Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.11 2000)

Deduced Amino Acid Sequence and Possible Catalytic Residues
of a Thermostable, Alkaline Cellulase from an Alkaliphilic Bacillus Strain・
Yoshihiro HAKAMADA,・・ Yuji HATADA, Kenzo KOIKE, Tadashi YOSHIMATSU,
Shuji KAWAI, Tohru KOBAYASHI, and Susumu ITO　p.2281

Effect of Dietary Linoleate/alpha-Linolenate Balance on the Brain Lipid
Composition, Reproductive Outcome and Behavior of Rats during
Their Prenatal and Postnatal Development
Sang-Hee CHEON,・ Moon-Haeng HUH, Yoon-Bok LEE, Jeom-Seon PARK,
Heon-Soo SOHN, and Chai-Won CHUNG p.2290

Antigen Binding of an Ovomucoid-specific Antibody is Affected
by a Carbohydrate Chain Located on the Light Chain Variable Region
Yoshinori FUJIMURA,1 Hirofumi TACHIBANA,1,・ Nozomu ETO2, and Koji YAMADA1 p.2298

Isolation and Characterization of Thermotolerant Gluconobacter Strains Catalyzing Oxidative
Fermentation at Higher Temperatures・
Duangtip MOONMANGMEE,・・,・・・ Osao ADACHI, Yoshitaka ANO, Emiko SHINAGAWA,＊
Hirohide TOYAMA, Gunjana THEERAGOOL,＊＊ Napha LOTONG,＊＊ and Kazunobu MATSUSHITA p.2306

High P Diet Induces Acute Secretion of Parathyroidhormone
without Alteration of Serum Calcium Levels in Rats
Ritsuko MASUYAMA, Mariko UEHARA, and Kazuharu SUZUKI p.2316

Synthesis of Optically Active Olivil Type of Lignan from L-Arabinose Using
threo-Selective Aldol Condensation as a Key Reaction
Satoshi YAMAUCHI・ and Yoshiro KINOSHITA p.2320

Molecular Cloning and Sequence Analysis of an Endoinulinase Gene
from Penicillium sp. Strain TN-88
Hidetoshi AKIMOTO, Naoyuki KIYOTA, Takayuki KUSHIMA, Toyohiko NAKAMURA,
and Kazuyoshi OHTA・ p.2328

Role of Tyrosine 114 of L-Methionine γ-lyase from Pseudomonas putida
Hiroyuki INOUE,1,・ Kenji INAGAKI,1 Naoki ADACHI,1 Takashi TAMURA,1 Nobuyoshi ESAKI,2 Kenji SODA,3 and Hidehiko TANAKA1,＊ p.2336

Purification and Characterization of Novel Transglutaminase
from Bacillus subtilis Spores
Shun′ichi SUZUKI, Yuko IZAWA, Katusnori KOBAYASHI, Yuzuru ETO,
Shigeru YAMANAKA, Koji KUBOTA, and Kenzo YOKOZEKI p.2344

Characterization of O-Acetyl-L-serine Sulfhydrylase Purified
from an Alkaliphilic Bacterium
Yukiko SUGIHARA,＊ Shuzo YAMAGATA,＊,＊＊,・ Yasuko MIZUNO,＊ and Takayuki EZAKI＊＊＊ p.2352

Purification and Characterization of Thermostable Pectate Lyase
with Protopectinase Activity from Thermophilic Bacillus sp. TS 47
Makoto TAKAO, Tetsuko NAKANIWA, Kentaro YOSHIKAWA, Takao TERASHITA,
and Takuo SAKAI p.2360

Potent Inhibition of Macrophomate Synthase
by Reaction Intermediate Analogs
Hideaki OIKAWA, Kenji YAGI, Satoshi OHASHI, Kenji WATANABE, Takashi, MIE,
Akitami ICHIHARA, Mamoru HONMA, and Kimiko KOBAYASHI§ p.2368

Qualitative and Quantitative Analysis of Endogenous Jasmonoids
in Potato Plant (Solanum tuberosum L.)
Hideyuki MATSUURA, Fumihiro OHMORI, Masatomo KOBAYASHI,＊ Akira SAKURAI,＊
and Teruhiko YOSHIHARA・ p.2380

Gordonan, an Acidic Polysaccharide with Cell Aggregation-Inducing Activity
in Insect BM-N4 Cells, Produced by Gordonia sp.
Tatsuhiko KONDO, Daisuke YAMAMOTO, Akira YOKOTA,＊ Akinori SUZUKI,
Hiromichi NAGASAWA, and Shohei SAKUDA・ p.2388

Dietary Antioxidants Fail in Protection against Oxidative Genetic Damage
in In Vitro Evaluation
Mingzhou SUN, Hiroyuki SAKAKIBARA, Hitoshi ASHIDA, Gen-ichi DANNO,
and Kazuki KANAZAWA・ p.2395

Two Novel Diterpenoids, Erinacines H and I from the Mycelia
of Hericium erinaceum
Eun Woo LEE,1 Kazue SHIZUKI,1 Satoshi HOSOKAWA,1 Masami SUZUKI,1 Hiroyuki SUGANUMA,2
Takahiro INAKUMA,2 Jingxuan LI,3 Mayumi OHNISHI-KAMEYAMA,4 Tadahiro NAGATA,5
Shoei FURUKAWA,6 and Hirokazu KAWAGISHI1,・ p.2402

Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl-
Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,
and Isolation and Characterization of the Products
Sumio KITAHATA,a,・ Toshiko TANIMOTO,b Yasuyo OKADA,b Akiko IKUTA,b Keiko TANAKA,b
Hiromi MURAKAMI,a Hirofumi NAKANO,a and Kyoko KOIZUMIb p.2406

Gene Cloning, Purification, and Characterization of Two Cyanobacterial NifS Homologs Driving
Iron-Sulfur Cluster Formation
Shin-ichiro KATO,§ Hisaaki MIHARA,§ Tatsuo KURIHARA, Tohru YOSHIMURA,
and Nobuyoshi ESAKI・ p.2412

A Novel Lantibiotic, Nukacin ISK-1, of Staphylococcus warneri ISK-1:
Cloning of the Structural Gene and Identification of the Structure
Toshihiro SASHIHARA, Hirokazu KIMURA, Toshimasa HIGUCHI, Asaho ADACHI,
Hiromi MATSUSAKI, Kenji SONOMOTO, and Ayaaki ISHIZAKI・p.2420

Synthesis and Tumor-promoting Activities of 12-Epi-phorbol-12,13-dibutyrate
Kazuhiro IRIE,・ Akifumi NAKAHARA, Yasuyuki IKAWA, Minoru TANAKA, Yu NAKAGAWA,
Yoshimasa NAKAMURA, Hajime OHIGASHI, and Paul A. WENDER＃ p.2429

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris
and its Purification and Characterization
Eri CHATANI, Naoki TANIMIZU, Hiroshi UENO, and Rikimaru HAYASHI p.2437

Family 19 Chitinases from Streptomyces thermoviolaceus OPC-520:
Molecular Cloning and Characterization
Hiroshi TSUJIBO,・ Takashi OKAMOTO, Naoya HATANO, Katsushiro MIYAMOTO,
Takeshi WATANABE,＊ Masaru MITSUTOMI,＊＊ and Yoshihiko INAMORI p.2445

Note
Purification of Stearidonic Acid (18:4(n-3)) and Hexadecatetraenoic Acid
(16:4(n-3)) from Algal Fatty Acid with Lipase and Medium Pressure
Liquid Chromatography
Kenji ISHIHARA,・ Masakazu MURATA, Masaki KANENIWA, Hiroaki SAITO,
Wataru KOMATSU,＊ and Kazuki SHINOHARA＊＊ p.2454

Note
Inhibition of Alpha-glucosidase and Amylase by Luteolin, a Flavonoid
Jong-Sang KIM,・ Chong-Suk KWON,1 and Kun Ho SON1 p.2458

Note
Increases in 1,5-Anhydroglucitol Levels in Germinating Amaranth Seeds
and in Ripening Banana
Yotaro KONISHI,・ Keiko HASHIMA, and Kunihiro KISHIDA p.2462

Note
Influence of Exogenous Natural Oils on the ω-1 and ω-2 Hydroxy Fatty
Acid Moiety of Sophorose Lipid Produced by Candida bombicola
Shin OGAWA and Yasuhide OTA・ p.2466

Note
Dietary β- and γ-Cyclodextrins Stimulation of Hepatic Metallothionein
Gene Expression in Rats
Sarunya KAEWPRASERT,1 Minoru OKADA,2 and Yoritaka AOYAMA1 p.2469

Note
Simple Synthesis of 5,9-Dimethylated Long-Chain Alkanes,
the Sex Pheromones of Leaf Miner Moths
Ting LIANG, Shigefumi KUWAHARA,・ Morifumi HASEGAWA, and Osamu KODAMA p.2474

Note
Affinity of Placental Decorin for Collagen
Tumurbaatar BATBAYAR, Yoshihiro NOMURA, Yasuhiro ISHII, and Kunio SHIRAI p.2478

Note
Elongation Factor 2 in the Liver and Skeletal Muscle of Mice
is Decreased by Starvation
Fumiaki YOSHIZAWA, Yutaka MIURA,＊,・ Kazuki TSURUMARU,＊ Yukio KIMATA,＊＊
Kazumi YAGASAKI,＊ and Ryuhei FUNABIKI＊ p.2482

Note
Compilation and Characterization of Histidine-Containing
Phosphotransmitters Implicated in His-to-Asp Phosphorelay in Plants:
AHP Signal Transducers of Arabidopsis thaliana
Tomomi SUZUKI, Kensuke SAKURAI, Aya IMAMURA, Ayako NAKAMURA,
Chiharu UEGUCHI, and Takeshi MIZUNO・ p.2486

Note
Molecular Cloning and Characterization of a cDNA for a Rice Sec31p Homolog
Teruhisa KATO, Atsushi ISHIKAWA, Tadashi ASAHI, and Yukimoto IWASAKI＊ p.2490

Note
Identification and Characterization of a Novel Gene, hos2+, the Function
of Which Is Necessary for Growth under High Osmotic Stress in Fission Yeast
Norihito NAKAMICHI, Eiji YAMAMOTO, Hisami YAMADA, Hirofumi AIBA,・
and Takeshi MIZUNO p.2493

Note
Tributyrin Specifically Induces a Lipase with a Preference for the sn-2 Position
of Triglyceride in Geotrichum sp. FO401B
Yasuhide OTA,・ Takeshi SAWAMOTO, and Masaki HASUO p.2497

Note
Inhibition by (--)-Persenone A-related Compounds of Nitric Oxide
and Superoxide Generation from Inflammatory Leukocytes
Oe Kyung KIM,1 Akira MURAKAMI,2 Yoshimasa NAKAMURA,1 Ha Won KIM,1
and Hajime OHIGASHI1,・ p.2500

Note
An Avocado Constituent, Persenone A, Suppresses Expression of Inducible
Forms of Nitric Oxide Synthase and Cyclooxygenase in Macrophages,
and Hydrogen Peroxide Generation in Mouse Skin
Oe Kyung KIM,1 Akira MURAKAMI,2 Daisuke TAKAHASHI,2 Yoshimasa NAKAMURA,1
Koji TORIKAI,1 Ha Won KIM1 and Hajime OHIGASHI1,・ p.2504

Note
Regulation of Virulence Factors of Enterohemorrhagic Escherichia coli
O157:H7 by Self-Produced Extracellular Factors
Kyoko KANAMARU,1,・,＊ Kengo KANAMARU,2 Ichiro TATSUNO,1 Toru TOBE,1
and Chihiro SASAKAWA1 p.2508

Note
Molecular Cloning of the Gene Encoding an Outer-Membrane-Associated
β-N-Acetylglucosaminidase Involved in Chitin Degradation System
of Alteromonas sp. Strain O-7
Hiroshi TSUJIBO,・ Junko MIYAMOTO, Norihiko KONDO, Katsushiro MIYAMOTO,
Nao BABA, and Yoshihiko INAMORI p.2512

Note
Binding Affinity of the Methyl Ester of AK-toxin I to Membrane Fractions
from Japanese Pear Leaves
Masakazu OKADA, Hisashi MIYAGAWA, Yoshiaki NAKAGAWA, and Tamio UENO p.2517

-1-
Deduced Amino Acid Sequence and Possible Catalytic Residues
of a Thermostable, Alkaline Cellulase from an Alkaliphilic Bacillus Strain・

Yoshihiro HAKAMADA,・・ Yuji HATADA, Kenzo KOIKE, Tadashi YOSHIMATSU,
Shuji KAWAI, Tohru KOBAYASHI, and Susumu ITO

Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan

Received December 16, 1999; Accepted June 23, 2000
Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-β-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-amino-acid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.
Key words: Bacillus pseudofirmus; alkaliphile; cellulase; thermostability; site-directed mutagenesis

-2-
Effect of Dietary Linoleate/alpha-Linolenate Balance on the Brain Lipid
Composition, Reproductive Outcome and Behavior of Rats during
Their Prenatal and Postnatal Development

Sang-Hee CHEON,・ Moon-Haeng HUH, Yoon-Bok LEE, Jeom-Seon PARK,
Heon-Soo SOHN, and Chai-Won CHUNG

Department of Laboratory Animal Science, Central Research Institute, Dr. Chung′s Food Co. Ltd.,
1-25 Songjung-Dong, Heungduk-Gu, Chungjoo-Si, Choongchungbuk-Do, 361-290, Korea

Received March 1, 2000; Accepted June 5, 2000
The effect of the dietary linoleate (LA)/alpha-linolenate (LNA) balance during development on the brain lipid composition, reproductive outcome and behavior of rats was studied. Female rats were fed on experimental diets during pregnancy and the resulting pups for 16 weeks. The dietary LA/LNA ratios were 1.07 (LA1), 2.64 (LA2), 4.45 (LA3), 7.68 (LA4) and 10.35 (LA5). The relative content of docosahexaenoate (DHA) in the brain of pups tended to increase with decreasing LA/LNA ratio at 0 and 3 weeks, while the level of DHA was maintained constant at 16 weeks regardless of the dietary LA/LNA ratio. The learning ability was measured at 12 weeks of age, and there was no difference among the groups. In an open field test, the exploratory index was significantly lower in the LA1 group than in the LA2 group. The LA1 group had a smaller litter size and lower survival rate than the other groups. We conclude that if the diet contained appropriate amounts and balance of LA and LNA, it was possible for rats to synthesize an appropriate amount of DHA and have normal behavioral activity without DHA supplementation.
Key words: docosahexaenoate/arachidonate ratio; linoleate/α-linolenate ratio; brain lipid; behavior; reproductive outcome

-3-
Antigen Binding of an Ovomucoid-specific Antibody is Affected
by a Carbohydrate Chain Located on the Light Chain Variable Region

Yoshinori FUJIMURA,1 Hirofumi TACHIBANA,1,・ Nozomu ETO2, and Koji YAMADA1

1Division of Bioresources and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
2Applied Biochemistry Division, Department of Biological Resource Sciences, Faculty of Agriculture,
Miyazaki University, Miyazaki 889-2192, Japan

Received March 21, 2000; Accepted June 29, 2000
We cloned the variable regions of heavy and light chain genes of an anti-ovomucoid monoclonal antibody (MAb-OM21) produced by the mouse hybridoma cell line OM21. DNA sequence analysis showed that the light chain of the MAb-OM21 has only one potential N-glycosylation consensus sequence in the complementarity determining region 2 of the light chain. To find whether carbohydrate chains are located on the light chain, we assayed for the size of the light chain, after treatment with N-glycosidase, by western blotting, and also detection of the carbohydrate chains on the light chain was done using the lectin blot assay. A N-linked carbohydrate chain has been shown to bind to the light chain. To clarify the role of this carbohydrate chain in the light chain, we produced carbohydrate variant antibodies by N-deglycosylation using glycosidase or by expressing the antibody from different host cells. The N-deglycosylated variant antibody has greater antigen binding, and the antibody produced from the different host cells showed a reduced antigen binding activity and acquired the ability to react to ovalbumin. These results suggest that antigen binding of the ovomucoid specific antibody MAb-OM21 can be affected by the carbohydrate chain on the light chain variable region.
Key words: antigen binding; glycosylation; light chain variable region; human IgG; ovomucoid

-4-
Isolation and Characterization of Thermotolerant Gluconobacter Strains Catalyzing Oxidative
Fermentation at Higher Temperatures・

Duangtip MOONMANGMEE,・・,・・・ Osao ADACHI, Yoshitaka ANO, Emiko SHINAGAWA,＊
Hirohide TOYAMA, Gunjana THEERAGOOL,＊＊ Napha LOTONG,＊＊ and Kazunobu MATSUSHITA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
＊Department of Chemical and Biological Engineering, Ube National College of Technology,
Tokiwadai, Ube 755-8555, Japan
＊＊Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

Received March 27, 2000; Accepted June 20, 2000
Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1％ D-sorbitol or 1％ D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10°C to 37°C and had average optimum growth temperature between 30--33°C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37°C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36--99.79％. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37°C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37°C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100％ within 24 h at 30°C. Even oxidative fermentation of D-fructose done at 37°C, the strain CHM16 still accumulated D-fructose at 80％ within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37°C was superior to that observed at 30°C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.
acetic acid bacteria; Gluconobacter frateurii; oxidative fermentation; thermotolerant Gluconobacter

-5-
High P Diet Induces Acute Secretion of Parathyroidhormone
without Alteration of Serum Calcium Levels in Rats

Ritsuko MASUYAMA, Mariko UEHARA, and Kazuharu SUZUKI

Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received April 10, 2000; Accepted July 5, 2000
To find whether a high phosphorus (P) diet stimulate the secretion of PTH, a high-P diet was fed to rats and an increase in serum P levels has occurred. All rats were fed a control diet (0.5％ calcium (Ca), 0.5％ P) for 7 days, while they were being adapted, for 1 hour at 8:00 AM and again at 8:00 PM. Four groups were switched to the high-P diet (0.5％ Ca, 1.5％ P) at the time of their morning meal for 1 hour. The other 4 groups continued to receive the control diet. Blood samples were collected from the rats in the remaining group, which served as a pre-feeding control. Every 30 minutes after the start of feeding (30, 60, 90, 120 min), blood samples were collected from the rats in the groups fed the control and high-P diets. Serum P concentrations increased upon intake of the high P diet, within 30 minutes after the start of feeding. Serum PTH levels also increased upon intake of the high P diet, within 30 minutes after the start of feeding, and the levels were significantly higher in the high-P group than in the control group. However, no significant difference was observed in serum Ca levels between the two groups. From these results, our findings suggest that an increase in serum P concentration might be a trigger of PTH secretion without any changes of serum calcium levels.
Key words: calcium; phosphorus; parathyroid hormone

-6-
Synthesis of Optically Active Olivil Type of Lignan from L-Arabinose Using
threo-Selective Aldol Condensation as a Key Reaction

Satoshi YAMAUCHI・ and Yoshiro KINOSHITA

College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan

Received April 10, 2000; Accepted June 20, 2000
The threo-selective aldol condensation of (3R, 4S)-3-hydroxy-5-trityloxy-4-pentanolide, which was prepared from L-arabinose, with piperonal was applied to the stereoselective synthesis of the olivil type of lignan, (2R, 3R, 4R) - 4 - benzyl - 4 - hydroxy - 3 - hydroxymethyl - 2 - (3,4-methylenedioxyphenyl)tetrahydrofuran.
Key words: lignan; tetrahydrofuran lignan; olivil; threo selective aldol condensation

-7-
Molecular Cloning and Sequence Analysis of an Endoinulinase Gene
from Penicillium sp. Strain TN-88

Hidetoshi AKIMOTO, Naoyuki KIYOTA, Takayuki KUSHIMA, Toyohiko NAKAMURA,
and Kazuyoshi OHTA・

Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University,
1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan

Received April 17, 2000; Accepted July 19, 2000
A genomic DNA segment and cDNAs encoding an extracellular endoinulinase of Penicillium sp. strain TN-88 were cloned and sequenced. Southern blot analysis indicated that the endoinulinase gene (inuC) was present as a single copy in the genome. An open reading frame, consisting of 1,545 bp, was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. Three distinct transcriptional start points were observed at positions -242 (A), -215 (A), and -75 (C) from the start codon. The 5?-noncoding region had a putative TATA box at position -120 (TATATATA) and two contiguous CAAT sequences at -159 to -151. The deduced amino acid sequence showed 72 and 85％ identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct cluster from other members of the β-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase.
Key words: cDNA; endoinulinase; inulin; Penicillium sp.; phylogenetic analysis

-8-
Role of Tyrosine 114 of L-Methionine γ-lyase from Pseudomonas putida

Hiroyuki INOUE,1,・ Kenji INAGAKI,1 Naoki ADACHI,1 Takashi TAMURA,1 Nobuyoshi ESAKI,2 Kenji SODA,3 and Hidehiko TANAKA1,＊

1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama, Okayama 700-8530, Japan
2Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
3Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka, 564-8680, Japan

Received April 21, 2000; Accepted June 9, 2000
L-Methionine γ-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of γ-family pyridoxal 5?-phosphate dependent enzymes. A mutant form of L-methionine γ-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in kcat for α,γ-elimination of L-methionine, while the Km remained the same as the wild type enzyme. The Y114F mutant had the reduced kcat by only 28- and 16-fold for substrates with an electron-withdrawing group at the γ-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of kcat for α,β-elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that γ-elimination process for L-methionine is the rate-limiting determination step in α,γ-elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine γ-lyase is important in γ-elimination of the substrate.
Key words: general acid catalyst; L-methionine γ-lyase; reaction mechanism; site-directed mutagenesis

-9-
Purification and Characterization of Novel Transglutaminase
from Bacillus subtilis Spores

Shun′ichi SUZUKI, Yuko IZAWA, Katusnori KOBAYASHI, Yuzuru ETO,
Shigeru YAMANAKA, Koji KUBOTA, and Kenzo YOKOZEKI

Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki, Kanagawa 210-8681, Japan

Received April 26, 2000; Accepted June 22, 2000
Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as αs-casein and BSA.
Key words: Transglutaminase; Bacillus subtilis; spore; solubilization; ε-(γ-glutamyl)lysine isopeptide

-10-
Characterization of O-Acetyl-L-serine Sulfhydrylase Purified
from an Alkaliphilic Bacterium

Yukiko SUGIHARA,＊ Shuzo YAMAGATA,＊,＊＊,・ Yasuko MIZUNO,＊ and Takayuki EZAKI＊＊＊

＊United Graduate School of Agricultural Sciences, ＊＊Department of Biotechnology, Faculty of Agriculture,
Gifu University, Gifu 501-1193, Japan, ＊＊＊Department of Microbiology, School of Medicine, Gifu University,
Gifu 500-8705, Japan

Received April 26, 2000; Accepted June 15, 2000
O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55％ by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25％ and 12％ by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21％. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the β-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40°C (when tested at pH 7.5), and at pH 6.5 (at 40°C). The enzyme kept 90％ its activity after incubation at 65°C (at pH 7.5) for 30 min, and more than 90％ after 30 min incubation at pHs 7--12 at 30°C. The enzyme had a Km of 4 mM for O-acetyl-L-serine and a Vmax of 37.0 μmol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5％ total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5?-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.
Key words: cysteine; O-acetyl-L-serine; sulfhydrylase; alkaliphilic bacterium

-11-
Purification and Characterization of Thermostable Pectate Lyase
with Protopectinase Activity from Thermophilic Bacillus sp. TS 47

Makoto TAKAO, Tetsuko NAKANIWA, Kentaro YOSHIKAWA, Takao TERASHITA,
and Takuo SAKAI

Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, 3327-204,
Nakamachi, Nara 631-8505, Japan

Received May 8, 2000; Accepted June 13, 2000
A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50％ identity.
Key words: thermophili; Bacillus; pectate lyase; thermostable enzyme

-12-
Potent Inhibition of Macrophomate Synthase
by Reaction Intermediate Analogs

Hideaki OIKAWA, Kenji YAGI, Satoshi OHASHI, Kenji WATANABE, Takashi, MIE,
Akitami ICHIHARA, Mamoru HONMA, and Kimiko KOBAYASHI§

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
§Molecular Characterization Division, RIKEN (The Institute of Physical and Chemical Research),
Wako-shi, Saitama 351-0198, Japan

Received May 12, 2000; Accepted June 26, 2000
Potent inhibitors for macrophomate synthase, which has recently been found to catalyze a highly unusual five-step chemical transformation, were explored. Among 11 oxalacetate analogs tested, only three analogs had moderate to relatively strong inhibitory activities (I50 1.3--8.1 mM). On the other hand, among 35 bicyclic intermediate analogs synthesized, two diacids were found to be the most potent inhibitors (I50 0.80, 0.84 mM) which had a much higher affinity than that of the natural substrate 2-pyrone. (--)-Enantiomers of the diacids showed 30 times stronger activity (I50 0.34, 0.41 mM) than (+)-ones. The I50/Km values (0.20, 0.24) showed their potent inhibitions. Competitive inhibitions were observed in two representative inhibitors.
Key words: macrophomate synthase; inhibitors; 2-pyrone; oxalacetate

-13-
Qualitative and Quantitative Analysis of Endogenous Jasmonoids
in Potato Plant (Solanum tuberosum L.)

Hideyuki MATSUURA, Fumihiro OHMORI, Masatomo KOBAYASHI,＊ Akira SAKURAI,＊
and Teruhiko YOSHIHARA・

Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University,
Sapporo, 060-8589, Japan
＊The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama,
351-0198, Japan

Received May 15, 2000; Accepted July 5, 2000
Qualitative and quantitative analyses of endogenous jasmonoids were done by liquid chromatography/selected ion monitoring (LC-SIM) using deuterium-labeled compounds as internal standards. To prove the practicality of this way of analyzing the contents of endogenous jasmonoids in plants, the method was used for estimating jasmonoids in potato plants.
Key words: Solanum tuberosum L.; potato micro-tuber inducing stimuli; jasmonoids; liquid chromatography/selected ion monitoring (LC-SIM)

-14-
Gordonan, an Acidic Polysaccharide with Cell Aggregation-Inducing Activity
in Insect BM-N4 Cells, Produced by Gordonia sp.

Tatsuhiko KONDO, Daisuke YAMAMOTO, Akira YOKOTA,＊ Akinori SUZUKI,
Hiromichi NAGASAWA, and Shohei SAKUDA・

Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
＊Institute of Molecular and Cellular Biosciences. The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received May 17, 2000; Accepted June 28, 2000
An acidic polysaccharide, termed gordonan, was isolated from the culture medium of Gordonia sp. as an inducer of cell aggregation in an insect cell line, BM-N4. Gordonan had an average molecular weight of 5×106 and its structure was identified as →3)-4-O-(1-carboxyethyl) - β - D - Manp - (1→4)-β - D - GlcAp - (1→4)-β-D-Glcp-(1→ mainly by acid hydrolysis experiments and NMR analysis. It induces cell aggregation at the concentration of 4 μg/ml. A partially hydrolyzed polysaccharide derived from gordonan with a molecular weight of 5×105 showed weak activity, while any fragment molecules with lower molecular weights prepared from gordonan showed no activity.
Key words: Gordonia; polysaccharide; cell aggregation; insect cell line

-15-
Dietary Antioxidants Fail in Protection against Oxidative Genetic Damage
in In Vitro Evaluation

Mingzhou SUN, Hiroyuki SAKAKIBARA, Hitoshi ASHIDA, Gen-ichi DANNO,
and Kazuki KANAZAWA・

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

Received May 18, 2000; Accepted June 29, 2000
Carcinogenesis is believed to be induced through the oxidative damage of DNA, and antioxidants are expected to suppress it. So, the polyphenolic antioxidants in daily foods were investigated to see whether they protect against genetic damage by active oxygen. In the evaluation, we used a bioassay and a chemical determination, a Salmonella mutagenicity test for mutation by a N-hydroxyl radical from one of the dietary carcinogens 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the formation of 8-hydroxyl (8-OHdG) from 2?-deoxyguanosine (2?-dG) in a Fenton OH-radical generating system. Thirty-one antioxidants including flavonoids were compared in terms of radical-trapping activity with bacterial DNA and 2?-dG. Antioxidants inhibited the mutation but the IC50 values were in the mM order. Against 8-OHdG formation, only α-tocopherol had a suppressive effect with an IC50 of 1.5 μM. Thus, except α-tocopherol, the dietary antioxidants did not scavenge the biological radicals faster than bacterial DNA and intact 2?-dG, indicating that they failed to prevent oxidative gene damage and probably carcinogenesis.
Key words: flavonoid; 8-OHdG; hydrogen peroxide; antioxidant; genotoxicity

-16-
Two Novel Diterpenoids, Erinacines H and I from the Mycelia
of Hericium erinaceum

Eun Woo LEE,1 Kazue SHIZUKI,1 Satoshi HOSOKAWA,1 Masami SUZUKI,1 Hiroyuki SUGANUMA,2
Takahiro INAKUMA,2 Jingxuan LI,3 Mayumi OHNISHI-KAMEYAMA,4 Tadahiro NAGATA,5
Shoei FURUKAWA,6 and Hirokazu KAWAGISHI1,・

1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan
2Research Institute, Kagome Co., Ltd., 17 Nishitomiyama, Nishinasuno-machi, Tochigi 329-2762, Japan
3Jilin Institute of Biology, 6 Qian, Jin Street, Changchun, China
4National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba,
Ibaraki 305-8642, Japan
5Department of Animal Products, National Institute of Animal Industry-NIAI, 2 Ikenodai, Kukizaki,
Ibaraki 305-0901, Japan
6Department of Molecular Biology, Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi,
Gifu 502-8585, Japan

Revised May 19, 2000; Accepted June 27, 2000
Novel diterpenoids, erinacines H (1) and I (3), were isolated from the cultured mycelia of Hericium erinaceum. The structures of the compounds were determined by interpretation of the spectral data. Erinacine H showed stimulating activity of nerve growth factor (NGF)-synthesis.
Key words: Hericium erinaceum; nerve growth factor; stimulator; diterpenoid

-17-
Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl-
Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,
and Isolation and Characterization of the Products

Sumio KITAHATA,a,・ Toshiko TANIMOTO,b Yasuyo OKADA,b Akiko IKUTA,b Keiko TANAKA,b
Hiromi MURAKAMI,a Hirofumi NAKANO,a and Kyoko KOIZUMIb

aOsaka Municipal Technical Research Institute, 1-6-50, Morinomiya, Joto-ku, Osaka 536-0025, Japan
bSchool of Pharmaceutical Sciences, Mukogawa Women′s University, 11-68, Koshien, Kyuban-cho,
Nishinomiya 663-8179, Japan

Received May 22, 2000; Accepted June 22, 2000
α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)2-βCD were produced in about 20％ and 4％ yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)2-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(63-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)2-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)2-βCDs were identified as 61,62-, 61,63- and
61,64-di-O-(63-O-α-D-mannosyl-maltotriosyl)-βCD.
Key words: reverse action; pullulanase; heterobranched CD

-18-
Gene Cloning, Purification, and Characterization of Two Cyanobacterial NifS Homologs Driving
Iron-Sulfur Cluster Formation

Shin-ichiro KATO,§ Hisaaki MIHARA,§ Tatsuo KURIHARA, Tohru YOSHIMURA,
and Nobuyoshi ESAKI・

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

Received May 24, 2000; Accepted July 10, 2000
Iron-sulfur proteins are essential in the photosynthetic system and many other biological processes. We have isolated and characterized enzymes driving the formation of iron-sulfur clusters from Synechocystis sp. PCC6803. Two genes (slr0387 and sll0704), showing similarity to nifS of Azotobacter vinelandii, were cloned, and their gene products (SsCsd1 and SsCsd2) were purified. They catalyzed the desulfuration of L-cysteine. Reconstitution of a [2Fe-2S] cluster of cyanobacterial ferredoxin proceeded much faster in the presence of L-cysteine and either of these enzymes than when using sodium sulfide. These results suggest that SsCsd1 and SsCsd2 facilitate the iron-sulfur cluster assembly by producing inorganic sulfur from L-cysteine. Synechocystis sp. PCC6803 has no gene coding for a protein with similarity to the N-terminal domain of NifU of A. vinelandii, which is believed to cooperate with NifS to assemble iron-sulfur clusters. Thus, the cluster formation in the cyanobacterium probably proceeds through a mechanism that is different from that in A. vinelandii.
Key words: iron-sulfur cluster; cysteine desulfurase; NifS; cyanobacterium

-19-
A Novel Lantibiotic, Nukacin ISK-1, of Staphylococcus warneri ISK-1:
Cloning of the Structural Gene and Identification of the Structure

Toshihiro SASHIHARA, Hirokazu KIMURA, Toshimasa HIGUCHI, Asaho ADACHI,
Hiromi MATSUSAKI, Kenji SONOMOTO, and Ayaaki ISHIZAKI・

Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department
of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, 6-10-1,
Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received May 29, 2000; Accepted July 7, 2000
Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.
Key words: lantibiotic; nukacin ISK-1; Staphylococcus; cloning; bacteriocin

-20-
Synthesis and Tumor-promoting Activities of 12-Epi-phorbol-12,13-dibutyrate

Kazuhiro IRIE,・ Akifumi NAKAHARA, Yasuyuki IKAWA, Minoru TANAKA, Yu NAKAGAWA,
Yoshimasa NAKAMURA, Hajime OHIGASHI, and Paul A. WENDER＃

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
＃Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA

Received May 30, 2000; Accepted June 24, 2000
12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O−2) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the β-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.
Key words: 12-epi-phorbol-12,13-dibutyrate; Epstein-Barr virus; phorbol ester; protein kinase C; tumor promoter

-21-
Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris
and its Purification and Characterization

Eri CHATANI, Naoki TANIMIZU, Hiroshi UENO, and Rikimaru HAYASHI

Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School
of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received June 5, 2000; Accepted July 6, 2000
A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60--70％ of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.
Key words: RNase A; Pichia pastoris; expression; glycosylated RNase A

-22-
Family 19 Chitinases from Streptomyces thermoviolaceus OPC-520:
Molecular Cloning and Characterization

Hiroshi TSUJIBO,・ Takashi OKAMOTO, Naoya HATANO, Katsushiro MIYAMOTO,
Takeshi WATANABE,＊ Masaru MITSUTOMI,＊＊ and Yoshihiko INAMORI

Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
＊Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University,
Niigata 950-21, Japan
＊＊Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan

Received June 8, 2000; Accepted July 14, 2000
Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5--6, and the optimum temperature of Chi35 and Chi25 were 60 and 70°C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.
Key words: chitinase; antifungal activity; Streptomyces thermoviolaceus

-23-
Note
Purification of Stearidonic Acid (18:4(n-3)) and Hexadecatetraenoic Acid
(16:4(n-3)) from Algal Fatty Acid with Lipase and Medium Pressure
Liquid Chromatography

Kenji ISHIHARA,・ Masakazu MURATA, Masaki KANENIWA, Hiroaki SAITO,
Wataru KOMATSU,＊ and Kazuki SHINOHARA＊＊

Marine Biochemistry Division, National Research Institute of Fisheries Science, 2-12-4 Fukuura,
Kanazawa-ku, Yokohama 236-8648, Japan
＊Department of Life Science, Toita Women′s College, Hachioji 193-0802, Japan
＊＊National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-0856, Japan

Received December 9, 1999; Accepted July 3, 2000
Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40％) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465--1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95％ purity.
Key words: stearidonic acid; lipase; hexadecatetraenoic acid; marine alga; selective esterification

-24-
Note
Inhibition of Alpha-glucosidase and Amylase by Luteolin, a Flavonoid

Jong-Sang KIM,・ Chong-Suk KWON,1 and Kun Ho SON1

Department of Animal Science and Biotechnology, Kyungpook National University, Taegu 702-701, Korea
1Department of Food Science and Nutrition, Andong National University, Andong, 760-749, Korea

Received January 17, 2000; Accepted June 26, 2000
Twenty-one naturally occurring flavonoids were tested for inhibitory activities against alpha-glucosidase (EC 3.2.1.20) and alpha-amylase (EC 3.2.1.1). Luteolin, amentoflavone, luteolin 7-O-glucoside, and daidzein were the strongest inhibitors among the compounds tested. Luteolin inhibited alpha-glucosidase by 36％ at the concentration of 0.5 mg/ml and was stronger than acarbose, the most widely prescribed drug, in inhibitory potency, suggesting that it has the possibility to effectively suppress postprandial hyperglycemia in patients with non-insulin dependent diabetes mellitus. Luteolin also inhibited alpha-amylase effectively although it was less potent than acarbose. The clinical value of luteolin needs to be further evaluated.
Key words: alpha-glucosidase inhibitor; alpha-amylase; luteolin; flavonoids

-25-
Note
Increases in 1,5-Anhydroglucitol Levels in Germinating Amaranth Seeds
and in Ripening Banana

Yotaro KONISHI,・ Keiko HASHIMA, and Kunihiro KISHIDA

Faculty of Human Life Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi-ku,
Osaka 558-8585, Japan

Received March 21, 2000; Accepted July 14, 2000
To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700--5,000 times lower than those of total soluble carbohydrates. α-1,4-Glucan lyase activity, which is measured by the 1,5-
anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via α-1,4-glucan lyase/AF reductase exists in plant tissues.
Key words: 1,5-Anhydroglucitol; starch degradation; amaranth; banana

-26-
Note
Influence of Exogenous Natural Oils on the ω-1 and ω-2 Hydroxy Fatty
Acid Moiety of Sophorose Lipid Produced by Candida bombicola

Shin OGAWA and Yasuhide OTA・

Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

Received March 24, 2000; Accepted June 21, 2000
Candida bombicola can synthesize monohydroxy fatty acid as a moiety of sophorose lipids. The hydroxy fatty acids contained in a major lactone were identified by GC-MS, after culturing with natural oils such as coconut, rapeseed, olive, and soybean oils. Hydroxy fatty acids of C18 and C16 were always synthesized, but differences were observed among the oils regarding the positions of hydroxyl groups, unsaturation, and composition of the fatty acids. A new C17 hydroxy acid was found without addition of oil.
Key words: Candida bombicola; sophorose lipid; hydroxy fatty acid

-27-
Note
Dietary β- and γ-Cyclodextrins Stimulation of Hepatic Metallothionein
Gene Expression in Rats

Sarunya KAEWPRASERT,1 Minoru OKADA,2 and Yoritaka AOYAMA1

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Nishi-9, Kita-9, Kita-Ku, Sapporo 060-8589, Japan
2Nihon Shokuhin Kako Co. Ltd., Fuji, Shizuoka 417-8530, Japan

Received April 10, 2000; Accepted June 19, 2000
This study investigated whether hepatic metallothionein gene expression is affected by dietary cyclodextrins. Young male Wistar rats were fed a basal diet or cyclodextrin-supplemented (50 g of cyclodextrin per kg diet) diets for 7 d. Copper content in the liver did not show any significant changes among rats fed the basal, β- and γ-cyclodextrin diets. There were no differences in liver or serum zinc among groups. Copper content in serum was markedly decreased in rats fed the γ-cyclodextrin-supplemented diet. Liver metallothionein mRNA levels were significantly elevated in both β- and γ-cyclodextrins-fed rats, but not in α-cyclodextrin-fed rats. Thus, the increase in hepatic metallothionein mRNA levels might be due to this mechanism except for the contents of copper and zinc in the liver.
Key words: cyclodextrin; metallothionein mRNA; copper; zinc

-28-
Note
Simple Synthesis of 5,9-Dimethylated Long-Chain Alkanes,
the Sex Pheromones of Leaf Miner Moths

Ting LIANG, Shigefumi KUWAHARA,・ Morifumi HASEGAWA, and Osamu KODAMA

Laboratory of Agricultural Chemicals, Faculty of Agriculture, Ibaraki University, Ami-machi,
Inashiki-gun, Ibaraki 300-0393, Japan

Received May 1, 2000; Accepted June 13, 2000
Each stereoisomeric mixture of 5,9-dimethylpentadecane and 5,9-dimethylhexadecane, the major and the minor sex pheromone components of Perileucoptera coffeella, respectively, was synthesized in about 25％ overall yield through 6 steps from β-citronellol. 5,9-Dimethylheptadecane, the major sex pheromone component of Leucoptera malifoliella, was also synthesized analogously as a stereoisomeric mixture in a 22％ overall yield.
Key words: sex pheromone; Perileucoptera coffeella; 5,9-dimethylpentadecane; 5,9-dimethylheptadecane; 5,9-dimethylhexadecane

-29-
Note
Affinity of Placental Decorin for Collagen

Tumurbaatar BATBAYAR, Yoshihiro NOMURA, Yasuhiro ISHII, and Kunio SHIRAI

Applied Protein Chemistry, Faculty of Agriculture, Tokyo University of Agriculture and Technology,
3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

Received May 1, 2000; Accepted July 3, 2000
Decorin was isolated from 7 M urea extract of bovine placental cotyledons by ion-exchange and hydrophobic chromatography. Decorin and its core protein showed a broad band at about 115 kDa and a single band at 47 kDa, respectively by SDS-PAGE. Anti-decorin core protein antiserum from pig skin was reacted with placental decorin and its core protein in western blotting. The NH2-terminal amino acid sequence of core protein from placental cotyledons was not different from that of core protein from skin and bone. Glycosaminoglycan of decorin was identified as dermatan sulfate by electrophoresis on a cellulose-acetate membrane and chondroitinase digestivity. Decorin bound to collagen in the order for type III, I, and V.
Key words: decorin; core protein; dermatan sulfate; collagen; placenta

-30-
Note
Elongation Factor 2 in the Liver and Skeletal Muscle of Mice
is Decreased by Starvation

Fumiaki YOSHIZAWA, Yutaka MIURA,＊,・ Kazuki TSURUMARU,＊ Yukio KIMATA,＊＊
Kazumi YAGASAKI,＊ and Ryuhei FUNABIKI＊

Department of Science of Living, Morioka Junior College, Iwate Prefectural University,
Takizawa, Iwate 020-0173, Japan
＊Department of Applied Biological Science, Tokyo Noko University, Fuchu, Tokyo 183-8509, Japan
＊＊Research and Education Center for Genetic Information, Nara Institute of Science and Technology,
Ikoma, Nara 630-0101, Japan

Received May 8, 2000; Accepted July 7, 2000
We examined whether starvation affected the amount of EF-2 protein as well as the level of its mRNA in the liver and skeletal muscle of mice, to understand the molecular mechanism for nutritional adaptation of protein-turnover. Although the amount of EF-2 was diminished by starvation in each of the tissues examined, the amount of EF-2 mRNA did not decrease in parallel with the protein.
Key words: elongation factor 2; p70 S6 kinase; starvation; protein synthesis; mice

-31-
Note
Compilation and Characterization of Histidine-Containing
Phosphotransmitters Implicated in His-to-Asp Phosphorelay in Plants:
AHP Signal Transducers of Arabidopsis thaliana

Tomomi SUZUKI, Kensuke SAKURAI, Aya IMAMURA, Ayako NAKAMURA,
Chiharu UEGUCHI, and Takeshi MIZUNO・

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

Received May 12, 2000; Accepted June 8, 2000
Histidine (His)-to-Aspartate (Asp) phosphorelay signal transduction systems are generally made up of a ``sensor histidine (His)-kinase′′, a ``response regulator′′, and a ``histidine-containing phosphotransmitter (HPt)′′. In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that the His-to-Asp phosphorelay mechanism is at least partly responsible for propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we compiled the members of the HPt family of phosphotransmitters in Arabidopsis thaliana (AHP-
series, Arabidopsis HPt phosphotransmitters), based on both database and experimental analyses, in order to provide a comprehensive basis at the molecular level for understanding the function of the AHP phosphotransmitters that are implicated in the His-to-Asp phosphorelay of higher plants.
Key words: Arabidopsis thaliana; His-to-Asp phosphorelay; HPt signal transducers; response regulators; His-kinases

-32-
Note
Molecular Cloning and Characterization of a cDNA for a Rice Sec31p Homolog

Teruhisa KATO, Atsushi ISHIKAWA, Tadashi ASAHI, and Yukimoto IWASAKI＊

Department of Bioscience, Faculty of Biotechnology, Fukui Prefectural University, 4-1-1, Kenjyojima,
Matsuoka-cho, Yoshida-gun, Fukui 910-1195, Japan

Received May 17, 2000; Accepted June 27, 2000
A cDNA for a putative Sec31p in rice has been cloned and sequenced. In yeast, Sec31p is a component of a protein-coated vesicle, COPII, which functions in the transport of cargo proteins from the endoplasmic reticulum to the cis-Golgi network. Structural similarities between yeast Sec31p and the rice putative homolog are discussed.
Key words: Sec31p; COPII; heterotrimeric G protein; rice

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Identification and Characterization of a Novel Gene, hos2+, the Function
of Which Is Necessary for Growth under High Osmotic Stress in Fission Yeast

Norihito NAKAMICHI, Eiji YAMAMOTO, Hisami YAMADA, Hirofumi AIBA,・
and Takeshi MIZUNO

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

Received May 22, 2000; Accepted July 2, 2000
hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2Δ) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2Δ mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.
Key words: osmoregulation; Schizosaccharomyces pombe

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Tributyrin Specifically Induces a Lipase with a Preference for the sn-2 Position
of Triglyceride in Geotrichum sp. FO401B

Yasuhide OTA,・ Takeshi SAWAMOTO, and Masaki HASUO

Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

Received May 24, 2000; Accepted June 26, 2000
The extracellular Lipases A and C produced by Geotrichum sp. FO401B have a preference for the sn-1,3 and sn-2 positions of triglyceride, respectively. Total production of these lipases was increased by plant oils and tributyrin. Butyl Toyopearl column chromatography demonstrated that only Lipase C was produced in the presence of tributyrin. Lipase C hydrolysed natural fats except sardine oil preferentially at the sn-2 position, but it showed little stereoselectivity for triolein.
Key words: Geotrichum; lipase; positional specificity; stereoselectivity; tributyrin

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Inhibition by (--)-Persenone A-related Compounds of Nitric Oxide
and Superoxide Generation from Inflammatory Leukocytes

Oe Kyung KIM,1 Akira MURAKAMI,2 Yoshimasa NAKAMURA,1 Ha Won KIM,1
and Hajime OHIGASHI1,・

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology,
Kinki University, Iwade-Uchita, Wakayama, 649-6493, Japan

Received June 2, 2000; Accepted July 17, 2000
We have previously reported that persenone A, isolated from avocado fruit, is an effective inhibitor of both nitric oxide (NO) and superoxide (O--2) generation in cell culture systems. In this study, we have prepared four persenone A-related compounds and examined their inhibition of NO and O--2 generation from inflammatory leukocytes. Some structural importance in persenone A to attenuate free radical generation is discussed.
Key words: nitric oxide; superoxide; persenone A

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An Avocado Constituent, Persenone A, Suppresses Expression of Inducible
Forms of Nitric Oxide Synthase and Cyclooxygenase in Macrophages,
and Hydrogen Peroxide Generation in Mouse Skin

Oe Kyung KIM,1 Akira MURAKAMI,2 Daisuke TAKAHASHI,2 Yoshimasa NAKAMURA,1
Koji TORIKAI,1 Ha Won KIM1 and Hajime OHIGASHI1,・

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology,
Kinki University, Iwade-Uchita, Wakayama 649-6493, Japan

Received June 2, 2000; Accepted July 17, 2000
We investigated the suppressive effects of an avocado constituent, persenone A, on lipopolysaccharide- and interferon-γ-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in a mouse macrophage cell line RAW 264.7. Persenone A at concentration of 20 μM almost completely suppressed both iNOS and COX-2 protein expression. In mouse skin, double treatments with persenone A (810 nmol) significantly suppressed double 12-O-tetradecanoylphorbol-13-acetate (TPA, 8.1 nmol) application-induced hydrogen peroxide (H2O2) generation. Treatment with persenone A before the second TPA treatment was sufficient to inhibit H2O2 generation, while the first treatment was not. This study thus suggests that persenone A is a possible agent to prevent inflammation-associated diseases including cancer.
Key words: persenone A; inducible nitric oxide synthase; cyclooxygenase-2; hydrogen peroxide

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Regulation of Virulence Factors of Enterohemorrhagic Escherichia coli
O157:H7 by Self-Produced Extracellular Factors

Kyoko KANAMARU,1,・,＊ Kengo KANAMARU,2 Ichiro TATSUNO,1 Toru TOBE,1
and Chihiro SASAKAWA1

1Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
2Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-0032, Japan

Received June 13, 2000; Accepted July 11, 2000
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes serious diarrhea and hemolytic uremic syndrome in humans. The expressions of EspD and intimin by O157:H7 have now been shown to be down-regulated by medium conditioned by O157:H7 grown at stationary phase. Preparation of conditioned medium showing the effect on the amount of EspD was not dependent on temperature or growth medium, but was dependent on growth phase. Inhibition of EspD and intimin expression was also induced by medium conditioned by E. coli K-12 strains and homoserine lactone, a signal molecule of the quorum-sensing system in Gram-negative bacteria. These results suggest the possibility that the quorum-sensing system mediated by self-produced extracellular factors plays an important role in control of colonization of EHEC O157:H7.
Key words: enterohemorrhagic Escherichia coli (EHEC) O157:H7; virulence factors; gene expression; quorum-sensing system; auoinducer

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Molecular Cloning of the Gene Encoding an Outer-Membrane-Associated
β-N-Acetylglucosaminidase Involved in Chitin Degradation System
of Alteromonas sp. Strain O-7

Hiroshi TSUJIBO,・ Junko MIYAMOTO, Norihiko KONDO, Katsushiro MIYAMOTO,
Nao BABA, and Yoshihiko INAMORI

Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 560-1094, Japan

Received July 3, 2000; Accepted July 17, 2000
The gene encoding β-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97 kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial β-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.
Key words: β-N-acetylglucosaminidase; Alteromonas sp. O-7; chitin degradation; lipoprotein

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Binding Affinity of the Methyl Ester of AK-toxin I to Membrane Fractions
from Japanese Pear Leaves

Masakazu OKADA, Hisashi MIYAGAWA, Yoshiaki NAKAGAWA, and Tamio UENO

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 6068502, Japan

Received July 3, 2000; Accepted July 18, 2000
The binding site of AK-toxin, a host-specific toxin against Japanese pear, was searched for in the membrane fractions of the pear leaves, using 3H-labeled AK-toxin I methyl ester. Binding activity, which was displaceable by the unlabeled ligand, was observed for microsomal fraction from a toxin-susceptible cultivar, Nijisseiki. However, the binding was also observed for those from toxin-resistant cultivars, Kosui and Hosui. Detection of the specific binding failed for the plasma membrane fraction which was prepared from microsomal fraction of the toxin-susceptible cultivar by aqueous two-phase separation, and the hitherto presumed model of the AK-toxin receptor in the plasma membrane could not be verified.
Key words: Japanese pear; host-specific toxin; AK-toxin; plasma membrane; ligand binding assay