Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.12 2002)

Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a Quinone Solution
Masatsune MURATA, Makie SUGIURA, Yuko SONOKAWA, Tomomi SHIMAMURA, and Seiichi HOMMA
p.2525

Cystatin C in Milk Basic Protein (MBP) and Its Inhibitory Effect on Bone Resorption in Vitro
Yasuhiro MATSUOKA,1, Atsushi SERIZAWA,1 Tomoe YOSHIOKA,2 Jun-ichi YAMAMURA,1 Yoshikazu MORITA,1 Hiroshi KAWAKAMI,1 Yasuhiro TOBA,1 Yukihiro TAKADA,1 and Masayoshi KUMEGAWA3 p.2531

Enzyme Inhibitors to Increase Poly-3-hydroxybutyrate Production by Transgenic Tobacco
Yoshikatsu SUZUKI,1, Minoru KURANO,1,2 Yuko ARAI,1 Hideo NAKASHITA,1 Yoshiharu DOI,1 Ron USAMI,2 Kohki HORIKOSHI,2 and Isamu YAMAGUCHI1 p.2537

New Estimation Method for Fatty Acid Composition in Oil Using Near Infrared Spectroscopy
Tetsuo SATO p.2543

Induced Volatiles in Elicitor-treated and Rice Blast Fungus-inoculated Rice Leaves
Naomi OBARA,1,2 Morifumi HASEGAWA,1,3, and Osamu KODAMA1,3 p.2549

Comparison of C-S Lyase in Lentinus edodes and Allium sativum
Hitomi KUMAGAI,1, Hidetoshi KONO,1 Hidetoshi SAKURAI,1 and Keisuke TOKIMOTO2 p.2560

Transcriptional Regulators of the 51D Surface Protein Gene of Paramecium tetraurelia
Hiroyuki TANABE, Isao HARAGUCHI, and Sachiko YOSHIOKA p.2567

Oxidative Stability of Polyunsaturated Fatty Acid in Phosphatidylcholine Liposomes
Mina ARASEKI, Kanako YAMAMOTO, and Kazuo MIYASHITA p.2573

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium, Thermotoga maritima MSB8
Eranna RAJASHEKHARA, Motomitsu KITAOKA, Yeon-Kye KIM, and Kiyoshi HAYASHI p.2578

Mutant Luciferase Enzymes from Fireflies with Increased Resistance to Benzalkonium Chloride
Noriaki HATTORI,1, Naoki KAJIYAMA,1 Masako MAEDA,2 and Seiji MURAKAMI1 p.2587

Cloning of the Maltose Phosphorylase Gene from Bacillus sp. Strain RK-1 and Efficient Production of the Cloned Gene and the Trehalose Phosphorylase Gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis
Yasushi INOUE, Nozomu YASUTAKE, Yoshie OSHIMA, Yoshie YAMAMOTO, Tetsuji TOMITA, Shinsuke MIYOSHI, and Tsuneya YATAKE p.2594

Cloning and Nucleotide Sequence of the Glutamate Decarboxylase-encoding Gene gadA from Aspergillus oryzae
Yuki KATO, Yoko KATO, Keiji FURUKAWA, and Shodo HARA p.2600

Synthesis of 27-Oxo, 27-Hydroxymilbemycins A3 and A4 and Novel 27-Alkoxymilbemycins A3 and A4 from Milbemycins A3 and A4 and Their Acaricidal Activities
Takahiro TSUKIYAMA,1, Yoshihisa TSUKAMOTO,1 Hitomi SUEMOTO,1 Yoshihiro OKAMOTO,2 Yasushi TAMAGAWA,1 Ayako KINOSHITA,1 Reiji ICHINOSE,2 and Kazuo SATO1 p.2606

Novel Enzymatic Method for the Production of Xylitol from D-Arabitol by Gluconobacter oxydans
Shun-ichi SUZUKI, Masakazu SUGIYAMA, Yasuhiro MIHARA, Ken-ichi HASHIGUCHI, and Kenzo YOKOZEKI p.2614

A New Polypeptide Toxin from the Nematocyst Venom of an Okinawan Sea Anemone Phyllodiscus semoni (Japanese name <Oh><0192><0192><Wa>unbachi-isoginchaku′′)
Hiroshi NAGAI,1, Naomasa OSHIRO,2 Kyoko TAKUWA-KURODA,1 Setsuko IWANAGA,2 Masatoshi NOZAKI,2 and Terumi NAKAJIMA1 p.2621

Isoflavone Aglycon Produced by Culture of Soybean Extracts with Basidiomycetes and Its Anti-angiogenic Activity
Takehito MIURA, Lan YUAN, Buxiang SUN, Hajime FUJII, Mayumi YOSHIDA, Koji WAKAME, and Ken-ichi KOSUNA p.2626

Incorporation and Accumulation of Docosahexaenoic Acid from the Medium by Pichia methanolica HA-32
Hideyuki AOKI,1 Noriko MIYAMOTO,1 Yuji FURUYA,1 Mitsumasa MANKURA,1 Yasushi ENDO,2 and Kenshiro FUJIMOTO2, p.2632

Gene Cloning, Purification, and Characterization of 2,3-Diaminopropionate Ammonia-lyase from Escherichia coli
Takuma UO, Tohru YOSHIMURA, Tozo NISHIYAMA, and Nobuyoshi ESAKI p.2639

Zinc is Involved in the Expression of a Nuclear-encoded Alternative Oxidase Gene in the Yeast Hansenula anomala
Nobuko MINAGAWA,1, Harumi KARIYA,1 Natarajan Selvamuthu KUMARASWAMI,1 Ikuko KAMIMURA,1 Shigeru SAKAJO,2 and Akio YOSHIMOTO1 p.2645

Old Yellow Enzyme from Candida macedoniensis Catalyzes the Stereospecific Reduction of the C＝C Bond of Ketoisophorone
Michihiko KATAOKA,1, Atsushi KOTAKA,1 Akiko HASEGAWA,1 Masaru WADA,2 Ayumi YOSHIZUMI,2 Shigeru NAKAMORI,2 and Sakayu SHIMIZU1 p.2651

Novel S-Benzylisothiourea Compound That Induces Spherical Cells in Escherichia coli Probably by Acting on a Rod-shape-determining Protein(s) Other Than Penicillin-binding Protein 2
Noritaka IWAI,1, Kazuo NAGAI,2 and Masaaki WACHI1, p.2658

His-to-Asp Phosphorelay Circuitry for Regulation of Sexual Development in Schizosaccharomyces pombe
Norihito NAKAMICHI, Hisami YAMADA, Keisuke AOYAMA, Ryusuke OHMIYA, Hirofumi AIBA, and Takeshi MIZUNO p.2663

Note
Use of Shark Collagen for Cell Culture and Zymography
Yoshihiro NOMURA and Nobuhide KITAZUME p.2673

Note
Antifungal Activities of N-Arylbenzenesulfonamides against Phytopathogens and Control Efficacy on Wheat Leaf Rust and Cabbage Club Root Diseases
Jae Gon KANG,1 Jong Hyun HUR,1 Sung Jun CHOI,1 Gyung Ja CHOI,2 Kwang Yun CHO,2 Leonid N. TEN,3 Ki Hun PARK,1 and Kyu Young KANG1,　p.2677

Note
Inhibition by Theanine of Binding of [3H]AMPA, [3H]Kainate, and [3H]MDL 105,519 to Glutamate Receptors
Takami KAKUDA, Ayumu NOZAWA, Akio SUGIMOTO, and Hitoshi NIINO p.2683

Note
Identification of a Toxic Mechanism of the Plasticizers, Phtahlic Acid Esters, which are Putative Endocrine Disrupters: Time-dependent Increase in Quinolinic Acid and Its Metabolites in Rats Fed di(2-ethylhexyl)phthalate
Tsutomu FUKUWATARI, Yuko SUZUKI, Etsuro SUGIMOTO, and Katsumi SHIBATA p.2687

Note
Identification of (3S, 9R)- and (3S, 9S)-Megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides as Damascenone Progenitors in the Flowers of Rosa damascena Mill.
Masayuki SUZUKI,1 Shigetaka MATSUMOTO,1 Masaya MIZOGUCHI,1 Satoshi HIRATA,1 Kazuteru TAKAGI,1 Ikue HASHIMOTO,1 Yumiko YAMANO,2 Masayoshi ITO,2 Peter FLEISCHMANN,3 Peter WINTERHALTER,3 Tetuichiro MORITA,4 and Naoharu WATANABE1, p.2692

Note
Labdane-type Diterpene Dialdehyde, Pungent Principle of Myoga, Zingiber mioga Roscoe
Masako ABE,1 Yoshio OZAWA,1, Yasushi UDA,2 Yoichi YAMADA,3 Yasujiro MORIMITSU,4 Yoshimasa NAKAMURA,5 and Toshihiko OSAWA5 p.2698

Note
Effects of Added Dietary Taurine on Erythrocyte Lipids and Oxidative Stress in Rabbits Fed a High Cholesterol Diet
Jale BALKAN, Serdar <Oh><0196><Wa>OZTEZCAN, G<Oh><0228><Wa>ul<Oh><0231><Wa>in AYKA<Oh><0199><Wa>-TOKER, and M<Oh><0228><Wa>ujdat UYSAL p.2701

Note
Substrate Inhibition of L-Cysteine α,β-Elimination Reaction Catalyzed by L-Cystathionine γ-Lyase of Saccharomyces cerevisiae
Shuzo YAMAGATA,1, Motomi ISAJI,2 Tomoko YAMANE,2 and Tomonori IWAMA1 p.2706

Note
Effects of Voluntary Resistance Exercise on Heme Biosynthesis in Rats Given Glucocorticoid-injections
Hyung-Sook KANG,1 Tatsuhiro MATSUO,2, Ryo-Won CHOUE,1 and Masashige SUZUKI3 p.2710

Note
Isolation of Three Main Sericin Components from the Cocoon of the Silkworm, Bombyx mori
Yoko TAKASU, Hiromi YAMADA, and Kozo TSUBOUCHI p.2715

Note
Molecular Characterization of the Gene Encoding Rice Allene Oxide Synthase and Its Expression
Suk-Bong HA,1 Bong-Choon LEE,1 Da-Eun LEE,1 Yong In KUK,2 A-Young LEE,1 Oksoo HAN,1 and Kyoungwhan BACK1, p.2719

Note
Biotransformation of the Mycotoxin, Zearalenone, to a Non-estrogenic Compound by a Fungal Strain of Clonostachys sp.
Hideaki KAKEYA,1 Naoko TAKAHASHI-ANDO,2 Makoto KIMURA,2 Rie ONOSE,1 Isamu YAMAGUCHI,2 and Hiroyuki OSADA1, p.2723

Note
OsRALyase1, a Putative F-Box Protein Identified in Rice, Oryza sativa, with Enzyme Activity Identical to That of Wheat RALyase
Yasuhiro ITO,1 Akihiko OZAWA,2 Tatsuya SAWASAKI,2 Yaeta ENDO,2 Kozo OCHI,1 and Yuzuru TOZAWA1, p.2727

Note
Design of a Fluorescent Electrophoretic Mobility Shift Assay Improved for the Quantitative and Multiple Analysis of Protein-DNA Complexes
Takuo ONIZUKA, Sumiyo ENDO, Masahiko HIRANO, Shozo KANAI, and Hideo AKIYAMA p.2732

Note
Identification of an Indispensable Amino Acid for ppGpp Synthesis of Escherichia coli SpoT Protein
Chizuko FUJITA, Maki MAEDA, Takako FUJII, Ryoko IWAMOTO, and Kenji IKEHARA p.2735

Note
Further Stabilization of Earthworm Serine Protease by Chemical Modification and Immobilization
Nobuyoshi NAKAJIMA,1, Kohji ISHIHARA,2 Manabu SUGIMOTO,3 Takashi NAKAHARA,4 and Hideaki TSUJI1 p.2739

Note
Antisense Suppression of Collagen VI Synthesis Results in Reduced Expression of Collagen I in Normal Human Osteoblast-like Cells
Satoru HARUMIYA,1,, Mark A. GIBSON,2 and Yasuko KOSHIHARA1, p.2743

Note
Novel Hydroquinone as a Matrix Metallo-proteinase Inhibitor from the Mushroom, Piptoporus betulinus
Hirokazu KAWAGISHI,1, Keiko HAMAJIMA,1 and Yoshimasa INOUE2 p.2748

Note
Inhibition of the Bacterial Surface Protein Anchoring Transpeptidase Sortase by Medicinal Plants
Soo-Whan KIM,1 Il-Moo CHANG,1 and Ki-Bong OH1,2 p.2751

Note
Cloning, Sequencing, and Overexpression in Escherichia coli of a Phenylserine Dehydratase Gene from Ralstonia pickettii PS22
Heiwa OKUDA, Shinji NAGATA, and Haruo MISONO p.2755

-1-
Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a Quinone Solution

Masatsune MURATA, Makie SUGIURA, Yuko SONOKAWA, Tomomi SHIMAMURA, and Seiichi HOMMA

Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan

Received February 20, 2002; Accepted August 9, 2002
Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7×103 for 325 nm and 9.7×102 for 400 nm in an acidic aqueous solution. Chlorogenic acid and H2O2 were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H2O2 spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.
Key words: chlorogenic acid; chlorogenic acid quinone; o-quinone; enzymatic browning; H2O2

-2-
Cystatin C in Milk Basic Protein (MBP) and Its Inhibitory Effect on Bone Resorption in Vitro

Yasuhiro MATSUOKA,1, Atsushi SERIZAWA,1 Tomoe YOSHIOKA,2 Jun-ichi YAMAMURA,1 Yoshikazu MORITA,1 Hiroshi KAWAKAMI,1 Yasuhiro TOBA,1 Yukihiro TAKADA,1 and Masayoshi KUMEGAWA3

1Technology and Research Institute, Snow Brand Milk Products Co. Ltd., 1-1-2 Minamidai, Kawagoe, Saitama 350-1165, Japan 2Sapporo Research Laboratory, Snow Brand Milk Products Co. Ltd., 6-1-1-Naebocho, Higashiku, Sapporo, Hokkaido 065-0043, Japan 3Department of Oral Anatomy, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283, Japan

Received April 12, 2002; Accepted August 9, 2002
A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.
Key words: milk basic protein; bone resorption; cystein protease inhibitor; cystatin; osteoclasts

-3-
Enzyme Inhibitors to Increase Poly-3-hydroxybutyrate Production by Transgenic Tobacco

Yoshikatsu SUZUKI,1, Minoru KURANO,1,2 Yuko ARAI,1 Hideo NAKASHITA,1 Yoshiharu DOI,1 Ron USAMI,2 Kohki HORIKOSHI,2 and Isamu YAMAGUCHI1

1RIKEN Institute (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 2Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan

Received April 25, 2002; Accepted July 19, 2002
Chemical regulation of secondary-metabolite synthesis was investigated through the improvement of poly-3-hydroxybutyrate (PHB) production in transgenic tobacco plants by the use of enzyme inhibitors. Two tobacco lines, BC3 and rCAB8, that produce PHB in both the cytosol and plastids were used. An acetyl-CoA carboxylase inhibitor, D-(＋)-Quizalofop-ethyl, increased PHB accumulation in both lines 2-fold. The accumulation rate of plastidial PHB in the rCAB8 line was 2.5-fold higher than that of cytosolic PHB in the BC3 line. A specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, mevastatin, also increased PHB accumulation but only in the BC3 line. These results indicated that chemical regulation of the native metabolic flows by the specific enzyme inhibitors increased secondary-metabolite production in the transgenic tobacco plants we used.
Key words: ACCase; HMG-CoA reductase; poly-3-hydroxybutyrate; Quizalofop; mevastatin

-4-
New Estimation Method for Fatty Acid Composition in Oil Using Near Infrared Spectroscopy

Tetsuo SATO
National Agricultural Research Center for Kyushu Okinawa Region (KONARC), National Agricultural Research Organization (NARO), Nishigoshi-machi, Kikuchi-gun, Kumamoto-ken 861-1192, Japan

Received May 2, 2002; Accepted August 14, 2002
The absorption bands of cis-unsaturation and the carbon chain length of the fatty acid moieties in oil appear in the near infrared (NIR) wavelength region, especially around 1600--1800 nm. Using this region, a new estimation method for fatty acid composition analysis is proposed. Because the differences of the original NIR spectra are miniscule even in this region, the second derivative NIR spectra were examined in order to estimate the fatty acid composition in oil exclusively from the spectral patterns obtained. The parameters for calculating the second derivative NIR spectra were examined to make the spectral difference clearer. In any parameter, the absorption band was shifted to the shorter wavelength region when the unsaturation in fatty acid moieties increased, and it was shifted to the longer wavelength region when the carbon chain length increased. When the parameters were correct, this NIR method can estimate the fatty acid composition roughly, but simply, easily, and sometimes nondestructively.
Key words: analysis; fatty acid composition; near infrared; second derivative; spectroscopy

-5-
Induced Volatiles in Elicitor-treated and Rice Blast Fungus-inoculated Rice Leaves

Naomi OBARA,1,2 Morifumi HASEGAWA,1,3, and Osamu KODAMA1,3

1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-city, Tokyo 183-8509, Japan 2Vista Co. Ltd. 528 Imai-cho, Hodogaya-ku, Yokohama, Kanagawa 240-0035, Japan 3Laboratory of Phytochemical Ecology, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan

Received May 7, 2002; Accepted July 22, 2002
The volatiles released from elicitor (copper chloride, jasmonic acid, UV, L-methionine and chitosan oligomer)-treated and rice blast fungus-inoculated rice leaves were collected by the solid-phase microextraction technique and analyzed by GC-MS. (Z)-3-Hexen-1-ol, monoterpenes, methyl salicylate, and sesquiterpenes were identified as elicitor-induced volatiles by a comparison of their GC retention times and mass spectra with those of authentic compounds. The different elicitors resulted in some qualitative and quantitative differences in the production of volatiles. Monoterpenes and sesquiterpenes were identified as the rice blast fungus-induced volatiles.
Key words: volatile; elicitor; phytoalexin; rice blast fungus; jasmonic acid

-6-
Comparison of C-S Lyase in Lentinus edodes and Allium sativum

Hitomi KUMAGAI,1, Hidetoshi KONO,1 Hidetoshi SAKURAI,1 and Keisuke TOKIMOTO2

1Department of Agricultural and Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi 252-8510, Japan 2The Tottori Mycological Institute, 211 Kokoge, Tottori 689-1125, Japan

Received May 7, 2002; Accepted July 29, 2002
The characteristics of C-S lyase in Lentinus edodes (shiitake) were compared with those in Allium sativum (garlic). C-S lyase mRNA from shiitake was hybridized with the garlic C-S lyase cDNA fragment, being almost the same length as that from garlic. The isoelectric point of the C-S lyase from shiitake was between pH 4 and 5, while that from garlic was over a wider range between pH 4 and 8. Different from the C-S lyase from garlic, that from shiitake was not a glycoprotein without being stained by PAS, and was not bound to the anti-garlic C-S lyase antibody. Similar to garlic C-S lyase, shiitake C-S lyase comprised a homodimer, and its molecular mass was 84 kDa. However, the N-terminal amino acid sequences of each subunit of shiitake C-S lyase were totally different from those of garlic C-S lyase.
Key words: C-S lyase; Lentinus edodes; shiitake; Allium sativum; garlic

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Transcriptional Regulators of the 51D Surface Protein Gene of Paramecium tetraurelia

Hiroyuki TANABE, Isao HARAGUCHI, and Sachiko YOSHIOKA

Laboratory of Biochemistry, Faculty of Agriculture, Kinki University, Nara 631-8505, Japan

Received May 15, 2002; Accepted August 6, 2002
The α-51D gene encodes surface protein 51D, which specifies serotype 51D of Paramecium tetraurelia stock 51. Previously, we isolated the gene as one expressed at much higher levels in a short-lived mutant than in its parental wild-type stock and characterized its expression to be up-regulated with increased clonal age of the wild-type stock. Here we investigated the transcriptional mechanism for the serotype expression in light of its possible causal relationship to the Paramecium clonal life span. DNA-protein binding analyses of the upstream of the α-51D gene identified a stretch of DNA sequence that interacted specifically with macronuclear proteins prepared from the 51D-expressing mutant. The DNA sequence was mapped to the 23 bp between −310 and −288, counting from the initiation position of 51D transcription. A protein with a binding ability for this DNA element was purified to homogeneity from the macronuclear proteins by chromatography using the specific DNA-protein interaction. In vitro transcriptional analyses showed that both the purified protein and its target domain are essential for increased synthesis of the 51D transcript. These results showed that the DNA-protein interaction is required for induction of the 51D expression in the mutant. However, aged wild-type paramecia, in which the serotype is also highly expressed, lacked the same specific transcriptional activity as detected in the mutant, suggesting another DNA-protein interaction involved in the 51D up-regulation.
Key words: life span; aging; surface protein 51D; Paramecium tetraurelia

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Oxidative Stability of Polyunsaturated Fatty Acid in Phosphatidylcholine Liposomes

Mina ARASEKI, Kanako YAMAMOTO, and Kazuo MIYASHITA

Laboratory of Biofunctional Material Chemistry, Division of Marine Bioscience, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan

Received May 24, 2002; Accepted August 17, 2002
Synthesized PCs containing docosahexaenoic acid (DHA), arachidonic acid (AA), linoleic acid (LA), and palmitic acid (PA) at known positions in the glycerol moiety were oxidized in liposomes, bulk, and organic solvent. In bulk and organic solvent, the oxidative stability of PC decreased with increasing degrees of unsaturation. However, the degree of unsaturation had little effect on the stability of PC in liposomes. The oxidative stability of PC in liposomes would be affected by the chemical reactivity based on the degree of unsaturation and by the conformation of fatty acyl component in PC bilayers. When the oxidative stability of 1-PA-2-LA-PC or 1-PA-2-AA-PC was compared with that of a 1:1 (mol ratio) mixture of 1,2-diPA-PC＋1,2-diLA-PC, or 1,2-diPA-PC＋1,2-diAA-PC, respectively, the former PC was more oxidatively stable than that of the latter PC mixture in all oxidation systems, although the degree of unsaturation of 1-PA-2-PUFA-PC was the same as that of the corresponding mixture of diPA-PC＋diPUFA-PC. The higher oxidative stability of 1-PA-2-PUFA-PC than that of a corresponding mixture of diPA-PC＋diPUFA-PC in liposomes was suggested to be due to the different conformation of PC bilayers and the different rate of hydrogen abstraction by free radicals from intermolecular and intramolecular acyl groups.
Key words: oxidative stability; PC liposome; fatty acid composition; vesicle size

-9-
Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium, Thermotoga maritima MSB8

Eranna RAJASHEKHARA, Motomitsu KITAOKA, Yeon-Kye KIM, and Kiyoshi HAYASHI

Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received May 27, 2002; Accepted July 25, 2002
The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6--8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s−1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s−1) kcat followed by 6-deoxy-D-glucose (17 s−1) and 2-deoxy-D-glucose (16 s−1). The natural substrate, D-glucose with the kcat of 8.0 s−1 had the highest (1.1×104 M−1 s−1) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.
Key words: hyperthermostable enzyme; cellobiose phosphorylase; Thermotoga maritima; phosphorolytic reaction; synthetic reaction

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Mutant Luciferase Enzymes from Fireflies with Increased Resistance to Benzalkonium Chloride

Noriaki HATTORI,1, Naoki KAJIYAMA,1 Masako MAEDA,2 and Seiji MURAKAMI1

1Research and Development Division, Kikkoman Corporation, 399 Noda, Noda city, Chiba pref. 278-0037, Japan 2School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Received June 4, 2002; Accepted August 5, 2002
Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4％ and a decay rate per minute of 29.6％ in the presence of 0.1％ BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.
Key words: ATP-bioluminescence; firefly luciferase; benzalkonium chloride; hygiene monitoring; biomass assay

-11-
Cloning of the Maltose Phosphorylase Gene from Bacillus sp. Strain RK-1 and Efficient Production of the Cloned Gene and the Trehalose Phosphorylase Gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

Yasushi INOUE, Nozomu YASUTAKE, Yoshie OSHIMA, Yoshie YAMAMOTO, Tetsuji TOMITA, Shinsuke MIYOSHI, and Tsuneya YATAKE

Showa Sangyo Co., Ltd., 1-16 Sakura, Tsukuba, Ibaraki 305-0003, Japan

Received June 7, 2002; Accepted August 4, 2002
The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens α-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60％ of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.
Key words: maltose phosphorylase; trehalose phosphorylase; intracellular expression; Bacillus subtilis

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Cloning and Nucleotide Sequence of the Glutamate Decarboxylase-encoding Gene gadA from Aspergillus oryzae

Yuki KATO, Yoko KATO, Keiji FURUKAWA, and Shodo HARA

General Research Laboratories of Kiku-Masamune Sake Brewing Co., Ltd., 1-8-6 Uozakinishi-machi, Higashinada-ku, Kobe, Hyogo 658-0026, Japan

Received June 12, 2002; Accepted August 28, 2002
We cloned a genomic DNA encoding the glutamate decarboxylase (GAD) from Aspergillus oryzae using a 200-bp DNA fragment as the probe. This DNA fragment was amplified by the reverse transcription polymerase chain reaction with mRNA of A. oryzae as the template and degenerate primers designed from the conserved amino acid sequence of Escherichia coli GAD and Arabidopsis thaliana GAD. Nucleotide sequencing analysis showed that the cloned gene (designated gadA) encoded 514 amino acid residues and contained three introns. Southern hybridization showed that the gadA gene was on a 6.0-kb SacI fragment and that there was a single copy in the A. oryzae chromosome. The cloned gene was functional, because one transformant of A. oryzae containing multiple copies of the gadA gene had 10-fold the GAD activity and a 12-fold increase in gamma-aminobutyric acid production compared with the control strain.
Key words: glutamate decarboxylase; gamma-aminobutyric acid; A. oryzae; gadA

-13-
Synthesis of 27-Oxo, 27-Hydroxymilbemycins A3 and A4 and Novel 27-Alkoxymilbemycins A3 and A4 from Milbemycins A3 and A4 and Their Acaricidal Activities

Takahiro TSUKIYAMA,1, Yoshihisa TSUKAMOTO,1 Hitomi SUEMOTO,1 Yoshihiro OKAMOTO,2 Yasushi TAMAGAWA,1 Ayako KINOSHITA,1 Reiji ICHINOSE,2 and Kazuo SATO1

1Agroscience Research Laboratories, Crop Protection Company, Sankyo Co., Ltd., 1041 Yasu, Yasu-cho, Yasu-gun, Shiga 520-2342, Japan 2Crop Protection Department, Crop Protection Company, Sankyo Co., Ltd., 7-12 Ginza 2-Chome, Chuo-ku, Tokyo 104-8113, Japan

Received June 13, 2002; Accepted July 18, 2002
27-Oxomilbemycins A3 and A4 and 27-hydroxymilbemycins A3 and A4 were identified as metabolites in soil metabolism studies of milbemycins A3 and A4. Chemical derivation methods were developed to synthesize 27-oxomilbemycins A3 and A4 and 27-hydroxymilbemycins A3 and A4 from milbemycins A3 and A4. In addition, 27-alkoxymilbemycin derivatives were also synthesized from the same precursors. Some of the synthesized compounds displayed satisfactory acaricidal activity against the organophosphorus-sensitive two-spotted spider mite (Tetranychus urticae), but did not have superior activity to corresponding milbemycins A3 and A4.
Key words: milbemycin; acaricide

-14-
Novel Enzymatic Method for the Production of Xylitol from D-Arabitol by Gluconobacter oxydans

Shun-ichi SUZUKI, Masakazu SUGIYAMA, Yasuhiro MIHARA, Ken-ichi HASHIGUCHI, and Kenzo YOKOZEKI

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

Received June 14, 2002; Accepted August 23, 2002
Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5％ (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98％, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.
Key words: xylitol; NADH regeneration; Gluconobacter; membrane-bound dehydrogenase

-15-
A New Polypeptide Toxin from the Nematocyst Venom of an Okinawan Sea Anemone Phyllodiscus semoni (Japanese name <Oh><0192><0192><Wa>unbachi-isoginchaku′′)

Hiroshi NAGAI,1, Naomasa OSHIRO,2 Kyoko TAKUWA-KURODA,1 Setsuko IWANAGA,2 Masatoshi NOZAKI,2 and Terumi NAKAJIMA1

1Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan 2Okinawa Prefectural Institute of Health and Environment, 2085 Ozato, Ozato-son, Okinawa 901-1202, Japan

Received June 18, 2002; Accepted August 1, 2002
The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD50, 50 μg/kg) and hemolytic activity toward 0.8％ sheep red blood cells (ED50, 80 ng/ml).
Key words: sea anemone; Phyllodiscus semoni; polypeptide; toxin; hemolysis

-16-
Isoflavone Aglycon Produced by Culture of Soybean Extracts with Basidiomycetes and Its Anti-angiogenic Activity

Takehito MIURA, Lan YUAN, Buxiang SUN, Hajime FUJII, Mayumi YOSHIDA, Koji WAKAME, and Ken-ichi KOSUNA

Department of Biochemistry, Amino Up Chemical Co., Ltd., 363-32 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan

Received June 24, 2002; Accepted August 16, 2002
Soybean extracts (SBE) containing isoflavone glycosides were cultured with Ganoderma lucidum mycelia producing β-glucosidase. The anti-angiogenic effects of the cultivated product, containing rich in genistein, named GCP (genistein combined polysaccharide), were assessed with chick chorioallantoic membranes (CAM) and a mouse dorsal air-sac model. β-Glucosidase produced by the mycelia converted the isoflavone glycosides into aglycons. A test of volunteers showed that serum concentrations of genistein in the subjects treated with GCP (n＝4) at 3 h after administration were significantly higher than those in the subjects treated with SBE (n＝4). GCP inhibited angiogenesis in CAM, and the activity of GCP was greater than that of SBE. GCP inhibited the formation of new vessels induced by colon carcinoma cells in vivo.
Key words: genistein; angiogenesis; β-glucosidase; basidiomycetes; isoflavone aglycon

-17-
Incorporation and Accumulation of Docosahexaenoic Acid from the Medium by Pichia methanolica HA-32

Hideyuki AOKI,1 Noriko MIYAMOTO,1 Yuji FURUYA,1 Mitsumasa MANKURA,1 Yasushi ENDO,2 and Kenshiro FUJIMOTO2,

1Research Laboratory, Ikeda Food Research Co., Ltd., 95-7 Minooki-Cho, Fukuyama, Hiroshima 721-0956, Japan 2Department of Science of Biological Function, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan

Received July 4, 2002; Accepted August 27, 2002
Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5％ free fatty acid prepared from fish oil (DHA, 28％ of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5％ glucose, 20％ yeast extract, and 3％ free fatty acid in the medium, at pH 6.0 and with incubated at 25°C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K2HPO4, which caused browning of the medium, promoted the accumulation of DHA in cells.
Key words: incorporation; docosahexaenoic acid; Pichia methanolica

-18-
Gene Cloning, Purification, and Characterization of 2,3-Diaminopropionate Ammonia-lyase from Escherichia coli

Takuma UO, Tohru YOSHIMURA, Tozo NISHIYAMA, and Nobuyoshi ESAKI

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Received July 5, 2002; Accepted August 28, 2002
2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5％ that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.
Key words: 2,3-diaminopropionate; 2,3-diaminopropionate ammonia-lyase; D-serine; Escherichia coli

-19-
Zinc is Involved in the Expression of a Nuclear-encoded Alternative Oxidase Gene in the Yeast Hansenula anomala

Nobuko MINAGAWA,1, Harumi KARIYA,1 Natarajan Selvamuthu KUMARASWAMI,1 Ikuko KAMIMURA,1 Shigeru SAKAJO,2 and Akio YOSHIMOTO1

1Department of Biochemistry, School of Pharmacy, Niigata University of Pharmacy and Applied Life Sciences, 5-13-2 Kamishin′ei-cho, Niigata 950-2081, Japan 2Department of Food and Nutrition, Koriyama Women′s University, Koriyama 963-8503, Japan

Received July 8, 2002; Accepted August 20, 2002
When Hansenula anomala cells were treated by the combined addition of pyrithione, a zinc ionophore, and metal chelating agents such as EDTA and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, the antimycin A3-dependent induction of cyanide-resistant respiratory activity was suppressed. Among the chelators we tested, Zn-saturated EDTA failed to sustain the inhibitory effect, and added zinc ions restored the induction in the treated cells. Further, the antimycin A3-inducible mRNA level of the nuclear-encoded alternative oxidase gene detected by reverse transcriptase-PCR was significantly decreased by the treatment, and recovered to the level of untreated cells upon the addition of zinc ions. These results suggest that the treatment with pyrithione plus chelator resulted in an intracellular zinc-deficiency, which suppressed the expression of the nuclear-encoded alternative oxidase gene. The added zinc ions reversibly restored the expression, indicating that zinc is involved in the alternative oxidase gene expression.
Key words: Hansenula anomala; alternative oxidase; gene expression; zinc; pyrithione

-20-
Old Yellow Enzyme from Candida macedoniensis Catalyzes the Stereospecific Reduction of the C＝C Bond of Ketoisophorone

Michihiko KATAOKA,1, Atsushi KOTAKA,1 Akiko HASEGAWA,1 Masaru WADA,2 Ayumi YOSHIZUMI,2 Shigeru NAKAMORI,2 and Sakayu SHIMIZU1

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 2Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho, Fukui 910-1195, Japan

Received July 10, 2002; Accepted September 5, 2002
Microorganisms were screened for ones that reduced 3,5,5-trimethyl-2-cyclohexene-1,4-dione (ketoisophorone; KIP), and several strains were found to produce (6R)-2,2,6-trimethylcyclohexane-1,4-dione (levodione). The enzyme catalyzing the reduction of the C＝C bond of KIP to yield (6R)-levodione was isolated from Candida macedoniensis AKU4588. The results of primary structural analysis and its enzymatic properties suggested that the enzyme might be an Old Yellow Enzyme family protein.
Key words: Old Yellow Enzyme; ketoisophorone; enone reductase; Candida macedoniensis

-21-
Novel S-Benzylisothiourea Compound That Induces Spherical Cells in Escherichia coli Probably by Acting on a Rod-shape-determining Protein(s) Other Than Penicillin-binding Protein 2

Noritaka IWAI,1, Kazuo NAGAI,2 and Masaaki WACHI1,

1Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan 2Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501, Japan

Received July 22, 2002; Accepted August 26, 2002
Random screening for inhibitors of chromosome partitioning in Escherichia coli was done by the anucleate cell blue assay. A novel S-benzylisothiourea derivative, S-(3,4-dichlorobenzyl)isothiourea, tentatively named A22, was found to induce spherical cells and spherical anucleate cells in E. coli. Mecillinam, a specific inhibitor of penicillin-binding protein 2, which induces spherical cells in E. coli, also caused anucleate cell production. Spherical cells induced by treatment with either A22 or mecillinam varied in size, and anucleate cells seemed to be more frequent among the smaller cells. These results suggest that loss of the rod shape in E. coli leads to asymmetric cell division that results in production of anucleate cells. No competition was observed even in the presence of a 10-fold excess A22 in an in vitro assay of 14C-penicillin G binding, but mecillinam specifically inhibited binding of 14C-penicillin G to penicillin-binding protein 2. Simultaneous treatment with mecillinam and cephalexin, a specific inhibitor of penicillin-binding protein 3, induced lysis of E. coli cells, but a combination of A22 and cephalexin did not. These results suggest that the target molecule(s) of A22 was not penicillin-binding protein 2. A22 may act on a rod-shape-determining protein(s) other than penicillin-binding protein 2, such as RodA or MreB.
Key words: benzylisothiourea; chromosome partitioning; mecillinam; PBP2; rod-shape determination

-22-
His-to-Asp Phosphorelay Circuitry for Regulation of Sexual Development in Schizosaccharomyces pombe

Norihito NAKAMICHI, Hisami YAMADA, Keisuke AOYAMA, Ryusuke OHMIYA, Hirofumi AIBA, and Takeshi MIZUNO

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

Received July 24, 2002; Accepted August 22, 2002
The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), and two response regulators (Mcs4 and Prr1). The results of recent extensive studies on the S. pombe His-to-Asp phosphorelay circuitry suggested that it is involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. The functions of these histidine kinases have not yet been fully characterized. Here we characterize a homothallic (h90) mutant lacking the genes for all the histidine kinases, with special reference to sexual development. Homothallic phk1/2/3Δ cells underwent mating precociously in a nitrogen-deficient medium. Surprisingly, the mutant cells underwent mating even in a nitrogen-sufficient medium, under which conditions wild-type cells did so rarely if at all. Under anaerobic (or microaerobic) growth conditions, wild-type cells did not undergo sexual development even in a nitrogen-deficient medium, but the homothallic phk1/2/3Δ cells mated efficiently. Oxidative reagents such as H2O2 induced sexual development in wild-type cells grown anaerobically. On the basis of these results, we propose the novel view that the S. pombe His-to-Asp phosphorelay, initiated by the Phk histidine kinases, is crucial for regulation of sexual development. This Phk-mediated signaling pathway is linked to the well-documented canonical pathway for induction of the sexual development, in that both converge at the initiation of meiosis through activation of ste11＋, mam2＋, and mei2＋ transcription.
Key words: fission yeast; His-kinase; His-to-Asp phosphorelay; meiosis

-23-
Note
Use of Shark Collagen for Cell Culture and Zymography

Yoshihiro NOMURA and Nobuhide KITAZUME

Department of Applied Protein Chemistry, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan

Received February 28, 2002; Accepted August 24, 2002
The uses of shark collagen as a matrix for cell culture and as a substrate for zymography were investigated. Fibroblasts were cultured on a gel matrix of shark type I collagen at 30°C. The collagen gel had contracted by 4 days of incubation. Individual fibroblasts were visible against the transparent background of the contracted collagen as long, lean star-shaped cells. The matrix metalloproteinases (MMPs) from fibroblasts secreted from the medium more easily digested shark gelatin than pig gelatin. MMP-2, -9, and that of potential form were recognizable in the zymographic gel of shark gelatin.
Key words: shark collagen; gel contraction; cell culture matrix; matrix metalloproteinase; gelatin zymography

-24-
Note
Antifungal Activities of N-Arylbenzenesulfonamides against Phytopathogens and Control Efficacy on Wheat Leaf Rust and Cabbage Club Root Diseases

Jae Gon KANG,1 Jong Hyun HUR,1 Sung Jun CHOI,1 Gyung Ja CHOI,2 Kwang Yun CHO,2 Leonid N. TEN,3 Ki Hun PARK,1 and Kyu Young KANG1,

1Department of Agricultural Chemistry, Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, Korea 2Screening Division, Korea Research Institute of Chemical Technology, Yusong-Gu, Taejon 305-600, Korea 3National University of Uzbekistan, VUZ Gorodok, Tashkent 700179, Uzbekistan

Received April 15, 2002; Accepted July 25, 2002
A set of N-arylbenzenesulfonamides with various substituents at the arylamine and benzenesulfonyl positions were prepared, and their antifungal properties were measured in vitro against such plant pathogenic fungi as Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, and Botrytis cinerea. Compounds 3, 4, 8, 9, 10, 14, 16, 18, 20, 21, 24 and 27 had antifungal activity over a broad spectrum of the phytopathogenic fungi tested, where 50％ of inhibition (ED50) was in the range of 3--15 μg/ml. Based on the in vitro activity, six derivatives (3, 4, 10, 18, 21 and 27) were selected and tested further for their fungicidal efficacy in vivo. The fungicidal efficacy of 10, 21 and 27 had a disease control value of over 85％ at 50 μg/ml against wheat leaf rust, while that of 4 was selective against cabbage club root disease.
Key words: N-arylbenzenesulfonamide; fungicidal activity; wheat leaf rust; cabbage club root

-25-
Note
Inhibition by Theanine of Binding of [3H]AMPA, [3H]Kainate, and [3H]MDL 105,519 to Glutamate Receptors

Takami KAKUDA, Ayumu NOZAWA, Akio SUGIMOTO, and Hitoshi NIINO

Central Research Institute, Itoen Ltd., 21 Mekami, Sagara-cho, Haibara-gun, Shizuoka 421-0516, Japan

Received April 22, 2002; Accepted August 23, 2002
In an investigation of the mechanisms of the neuroprotective effects of theanine (γ-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-
phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-
carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.
Key words: theanine; glutamate receptor; neuroprotection; tea

-26-
Note
Identification of a Toxic Mechanism of the Plasticizers, Phtahlic Acid Esters, which are Putative Endocrine Disrupters: Time-dependent Increase in Quinolinic Acid and Its Metabolites in Rats Fed di(2-ethylhexyl)phthalate

Tsutomu FUKUWATARI, Yuko SUZUKI, Etsuro SUGIMOTO, and Katsumi SHIBATA

Laboratory of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures, The University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan

Received May 7, 2002; Accepted August 13, 2002
We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20％ casein diet or the same diet supplemented with 0.1％ DEHP or 0.043％ phthalic acid and 0.067％ 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.
Key words: phthalate ester; di(2-ethylhexyl)phthalate; quinolinic acid; endocrine disrupters; tryptophan metabolism

-27-
Identification of (3S, 9R)- and (3S, 9S)-Megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides as Damascenone Progenitors in the Flowers of Rosa damascena Mill.

Masayuki SUZUKI,1 Shigetaka MATSUMOTO,1 Masaya MIZOGUCHI,1 Satoshi HIRATA,1 Kazuteru TAKAGI,1 Ikue HASHIMOTO,1 Yumiko YAMANO,2 Masayoshi ITO,2 Peter FLEISCHMANN,3 Peter WINTERHALTER,3 Tetuichiro MORITA,4 and Naoharu WATANABE1,

1Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan 2Kobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada-ku, Kobe, Hyogo 658-8558, Japan 3Institut f<Oh><0228><Wa>ur Lebensmittel Chemie, TU-Braunschweig, Schleinitzstr. 20, Braunschweig D-38106, Germany 4Jeol Co., Ltd., Applied Research Center, 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan

Received May 7, 2002; Accepted July 15, 2002
The progenitors of damascenone (1), the most intensive C13-norisoprenoid volatile aroma constituent of rose essential oil, were surveyed in the flowers of Rosa damascena Mill. Besides 9-O-β-D-glucopyranosyl-3-hydroxy-7,8-didehydro-β-ionol (4b), a stable progenitor already isolated from the residual water after steam distillation of flowers of R. damascena Mill., two labile progenitors were identified to be (3S, 9R)- and (3S, 9S)-megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides (2b) based on their synthesis and HPLC-MS analytical data. Compound 2b gave damascenone (1), 3-hydroxy-β-damascone (3) and 4b upon heating under acidic conditions.
Key words: rose flower; damascenone; C13-norisoprenoid; progenitor; megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranoside

-28-
Note
Labdane-type Diterpene Dialdehyde, Pungent Principle of Myoga, Zingiber mioga Roscoe

Masako ABE,1 Yoshio OZAWA,1, Yasushi UDA,2 Yoichi YAMADA,3 Yasujiro MORIMITSU,4 Yoshimasa NAKAMURA,5 and Toshihiko OSAWA5

1Department of Health and Nutrition, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan 2Department of Bioproductive Sciences, Utsunomiya University, Utsunomiya 321-8505, Japan 3Department of Chemistry, Faculty of Education, Utsunomiya University, Utsunomiya 321-8505, Japan 4Laboratory of Food Chemistry, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan 5Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan

Received May 21, 2002; Accepted August 23, 2002
The pungent principle of myoga (Zingiber mioga Roscoe) was identified as (E)-8β(17)-epoxylabd-12-ene-15,16-dial (miogadial) on the basis of its physical and spectroscopic properties (MS, NMR, IR, and UV). Galanal A and B, isolated as well as miogadial, had no hot taste. Reduced miogadial also was tasteless. The pungency of miogadial depended on the presence of αβ-unsaturated-1,4-dialdehyde group.
Key words: Zingiber mioga Roscoe; pungent principle; labdane-type diterpene; miogadial

-29-
Note
Effects of Added Dietary Taurine on Erythrocyte Lipids and Oxidative Stress in Rabbits Fed a High Cholesterol Diet

Jale BALKAN, Serdar <Oh><0196><Wa>OZTEZCAN, G<Oh><0228><Wa>ul<Oh><0231><Wa>in AYKA<Oh><0199><Wa>-TOKER, and M<Oh><0228><Wa>ujdat UYSAL

Department of Biochemistry, Istanbul Medical Faculty, University of <Oh>_<Wa>Istanbul, <Oh><0199><Wa>apa 34390 <Oh>_<Wa>Istanbul, Turkey

Received May 21, 2002; Accepted August 13, 2002
Lipid peroxidation leads to damage of polyunsaturated fatty acids of membrane phospholipids. The contribution of oxidative stress to hypercholesterolemia-induced hemolytic anemia and the effects of addition of taurine on erythrocyte lipid composition, oxidative stres, and hematological data were studied in rabbits fed on a high cholesterol (HC) diet (1％, w/w) for 2 months. The effects of taurine on erythrocyte hemolysis and H<Oh>_<Wa>{2}O<Oh>_<Wa>{2}-induced lipid peroxidation were investigated in normal rabbit erythrocytes in vitro. The HC diet resulted in increases in plasma lipids and lipid peroxide levels as well as increases in cholesterol levels and the cholesterol:phospholipid ratio in the erythrocytes. This diet caused a hemolytic anemia, but lipid peroxide levels remained unchanged in the erythrocytes of the rabbits. Taurine (2.5％, w/w) added to the food has an ameliorating effect on plasma lipids and lipid peroxide levels in rabbits fed on a HC diet. This treatment also caused decreases in elevated erythrocyte cholesterol levels and cholesterol:phospholipid ratio due to the HC diet, but it did not prevent the hemolytic anemia and did not change erythrocyte lipid peroxide levels. In addition, in an in vitro study, taurine did not protect erythrocytes against H<Oh>_<Wa>{2}O<Oh>_<Wa>{2}-induced hemolysis or lipid peroxidation. These results show that the HC diet causes hemolytic anemia without any changes in erythrocyte lipid peroxidation, and taurine treatment was not effective against hemolytic anemia caused by the HC diet.
Key words: taurine; lipid peroxidation; erythrocyte; high cholesterol diet; rabbit

-30-
Note
Substrate Inhibition of L-Cysteine α,β-Elimination Reaction Catalyzed by L-Cystathionine γ-Lyase of Saccharomyces cerevisiae

Shuzo YAMAGATA,1, Motomi ISAJI,2 Tomoko YAMANE,2 and Tomonori IWAMA1

1Department of Biotechnology and 2Division of Veterinary Science, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received May 24, 2002; Accepted July 29, 2002
The α,β-elimination of L-cysteine catalyzed by Saccharomyces cerevisiae L-cystathionine γ-lyase (EC 4.4.1.1) was inhibited by the substrate. The absorption spectrum of the holoenzyme in the presence of L-cysteine showed that the substrate inhibition observed in this reaction was due mainly to removal of the cofactor.
Key words: cysteine β-elimination; cystine β-elimination; cystathionine γ-lyase; pyridoxal 5′-phosphate; Saccharomyces cerevisiae

-31-
Note
Effects of Voluntary Resistance Exercise on Heme Biosynthesis in Rats Given Glucocorticoid-injections

Hyung-Sook KANG,1 Tatsuhiro MATSUO,2, Ryo-Won CHOUE,1 and Masashige SUZUKI3

1Graduate School of East-West Medical Science, Kyung-Hee University, Seoul, Korea 2Faculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan 3Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8574, Japan

Received June 6, 2002; Accepted September 9, 2002
To identify the effects of a tower-climbing exercise on bone marrow heme biosynthesis and hematological status in rats given glucocorticoid-injections as a model of aging, 29 male Sprague-Dawley rats, 10 weeks of age, were assigned to three groups: a saline control (C, n＝9), a glucocorticoid-sedentary (GS, n＝10) group, and a glucocorticoid-exercise (GE, n＝10) group. The GS and GE groups were given 2 mg/kg prednisolone daily, and the C group was given 2 ml of saline daily, subcutaneously. Each group was meal-fed commercial rat chow isoenergetically and given free access to water for 8 weeks. The GE group were allowed to climb a 200-cm tower to drink water from a bottle set at the top of it. Weight gain during the 8-week experimental period was greater in the C group than in the GS and GE groups. The gastrocnemius and tibialis anterior muscles were heavier in the C group than in other groups. The hematological parameters were not influenced by glucocorticoid administration with or without climbing exercise. Bone marrow δ-aminolevulinic acid dehydratase activity was significantly higher in the GE group than in the C group. These results suggest that 8 weeks of climbing exercise increases heme biosynthesis without alteration of hematological status in rats given glucocorticoid-injections. Resistance exercise may be a preventive therapy for iron-deficiency anemia associated with aging.
Key words: resistance exercise; glucocorticoid; heme biosynthesis; δ-aminolevulinic acid dehydratase; rat

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Isolation of Three Main Sericin Components from the Cocoon of the Silkworm, Bombyx mori

Yoko TAKASU, Hiromi YAMADA, and Kozo TSUBOUCHI

National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan

Received June 7, 2002; Accepted July 31, 2002
To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.
Key words: sericin; Bombyx mori; SDS-PAGE; cocoon; middle silk gland

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Molecular Characterization of the Gene Encoding Rice Allene Oxide Synthase and Its Expression

Suk-Bong HA,1 Bong-Choon LEE,1 Da-Eun LEE,1 Yong In KUK,2 A-Young LEE,1 Oksoo HAN,1 and Kyoungwhan BACK1,

1Department of Genetic Engineering, Biotechnology Research Institute, Chonnam National University, Gwangju 500-757, Korea 2Biotechnology Research Institute, Chonnam National University, Gwangju 500-757, Korea

Received June 7, 2002; Accepted September 6, 2002
The gene encoding rice allene oxide synthase, OsAOS, was intronless and had nucleotide sequences with the high GC content of 67％. Deduced amino acid sequences had very high similarity with other AOS proteins, in particular 74％ similarity to barley, characterized by the conserved motifs of P450 cytochrome of the CYP74A family. Purified recombinant rice AOS protein expressed in Escherichia coli converted 13-hydroperoxylinolenic acid to allene oxide. Several restriction enzyme digestions and Southern analysis showed that OsAOS was likely to have two copies in its genome. The basal level of OsAOS expression was detected in various tissues and the transcription level was increased by jasmonate treatment.
Key words: allene oxide synthase; jasmonate; rice

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Biotransformation of the Mycotoxin, Zearalenone, to a Non-estrogenic Compound by a Fungal Strain of Clonostachys sp.

Hideaki KAKEYA,1 Naoko TAKAHASHI-ANDO,2 Makoto KIMURA,2 Rie ONOSE,1 Isamu YAMAGUCHI,2 and Hiroyuki OSADA1,

1Antibiotics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 2Laboratory for Remediation Research, Plant Science Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Received June 10, 2002; Accepted August 7, 2002
Zearalenones are mycotoxins with estrogenic activity consisting of a resorcinol moiety fused to a 14-membered macrocyclic lactone and are produced by various Fusarium species. We found that Clonostachys rosea IFO 7063 was effectively capable of converting zearalenone (1) to cleavage product (2), 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1′E-undecene-6′-one. Moreover, cleavage product 2 did not show potent estrogenic activity like that of 1 and 17β-estradiol in the human breast cancer MCF-7 cell proliferation assay.
Key words: biotransformation; zearalenone; mycotoxins; microorganisms; detoxification

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OsRALyase1, a Putative F-Box Protein Identified in Rice, Oryza sativa, with Enzyme Activity Identical to That of Wheat RALyase

Yasuhiro ITO,1 Akihiko OZAWA,2 Tatsuya SAWASAKI,2 Yaeta ENDO,2 Kozo OCHI,1 and Yuzuru TOZAWA1,

1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 2Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan

Received June 13, 2002; Accepted August 18, 2002
A rice gene, OsRALyase1, encoding a product similar to wheat ribosomal RNA apurinic site specific lyase (RALyase), was isolated and expressed in vitro. An open reading frame of the gene predicted a protein of 476 amino acid residues with 75％ identity to RALyase and contained an F-box-like motif in its amino terminal region. The rice gene product expressed in a wheat-germ protein expression system had the same characteristics as its wheat counterpart, cleaving a specific depurinated site of the 28S rRNA sarcin-ricin domain.
Key words: rice (Oryza sativa); RALyase; sarcin-ricin (S/R) domain; F-box; wheat-germ protein expression system

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Design of a Fluorescent Electrophoretic Mobility Shift Assay Improved for the Quantitative and Multiple Analysis of Protein-DNA Complexes

Takuo ONIZUKA, Sumiyo ENDO, Masahiko HIRANO, Shozo KANAI, and Hideo AKIYAMA

Biological Sciences Department, Toray Research Center, Inc., 1111 Tebiro, Kamakura, Kanagawa 248-8555, Japan

Received June 17, 2002; Accepted July 30, 2002
We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.
Key words: electrophoresis; fluorescence; DNA-binding proteins

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Identification of an Indispensable Amino Acid for ppGpp Synthesis of Escherichia coli SpoT Protein

Chizuko FUJITA, Maki MAEDA, Takako FUJII, Ryoko IWAMOTO, and Kenji IKEHARA

Department of Chemistry, Faculty of Science, Nara Women′s University, Kita-uoya-nishi-machi, Nara 630-8506, Japan

Received June 20, 2002; Accepted August 1, 2002
Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coli SpoT protein. SpoT protein changed from Asp to Ala at the 293rd position did not restore cell growth of E. coli CF8295 (ΔrelA, ΔspoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein.
Key words: stringent response; ppGpp synthesis; SpoT protein; catalytic center; amino acid substitution

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Further Stabilization of Earthworm Serine Protease by Chemical Modification and Immobilization

Nobuyoshi NAKAJIMA,1, Kohji ISHIHARA,2 Manabu SUGIMOTO,3 Takashi NAKAHARA,4 and Hideaki TSUJI1

1Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan 2Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612-8522, Japan 3Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan 4Graduate School of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan

Received June 26, 2002; Accepted September 4, 2002
Earthworm serine protease is more stable and is less affected by organic solvents and detergent than other proteases. However, it is inactivated, probably by autolysis, at 60°C or above under alkaline conditions. Further stabilization was managed by chemical modification of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and phenylglyoxal to protect the activity from the autolytic inactivation. Stabilization was possible also under acidic conditions, in which the stability of the enzyme was rather low, by immobilization with folded sheet mesoporous material. Thus, further stabilization of the enzyme has been achieved by chemical modification or immobilization.
Key words: serine protease; earthworm; stabilization; chemical modification; immobilization

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Antisense Suppression of Collagen VI Synthesis Results in Reduced Expression of Collagen I in Normal Human Osteoblast-like Cells

Satoru HARUMIYA,1,, Mark A. GIBSON,2 and Yasuko KOSHIHARA1,

1Department of Nutrition, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan 2Department of Pathology, University of Adelaide, Adelaide, South Australia 5005, Australia

Received June 26, 2002; Accepted August 5, 2002
A transient increase in collagen VI expression precedes the accumulation of collagen I associated with interleukin-4 (IL-4)-induced mineralization in human osteoblast-like cells. Transfection with an antisense oligonucleotide specific for α1(VI) collagen mRNA was shown to attenuate mRNA levels of collagens VI and I. Incubating IL-4 treated cells with anti-collagen VI antiserum decreased expression of α1(I) mRNA. The results suggest that collagen VI may regulate collagen I expression in the early phase of IL-4-induced mineralization.
Key words: osteoblast; collagen VI; collagen I; human; antisense oligonucleotide

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Novel Hydroquinone as a Matrix Metallo-proteinase Inhibitor from the Mushroom, Piptoporus betulinus

Hirokazu KAWAGISHI,1, Keiko HAMAJIMA,1 and Yoshimasa INOUE2

1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan 2Target Discovery Group 2, R ＆ D Laboratories, Nippon Organon K. K., 5-90 Tomobuchi-cho 1-chome, Miyakojima-ku, Osaka 534-0016, Japan

Received July 4, 2002; Accepted August 7, 2002
The novel hydroquinone, (E)-2-(4-hydroxy-3-methyl-2-butenyl)-hydroquinone, and known compound, polyporenic acid C, were isolated as matrix metallo-proteinase inhibitors from the mushroom, Piptoporus betulinus.
Key words: matrix metallo-proteinase; inhibitor; mushroom; Piptoporus betulinus

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Inhibition of the Bacterial Surface Protein Anchoring Transpeptidase Sortase by Medicinal Plants

Soo-Whan KIM,1 Il-Moo CHANG,1 and Ki-Bong OH1,2

1Natural Products Research Institute, Seoul National University, 28 Yungun, Jongro, Seoul 110-460, Korea 2Graduate School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea

Received July 11, 2002; Accepted August 7, 2002
Inhibition by medicinal plant extracts of a recombinant sortase was evaluated for antibacterial drug discovery. The coding region of sortase, a transpeptidase that cleaves surface proteins of Gram-positive bacteria, was amplified by PCR from the chromosome of Staphylococcus aureus ATCC 6538p with the exception of an N-terminal membrane anchor sequence, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The purified sortase had maximum activity at pH 7.5 and was stable at 20--45°C for the cleavage of a synthetic fluorophore substrate. The enzyme inhibitory activity in medicinal plants was also evaluated for antibacterial drug discovery. Among 80 medicinal plants tested, Cocculus trilobus, Fritillaria verticillata, Liriope platyphylla, and Rhus verniciflua had strong inhibitory activity. The extract with the greatest activity was the ethyl acetate fraction derived from the rhizome of Cocculus trilobus (IC50＝1.52 μg/ml).
Key words: Staphylococcus aureus; sortase expression; medicinal plants; inhibitory activity

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Cloning, Sequencing, and Overexpression in Escherichia coli of a Phenylserine Dehydratase Gene from Ralstonia pickettii PS22

Heiwa OKUDA, Shinji NAGATA, and Haruo MISONO

Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan

Received July 15, 2002; Accepted August 7, 2002
The structural gene coding for phenylserine dehydratase from Ralstonia pickettii PS22 was cloned into Escherichia coli cells, and the nucleotide sequence was identified. The predicted amino acid sequence had high sequence similarity to biodegradative and biosynthetic threonine dehydratases from E. coli and serine dehydratase from human liver. Transformed E. coli cells overproduced phenylserine dehydratase, and the recombinant enzyme was purified to homogeneity with a high yield and characterized.
Key words: phenylserine dehydratase; Ralstonia pickettii; D-threo-3-phenylserine; dehydratase; nucleotide sequence