(Vol.68 No.6 2004)
Review
Plant Acetyl-CoA Carboxylase: Structure, Biosynthesis, Regulation, and Gene
Manipulation for Plant Breeding
Yukiko Sasaki1,† and Yukio Nagano2 p.1175
Potent Hypocholesterolemic Activity of the Yeast Kluyveromyces
marxianus YIT 8292 in Rats Fed a High Cholesterol Diet
Yasuto Yoshida,† Wakae Yokoi, Yasue Wada, Kenji Ohishi, Masahiko Ito, and Haruji
Sawada p.1185
Agrobacterium-mediated Transformation of Javanica Rice cv. Rojolele
Diah Rachmawati,1,2 Takehiko Hosaka,1 Eiichi Inoue,1 and Hiroyuki Anzai1,†
p.1193
Analysis of Differentially Regulated Proteins in TM4 Cells Treated
with Bisphenol A
Do-Youn Lee, Sang-Soo Lee, Won-A Joo, Eun-Ju Lee, and Chan-Wha Kim† p.1210
A Novel Genetic System for Analysis of Co-activators for the N-Terminal
Transactivation Function Domain of the Human Androgen Receptor
Ken-ichi Takeyama,1,2 Saya Ito,1 Shun Sawatsubashi,1 Yuko Shirode,1,2 Ayako
Yamamoto,1 Eriko Suzuki,1 Akio Maki,1 Kaoru Yamagata,1,2 Yue Zhao,1 Alexandre
Kouzmenko,1,2 Tetsuya Tabata,1 and Shigeaki Kato1,2,† p.1209
ATP Amplification for Ultrasensitive Bioluminescence Assay: Detection
of a Single Bacterial Cell
Tetsuya Satoh,1 Junichi Kato,1 Noboru Takiguchi,1 Hisao Ohtake,1 and Akio Kuroda1,2,†
p.1216
Effects of Alcoholic Solvents on Antiradical Abilities of Protocatechuic
Acid and Its Alkyl Esters
Shizuka Saito, Yasuko Okamoto, and Jun Kawabata† p.1221
Influence of Low-temperature Processing of the Brackish-water Bivalve,
Corbicula japonica, on the Ornithine Content of Its Extract
Hidemitsu Uchisawa,1,5,† Arata Sato,2 Junji Ichita,3 Hajime Matsue,4 and Tomotada
Ono5 p.1228
Molecular Cloning of a Cysteine Proteinase cDNA from the Cotton
Boll Weevil Anthonomus grandis (Coleoptera: Curculionidae)
Osmundo Brilhante de oliveira neto,1,2 João Aguiar Nogueira Batista,1 Daniel
John Rigden,1 Octávio Luiz Franco,1,3 Rodrigo Rocha Fragoso,1,2 Ana Carolina
Santos Monteiro,1 Rose Gomes Monnerat,1 and Maria Fátima Grossi-de-sá1,† p.1235
Accumulation and Degradation of Thiamin-binding Protein and Level
of Thiamin in Wheat Seeds during Seed Maturation and Germination
Katsumi Watanabe,† Naoko Nishida, Takashi Adachi, Motoko Ueda, Toshio Mitsunaga,
and Yukio Kawamura p.1243
Biphenyl-inducible Promoters in a Polychlorinated Biphenyl-degrading
Bacterium, Rhodococcus sp. RHA1
Hisashi Takeda, Naho Hara, Masayuki Sakai,* Akihiro Yamada, Keisuke Miyauchi,
Eiji Masai, and Masao Fukuda† p.1249
Synthesis of (3R,4S)-3,4,5-Trihydroxy-4-methylpentylphosphonic
Acid as a Potential Inhibitor of the Nonmevalonate Pathway
Toshiaki Yoshioka, Hiroki Shimizu, and Hiroshi Ohrui† p.1259
Cell-lytic Activity of Tobacco BY-2 Induced by a Fungal Elicitor
from Alternaria alternata Attributed to the Expression of a Class I β-1,3-Glucanase
Gene
Tomonori Shinya, Shinobu Gondo, Hiroshi Iijima, Kazunari Hanai, Hideaki Matsuoka,
and Mikako Saito† p.1265
Structural and Functional Properties of Chicken Lysozyme Fused
Serine-rich Heptapeptides at the C-Terminus
Xiaohua Xu, Orie Kashima, Akira Saito, Hiroyuki Azakami, and Akio Kato†
p.1273
Multigene Family for Bowman–Birk Type Proteinase Inhibitors of
Wild Soja and Soybean: The Presence of Two BBI-A Genes and Pseudogenes
Masanobu Deshimaru,† Shingo Yoshimi, Seijiro Shioi, and Shigeyuki Terada
p.1279
Synthesis, Absolute Configuration and Biological Activities of
Both Enantiomers of 2-(5,7-Dichloro-3-indolyl)propionic Acid: a Novel Dichloroindole
Auxin and Antiauxin
Masato Katayama,1,† Yasuhito Kato,2 and Shingo Marumo3 p.1287
Fluorometric Study of the Acid-induced Denaturation of Staphylococcal
Nuclease and Its Mutant Forms
Akiyoshi Tanaka† p.1293
A Chitinase Indispensable for Formation of Protoplast of Schizophyllum
commune in Basidiomycete-lytic Enzyme Preparation Produced by Bacillus circulans
KA-304
Shigekazu Yano, Nopakarn Rattanakit, Mamoru Wakayama, and Takashi Tachiki†
p.1299
Molecular Characterization of an Intracellular β-N-Acetylglucosaminidase
Involved in the Chitin Degradation System of Streptomyces thermoviolaceus OPC-520
Takahiro Kubota, Katsushiro Miyamoto, Masahide Yasuda, Yoshihiko Inamori, and
Hiroshi Tsujibo† p.1306
cDNA Microarray Analysis of Rice Anther Genes under Chilling Stress
at the Microsporogenesis Stage Revealed Two Genes with DNA Transposon Castaway
in the 5’-Flanking Region
Tomoya Yamaguchi,1,† Katsuhiro Nakayama,1 Takami Hayashi,1 Junshi Yazaki,2 Naoki
Kishimoto,2 Shoshi Kikuchi,2 and Setsuo Koike1 p.1315
Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the
Fenton Reaction
Shin-ichi Ishikawa,† Yuki Yano, Keizo Arihara, and Makoto Itoh p.1324
Short-step Synthesis of Chenodiol from Stigmasterol
Toru Uekawa, Ken Ishigami, and Takeshi Kitahara† p.1332
Kinetic Analysis of a Chitinase from Red Sea Bream, Pagrus major
Shuji Karasuda,1 Kosuke Yamamoto,1 Michiko Kono,2 Shohei Sakuda,3 and Daizo
Koga1,† p.1338
Identification and Cloning of the Gene Involved in the Final Step
of Chlortetracycline Biosynthesis in Streptomyces aureofaciens
Tetsuo Nakano,1,† Koichiro Miyake,1 Hirofumi Endo,1 Tohru Dairi,2 Toru Mizukami,1
and Ryoichi Katsumata3 p.1345
Total Degradation of Pentachloroethane by an Engineered Alcaligenes
Strain Expressing a Modified Camphor Monooxygenase and a Hybrid Dioxygenase
Ryo Iwakiri, Kunichika Yoshihira, Ngadiman, Taiki Futagami, Masatoshi Goto,
and Kensuke Furukawa† p.1353
Note
Comparative Characterization of the oah2 Gene Homologous to the oah1 of Thermus
thermophilus HB8
Tomonori Iwama,† Hideo Hosokawa, Weimin Lin, Hideaki Shimizu, Keiichi Kawai,
and Shuzo Yamagata p.1357
Note
Anti-inflammatory Cyathane Diterpenoids from Sarcodon scabrosus
Tsunashi Kamo,1 Yuki Imura,1 Tomomi Hagio,1 Hidefumi Makabe,2 Hisao Shibata,1
and Mitsuru Hirota1,† p.1362
Note
Antifungal Compounds from the Root and Root Exudate of Zea mays
Sun Park, Yuuko Takano, Hideyuki Matsuura, and Teruhiko Yoshihara† p.1366
Note
The Role of the DRY Motif of Human MC4R for Receptor Activation
Yoshiaki Yamano,1,† Rio Kamon,1 Takao Yoshimizu,2 Yoshihisa Toda,2 Yuichi Oshida,2
Shigeyuki Chaki,2 Masanobu Yoshioka,1 and Isao Morishima1 p.1369
Note
Isolation of 8-Hydroxyglycitein and 6-Hydroxydaidzein from Soybean Miso
Akira Hirota,1,† Miyuki Inaba,1 Yu-Chi Chen,1 Naoki Abe,1 Shoji Taki,1 Masamichi
Yano,2 and Satoru Kawaii3 p.1372
Note
Effects of Isothiocyanates on Tumor Necrosis Factor- α Production by J774A.1
(BALB/c macrophage) Cells
Yoshio Ohta,1,† Kohji Yamaki,2 Toshihiko Osawa,3 and Shunro Kawakishi4 p.1375
Note
Effect of Dietary Level of Phytic Acid on Hepatic and Serum Lipid Status in
Rats Fed a High-sucrose Diet
Shoko Onomi, Yukako Okazaki, and Tetsuyuki Katayama† p.1379
Note
Region Dependent Efficiency for Recombinational Transfer of the Bacillus subtilis
168 Genome
Satoshi Tomita,1 Kenji Tsuge,2 Yo Kikuchi,1 and Mitsuhiro Itaya2,† p.1382
Note
Inhibition of Osteoporosis Induced by Protein Deficient (PD) Food Intake by
Active Vitamin D3 and Vitamin K2 in Rats
Liman Sun,1 Hajime Tamaki,1 Yutaka Ohta,2 Naofumi Katsuyama,2 Tetsuji Ishimaru,3
and Isao Chinen1,† p.1385
Note
Examining the Bases of the J3/4 Domain of Escherichia coli Ribonuclease P
Terumichi Tanaka,† Tomoaki Ando, Shinnosuke Haga, and Yo Kikuchi p.1388
Note
Potent Antioxidative Activity of Cacalol, a Sesquiterpene Contained in Cacalia
delphiniifolia Sleb et Zucc
Kazutoshi Shindo,† Mari Kimura, and Mayuko Iga p.1393
Note
Purification and Characterization of a Novel Prolyl Aminopeptidase from Maitake
(itGrifola frondosa)
Kazuyuki Hiwatashi,1 Kazuyuki Hori,1 Keitaro Takahashi,1 Akira Kagaya,2 Shunzo
Inoue,2 Toshihiro Sugiyama,3 and Saori Takahashi1,† p.1395
Communication
Absorption and Metabolism of Bisphenol A, a Possible Endocrine Disruptor, in
the Aquatic Edible Plant, Water Convolvulus (Ipomoea aquatica)
M. Imadeddin Noureddin,1 Toshio Furumoto,1 Yutaka Ishida,2 and Hiroshi Fukui1,†
p.1398
Communication
Neoculin as a New Taste-modifying Protein Occurring in the Fruit of Curculigo
latifolia
Yukako Shirasuka,1,* Ken-ichiro Nakajima,1,* Tomiko Asakura,2 Haruyuki Yamashita,3
Atsuko Yamamoto,4 Shoji Hata,1 Shinji Nagata,5 Mitsuru Abo,6 Hiroyuki Sorimachi,1
and Keiko Abe1,† p.1403
-1-
Plant Acetyl-CoA Carboxylase: Structure, Biosynthesis, Regulation, and Gene
Manipulation for Plant Breeding
Yukiko Sasaki1,† and Yukio Nagano2
1Genesis Research Institute, Inc., 4-1-35 Noritake-shinmachi, Nishi-ku, Nagoya 451-0051, Japan
2Analytical Research Center for Experimental Sciences, Saga University, Saga 840-8502, Japan
Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Heteromeric ACCase composed of four subunits is usually found in prokaryotes, and homomeric ACCase composed of a single large polypeptide is found in eukaryotes. Most plants have both forms, the heteromeric form in plastids, in which de novo fatty acids are synthesized, and the homomeric form in cytosol. This review focuses on the structure and regulation of plant heteromeric ACCase and its manipulation for plant breeding.
-2-
Potent Hypocholesterolemic Activity of the Yeast Kluyveromyces marxianus YIT
8292 in Rats Fed a High Cholesterol Diet
Yasuto Yoshida,† Wakae Yokoi, Yasue Wada, Kenji Ohishi, Masahiko Ito, and Haruji Sawada
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan
The hypocholesterolemic activities of 81 yeast strains were examined in rats fed a high cholesterol diet (HCD). Male Wistar rats were fed an HCD or an HCD supplemented with 10% yeast for 7 d. It was found that the hypocholesterolemic activities of the yeasts varied remarkably between strains. Kluyveromyces marxianus YIT 8292 exhibited the most potent hypocholesterolemic activity among the yeasts that were tested. K. marxianus YIT 8292 significantly decreased not only plasma total cholesterol but also liver total cholesterol when administered as a dietary admixture at a concentration of 3%. In contrast, brewer’s yeast and baker’s yeast, which have been predominantly used for food, did not exhibit hypocholesterolemic activity even when administered at a concentration of 10%. These results suggest that K. marxianus YIT 8292 may be utilized as a novel food material with the ability to contribute to the prevention of hypercholesterolemia.
-3-
Agrobacterium-mediated Transformation of Javanica Rice cv. Rojolele
Diah Rachmawati,1,2 Takehiko Hosaka,1 Eiichi Inoue,1 and Hiroyuki Anzai1,†
1Gene Research Center, Ibaraki University, Ami, Ibaraki 300-0393, Japan
2Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia
The Agrobacterium-mediated transformation system was extended to a famous Javanica rice variety, Rojolele, that is cultivated in Indonesia now. Efficient callus induction from immature and mature seeds of Rojolele did not succeed by any previous method for any rice cultivar. In this study, the callus from mature seeds of Rojolele exhibited a compact and nodular appearance on C medium after the carbon source and medium pH was modified. Scutellum-derived calli from mature seeds were co-cultivated with Agrobacterium tumefaciens strains EHA101 or LBA4404 that carried plasmid pAFT14, which contained the genes for β-glucuronidase (gus) and hygromycin resistance (hpt). Finally, the transformation efficiency of Rojolele variety using A. tumefaciens strain EHA101 (pAFT14) was improved to about 23%, similar to that of the Japonica rice variety Nipponbare. The seed fertility of transgenic Rojolele was more than 90%. The copy number of the transgene varied from one to three copies in the T0 transgenic lines. Both the gus and the hpt genes were inherited and expressed in the progeny.
-4-
Analysis of Differentially Regulated Proteins in TM4 Cells Treated with Bisphenol
A
Do-Youn Lee, Sang-Soo Lee, Won-A Joo, Eun-Ju Lee, and Chan-Wha Kim†
Graduate School of Life Sciences and Biotechnology, Korea University, 1, 5-ka Anam-dong, Sungbuk-ku, Seoul 136-701, Korea
BPA, bisphenol A, a monomer of epoxy resins and polycarbonate plastic, is used in many consumer products including the plastic linings of cans for food and babies’ bottles. BPA has been reported to cause reproductive toxicity and affects cells in rats and mice at high doses. In this study, the effect of BPA on protein expression in TM4 cells (a mouse Sertoli cell line) known to play an essential role in Spermatogenesis was investigated by two-dimensional electrophoresis (2-DE). After 16 h exposure to 50, 100, 150, 200, and 250 μM of BPA, the viability of TM4 cells decreased to about 90, 85, 78, 55, and 30% of control respectively. Approximately 800 protein spots in TM4 cells were analyzed by 2-DE with pH 4–7 linear immobilized pH gradient (IPG) Dry Strip, and 11 proteins which showed significantly different expression levels were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Among these, HSP 27 and placental calcium binding protein may be proteins differentially expressed by BPA exposure.
-5-
A Novel Genetic System for Analysis of Co-activators for the N-Terminal Transactivation
Function Domain of the Human Androgen Receptor
Ken-ichi Takeyama,1,2 Saya Ito,1 Shun Sawatsubashi,1 Yuko Shirode,1,2 Ayako Yamamoto,1 Eriko Suzuki,1 Akio Maki,1 Kaoru Yamagata,1,2 Yue Zhao,1 Alexandre Kouzmenko,1,2 Tetsuya Tabata,1 and Shigeaki Kato1,2,†
1Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2Solution Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Androgen receptor (hAR) regulates transcription of target genes in a ligand-dependent manner and recruits a number of co-activators for the ligand-induced transactivation via the N-terminal, activation function-1 (AF-1), and C-terminal, AF-2, transactivation domains. But the co-regulator functions on each of AR domains have not yet been fully understood. We have established a Drosophila transgenic system in which hAR and its deletion mutants are ectopically expressed in fly tissues together with an AR response element (ARE)-GFP reporter gene, and have confirmed that hAR was functional in ARE transactivation without affecting the expression of endogenous genes. We found that transcriptional activity of the hAR AF-1 domain was markedly reduced in Drosophila deficiency mutants of homologs for known mammalian co-activators of the AR ligand-dependent AF-2 domain. This suggests that hAR AF-1 recruits co-activators previously known only to interact with the AF-2 domain. Therefore, Drosophila with the hAR AF-1 transgene provides a relevant genetic system in which to uncover novel functions of vertebrate steroid hormone receptors and to screen for novel AF-1 co-regulators.
-6-
ATP Amplification for Ultrasensitive Bioluminescence Assay: Detection of a Single
Bacterial Cell
Tetsuya Satoh,1 Junichi Kato,1 Noboru Takiguchi,1 Hisao Ohtake,1 and Akio Kuroda1,2,†
1Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan
2PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.
-7-
Effects of Alcoholic Solvents on Antiradical Abilities of Protocatechuic Acid
and Its Alkyl Esters
Shizuka Saito, Yasuko Okamoto, and Jun Kawabata†
Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
DPPH radical scavenging reactions of protocatechuic acid and its methyl ester were investigated in various solvents. In alcoholic solvents, methyl protocatechuate rapidly scavenged more than four equivalents of the radical, whereas approximately two equivalents were consumed in aprotic solvents. Methyl, ethyl, butyl, isopropyl, and tert-butyl protocatechuates were examined for their DPPH radical scavenging abilities in methanol or ethanol. As a result, the radical scavenging equivalence of sterically bulky esters tended to decrease compared to that of methyl or ethyl ester. The ABTS radical scavenging ability of those esters in water also showed the same tendency. Since 2-methoxy derivatives were detected in the reaction mixture of methyl protocatechuate and DPPH radical in methanol, a nucleophilic attack of an alcoholic molecule on the o-quinone intermediate, which is sensitive to steric hindrance from alkyl groups of both esters and alcoholic solvents, must be crucial for total radical scavenging abilities.
-8-
Influence of Low-temperature Processing of the Brackish-water Bivalve, Corbicula
japonica, on the Ornithine Content of Its Extract
Hidemitsu Uchisawa,1,5,† Arata Sato,2 Junji Ichita,3 Hajime Matsue,4 and Tomotada Ono5
1Aomori Industrial Research Center, 11-6, 4-chome, Dainitonyamachi, Aomori 030-0113, Japan
2Fukushimasyouten Co., Ltd., 2-13, 1-chome, Tonyamachi, Aomori 030-0131, Japan
3Hirosaki Technical Laboratory, Aomori Industrial Research Center, 80 Fukuromachi, Hirosaki 036-8363, Japan
4Aomari University of Health and Welfare, 58-1 Mase, Hamadate, Aomori 030-8505, Japan
5The United Graduate School of Agricultural Sciences, Iwate University, 18-8, 3-chome, Ueda, Morioka 020-8550, Japan
The ornithine content of an extract of the brackish-water bivalve, Corbicula japonica, increased when the bivalve was frozen. It was not influenced by the period of freezing. This phenomenon was not apparent in the scallop, little-neck clam, or hard clam. We applied various low-temperature conditions for processing the bivalve from 4 °C to -10 °C and measured the ornithine content of each extract. The ornithine content was maximized by processing at -4 °C. The increase in this ornithine content was reduced when the bivalve was stored at 5 °C or 15 °C after processing at -4 °C, this decrease being reversed when the bivalve was again processed at -4 °C after warming. Low-temperature processing of the brackish-water bivalve therefore increased the ornithine content of the extract.
-9-
Molecular Cloning of a Cysteine Proteinase cDNA from the Cotton Boll Weevil
Anthonomus grandis (Coleoptera: Curculionidae)
Osmundo Brilhante de oliveira neto,1,2 João Aguiar Nogueira Batista,1 Daniel John Rigden,1 Octávio Luiz Franco,1,3 Rodrigo Rocha Fragoso,1,2 Ana Carolina Santos Monteiro,1 Rose Gomes Monnerat,1 and Maria Fátima Grossi-de-sá1,†
1Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Final W5, Asa Norte, 70770-900, Brasília, DF, Brazil
2Departamento de Biologia Celular, Universidade de Brasília, Asa Norte, 70.910-900, Brasília, DF, Brazil
3Universidade Católica de Brasília, Asa Norte, 70.790-160, Brasília, DF, Brazil
The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.
-10-
Accumulation and Degradation of Thiamin-binding Protein and Level of Thiamin
in Wheat Seeds during Seed Maturation and Germination
Katsumi Watanabe,† Naoko Nishida, Takashi Adachi, Motoko Ueda, Toshio Mitsunaga, and Yukio Kawamura
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.
-11-
Biphenyl-inducible Promoters in a Polychlorinated Biphenyl-degrading Bacterium,
Rhodococcus sp. RHA1
Hisashi Takeda, Naho Hara, Masayuki Sakai,* Akihiro Yamada, Keisuke Miyauchi, Eiji Masai, and Masao Fukuda†
Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan
Five transcriptional promoters of biphenyl-degradation genes in Rhodococcus sp. RHA1 were characterized. We newly identified the etbA4 promoter region, which was located adjacent upstream from a ferredoxin reductase gene, etbA4 and a dihydrodiol dehydrogenase gene, bphB2. The etbA4 promoter activity was determined in RHA1 using a promoter probe vector with a luxAB luciferase reporter gene, and was induced by a variety of aromatic compounds as well as the bphA1, ebdA1, etbA1, and etbD1 promoters. All these promoters were induced by aromatic compounds in a closely related heterologous host, R. erythropolis IAM1399 in the presence of RHA1 bphST genes, suggesting that these five promoters are under the control of bphST-coding two-component regulatory system. Sequence comparison of the bphA1 promoter with the ebdA1 and etbA1 promoters, whose transcription starts were determined by primer extension analysis, revealed a consensus sequence centering 42-bp upstream from the transcription start. This consensus was also conserved in the etbA4 and etbD1 promoters, and deletions of the bphA1 promoter affecting the consensus impaired inducible promoter activity. These results suggest that this consensus plays a role in transcription induction and/or the promotion of biphenyl degradation genes in RHA1.
-12-
Synthesis of (3R,4S)-3,4,5-Trihydroxy-4-methylpentylphosphonic Acid as a Potential
Inhibitor of the Nonmevalonate Pathway
Toshiaki Yoshioka, Hiroki Shimizu, and Hiroshi Ohrui†
Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan
The nonmevalonate pathway is widely found in higher plants and in many eubacteria, including pathogenic ones, but not in mammals. Identifying a nonmevalonate pathway inhibitor would greatly contribute to the search for new herbicides and antibacterial drugs to treat, for example, malaria. We describe here the synthesis of (3R,4S)-3,4,5-trihydroxy-4-methylpentylphosphonic acid as a candidate for inhibiting MEP cytidylyltransferase, which is the third step on the nonmevalonate pathway.
-13-
Cell-lytic Activity of Tobacco BY-2 Induced by a Fungal Elicitor from Alternaria
alternata Attributed to the Expression of a Class I β-1,3-Glucanase Gene
Tomonori Shinya, Shinobu Gondo, Hiroshi Iijima, Kazunari Hanai, Hideaki Matsuoka, and Mikako Saito†
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of β-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic β-1,3-glucanases. A class I β-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60–72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.
-14-
Structural and Functional Properties of Chicken Lysozyme Fused Serine-rich Heptapeptides
at the C-Terminus
Xiaohua Xu, Orie Kashima, Akira Saito, Hiroyuki Azakami, and Akio Kato†
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan
Two serine-rich heptapeptides, Ser-Ser-Ser-Lys-Ser-Ser-Ser (S6K) and Ser-Ser-Ser-Ser-Ser-Ser-Ser (S7), were fused to the C-terminus of chicken lysozyme (Lz) by genetic modification to improve the functional properties of lysozyme. The cDNAs of S6K-lysozyme (S6K-Lz) and S7-lysozyme (S7-Lz) were inserted into the expression vector of Pichia pastoris and secreted in yeast cultivation medium. The secretion amounts of S6K-Lz and S7-Lz were about 60% of that of wild-type lysozyme (Wt-Lz). The CD spectra showed that the conformation of S6K-Lz and S7-Lz was conserved regardless of the attachment of serine-rich peptides. The denaturation curves of S6K-Lz and S7-Lz also showed that the conformational changes were very small. The lytic activity of S6K-Lz and S7-Lz was almost the same as that of Wt-Lz, while the bactericidal activity against Escherichia coli of S6K-Lz and S7-Lz was greatly increased. The acetic acid-urea PAGE of phosphatase-treated S6K-Lz and S7-Lz indicated the possibility of phosphorylation of the fused serine-rich heptapeptides.
-15-
Multigene Family for Bowman–Birk Type Proteinase Inhibitors of Wild Soja and
Soybean: The Presence of Two BBI-A Genes and Pseudogenes
Masanobu Deshimaru,† Shingo Yoshimi, Seijiro Shioi, and Shigeyuki Terada
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
Genes for Bowman–Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.
-16-
Synthesis, Absolute Configuration and Biological Activities of Both Enantiomers
of 2-(5,7-Dichloro-3-indolyl)propionic Acid: a Novel Dichloroindole Auxin and
Antiauxin
Masato Katayama,1,† Yasuhito Kato,2 and Shingo Marumo3
1Cooperative Research Laboratory for Plant Growth Regulator Development, National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan
2Agro & Fine Chemicals Research Laboratory, Nippon Kayaku Co., Ltd., 225-1 Koshikiya, Ageo, Saitama 362-0064, Japan
3Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Racemic 2-(5,7-dichloro-3-indolyl)propionic acid (5,7-Cl2-2-IPA) was synthesized from 5,7-dichloroindole-3-acetic acid by successive esterification, methoxycarbonylation, methylation, and double hydrolysis. The racemate was converted to diastereomeric esters of l-menthol; these were separated by recycling HPLC into two optically active diastereomers that were then hydrolyzed with p-TsOH to two optically active enantiomers of 5,7-Cl2-2-IPA. The absolute configurations of both these enantiomers were determined by comparing the 1H-NMR spectra of their diastereomeric l-menthyl esters with those of the diastereomeric l-menthyl esters of 2-(3-indolyl)propionic acid (2-IPA) of known absolute configurations.
An assay by the coleoptile elongation of Avena sativa showed the (S)-(+)-enantiomer of 5,7-Cl2-2-IPA to have weak auxin activity, whereas the (R)-(-)-antipode had no auxin activity at any concentration tested. Interestingly, the (R)-(-)-enantiomer had antiauxin activity very close to that of 2-(5,7-dichloro-3-indolyl)isobutyric acid (5,7-Cl2-IIBA), a strong antiauxin. These data indicate that, of the two methyl groups in its molecule, the antiauxin activity of 5,7-Cl2-IIBA was due only to the (R)-methyl group.
-17-
Fluorometric Study of the Acid-induced Denaturation of Staphylococcal Nuclease
and Its Mutant Forms
Akiyoshi Tanaka†
Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
The acid-induced denaturation of wild-type Staphylococcal nuclease (WT) and its eight mutant forms, L25A, V66L, G79S, A90S, G88V, H124L, V66L/G88V, and V66L/G79S/G88V, was investigated using Trp140 fluorescence as a probe at 30 °C. The values of pH1/2, at which the denaturation is half completed, and n, the apparent number of protons which trigger the denaturation and are taken up by the proteins upon denaturation at pH1/2, were evaluated from the pH dependence of the fluorescence intensity. The values of pH1/2 and n for WT were 3.8 and 1.8 respectively. The amino acid replacements changed the pH1/2 values to a range between 3.0 (H124L) and 4.4 (G79S) and also changed the n values to a range between 1.0 (A90S) and 3.0 (G88V). There was a negative correlation between the values of pH1/2 and n. It was suggested that the amino acid replacements may change the energy levels of the native state and/or the denatured state mainly in the neutral (stable) pH region, not in the acidic (unstable) region, resulting in the correlative changes in pH1/2 and n.
-18-
A Chitinase Indispensable for Formation of Protoplast of Schizophyllum commune
in Basidiomycete-lytic Enzyme Preparation Produced by Bacillus circulans KA-304
Shigekazu Yano, Nopakarn Rattanakit, Mamoru Wakayama, and Takashi Tachiki†
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu 525-8577, Japan
KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. α-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0–30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30–50% saturation of KA-prep, which contained chitinase(s) and β-glucanase(s) but was inactive in the protoplast formation.
Chitinase(s) and β-glucanase(s) in the ammonium sulfate fraction of 30–50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the α-1,3-glucanase. The β-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of α-1,3-glucanase and the chitinase preparation.
The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with α-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N’-diacetylchitobiose and N,N’,N”-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-β-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.
-19-
Molecular Characterization of an Intracellular β-N-Acetylglucosaminidase Involved
in the Chitin Degradation System of Streptomyces thermoviolaceus OPC-520
Takahiro Kubota, Katsushiro Miyamoto, Masahide Yasuda, Yoshihiko Inamori, and Hiroshi Tsujibo†
Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
We purified and characterized an intracellular β-N-acetylglucosaminidase (NagC) from a cytoplasmic fraction of Streptomyces thermoviolaceus OPC-520. The molecular mass of NagC was estimated to be 60 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the enzyme were 6.0 and 50 °C respectively. Purified NagC hydrolyzed chitin oligosaccharides from N,N’-diacetylchitobiose (GlcNAc)2 to chitopentaose (GlcNAc)5, hydrolyzed N,N’-diacetylchitobiose especially rapidly, and showed a tendency to decrease with increases in the degree of polymerization. But, NagC didn’t hydrolyze chitohexaose (GlcNAc)6. The gene encoding NagC was cloned and sequenced. The open reading frame of nagC encoded a protein of 564 amino acids with a calculated molecular mass of 62,076 Da. The deduced amino acid sequence of NagC showed homology with several β-N-acetylglucosaminidases belonging to glycosyl hydrolase family 20. The expression plasmid coding for NagC was constructed in Escherichia coli. The recombinant enzyme showed pH and temperature optima and substrate specificity similar to those of the native enzyme. The gene arrangement near the nagC gene of S. thermoviolaceus OPC-520 was compared with that of S. coelicolor A3(2). Three genes, which appear to constitute an ABC transport system for sugar, were missing in the vicinity of the nagC gene.
-20-
cDNA Microarray Analysis of Rice Anther Genes under Chilling Stress at the Microsporogenesis
Stage Revealed Two Genes with DNA Transposon Castaway in the 5’-Flanking Region
Tomoya Yamaguchi,1,† Katsuhiro Nakayama,1 Takami Hayashi,1 Junshi Yazaki,2 Naoki Kishimoto,2 Shoshi Kikuchi,2 and Setsuo Koike1
1Plant Physiology Laboratory, National Agricultural Research Center for Tohoku Region, Shimo-kuriyagawa, Morioka 020-0198, Japan
2Department of Molecular Genetics, National Institute of Agrobiological Sciences, Kannondai, Tsukuba 305-8602, Japan
Rice is most chilling sensitive at the onset of microspore release. Chilling treatment at this stage causes male sterility. The gene expression profile during the microspore development process under chilling stress was revealed using a microarray that included 8,987 rice cDNAs. As many as 160 cDNAs were up- or down-regulated by chilling during the microspore release stage. RT-PCR analysis of 5 genes confirmed the microarray results. We identified 3 novel genes whose expression levels were remarkably changed by chilling in rice anther. A new cis element that includes a DNA transposon Castaway sequence was found in the 5’ upstream region of two genes which were conspicuously down-regulated by chilling temperatures in rice anther.
-21-
Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the Fenton Reaction
Shin-ichi Ishikawa,† Yuki Yano, Keizo Arihara, and Makoto Itoh
School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan
Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical (•OH) formation from H2O2 in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited •OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the •OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H2O2. These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.
-22-
Short-step Synthesis of Chenodiol from Stigmasterol
Toru Uekawa, Ken Ishigami, and Takeshi Kitahara†
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
Chenodiol is an important bile acid widely used for gallstone dissolution and cholestatic liver diseases. We succeeded in a short-step synthesis of chenodiol, starting from the safer phytosterol, stigmasterol.
-23-
Kinetic Analysis of a Chitinase from Red Sea Bream, Pagrus major
Shuji Karasuda,1 Kosuke Yamamoto,1 Michiko Kono,2 Shohei Sakuda,3 and Daizo Koga1,†
1Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
2Fisheries Research Laboratory, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Maisaka, Shizuoka 431-0211, Japan
3Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Kinetic analysis was done on the 46-kDa chitinase (EC 3.2.1.14) purified from the stomach of red sea bream, Pagrus major, using glycolchitin and N-acetylchitooligosaccharides (GlcNAcn, n = 2–6) as substrates. High activity was observed at two pHs, such as 2.5 and 9.0, toward glycolchitin as seen in other insect chitinases, and also at both pH 2.5 and 5.0 even toward a short substrate, N-acetylchitopentasaccharide. Allosamidin competitively inhibited chitinase with Ki value of 0.0214 μM at pH 2.5 and 0.0024 μM at pH 9.0 in the reaction of glycolchitin. Substrate inhibition was observed in the reaction of N-acetylchitopentasaccharide. The anomeric forms of the products from N-acetylchitooligosaccharides were analyzed to be β anomer by the high pressure liquid chromatography (HPLC) method. The data for both β-anomer formation and allosamidin inhibition suggest that red sea bream chitinase belongs to family 18 of glycosyl hydrolases. This suggestion is also supported by the results for the N-terminal amino acid sequence.
-24-
Identification and Cloning of the Gene Involved in the Final Step of Chlortetracycline
Biosynthesis in Streptomyces aureofaciens
Tetsuo Nakano,1,† Koichiro Miyake,1 Hirofumi Endo,1 Tohru Dairi,2 Toru Mizukami,1 and Ryoichi Katsumata3
1Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo 194-8533, Japan
2Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan
3Graduate school of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.
-25-
Note
Total Degradation of Pentachloroethane by an Engineered Alcaligenes Strain Expressing
a Modified Camphor Monooxygenase and a Hybrid Dioxygenase
Ryo Iwakiri, Kunichika Yoshihira, Ngadiman, Taiki Futagami, Masatoshi Goto, and Kensuke Furukawa†
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.
-26-
Note
Comparative Characterization of the oah2 Gene Homologous to the oah1 of Thermus
thermophilus HB8
Tomonori Iwama,† Hideo Hosokawa, Weimin Lin, Hideaki Shimizu, Keiichi Kawai, and Shuzo Yamagata
Department of Biotechnology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan
The oah2 gene homologous to the oah1 of Thermus thermophilus HB8 was cloned and sequenced. It comprised 1,236 bp encoding a protein of 412 amino acid residues and was overexpressed. The gene product, also having O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity, was purified to homogeneity and characterized comparatively with the oah1 product. The two proteins shared many characteristics.
-27-
Note
Anti-inflammatory Cyathane Diterpenoids from Sarcodon scabrosus
Tsunashi Kamo,1 Yuki Imura,1 Tomomi Hagio,1 Hidefumi Makabe,2 Hisao Shibata,1 and Mitsuru Hirota1,†
1Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
2Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
Four novel diterpenoids were isolated from the fruiting bodies of Sarcodon scabrosus (Fr.) Karst. (Boraginaceae) together with neosarcodonin A. One of the novel compounds was elucidated to be a cyathane diterpenoid, namely neosarcodonin O, by its spectral data. The others were characterized as 19-O-linoleoyl, 19-O-oleoyl and 19-O-stearoyl derivatives of sarcodonin A, after comparison with the authentic samples synthetically prepared from sarcodonin A. These compounds, together with the five 19-O-acyl derivatives synthesized from sarcodonin A, each exhibited inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears by topical application.
-28-
Note
Antifungal Compounds from the Root and Root Exudate of Zea mays
Sun Park, Yuuko Takano, Hideyuki Matsuura, and Teruhiko Yoshihara†
Laboratory of Bio-organic Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kitaku, Sapporo 060-8589, Japan
A maize plant (Zea mays) planted in a test tube was found to inhibit the growth of the soil-borne plant pathogen, Fusarium oxysporum f. sp. melongenae. The antifungal compounds, 6-methoxybenzoxazolinone and 6,7-dimethoxybenzoxazolinone, were isolated from an ethanol extract of Zea mays roots, and (6R)-7,8-dihydro-3-oxo- α -ionone and (6R,9R)-7,8-dihydro-3-oxo- α -ionol were isolated from the root exudate.
-29-
Note
The Role of the DRY Motif of Human MC4R for Receptor Activation
Yoshiaki Yamano,1,† Rio Kamon,1 Takao Yoshimizu,2 Yoshihisa Toda,2 Yuichi Oshida,2 Shigeyuki Chaki,2 Masanobu Yoshioka,1 and Isao Morishima1
1Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
2Medicinal Pharmacology Laboratory, Taisho Pharmaceutical Co., Ltd., Saitama 331-9530, Japan
We constructed several mutant human MC4R cDNAs by site directed mutagenesis and expressed these receptors in COS-1 cells. The conserved DRY motif among GPCRs was mutated to generate eight mutants. While no MC4R ligand binding was detected in any of the mutants, one mutant, D146A, resulted in higher cAMP production in cells than the wild-type receptor without ligand stimulation.
-30-
Note
Isolation of 8-Hydroxyglycitein and 6-Hydroxydaidzein from Soybean Miso
Akira Hirota,1,† Miyuki Inaba,1 Yu-Chi Chen,1 Naoki Abe,1 Shoji Taki,1 Masamichi Yano,2 and Satoru Kawaii3
1Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
2National Institute of Fruit Tree Science, Okitsu-naka, Shimizu 424-0204, Japan
3Faculty of Science and Engineering, Tokyo Denki University, Hatoyama, Saitama 350-0394, Japan
We isolated from soybean miso 8-hydroxyglycitein and 6-hydroxydaidzein as DPPH-radical scavengers, and elucidated their chemical structures by mass spectrometric, and 1H- and 13C-NMR spectrosopic analyses. These compounds showed DPPH-radical scavenging activity as high as that of α -tocopherol, 8-hydroxygenistein and 8-hydroxydaidzein. This is the first report of the isolation of 8-hydroxyglycitein from a natural source.
-31-
Note
Effects of Isothiocyanates on Tumor Necrosis Factor- α Production by J774A.1
(BALB/c macrophage) Cells
Yoshio Ohta,1,† Kohji Yamaki,2 Toshihiko Osawa,3 and Shunro Kawakishi4
1Hiroshima Prefectural Food Technology Research Center, Hijiyamahon-machi, Minami-ku, Hiroshima 732-0816, Japan
2National Agricultual Research Center for Tohoku Region, Harajuku-Minami, Arai, Fukushima 960-2156, Japan
3Nagoya University, Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
4Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
The effects of isothiocyanates (ITCs) on tumor necrosis factor- α (TNF- α ) production by the J774A.1 mouse macrophage cell line stimulated with lipopolysaccharide (LPS) were examined. Some individual ITCs examined showed a priming effect, which was expressed as pre-activation, but also inhibited the production of TNF- α with concomitant stimulation by LPS, as a triggering stimulant of TNF production. These results suggest that ITCs exert opposing activities that can enhance or inhibit the host defense system.
-32-
Note
Effect of Dietary Level of Phytic Acid on Hepatic and Serum Lipid Status in
Rats Fed a High-sucrose Diet
Shoko Onomi, Yukako Okazaki, and Tetsuyuki Katayama†
Department of Human Life Sciences Education, Graduate School of Education, Hiroshima University, Higashi-Hiroshima 739-8524, Japan
The effect of dietary 0.02–10% sodium phytate on the hepatic and serum lipid status of rats fed a high-sucrose diet for 14 d was investigated. Hepatic levels of triglyceride and cholesterol and lipogenic enzymes activity were reduced with increasing dietary phytate level. The addition of 10% sodium phytate drastically depressed growth, food intake, and serum triglyceride and cholesterol levels.
-33-
Note
Region Dependent Efficiency for Recombinational Transfer of the Bacillus subtilis
168 Genome
Satoshi Tomita,1 Kenji Tsuge,2 Yo Kikuchi,1 and Mitsuhiro Itaya2,†
1Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
2Mitsubishi Kagaku Institute of Life Sciences, Minamiooya, Machida, Tokyo 194-8511, Japan
Submega-sized regions of the Bacillus subtilis genome were cloned to plasmid by the B. subtilis Recombinational Transfer (BReT) method. BReT efficiency depends not only on the genome location but also on the choice of sequences for simultaneous homologous recombination during BReT. In an extreme case, a 91-kb region that was unsuccessful on the first attempt was obtained when the slightly shifted 98-kb region was targeted.
-34-
Note
Inhibition of Osteoporosis Induced by Protein Deficient (PD) Food Intake by
Active Vitamin D3 and Vitamin K2 in Rats
Liman Sun,1 Hajime Tamaki,1 Yutaka Ohta,2 Naofumi Katsuyama,2 Tetsuji Ishimaru,3 and Isao Chinen1,†
1Laboratories of Applied Biochemistry, Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-01213, Japan
2Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0123, Japan
3Musashi Junior High School, Nariyoshi 810, Musashi-machi, Higashikunisaki-gun, Oita 873-0404, Japan
Independent use of K2 and D3 and simultaneous application of K2 and D3 inhibited the development of osteoporosis caused by PD food intake. The ALP activity of urine as a marker of bone formation osteoporosis did not rise in rats fed PD foods containing D3, K2 or both together. Body and womb weights fell in rats fed PD foods with D3 K2 and both D3, K2. Osteoporosis caused by PD food intake found to be very similar to type II osteoporosis in respects of inhibition by D3 and K2 and rising urinary ALP activity.
-35-
Note
Examining the Bases of the J3/4 Domain of Escherichia coli Ribonuclease P
Terumichi Tanaka,† Tomoaki Ando, Shinnosuke Haga, and Yo Kikuchi
Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempakucho, Toyohashi, Aichi 441-8580, Japan
We prepared several mutants of the J3/4 and P4 domains of Escherichia coli ribonuclease P (RNase P): A62G, A62U, G63C/G64C, A65G, A67G, U69A, U69G, U69C, U69 Δ , and U69UU. Comparison of the ribozyme and holo enzyme reactions at various concentrations of magnesium ions showed that the presence of a bulge at U69 in the P4 domain was important in the holo enzyme. The results also showed that the conserved bases G63 and G64 in the J3/4 domain were important for efficient ribozyme reactions but were replaceable in the presence of the protein component. Our data showed that the bases in the J3/4 and P4 domains displayed different responses to the metal ions that were affected by the presence of the protein component.
-36-
Note
Potent Antioxidative Activity of Cacalol, a Sesquiterpene Contained in Cacalia
delphiniifolia Sleb et Zucc
Kazutoshi Shindo,† Mari Kimura, and Mayuko Iga
Department of Food and Nutrition, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
The potent antioxidative sesquiterpene, cacalol, was isolated from Cacalia delphiniifolia Sleb et Zucc, and identified by an analysis of the MS and NMR spectral data. Cacalol showed potent antioxidative activity in a rat brain homogenate model (IC50 of 40 nM). Cacalol also proved to be a potent neuroprotective substance which could protect neuronal hybridoma N18-RE-105 cells from L-glutamate toxicity.
-37-
Note
Purification and Characterization of a Novel Prolyl Aminopeptidase from Maitake
(itGrifola frondosa)
Kazuyuki Hiwatashi,1 Kazuyuki Hori,1 Keitaro Takahashi,1 Akira Kagaya,2 Shunzo Inoue,2 Toshihiro Sugiyama,3 and Saori Takahashi1,†
1Department of Bioengineering, Akita Research Institute of Food and Brewing (ARIF), 4-26 Sanuki, Araya-machi, Akita 010-1623, Japan
2Akita Jujo Chemicals Co., Ltd., 1-1 Toriki-cho, Araya, Akita 010-1633, Japan
3Department of Biochemistry, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan
We have found a novel prolyl aminopeptidase in Grifola frondosa. The enzyme was purified by DEAE-Sepharose CL-6B, Butyl-Toyopearl, Sephacryl S-100, and Mono-Q column chromatographies. The purified enzyme exists as a dimer and gives high activity toward L-proline-p-nitroanilide. The enzyme was strongly inhibited by p-chloromercuribenzoic acid and iodoacetic acid and markedly inhibited by phenylmethylsulfonyl fluoride and arphamenin A.
-38-
Communication
Absorption and Metabolism of Bisphenol A, a Possible Endocrine Disruptor, in
the Aquatic Edible Plant, Water Convolvulus (Ipomoea aquatica)
M. Imadeddin Noureddin,1 Toshio Furumoto,1 Yutaka Ishida,2 and Hiroshi Fukui1,†
1Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan
2Biotechnology Department, Shikoku Research Institute, Inc., Takamatsu, Kagawa 761-0192, Japan
Water convolvulus, a vegetable, absorbed bisphenol A (BPA), an endocrine disruptor, from the medium. One week later, no BPA could be detected in the plant, indicating that BPA had been metabolized in the plant. BPA monoglucoside was detected as the BPA base at ca. 10% in the roots, some in the stems, but none in the leaves. 2H-NMR analyses of MeOH extracts and hydrolyzates of the plant treated with BPA-d16 showed the presence of metabolites (ca. 7% and 26%, respectively, as BPA equivalents) other than the glucoside. Over 50% of BPA might be polymerized and/or tightly bound in the plant residues.
-39-
Communication
Neoculin as a New Taste-modifying Protein Occurring in the Fruit of Curculigo
latifolia
Yukako Shirasuka,1,* Ken-ichiro Nakajima,1,* Tomiko Asakura,2 Haruyuki Yamashita,3 Atsuko Yamamoto,4 Shoji Hata,1 Shinji Nagata,5 Mitsuru Abo,6 Hiroyuki Sorimachi,1 and Keiko Abe1,†
1Laboratory of Biological Function, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Laboratory of Food Science, Atomi Junior College, Bunkyo-ku, Tokyo 112-8687, Japan
3Basic Research Laboratory, Asahi Denka Kogyo K.K., 235 Higashi-ogu, 7 Arakawa, Tokyo 116-8553, Japan
4Mitsukan Group Corporation, Central Research Institute, 2-6 Nakamura-cho, Hada, Aichi 475-8585, Japan
5Laboratory of Bioorganic Chemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
6Laboratory of Analytical Chemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.