhydride III (DFAIII) in humans. A randomized controlled single-blind crossover study with thirteen subjects was conducted. Each subject ingested 5 g of DFAIII or palatinose daily for 12 days, before and after which the subject was loaded with 10 g of DFAIII and had breath hydrogen measured from 0 to 9 h (DL test) to evaluate the fermentability of DFAIII. The defecation frequency and abdominal symptom score were the same between each ingestion period. Moreover, DFAIII ingestion had no influence on blood test results. Only the breath hydrogen excretion in post-DFAIII ingestion was slightly higher at h 8 than the pre-ingestion. Consequently, repeated ingestion of DFAIII for 12 days was as safe as palatinose ingestion, especially with respect to abdominal symptoms and blood test results, and its high resistance to enterobacterial fermentation in humans was not impaired.

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Purification and Characterization of Glutamine Synthetase of Pseudomonas taetrolens Y-30: An Enzyme Usable for Production of Theanine by Coupling with the Alcoholic Fermentation System of Bakers Yeast

Sachiko Yamamoto, Kousuke Uchimura, Mamoru Wakayama, and Takashi Tachiki

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

Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed ³-glutamylethylamide (theanine) from glutamic acid and ethylamine in a mixture containing the alcoholic fermentation system of bakers yeast for ATP-regeneration. Glutamine synthetase (GS), probably responsible for theanine formation, was isolated from the extract of the organism grown on a medium containing 1% methylamine, 1% glycerol, 0.5% yeast extract, and 0.2% polypepton as carbon and nitrogen sources. The molecular mass was estimated to be 660 kDa by gel filtration and 55 kDa by SDS-polyacrylamide gel electrophoresis, suggesting that Ps. taetrolens Y-30 GS consists of 12 identical subunits. The enzyme required Mg2+ or Mn2+ for its activity. Under the standard reaction condition for glutamine formation (pH 8.0 with 30 mM Mg2+), GS showed 7% and 1% reactivity toward methylamine and ethylamine respectively of that to ammonia. Reactivity to the alkylamines varied with optimum pH of the reaction in response to divalent cation in the mixture: pH 11.0 was the optimum for the Mg2+-dependent reaction with ethylamine, and pH 8.5 was the optimum for the Mn2+-dependent reaction. In a mixture of an optimum reaction condition with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn2+), reactivity increased up to 7% of the reactivity to ammonia in the standard reaction condition. The isolated GS formed theanine in the mixture with the yeast fermentation system.

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Target Genes of the Developmental Regulator PRIB of the Mushroom Lentinula edodes

Yasumasa Miyazaki, Yuta Sakuragi, Takashi Yamazaki, and Kazuo Shishido

Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

Using the method of genomic binding-site cloning, we identified three target genes of the developmental regulator, the product of priB gene (PRIB) in Lentinula edodes: the previously cloned priB and uck1 (UMP-CMP kinase gene) and a new gene, which we named mfbC. Identification of the former two genes was expected, because the promoter regions of priB and the gene encoding UMP-CMP kinase (uck1) have been shown to contain four or two consensus-like sequences of PRIB binding respectively. The mfbC gene contained two consensus-like sequences of PRIB binding in its promoter region and the PRIB protein bound them. The deduced 330 amino acid sequence of the product of mfbC gene (MFBC) was highly homologous to the 325 amino acid sequence of S. cerevisiae YJR070C/Lia1, the protein interacting with a putative translation initiation factor. Only the mature fruiting body of L. edodes was shown to contain the transcript of the mfbC gene almost exclusively, suggesting that mfbC may play a role in the final stage of fruiting-body formation.

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Mode of AmyR Binding to the CGGN8AGG Sequence in the Aspergillus oryzae taaG2 Promoter

Tatsuo Ito, Shuji Tani, Tomoyuki Itoh, Norihiro Tsukagoshi, Masashi Kato, and Tetsuo Kobayashi

Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

AmyR is a transcriptional activator in Aspergillus spp. necessary for induction of the amylolytic enzyme genes. It recognizes 5-CGGN8CGG-3 conserved in a number of the amylolytic gene promoters, and in addition 5-CGGAAATTTAA-3 in the A. oryzae ±-amylase promoter. In this report, interaction of AmyR with the 5-CGGAAATTTAA-3 type binding site in the Taka-amylase gene (taaG2) promoter was precisely characterized by DNase I footprinting analysis and electrophoretic mobility shift assay in vitro, and also by examination of the in vivo activity of the mutated promoters. The in vitro and in vivo analyses indicated that two AmyR molecules bind cooperatively to the 5-CGGAAATTTAA-3 sequence by recognizing the CGG triplet at the 5-end and the AGG triplet just downstream of the sequence.

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Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

Kazumi Funane1, Tadashi Ishii2, Kazue Terasawa1, Tomoko Yamamoto1, and Mikihiko Kobayashi3

1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2Wood Biochemistry Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-0903, Japan
3National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making ±-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.

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Purification and Characterization of Aromatic Amine Dehydrogenase from Alcaligenes xylosoxidans

Tetsuya Kondo1, Emi Kondo2, Hitomi Maki2, Kyoden Yasumoto2, Kazuyoshi Takagi3, Kenji Kano4, and Tokuji Ikeda4

1Food Research Center, Aichi Industrial Technology Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji-cho, Nishi-ku, Nagoya 451-0083, Japan
2Department of Food and Nutrition, School of Life Studies, Sugiyama Jogakuen University, Chikusa-ku, Nagoya 464-8662, Japan
3Department of Applied Chemistry, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
4Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on ²-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (±2²2) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the ±-subunit (42.3-kDa subunit) and the ²-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the ²-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and ²-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 °C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, ²-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 ¼M respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 °C for one month at least in phosphate buffer (pH 7.0).

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A Soluble Flavonoid-glycoside, ±G-Rutin, Is Absorbed as Glycosides in the Isolated Gastric and Intestinal Mucosa

Megumi Matsumoto1,2, Noriko Matsukawa2, Hitoshi Mineo2, Hideyuki Chiji2, and Hiroshi Hara1

1Laboratory of Nutritional Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan
2Department of Food Science and Human Nutrition, Faculty of Human Life Science, Fuji Womens University, Ishikari, Hokkaido 061-3204, Japan

We investigated the absorption and metabolism of the highly soluble quercetin glycoside ±G-rutin, a glucose adduct of insoluble rutin, using the isolated mucosa of the rat stomach and intestines equipped with the Ussing chamber. ±G-rutin and rutin appeared in the serosal sides of the gastric body and all the intestinal mucosa after the addition of ±G-rutin (1 mM) to the mucosal fluid. The degree of ±G-rutin appearance was much lower in the gastric fundus than in the other parts. Quercetin was not found in the mucosal fluid of any mucosal specimen. The concentrations (¼M) of ±G-rutin and rutin in the serosal fluid as a result of transport from the mucosal side increased time-dependently and linearly with mucosal ±G-rutin concentration (1, 10 or 100 mM). The highest transport was shown in the ileal mucosa. These results indicate that ±G-rutin is partly hydrolyzed to rutin through the intestine and absorbed as such.

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Molecular Characterization of Two Highly Homologous Receptor-like Kinase Genes, RLK902 and RKL1, in Arabidopsis thaliana

Yoshiaki Tarutani1, Takashi Morimoto1, Akiko Sasaki1, Michiko Yasuda2, Hideo Nakashita2, Shigeo Yoshida2, Isomaro Yamaguchi1, and Yoshihito Suzuki1

1Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Plant Functions Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Receptor-like kinases (RLKs) constitute a large family of signal perception molecules. We characterized two highly homologous RLK genes, RLK902 and RKL1, in Arabidopsis. RLK902 and RKL1 showed a 75% amino acid sequence identity over their entire regions. In the RLK902 pro::GUS transgenic lines, GUS activity was strong in the root tips, lateral root primordia, stipules, and floral organ abscission zones, while the RKL1 promoter activity was dominant in the stomata cells, hydathodes and trichomes of young rosette leaves, and floral organ abscission zones. Neither the rlk902 mutant line, rkl1 mutant line nor rlk902/rkl1 double-knockout mutant line showed any significant phenotypes under normal growth conditions. These results suggest that RLK902 and RKL1 might mediate the signal transduction pathway in which at least one other complementary signaling pathway to these two RLKs might exist.

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Functional Expression and Characterization of a Bacterial Light-harvesting Membrane Protein in Escherichia coli and Cell-free Synthesis Systems

Yuichiro Shimada, Zheng-Yu Wang, Yushi Mochizuki, Masayuki Kobayashi, and Tsunenori Nozawa

Department of Biomolecular Engineering, Graduate School of Engineering, Center for Interdisciplinary Research, Tohoku University, Sendai 980-8579, Japan

Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E. coli extracts. The ±-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His6) tag added to the carboxyl terminus. Both of the expression systems produced ±-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native ²-apoprotein and the pigment molecule bacteriochlorophyll a. The expression product in E. coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100. Circular dichroism measurement indicated that the expressed ±-apoproteins from both systems had ±-helical contents essentially identical with that of the native one. About two thirds of the ±-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the ±-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue. Functional expression of the ±-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis.

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Characterization of Starch Synthase I and II Expressed in Early Developing Seeds of Kidney Bean (Phaseolus vulgaris L.)

Takeshi Senoura, Naoto Isono, Motoyo Yoshikawa, Ayako Asao, Shigeki Hamada, Kenji Watanabe, Hiroyuki Ito, and Hirokazu Matsui

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

Plant starch synthase (SS) contributes to the elongation of glucan chains during starch biosynthesis and hence plays an essential role in determining the fine structure of amylopectin. To elucidate the role of SS activity in the formation of amylopectin in kidney bean (Phaseolus vulgaris L.), a study was undertaken to isolate cDNA clones for SS and to characterize the enzymatic properties of the coded recombinant enzymes. Two SS cDNAs, designated pvss1 and pvss21, which were isolated from early developing seeds, encoded SSI and SSII (designated PvSSI and PvSSII-1) that displayed significant identity (more than 65%) with other SSI and SSII members, respectively. RNA gel blot analysis indicated that both transcripts accumulate in leaves and developing seeds at the early stage. Immunoblot analysis with antisera raised against both recombinant proteins (rPvSSI and rPvSSII-1) showed that the accumulation of both proteins parallels the gene expression profiles, although both were detectable only in starch-granule fractions. Recombinant enzymes expressed by Escherichia coli cells showed distinct chain-length specificities for the extension of glucan chains. Our results suggest that these SS isozymes for synthesis of transitory starch are also responsible for synthesis of storage starch in early developing seeds of kidney bean.

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First Synthesis of (±)-Robinlin

Hirosato Takikawa, Shintaro Hosoe, Keiko Ueda, and Mitsuru Sasaki

Department of Biosystems Science, Graduate School of Science and Technology, Kobe University, Rokkodai 1-1, Nada-ku, Kobe 657-8501, Japan

The first synthesis of (±)-robinlin (1), a novel homo-monoterpene with strong bioactivity in the brine shrimp lethality test, was achieved by starting from 3-isobutyloxy-2,6,6-trimethyl-2-cyclohexen-1-one (2).

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Rapid Response of Arabidopsis T87 Cultured Cells to Cytokinin through His-to-Asp Phosphorelay Signal Transduction

Hisami Yamada, Nobuya Koizumi, Norihito Nakamichi, Takatoshi Kiba, Takafumi Yamashino, and Takeshi Mizuno

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

According to the current consistent model for the higher plant Arabidopsis thaliana, the scheme for an immediate early response to the plant hormone cytokinin can be formulated as Arabidopsis histidine kinase (AHK) cytokinin receptor-mediated His ’ Asp phosphorelay signal transduction. Nonetheless, clarification of the comprehensive picture of cytokinin-mediated signal transduction in this higher plant is at a very early stage. As a new approach to this end, we studied whether or not a certain Arabidopsis cell line (named T87) would be versatile for such work on cytokinin signal transduction. We show that T87 cells had the ability to respond to cytokinin, displaying the immediate early induction of type-A Arabidopsis response regulator (ARR) family genes (e.g., ARR6) at the transcriptional level. This event was further confirmed by employing the stable transgenic lines of T87 cells with a set of ARR::LUC reporter transgenes. We also show that T87 cells had the ability to respond to auxin when the expression of a set of AUX/IAA genes (e.g., IAA5) was examined. As postulated for intact plants, in T87 cells too, the induction of IAA5 by auxin was selectively inhibited in the presence of a proteasome inhibitor, while the induction of ARR6 by cytokinin was not significantly affected under the same conditions. Through transient expression assays with T87 protoplasts, it is shown that the intracellular localization profiles of the phosphorelay intermediate Arabidopsis histidine-containing phosphotransfer factor (AHPs; e.g., AHP1 and AHP4) were markedly affected in response to cytokinin, but those of type-A ARRs were not (e.g., ARR15 and ARR16). Taken together, we conclude that, in T87 cells, the AHK-dependent His \to Asp phosphorelay circuitry appears to be propagated in response to cytokinin, as in the case of plants, as far as the immediate early responses were concerned. This cultured cell system might therefore provide us with an alternative means to further characterize the mechanisms underlying cytokinin (and also auxin) responses at the molecular level.

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Note
Effects of Green Tea Polyphenol on Cognitive and Acetylcholinesterase Activities

Hye Kyung Kim1, Mija Kim2, Sunki Kim3, Mujo Kim4, and Joo Ho Chung5

1Department of Food and Biotechnology, Hanseo University, Seosan 356-706, Korea
2Graduate School of Obesity Science, Dongduk Womens University, Seoul 136-714, Korea
3Doosan Biotechnology, BU, Sooji, Yonginsi, Kyunggido, 449-844, Korea
4Pharma Foods International, Minami-ku, Kyoto 601-8357, Japan
5Department of Pharmacology, College of Medicine, Kyung Hee University, Seoul 130-701, Korea

The effect of tea polyphenol (TP) on cognitive and anti-cholinesterase activity was examined in scopolamine-treated mice. Chronic administration of TP significantly reversed scopolamine-induced retention deficits in both step-through passive avoidance and spontaneous alternation behavior tasks. Furthermore, TP exhibited a dramatic inhibitory effect on acetylcholinesterase activity. This finding suggests that TP might be useful in the treatment of Alzheimers disease.

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Note
Improved Bile Acid-binding Ability of Soybean Glycinin A1a Polypeptide by the Introduction of a Bile Acid-binding Peptide (VAWWMY)

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

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 23952401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.

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Note
Tyrosinase Inhibitor Isolated from the Leaves of Zanthoxylum piperitum

Chang Ho Jeong and Ki Hwan Shim

Division of Applied Life Science, Graduate School and Institute of Agricultural Life Sciences, Gyeongsang National University, JinJu 660-701, Korea

Two flavonols, quercetin (1) and quercitrin (2), were isolated from the leaves of Zanthoxylum piperitum. Their structures were established by UV, one- and two-dimensional NMR, and mass spectroscopic methods. Quercetin showed significant inhibition against mushroom tyrosinase with an IC50 value of 3.8 ¼g/ml, and appeared to inhibit the polyphenol oxidase activity of tyrosinase in a competitive manner (Ki = 10 ± 0.20 ¼M) when L-tyrosine was used as a substrate, although it did not inhibit the melanin production of Streptomyces bikiniensis.

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Note
Biosynthetic Pathway for the C45 Polyprenol, Solanesol, in Tobacco

Ei-ichiro Fukusaki1, Shinya Takeno1, Takeshi Bamba1, Hiroshi Okumoto2, Hiroko Katto2, Shinichiro Kajiyama1, and Akio Kobayashi1

1Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2Department of Chemistry and Bioscience, Kurashiki University of Science and the Arts, 2640 Nishinoura, Tsurajima, Kurashiki 712-8505, Japan

Feeding experiments were independently performed with [1-13C]deoxy-D-xylulose triacetate and (RS)-[2-13C]mevalonolactone in the tobacco plant. The labeling pattern for solanesol was elucidated to reveal that the isoprene moiety of solanesol would be derived from deoxy-xylulose. The result strongly suggests that tobacco solanesol is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

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Note
Soybean Glycinin A1aB1b Subunit Has a Molecular Chaperone-like Function to Assist Folding of the Other Subunit Having Low Folding Ability

Seon-kang Choi1, Motoyasu Adachi1, Masaaki Yoshikawa2, Nobuyuki Maruyama1, and Shigeru Utsumi1

1Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan
2Laboratory of Physiological Function of Food, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.

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Note
Synthesis of Microfolicoumarin, a Constituent of Cedrelopsis microfoliata

Marellapudi S. Ramachandra, Somepalli Venkateswarlu, and Gottumukkala V. Subbaraju

Laila Impex R & D Centre, Unit 1, Phase III, Jawahar Autonagar, Vijayawada 520 007, India

Microfolicoumarin (1), a prenylcoumarin from Cedrelopsis microfoliata, was synthesized from 2,4,5-trimethoxybenzaldehyde in five steps. 1 did not show significant antioxidative activity, but the key intermediates, esculetin (3) and 5-prenylesculetin (6), exhibited strong antioxidative activity in both the superoxide-radical and 1,1-diphenyl-2-picrylhydrazyl radical-scavenging models.

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Note
The Reversible Change in the Redox State of Type I Cu in Myrothecium verrucaria Bilirubin Oxidase Depending on pH

Giorgio Zoppellaro1, Nobuhiko Sakurai2, Kunishige Kataoka1, and Takeshi Sakurai1

1Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan
2Institute of Natural Sciences, Nagoya City University, Yamanohata 1, Mizuho, Nagoya 467-8501, Japan

The redox state of type I Cu in Myrothecium verrucaria bilirubin oxidase (BO), a multicopper oxidase utilized in the clinical investigation of liver, is an equilibrium state of the oxidized and reduced forms, reflected in the reversible absorption and electron paramagnetic resonance (EPR) spectral changes depending on pH.

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Communication
Diterpene Cyclases Responsible for the Biosynthesis of Phytoalexins, Momilactones A, B, and Oryzalexins AF in Rice

Kazuko Otomo1, Yuri Kanno1, Akihiro Motegi1, Hiromichi Kenmoku1, Hisakazu Yamane2, Wataru Mitsuhashi1, Hideaki Oikawa3, Hiroaki Toshima4, Hironori Itoh5, Makoto Matsuoka5, Takeshi Sassa1, and Tomonobu Toyomasu1

1Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan
2Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
3Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-8589, Japan
4Department of Bioresource Science, College of Agriculture, Ibaraki University, Inashiki, Ibaraki 300-0393, Japan
5Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan

Rice (Oryza sativa L.) produces diterpene phytoalexins, such as momilactones, oryzalexins, and phytocassanes. Using rice genome information and in vitro assay with recombinant enzymes, we identified genes (OsKS4 and OsKS10) encoding the type-A diterpene cyclases 9²-pimara-7,15-diene synthase and ent-sandaracopimaradiene synthase which are involved in the biosynthesis of momilactones A, B and oryzalexins AF respectively. Transcript levels of these two genes increased remarkably after ultraviolet (UV) treatment, which is consistent with elevated production of phytoalexins by UV. These two genes might prove powerful tools for understanding plant defense mechanisms in rice.