Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.5 2002)

Hypoglycemic Effect of a Lentinus edodes Exo-polymer Produced
from a Submerged Mycelial Culture
Byung-Keun YANG, Dong-Hyun KIM, Sang-Chul JEONG, Surajit DAS, Young-Sun CHOI,1
Joon-Shik SHIN,2 Sang-Chul LEE,2 and Chi-Hyun SONG† p.937

Biogenesis of 2-Phenylethanol in Rose Flowers: Incorporation of
[2H8]L-Phenylalanine into 2-Phenylethanol and its β-D-Glucopyranoside
during the Flower Opening of Rosa <Oh><0192><Wa>Hoh-Jun′ and Rosa damascena Mill.
Shuzo WATANABE,1 Kentaro HAYASHI,2 Kensuke YAGI,2 Tatsuo ASAI,3 Hazel MACTAVISH,4,†,Joanne PICONE,4 Colin TURNBULL,4 and Naoharu WATANABE2,＊ p.943

Identification of a Novel Anti-ice-nucleating Polysaccharide
from Bacillus thuringiensis YY529
Yasuhiro YAMASHITA†, Hidehisa KAWAHARA, and Hitoshi OBATA p.948

Purification, Characterization, and Gene Sequencing of a Catalase
from an Alkali- and Halo-tolerant Bacterium, Halomonas sp. SK1
Krisana PHUCHAROEN,1 Kiichi HOSHINO,2 Yuuki TAKENAKA,1 and Takao SHINOZAWA1,＊ p.955

Antigen Presentation by Peyer′s Patch Cells Can Induce both Th1- and
Th2-type Responses Depending on Antigen Dosage, but a Different
Cytokine Response Pattern from That of Spleen Cells
Tadashi YOSHIDA,1,†† Satoshi HACHIMURA,1,† Mina ISHIMORI,1 Fumitaka KINUGASA,1
Wataru ISE,1 Mamoru TOTSUKA,1 Akio AMETANI,1,2 and Shuichi KAMINOGAWA1 p.963

Antifungal Activity of Rye (Secale cereale) Seed Chitinases: the Different
Binding Manner of Class I and Class II Chitinases to the Fungal Cell Walls
Toki TAIRA,†,1 Takayuki OHNUMA,2 Takeshi YAMAGAMI,2 Yoichi ASO,2
Masatsune ISHIGURO,2 and Masanobu ISHIHARA1 p.970

Purification and Characterization of a Lipase from the
Glycolipid-Producing Yeast Kurtzmanomyces sp. I-11
Koji KAKUGAWA,1,2,3,† Megumi SHOBAYASHI,2 Osamu SUZUKI,1 and Tokichi MIYAKAWA1
p.978

Gene Cloning and Biochemical Analysis of Thermostable Chitosanase
(TCH-2) from Bacillus coagulans CK108
Ho-Geun YOON, Kyung-Han LEE, Hee-Yun KIM,＊ Hye-Kyung KIM,＊＊
Dong-Hoon SHIN, Bum-Shik HONG, and Hong-Yon CHO† p.986

Isolation and Culture Conditions of a Klebsiella pneumoniae Strain
That Can Utilize Ammonium and Nitrate Ions Simultaneously
with Controlled Iron and Molybdate Ion Concentrations
Young-Ju KIM,1 Makoto YOSHIZAWA,2 Shinji TAKENAKA,2 Shuichiro MURAKAMI,2
and Kenji AOKI2,† p.996

Molecular Cloning and Overexpression of fleA Gene Encoding
a Fucose-specific Lectin of Aspergillus oryzae
Hiroki ISHIDA,† Toshiyasu MORITANI, Yoji HATA, Akitsugu KAWATO,
Koji SUGINAMI, Yasuhisa ABE, and Satoshi IMAYASU p.1002

Rutin-enhanced Antibacterial Activities of Flavonoids against
Bacillus cereus and Salmonella enteritidis
Hidetoshi ARIMA, Hitoshi ASHIDA, and Gen-ichi DANNO† p.1009

Effect of Quercetin and Its Conjugated Metabolite on the Hydrogen
Peroxide-induced Intracellular Production of Reactive
Oxygen Species in Mouse Fibroblasts
Mutsuko SHIRAI, Rintaro YAMANISHI, Jae-Hak MOON, Kaeko MUROTA, and Junji TERAO†
p.1015

Purification, Molecular Cloning, and Characterization of Pyridoxine
4-Oxidase from Microbacterium luteolum
Yasuo KANEDA, Kouhei OHNISHI1, and Toshiharu YAGI† p.1022

Synthesis of the Four Stereoisomers of 3,12-Dimethylheptacosane,
(Z)-9-Pentacosene and (Z)-9-Heptacosene, the Cuticular
Hydrocarbons of the Ant, Diacamma sp.†
Yui MASUDA and Kenji MORI†† p.1032

Transcriptional Control of Nitric Oxide Reductase Gene (CYP55)
in the Fungal Denitrifier Fusarium oxysporum
Naoki TAKAYA,1 Hiromasa UCHIMURA,1 Yupeng LAI,1 and Hirofumi SHOUN2,† p.1039

Replication Timing Properties within the Mouse Distal Chromosome 7
Imprinting Cluster
Kazuhiro KAGOTANI,1 Shin-ichiro TAKEBAYASHI,1 Atsushi KOHDA,1 Hiroshi TAGUCHI,1
Martina PAULSEN,2,†† J<Oh><0228><Wa>orn WALTER,2,†† Wolf REIK,3 and Katsuzumi OKUMURA1,† p.1046

Evaluation of Sulfated Lentinus Edodes α-(1→3)-D-Glucan
as a Potential Antitumor Agent
Pingyi ZHANG† and Peter C. K. CHEUNG p.1052

Cloning and Sequencing of the Genes Encoding Cyclic Tetrasaccharide-
synthesizing Enzymes from Bacillus globisporus C11
Hajime AGA, Kazuhiko MARUTA, Takuo YAMAMOTO, Michio KUBOTA,
Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA p.1057

Phenylethylamine Induces an Increase in Cytosolic Ca2＋ in Yeast
Reinhard PINONTOAN,1,＊ Svetlana KRYSTOFOVA,1 Tomonori KAWANO,2,＊＊
Izumi C. MORI,1,＊＊＊ Frederick I. TSUJI,3 Hidetoshi IIDA,4,5 and Shoshi MUTO1,2,† p.1069

Chitinases A, B, and C1 of Serratia marcescens 2170 Produced
by Recombinant Escherichia coli: Enzymatic Properties and
Synergism on Chitin Degradation
Kazushi SUZUKI, Noriko SUGAWARA, Megumi SUZUKI, Taku UCHIYAMA,
Fuminori KATOUNO, Naoki NIKAIDOU, and Takeshi WATANABE† p.1075

Functional Analysis of the Chitin-binding Domain of a Family 19
Chitinase from Streptomyces griseus HUT6037: Substrate-binding
Affinity and cis-Dominant Increase of Antifungal Function
Yoshikane ITOH,1 Tomokazu KAWASE,1 Naoki NIKAIDOU,1 Harumi FUKADA,2
Masaru MITSUTOMI,3 Takeshi WATANABE,1† and Yoshifumi ITOH4 p.1084

Short-step Syntheses of All Stereoisomers of Preussin
and Their Bioactivities
Masayuki OKUE,1 Hidenori WATANABE,1 Koji KASAHARA,2 Minoru YOSHIDA,3
Sueharu HORINOUCHI,3 and Takeshi KITAHARA1† p.1093

Identification, Cloning, and Sequencing of the Genes Involved
in the Conversion of D,L-2-Amino-Δ2-Thiazoline-4-Carboxylic
Acid to L-Cysteine in Pseudomonas sp. Strain ON-4a
Tetsuo OHMACHI,1,＊ Mizuka NISHINO,1 Maki KAWATA,1 Namiko EDO,1 Hiroko FUNAKI,1,†
Megumi NARITA,1 Kazuyuki MORI,1,†† Yoshiharu TAMURA,2 and Yoshihiro ASADA1 p.1097

Note
Analysis of Transactivation Region of Elf-1 by Using
a Yeast One-hybrid System
Chiharu NISHIYAMA,1,2† Kyoko TAKAHASHI,2 Yasuyuki OHTAKE,2 Toyokazu YOKOTA,2
Ko OKUMURA,1 Hideoki OGAWA,1 and Chisei RA1 p.1105

Note
Preventive Effect of a Chicken Extract on the Development of Hypertension
in Stroke-prone Spontaneously Hypertensive Rats
Yasuo MATSUMURA,1,† Satomi KITA,1 Hiroyuki ONO,2 Yoshinobu KISO,3
and Takaharu TANAKA3 p.1110

Note
Selective Removal of Chymotrypsin Using Diphenyl α-Aminoalkylphosphonate
Immobilized on Sepharose Gel
Shin ONO,† Makiko UMEZAKI, Hajime NABARI, Ryutaro NAKAYAMA, Kumi HASEGAWA,
Satomi SAKO, Takayoshi FUJII, Isao YAMAZAKI,＊ and Toshiaki YOSHIMURA p.1111

Note
Characterization of Cloned Mouse Na＋/taurocholate Cotransporting
Polypeptide by Transient Expression in COS-7 Cells
Tohru SAEKI,† Noriki TAKAHASHI, Ryuhei KANAMOTO, and Kimikazu IWAMI p.1116

Note
Analysis of Hydrolytic Activity of a 65-kDa Chitinase
from the Silkworm, Bombyx mori
Babiker M. A. ABDEL-BANAT, Weiwen ZHOU, Shuji KARASUDA, and Daizo KOGA† p.1119

Note
Enantioselective Hydrolysis of o-Nitrostyrene Oxide by Whole
Cells of Aspergillus niger CGMCC 0496
Hao JIN and Zu-yi LI† p.1123

Note
Relationship between Conidial Enzymes and Germination
of the Apple Blue Mold, Penicillium expansum
Soichiro KIMURA,1 Seigo AMACHI,1 Nobuko OHNO,2 Haruo TAKAHASHI,3
Hirofumi SHINOYAMA,1 and Takaaki FUJII1,† p.1126

Note
Release of Hemorphin-5 from Human Hemoglobin by Pancreatic Elastase
Yunden JINSMAA and Masaaki YOSHIKAWA† p.1130

Note
Protocol for Measuring the Endurance Capacity of Mice
in an Adjustable-current Swimming Pool
Wataru MIZUNOYA, Shinichi OYAIZU, Kengo ISHIHARA, and Tohru FUSHIKI† p.1133

Note
Cloning and Sequencing of the Serine Dehydrogenase Gene
from Agrobacterium tumefaciens
Hisae FUJISAWA, Shinji NAGATA, Emran Kabir CHOWDHURY†,
Miki MATSUMOTO, and Haruo MISONO＊ p.1137

Note
Cloning and Characterization of cDNAs for the Jasmonic Acid-responsive
Genes RRJ1 and RRJ2 in Suspension-cultured Rice Cells
Miho SUGIMORI,1 Kyoko KIRIBUCHI,1 Chiharu AKIMOTO,2 Takeshi YAMAGUCHI,2
Eiichi MINAMI,2 Naoto SHIBUYA,2 Hiroyuki SOBAJIMA,1 Eun-Min CHO,1
Nobuyuki KOBASHI,1 Hideaki NOJIRI,1 Toshio OMORI,1 Makoto NISHIYAMA,1
and Hisakazu YAMANE1,† p.1140

Note
Functional Expression of Chitinase and Chitosanase, and Their Effects
on Morphologies in the Yeast Schizosaccharomyces pombe
Kumiko SHIMONO, Hideyuki MATSUDA, and Makoto KAWAMUKAI† p.1143

Note
Green Fluorescent Protein-labeled Recombinant Fluobody
for Detecting the Picloram Herbicide
In Seon KIM,1,† Jae Han SHIM,2 Yong Tack SUH,2 Kerrm Y. F. YAU,3
J. Christopher HALL,3 Jack T. TREVORS,3 and Hung LEE3 p.1148

Note
An Enzymatic Fluorometric Assay for Pyridoxal
with High Specificity and Sensitivity
Yanee TRONGPANICH, Mitsunori MITO, and Toshiharu YAGI† p.1152

Note
Analysis of the Molecular Construction of Xylogalacturonan Isolated
from Soluble Soybean Polysaccharides
Akihiro NAKAMURA,† Hitoshi FURUTA, Hirokazu MAEDA, Toshifumi TAKAO,1
and Yasunori NAGAMATSU2 p.1155

Note
Molecular Phylogeny of Symbiotic Basidiomycetes of Fungus-growing
Termites in Thailand and Their Relationship with the Host
Yaovapa TAPRAB,1,3 Moriya OHKUMA,1,2,† Toru JOHJIMA,1,2 Yoshimasa MAEDA,1
Shigeharu MORIYA,1,2,4 Tetsushi INOUE,1,2 Poonpilai SUWANARIT,3
Napavarn NOPARATNARAPORN,3 and Toshiaki KUDO1,2,4 p.1169

Note
Syntheses of Four Methyl-branched Secondary Acetates and a Methyl-branched
Ketone as Possible Candidates for the Female Pheromone
of the Screwworm Fly, Cochliomyia hominivorax†
Ayako FURUKAWA, Chi<Oh><0225><Wa>e SHIBATA and Kenji MORI†† p.1164

Note
Technical Improvement to 2D-PAGE of Rice Organelle Membrane Proteins
Satoshi MIKAMI, Tadashi KISHIMOTO, Hidetaka HORI, and Toshiaki MITSUI† p.1170

Note
Method for Purification of Fluorescence-Labeled Oligosaccharides
by Pyridylamination
Shunji NATSUKA,† Jiro ADACHI, Masahumi KAWAGUCHI, Akira ICHIKAWA, and Koji IKURA
p.1174

Note
The groESL Operon of the Halophilic Lactic Acid Bacterium
Tetragenococcus halophila
Daisuke FUKUDA, Maki WATANABE, Yuji ASO, Kenji SONOMOTO†, and Ayaaki ISHIZAKI
p.1176

Note
Purification and Characterization of Heparinase That Degrades
Both Heparin and Heparan Sulfate from Bacillus circulans
Eiichi YOSHIDA,1 Kazuya SAKAI,1 Shinji TOKUYAMA,1 Hirofumi MIYAZONO,2
Hiroshi MARUYAMA,2 Kiyoshi MORIKAWA,2 Keiichi YOSHIDA,2 and Yasutaka TAHARA1,†
p.1181

-1-
Hypoglycemic Effect of a Lentinus edodes Exo-polymer Produced
from a Submerged Mycelial Culture

Byung-Keun YANG, Dong-Hyun KIM, Sang-Chul JEONG, Surajit DAS, Young-Sun CHOI,1
Joon-Shik SHIN,2 Sang-Chul LEE,2 and Chi-Hyun SONG†

Department of Biotechnology, 1Department of Food ＆ Nutrition, Taegu University, Kyungsan,
Kyungbuk 712-714, South Korea
2Hospital of Jaseng Oriental Medicine ＆ Jaseng-Bio Corp., 635 Shinsa-dong, Kangnam-ku,
Seoul 135-896, South Korea

Received August 20, 2001; Accepted January 11, 2002
The hypoglycemic effect of an exo-polymer produced from a submerged mycelial culture of Lentinus edodes was investigated in streptozotocin-induced diabetic rats. The administration of the exo-polymer (200 mg/kg BW) reduced the plasma glucose level by as much as 21.5％, and increased plasma insulin by 22.1％ as compared to the control group. It also lowered the plasma total cholesterol and triglyceride levels by 25.1 and 44.5％, respectively. Gel chromatography of the exo-polymer revealed a single peak which is likely to have been a glycoprotein with a molecular weight of 52 kDa and was found to contain 83.5％ carbohydrate and 16.5％ protein. The Sugar and amino acid compositions of the exo-polymer were analyzed in detail.
Key words: exo-polymer; hypoglycemic effect; Lentinus edodes; submerged mycelial culture

-2-
Biogenesis of 2-Phenylethanol in Rose Flowers: Incorporation of
[2H8]L-Phenylalanine into 2-Phenylethanol and its β-D-Glucopyranoside
during the Flower Opening of Rosa <Oh><0192><Wa>Hoh-Jun′ and Rosa damascena Mill.

Shuzo WATANABE,1 Kentaro HAYASHI,2 Kensuke YAGI,2 Tatsuo ASAI,3 Hazel MACTAVISH,4,†
Joanne PICONE,4 Colin TURNBULL,4 and Naoharu WATANABE2,＊

1The United Graduate School of Agricultural Science, Gifu University (Shizuoka University),
836 Ohya, Shizuoka 422-8529, Japan
2Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
3University Farm, Faculty of Agriculture, Shizuoka University, 63 Kariyado, Fujieda,
Shizuoka 426-0001, Japan
4Department of Agricultural Sciences, Imperial College at Wye, University of London, Wye,
Ashford, Kent TN25 5AH, U.K.

Received September 6, 2001; Accepted January 17, 2002
To clarify the biosynthetic pathway to 2-phenylethanol (2), the deuterium-labeled putative precursor, [＾{2}H<Oh>_<Wa>{8}]L-phenylalanine ([＾{2}H<Oh>_<Wa>{8}-1]), was fed to the flowers of Rosa <Oh><0192><Wa>Hoh-Jun′ and R. damascena Mill. throughout maturation, ceasing feeding at the commencement of petal unfurling and at full bloom. Based on GC-MS analyses, [＾{2}H<Oh>_<Wa>{8}]-1 was incorporated into both 2 and 2-phenylethyl β-D-glucopyranoside (3) when the flowers were fed until full bloom, whereas no such incorporation into 2 was apparent when feeding was ceased earlier. In both species of rose, the labeling pattern for 2 was almost identical to that for 3, and indicated the presence of [＾{2}H<Oh>_<Wa>{6}]-, [＾{2}H<Oh>_<Wa>{7}]- and [＾{2}H<Oh>_<Wa>{8}]-2, and [＾{2}H<Oh>_<Wa>{6}]-, [＾{2}H<Oh>_<Wa>{7}]- and [＾{2}H<Oh>_<Wa>{8}]-3. This may be ascribed to the equilibrium between 2 and 3. The labeling pattern for 2 and 3 also indicated that these compounds were produced from 1 via several routes, the route involving phenylpyruvic acid being the major one.
Key words: rose flower; scent formation; biogenesis; 2-phenylethanol; 2-phenylethyl β-D-glucopyranoside

-3-
Identification of a Novel Anti-ice-nucleating Polysaccharide
from Bacillus thuringiensis YY529

Yasuhiro YAMASHITA†, Hidehisa KAWAHARA, and Hitoshi OBATA

Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho,
Suita city, Osaka 564-8680, Japan

Received September 25, 2001; Accepted January 8, 2002
Strain YY529, capable of producing some anti-ice-nucleating materials (ANM), was isolated from the surface of a camphor leaf. Strain YY529 was identified as Bacillus thuringiensis from its characteristics and taxonomy; the optimum temperature and pH for producing these ANMs were 30°C and 7.0, respectively. One of the ANM with the highest activities among them was purified from the culture. The molecular weight of the ANM was approximately 130 kDa based on a gel filtration analysis. We confirmed that this ANM was a polysaccharide based on the results of the treatment with a mannosidase and the molish reaction. In addition, the LCMS analysis showed that this anti-ice-nucleating polysaccharide (ANPS) had the polyacetyl-D-glucosamine moiety in its structure. Furthermore, this ANPS showed its ability as a non-freeze agent in a preservative solution for the cryopreservation of cock liver. This is the first report of ANPS as a novel ANM from Bacillus thuringiensis YY529.
Key words: Bacillus; anti-nucleating activity; ice-nucleating activity

-4-
Purification, Characterization, and Gene Sequencing of a Catalase
from an Alkali- and Halo-tolerant Bacterium, Halomonas sp. SK1

Krisana PHUCHAROEN,1 Kiichi HOSHINO,2 Yuuki TAKENAKA,1 and Takao SHINOZAWA1,＊

1Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University,
Kiryu, Gunma 376-8515, Japan
2Morin Chemical Industrial Co., Ltd., Tatebayashi, Gunma 374-0027, Japan

Received October 10, 2001; Accepted January 15, 2002
An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4％ yield of the catalase was obtained with 97％ purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH2OH, NaN3, and 3-amino-1,2,4-triazole, active at pH 5.0--11.0, thermo-sensitive, and KCl-
tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99％ identity with that of Vibrio rumoiensis S-1T.
Key words: alkali-tolerant; catalase; Halomonas; halotolerant

-5-
Antigen Presentation by Peyer′s Patch Cells Can Induce both Th1- and
Th2-type Responses Depending on Antigen Dosage, but a Different
Cytokine Response Pattern from That of Spleen Cells

Tadashi YOSHIDA,1,†† Satoshi HACHIMURA,1,† Mina ISHIMORI,1 Fumitaka KINUGASA,1
Wataru ISE,1 Mamoru TOTSUKA,1 Akio AMETANI,1,2 and Shuichi KAMINOGAWA1

1Department of Applied Biological Chemistry, University of Tokyo, Tokyo 113-8657, Japan
2Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, U.S.A.

Received October 16, 2001; Accepted December 28, 2001
The Th1 and Th2 preference induced by cells from the Peyer′s patch (PP) and spleen (SPL) with various doses of an antigen was examined. The same splenic T cell receptor-transgenic CD4＾{＋} T cells were first incubated with PP or SPL cells in the presence of various doses of an antigen, and the cytokine response was observed after secondary stimulation. A Th2-type pattern was only obtained for primary stimulation at 10 μM of the antigen with PP cells, whereas a Th1 pattern was induced at both higher and lower concentrations. SPL cells in the presence of 0.1 to 1 μM of the antigen induced the secretion of Th2-type cytokines. Ten and 100 μM of the antigen plus SPL cells did not induce the release of a large quantity of cytokines. PP cells induced a different cytokine pattern at the antigen concentration that induced a similar level of T cell proliferation with SPL cells. Our findings suggest that the antigen-dose dependent development of Th1/Th2 cells is differentially modulated by the antigen-presentation function of cells in PP and SPL.
Key words: antigen presentation; Peyer′s patch; Th1; Th2; antigen dosage

-6-
Antifungal Activity of Rye (Secale cereale) Seed Chitinases: the Different
Binding Manner of Class I and Class II Chitinases to the Fungal Cell Walls

Toki TAIRA,†,1 Takayuki OHNUMA,2 Takeshi YAMAGAMI,2 Yoichi ASO,2
Masatsune ISHIGURO,2 and Masanobu ISHIHARA1

1Department of Bioscience and Biotechnology, Faculty of Agriculture, Ryukyu University,
Okinawa 903-0213, Japan
2Laboratory of Protein Chemistry and Engineering, Graduate School of Genetic Resources Technology,
Kyushu University, Fukuoka 812-8581, Japan

Received October 17, 2001; Accepted January 8, 2002
The antifungal activities of rye seed chitinase-a (RSC-a, class I) and -c (RSC-c, class II) were studied in detail using two different bioassays with Trichoderma sp. as well as binding and degradation experiments with the cell walls prepared from its mycelia. RSC-a inhibited more strongly the re-extension of the hyphae, containing mainly mature cells, than RSC-c did. Upon incubation of the fungus with fluorescent chitinases, FITC-
labeled RSC-a was found to be located in the hyphal tips, lateral walls, and septa, while FITC-labeled RSC-c was only in the hyphal tip. RSC-a had a greater affinity for the cell walls than RSC-c. RSC-a liberated a larger amount of reducing sugar from the cell walls than RSC-c did. These results inferred that RSC-a first binds to the lateral walls and septa, consisting of the mature cell walls, and degrades mature chitin fiber, while RSC-c binds only to the hyphal tip followed by degradation of only nascent chitin. As a result, RSC-a inhibited fungal growth more effectively than RSC-c. Furthermore, it was suggested that the chitin-binding domain in RSC-a assists the antifungal action of RSC-a by binding to the fungal hypha.
Key words: chitinase; chitin-binding protein; rye seeds; antifungal activity; fungal cell wall

-7-
Purification and Characterization of a Lipase from the
Glycolipid-Producing Yeast Kurtzmanomyces sp. I-11

Koji KAKUGAWA,1,2,3,† Megumi SHOBAYASHI,2 Osamu SUZUKI,1 and Tokichi MIYAKAWA1

1Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan
2Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama,
Higashi-Hiroshima, Hiroshima 739-0046, Japan
3Hiroshima Prefectural Food Technology Research Center, 12-70 Hijiyama-honmachi, Minami-ku,
Hiroshima 734-0086, Japan

Received October 22, 2001; Accepted January 7, 2002
An extracellular lipase produced by the glycolipid-producing yeast Kurtzmanomyces sp. I-11 was purified by ammonium sulfate precipitation and column chromatographies on DEAE-Sephadex A-25, SP-Sephadex C-50, and Sephadex G-100. Based on the analysis of the purified lipase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified lipase was judged to be homogeneous and its molecular mass was estimated to be approximately 49 kDa. The optimum temperature for the activity was 75°C, and the activity was very stable at temperatures below 70°C. The active pH range of this lipase was 1.9--7.2, and the activity was stable at pH below 7.1. The lipase showed a preference for C18 acyl groups by measurements with p-nitrophenyl esters and triglycerides as substrates. The lipase was very stable in the presence of various organic solvents at a concentration of 40％. Although the N-
terminal sequence of the Kurtzmanomyces lipase was very similar to that of lipase A from Candida antarctica, the pH profiles of the two lipases were significantly different.
Key words: lipase; mannosylerythritol lipid; Kurtzmanomyces sp. yeast

-8-
Gene Cloning and Biochemical Analysis of Thermostable Chitosanase
(TCH-2) from Bacillus coagulans CK108

Ho-Geun YOON, Kyung-Han LEE, Hee-Yun KIM,＊ Hye-Kyung KIM,＊＊
Dong-Hoon SHIN, Bum-Shik HONG, and Hong-Yon CHO†

Department of Food and Biotechnology, Graduate School of Biotechnology, Korea University,
Seoul 136-701, Korea
＊＊Department of Food Additives, Korea Food and Drug Administration, Seoul 122-020, Korea
＊＊Department of Food and Biotechnology, Hanseo University, Chungnam 352-820, Korea

Received October 22, 2001; Accepted January 18, 2002
The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6％, 48.0％, and 12.6％ identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90°C. The purified protein was also highly stable, retaining above 50％ residual activities during treatment with denaturants such as urea (8 M) and guanidine・HCl (4 M) at 37°C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.
Key words: Thermostable chitosanase; Gene cloning; Nucleotide sequencing; Bacillus coagulans CK108; Chitosan oligosaccharide

-9-
Isolation and Culture Conditions of a Klebsiella pneumoniae Strain
That Can Utilize Ammonium and Nitrate Ions Simultaneously
with Controlled Iron and Molybdate Ion Concentrations

Young-Ju KIM,1 Makoto YOSHIZAWA,2 Shinji TAKENAKA,2 Shuichiro MURAKAMI,2
and Kenji AOKI2,†

1Division of Life Science, The Graduate School of Science and Technology and 2Laboratory of
Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture,
Kobe University, Rokko, Kobe 657-8501, Japan

Received October 29, 2001; Accepted January 8, 2002
A bacterial strain F-5-2, isolated from soil and identified as Klebsiella pneumoniae, removed NH<Oh>_<Wa>{4}＾{＋} completely in 24 h of aerobic cultivation in a medium containing 1 mg/ml of NH<Oh>_<Wa>{4}NO<Oh>_<Wa>{3}. However, 70％ of the NO<Oh>_<Wa>{3}＾{−} originally provided remained. When 100 μM Fe＾{2＋} was added to the medium, both NH<Oh>_<Wa>{4}＾{＋} and NO<Oh>_<Wa>{3}＾{−} were removed simultaneously and completely from the culture within 6 h of incubation. In addition, the amount of MoO<Oh>_<Wa>{4}＾{−} in the medium markedly affected the bacterial cell growth and utilization of NH<Oh>_<Wa>{4}＾{＋} and NO<Oh>_<Wa>{3}＾{−}. The bacterium could remove 4 mg/ml of NH<Oh>_<Wa>{4}NO<Oh>_<Wa>{3} completely in 48 h of aerobic cultivation in a medium containing 100 μM Fe＾{2＋} and 0.8 pM MoO<Oh>_<Wa>{4}＾{2−}. The total nitrogen in the culture containing its cells was decreased to 14％ of that in the NH<Oh>_<Wa>{4}NO<Oh>_<Wa>{3} originally provided. GC-MS analysis demonstrated that N<Oh>_<Wa>{2} was generated from the nitrogen atoms of both NH<Oh>_<Wa>{4}＾{＋} and NO<Oh>_<Wa>{3}＾{−}.
Key words: ammonium nitrate; iron ion; molybdate ion; heterotrophic nitrification; aerobic denitrification

-10-
Molecular Cloning and Overexpression of fleA Gene Encoding
a Fucose-specific Lectin of Aspergillus oryzae

Hiroki ISHIDA,† Toshiyasu MORITANI, Yoji HATA, Akitsugu KAWATO,
Koji SUGINAMI, Yasuhisa ABE, and Satoshi IMAYASU

Research Institute, Gekkeikan Sake Co. Ltd, 300 Katahara-chou, Fushimi-ku, Kyoto 612-8361, Japan

Received November 5, 2001; Accepted December 26, 2001
A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26％ similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.
Key words: fucose-specific lectin; Aspergillus oryzae; fleA; melO promoter; ferrichrysin

-11-
Rutin-enhanced Antibacterial Activities of Flavonoids against
Bacillus cereus and Salmonella enteritidis

Hidetoshi ARIMA, Hitoshi ASHIDA, and Gen-ichi DANNO†

Division of Life Science, Graduate School of Science and Technology, Kobe University,
Kobe 657-8501, Japan

Received November 12, 2001; Accepted January 22, 2002
The antibacterial activities of flavonoids were found by the paper disk method to be enhanced by combining or mixing them. The combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were much more active than either flavonoid alone. Although rutin did not show activity in itself, the antibacterial activities of quercetin and morin were enhanced in the presence of rutin. The antibacterial activities of flavonoids, in combination with morin and rutin, were evaluated, based on the minimum inhibition concentration (MIC) in a liquid culture, by using Salmonella enteritidis and Bacillus cereus as the test bacteria. The activities of galangin, kaempherol, myricetin and fisetin were each enhanced in the presence of rutin when S. enteritidis was used as the test bacterium. The MIC value for kaempherol was markedly decreased by the addition of rutin. Morin inhibited DNA synthesis, and this effect was promoted by rutin at a concentration of 25 μg/ml.
Key words: morin; rutin; antibacterial activity; synergistic effect; flavonoid

-12-
Effect of Quercetin and Its Conjugated Metabolite on the Hydrogen
Peroxide-induced Intracellular Production of Reactive
Oxygen Species in Mouse Fibroblasts

Mutsuko SHIRAI, Rintaro YAMANISHI, Jae-Hak MOON, Kaeko MUROTA, and Junji TERAO†

Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan

Received November 12, 2001; Accepted December 27, 2001
To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-β-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.
Key words: flavonoid; quercetin aglycon; quercetin 3-O-β-D-glucuronide; antioxidative effect; mouse fibroblast

-13-
Purification, Molecular Cloning, and Characterization of Pyridoxine
4-Oxidase from Microbacterium luteolum

Yasuo KANEDA, Kouhei OHNISHI1, and Toshiharu YAGI†

From the Department of Bioresources Science, Faculty of Agriculture, and the 1Research Institute
of Molecular Genetics, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan

Received November 14, 2001; Accepted December 25, 2001
Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (＝Aureobacterium luteolum YK-1).1 The purified enzyme required FAD for its catalytic activity and stability. The enzyme was a monomeric protein with the subunit molecular mass of 53,000±1,000 Da. PN was the only substrate as the hydrogen donor. Oxygen, 2,6-dichloroindophenol, and vitamin K3 were good substrates as the hydrogen acceptor. The gene (pno) encoding PN 4-oxidase was cloned. The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit. PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M. luteolum YK-1. Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family. M. luteolum YK-1 has four plasmids. The pno gene was found on a chromosomal DNA. Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.
Key words: vitamin B6; metabolic pathway; GMC oxidoreductase; flavoprotein

-14-
Synthesis of the Four Stereoisomers of 3,12-Dimethylheptacosane,
(Z)-9-Pentacosene and (Z)-9-Heptacosene, the Cuticular
Hydrocarbons of the Ant, Diacamma sp.†

Yui MASUDA and Kenji MORI††

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan

Received November 16, 2001; Accepted December 27, 2001
All of the four stereoisomers of 3,12-dimethylheptacosane were synthesized from the enantiomers of citronellal. (Z)-9-Pentacosene and (Z)-9-heptacosene were also synthesized. These hydrocarbons are the characteristic components of the cuticular hydrocarbons of the queen of the ant, Diacamma sp..
Key words: (Z)-alkene; cuticular hydrocarbon; Diacamma sp.; dimethyl-branched alkane; queen ant

-15-
Transcriptional Control of Nitric Oxide Reductase Gene (CYP55)
in the Fungal Denitrifier Fusarium oxysporum

Naoki TAKAYA,1 Hiromasa UCHIMURA,1 Yupeng LAI,1 and Hirofumi SHOUN2,†

1Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
2Department of Biotechnology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received November 16, 2001; Accepted January 5, 2002
Fungal denitrification is a dissimilating metabolic mechanism for nitrate and was first described in Fusarium oxysporum. Here we investigated regulatory systems of expression of CYP55, which encodes cytochrome P450 (P450nor) and is essential for the fungal denitrification. Promoter-reporter analysis of F. oxysporum CYP55 using Escherichia coli β-galactosidase showed that the region between nucleotides −526 and −515 was critical for induction by nitrate. It contained a nucleotide sequence similar to the binding consensus sequence of the pathway-specific transcriptional factor NirA, which induces expression of the nitrate-assimilatory genes of Aspergillus nidulans in the presence of nitrate. This indicates that expression of the nitrate dissimilatory gene (CYP55) is concomitantly regulated with the nitrate-assimilatory genes. The deletion studies also indicated that the nucleotide sequence between −118 and −107, which was similar to the binding consensus of the yeast Rox1p, which represses the anoxic genes under aerobic conditions, was responsible for repression of CYP55 under aerobic conditions. These results indicate that the fungus adapts to the denitrifying conditions by a combination of NirA- and Rox1-like transcription factors.
Key words: denitrification; nitric oxide reductase; cytochrome P450nor; nitrate respiration; Fusarium oxysporum

-16-
Replication Timing Properties within the Mouse Distal Chromosome 7
Imprinting Cluster

Kazuhiro KAGOTANI,1 Shin-ichiro TAKEBAYASHI,1 Atsushi KOHDA,1 Hiroshi TAGUCHI,1
Martina PAULSEN,2,†† J<Oh><0228><Wa>orn WALTER,2,†† Wolf REIK,3 and Katsuzumi OKUMURA1,†

1Laboratory of Molecular and Cellular Biology, Faculty of Bioresources, Mie University, 1515 Kamihama,
Tsu, Mie 514-8507, Japan
2Max-Planck-Institut f<Oh><0228><Wa>ur Molekulare Genetik, Ihnestr. 73, D-14195 Berlin, Germany
3Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge, CB2 4AT, UK

Received November 19, 2001; Accepted December 21, 2001
Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.
Key words: genomic imprinting; replication timing; fluorescence in situ hybridization; mouse chromosome 7; ES cells

-17-
Evaluation of Sulfated Lentinus Edodes α-(1→3)-D-Glucan
as a Potential Antitumor Agent

Pingyi ZHANG† and Peter C. K. CHEUNG

†Whistler Center for Carbohydrate Research, Purdue University, West Lafayette, Indiana 47907-1160, U.S.A.
Department of Biology, The Chinese University of Hong Kong, New Territories, Hong Kong

Received November 20, 2001; Accepted January 18, 2002
α-Glucan L-FV-II and β-glucan L-FV-I were shown to co-exist in the extract of fruiting bodies of Lentinus edodes with aq. 5％ NaOH/0.05％ NaBH4 in previous work. Water-insoluble α-(1→3)-D-glucan (L-FV-II) was treated with sulfur trioxide-pyridine complex at 25°C to synthesize the water-soluble sulfated derivative SL-FV-II with a degree of substitution (DS) 1.1 in non-selective sulfation. The weight-average molecular weight (Mw) of sulfated glucan SL-FV-II is 57％ of that of the original α-glucan L-FV-II. The α-glucan administered by gavaging at a dose of 50 mg/kg of body weight to BALB/C mice having implanted solid Sarcoma 180 was effective at an inhibition rate of 42％. In vitro experiments using human and murine tumor cell lines showed that SL-FV-II had antiproliferation activity at the concentration of 20 μg/mL towards four tumor cell lines. The sulfated α-(1→3)-D-glucan had potent antiproliferation action (52％) on human MCF-7 breast carcinoma cells.
Key words: α-(1→3)-D-glucan; Lentinus edodes; antitumor activity; sulfated derivative; molecular weight

-18-
Cloning and Sequencing of the Genes Encoding Cyclic Tetrasaccharide-
synthesizing Enzymes from Bacillus globisporus C11

Hajime AGA, Kazuhiko MARUTA, Takuo YAMAMOTO, Michio KUBOTA,
Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA

Amase Institute, Hayashibara Biochemical Laboratories, 7-7 Amase minami-machi,
Okayama 700-0834, Japan

Received December 3, 2001; Accepted January 5, 2002
The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from α-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.
Key words: 6-glucosyltransferase; isomaltosyltransferase; cyclic tetrasaccharide; Bacillus globisporus

-19-
Phenylethylamine Induces an Increase in Cytosolic Ca2＋ in Yeast

Reinhard PINONTOAN,1,＊ Svetlana KRYSTOFOVA,1 Tomonori KAWANO,2,＊＊
Izumi C. MORI,1,＊＊＊ Frederick I. TSUJI,3 Hidetoshi IIDA,4,5 and Shoshi MUTO1,2,†

1Nagoya University Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
3Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego,
La Jolla, CA 92093, U.S.A.
4Department of Biology, Tokyo Gakugei University, Koganei, Tokyo 184-8501, Japan
5CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

Received December 4, 2001; Accepted January 7, 2002
β-Phenylethylamine (PEA) induced an increase in cytosolic free calcium ion concentration ([Ca＾{2＋}]<Oh>_<Wa>{c}) in Saccharomyces cereviseae cells monitored with transgenic aequorin, a Ca＾{2＋}-dependent photoprotein. The PEA-induced [Ca＾{2＋}]<Oh>_<Wa>{c} increase was dependent on the concentrations of PEA applied, and the Ca＾{2＋} mostly originated from an extracellular source. Preceding the Ca＾{2＋} influx, H<Oh>_<Wa>{2}O<Oh>_<Wa>{2} was generated in the cells by the addition of PEA. Externally added H<Oh>_<Wa>{2}O<Oh>_<Wa>{2} also induced a [Ca＾{2＋}]<Oh>_<Wa>{c} increase. These results suggest that PEA induces the [Ca＾{2＋}]<Oh>_<Wa>{c} increase via H<Oh>_<Wa>{2}O<Oh>_<Wa>{2} generation. The PEA-induced [Ca＾{2＋}]<Oh>_<Wa>{c} increase occurred in the mid1 mutant with a slightly smaller peak than in the wild-type strain, indicating that Mid1, a stretch-activated nonselective cation channel, may not be mainly involved in the PEA-induced Ca＾{2＋} influx. When PEA was applied, the MATa mid1 mutant was rescued from α-factor-induced death in a Ca＾{2＋}-limited medium, suggesting that the PEA-induced [Ca＾{2＋}]<Oh>_<Wa>{c} increase can reinforce calcium signaling in the mating pheromone response pathway.
Key words: calcium signaling; cytosolic Ca＾{2＋}; hydrogen peroxide; β-phenylethylamine; Saccharomyces cerevisiae

-20-
Chitinases A, B, and C1 of Serratia marcescens 2170 Produced
by Recombinant Escherichia coli: Enzymatic Properties and
Synergism on Chitin Degradation

Kazushi SUZUKI, Noriko SUGAWARA, Megumi SUZUKI, Taku UCHIYAMA,
Fuminori KATOUNO, Naoki NIKAIDOU, and Takeshi WATANABE†

Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University,
8050 Ikarashi-2, Niigata 950-2181, Japan

Received December 7, 2001; Accepted January 23, 2002
To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAc)2 almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)6 to (GlcNAc)3, while ChiA exclusively generated (GlcNAc)2 and (GlcNAc)4. Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.
Key words: Serratia marcescens; chitinase; synergism

-21-
Functional Analysis of the Chitin-binding Domain of a Family 19
Chitinase from Streptomyces griseus HUT6037: Substrate-binding
Affinity and cis-Dominant Increase of Antifungal Function

Yoshikane ITOH,1 Tomokazu KAWASE,1 Naoki NIKAIDOU,1 Harumi FUKADA,2
Masaru MITSUTOMI,3 Takeshi WATANABE,1† and Yoshifumi ITOH4

1Department of Biosystem Science, Graduate School of Science and Technology, Niigata University,
8050 Ikarashi-2, Niigata 950-2181, Japan
2Laboratory of Biophysical Chemistry, Graduate School of Agriculture and Biological Sciences,
Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
3Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo-machi,
Saga 840-8502, Japan
4Division of Applied Microbiology, National Food Research Institute, Kannondai 2-1-12, Tsukuba,
Ibaraki 305-8642, Japan

Received December 7, 2001; Accepted January 23, 2002
Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBDChiC) differs from those of the plant enzymes. ChBDChiC and the catalytic domain (CatDChiC), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBDChiC binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBDChiC resulted in moderate (about 50％) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90％) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBDChiC itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatDChiC in trans.
Key words: Streptomyces griseus; chitinase; chitin-binding domain; antifungal activity

-22-
Short-step Syntheses of All Stereoisomers of Preussin
and Their Bioactivities

Masayuki OKUE,1 Hidenori WATANABE,1 Koji KASAHARA,2 Minoru YOSHIDA,3
Sueharu HORINOUCHI,3 and Takeshi KITAHARA1†

1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Science of Biological Supramolecular Systems, Graduate School of Integrated Science,
Yokohama City University, 1-7-29 Suehirocho, Tsurumi-ku, Yokohama 230-0045, Japan
3Department of Biotechnology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received December 21, 2001; Accepted Jannary 21, 2002
All the eight stereoisomers of (＋)-preussin (1b), an antifungal agent inhibiting the growth of fission yeast and human cancer cells, were synthesized in two steps by non-stereoselective reactions and chromatographic separation, starting from L- and D-N-protected-phenylalaninal (2). Their bioassay revealed all of the stereoisomers to be almost equally bioactive.
Key words: antifungal; antibacterial; cell growth inhibitor; apoptosis; preussin

-23-
Identification, Cloning, and Sequencing of the Genes Involved
in the Conversion of D,L-2-Amino-Δ2-Thiazoline-4-Carboxylic
Acid to L-Cysteine in Pseudomonas sp. Strain ON-4a

Tetsuo OHMACHI,1,＊ Mizuka NISHINO,1 Maki KAWATA,1 Namiko EDO,1 Hiroko FUNAKI,1,†
Megumi NARITA,1 Kazuyuki MORI,1,†† Yoshiharu TAMURA,2 and Yoshihiro ASADA1

1Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science,
Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan
2Research Center, Nippon Rikagakuyakuhin Co. Ltd., 2-2-8 Ohgi, Adachi, Tokyo 123-0873, Japan

Received December 25, 2001; Accepted December 27, 2001
The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-Δ2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-Δ2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5α harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5α carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-Δ2-thiazoline-4-
carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-Δ2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.
Key words: Pseudomonas species; gene cloning; D,L-2-amino-Δ2-thiazoline-4-carboxylicacid (D,L-ATC); N-carbamoyl-L-cysteine (NCC); L-cysteine

-24-
Note
Analysis of Transactivation Region of Elf-1 by Using
a Yeast One-hybrid System

Chiharu NISHIYAMA,1,2† Kyoko TAKAHASHI,2 Yasuyuki OHTAKE,2 Toyokazu YOKOTA,2
Ko OKUMURA,1 Hideoki OGAWA,1 and Chisei RA1

1Allergy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan
2Foods ＆ Pharmaceuticals Research ＆ Development Laboratory, Asahi Breweries, Ltd., 1-1-21 Midori,
Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan

Received August 3, 2001; Accepted January 9, 2002
The transcriptional regulatory region of Elf-1 was analyzed by the combination of a yeast one-hybrid system and site-directed mutagenesis. This analysis enabled us to map an activation region between 85-175 of Elf-1.
Key words: Elf-1; transcription factor; Ets; yeast one-hybrid

-25-
Note
Preventive Effect of a Chicken Extract on the Development of Hypertension
in Stroke-prone Spontaneously Hypertensive Rats

Yasuo MATSUMURA,1,† Satomi KITA,1 Hiroyuki ONO,2 Yoshinobu KISO,3
and Takaharu TANAKA3

1Department of Pharmacology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara,
Takatsuki, Osaka 569-1094, Japan
2Department of Research ＆ Development, Cerebos Pacific Ltd., 1 Kim Seng Promenade ＃11-01/06,
Singapore 237994
3Institute for Health Care Science, Suntory Ltd., Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan

Received August 6, 2001; Accepted December 25, 2001
The antihypertensive effect of Brand′s Essence of Chicken (BEC), a popular chicken extract used as a traditional health food, was examined with stroke-prone spontaneously hypertensive rats (SHRSPs). The animals were maintained from 6 to 25 weeks of age on drinking water with or without BEC. The BEC-fed group showed a significant reduction in the development of hypertension when compared with the control animals. The levels of blood urea nitrogen and plasma creatinine in the BEC-fed group were significantly lower than those in the control group, suggesting that the renal glomerular function had been improved by the daily administration of BEC. It thus seems likely that BEC would be useful as a prophylactic treatment against the development of hypertension and renal injury.
Key words: chicken extract; stroke-prone spontaneously hypertensive rat; antihypertensive effect; renoprotective effect

-26-
Note
Selective Removal of Chymotrypsin Using Diphenyl α-Aminoalkylphosphonate
Immobilized on Sepharose Gel

Shin ONO,† Makiko UMEZAKI, Hajime NABARI, Ryutaro NAKAYAMA, Kumi HASEGAWA,
Satomi SAKO, Takayoshi FUJII, Isao YAMAZAKI,＊ and Toshiaki YOSHIMURA

Department of System Engineering of Materials and Life Science, Faculty of Engineering,
Toyama University, Gofuku 3190, Toyama 930-8555, Japan
＊Yayoi Kagaku Kogyo Co., Ltd., Fukuoka-machi, Nishitonami-gun, Toyama 939-0135, Japan

Received August 13, 2001; Accepted January 16, 2002
A diphenyl α-aminoalkylphosphonate derivative, which is an irreversible inhibitor of chymotrypsin-like serine proteases, was immobilized on cyanogen bromide-activated Sepharose, and the selective binding of chymotrypsin to the obtained inhibitor-gel was evaluated using batch and column methods. Complete removal of chymotrypsin in an aqueous solution was done using the column method, while partial removal was done using the batch method.
Key words: covalent chromatography; diphenyl α-aminoalkylphosphonate; irreversible inhibitor of serine protease; selective removal of chymotrypsin

-27-
Note
Characterization of Cloned Mouse Na＋/taurocholate Cotransporting
Polypeptide by Transient Expression in COS-7 Cells

Tohru SAEKI,† Noriki TAKAHASHI, Ryuhei KANAMOTO, and Kimikazu IWAMI

Laboratory of Vital ＆ Molecular Nutrition, Department of Biological Resource Chemistry,
Faculty of Agriculture, Kyoto Prefectural University, Nakaragi, Shimogamo, Sakyo-ku,
Kyoto 606-8522, Japan

Received August 22, 2001; Accepted December 25, 2001
The mouse Na＋/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [3H]taurocholic acid with Km and Vmax values of 18 μM and 102 pmol/mg protein/min, respectively. This Km value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [3H]taurocholic acid transport.
Key words: Na＋/taurocholate cotransporting polypeptide; transient expression; kinetic analysis; RT-PCR

-28-
Note
Analysis of Hydrolytic Activity of a 65-kDa Chitinase
from the Silkworm, Bombyx mori

Babiker M. A. ABDEL-BANAT, Weiwen ZHOU, Shuji KARASUDA, and Daizo KOGA†

Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture,
Yamaguchi University, Yamaguchi 753-8515, Japan

Received October 15, 2001; Accepted January 21, 2002
The hydrolytic reactions of Bombyx mori 65-kDa chitinase with the short substrates, N-acetylchitooligosaccharides, were analyzed by HPLC. Analysis of the hydrolyzed products showed that the newly produced oligosaccharides are all β anomers, suggesting that, similar to other family 18 glycosyl hydrolases, the 65-kDa chitinase acts in the retaining mechanism. Furthermore, the enzyme cleaves the N-acetylchitooligosaccharides mainly at the linkage between the second and the third GlcNAc moieties from the non-reducing end, while the other sites were cleaved in smaller proportions. Moreover, the initial reaction rates of the enzyme with the longer N-acetylchitooligosaccharides were higher than those with shorter ones. These results suggest that the enzyme is an endo-cleaving type and more efficient on the longer substrates.
Key words: Bombyx mori; 65-kDa chitinase; N-acetylchitooligosaccharides; cleavage pattern; β anomer formation

-29-
Note
Enantioselective Hydrolysis of o-Nitrostyrene Oxide by Whole
Cells of Aspergillus niger CGMCC 0496

Hao JIN and Zu-yi LI†

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu,
Shanghai 200032, China

Received October 22, 2001; Accepted January 16, 2002
The asymmetric biohydrolysis is described of o-nitrostyrene oxide with high selectivity by whole cells of Aspergillus niger CGMCC 0496. Both the epoxide and diol could be obtained in a high state of optical purity (over 98％). Product inhibition was found when using a high ratio of substrate to cells.
Key words: enantioselective hydrolysis; epoxide hydrolase; Aspergillus niger; o-nitrostyrene oxide

-30-
Note
Relationship between Conidial Enzymes and Germination
of the Apple Blue Mold, Penicillium expansum

Soichiro KIMURA,1 Seigo AMACHI,1 Nobuko OHNO,2 Haruo TAKAHASHI,3
Hirofumi SHINOYAMA,1 and Takaaki FUJII1,†

1Department of Life and Bioresource Science, Chiba University, 648 Matsudo, Matsudo-city,
Chiba 271-8510, Japan
2Department of Health and Nutrition, School of Home Economics, Wayo Women′s University,
2-3-1 Konodai, Ichikawa-city 272-8533, Japan
3Public Health Laboratory of Chiba Prefecture, 666-2 Nitona-cho, Chuo-ku, Chiba-city 260-8715, Japan

Received October 25, 2001; Accepted January 7, 2002
The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.
Key words: Penicillium expansum; conidium; xylanase; pectinase; germination

-31-
Note
Release of Hemorphin-5 from Human Hemoglobin by Pancreatic Elastase

Yunden JINSMAA and Masaaki YOSHIKAWA†

Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received November 5, 2001; Accepted January 7, 2002
Opioid peptide hemorphin-5 (YPWTQ) was released from human hemoglobin by the action of pancreatic elastase. V-hemorphin-5 (VYPWTQ) was also released under the same conditions. The amount of hemorphin-5 was about 1/10 that of V-hemorphin-5. This is the first report showing release of hemorphin-5 only by the action of one enzyme.
Key words: hemoglobin; hemorphin; opioid; elastase

-32-
Note
Protocol for Measuring the Endurance Capacity of Mice
in an Adjustable-current Swimming Pool

Wataru MIZUNOYA, Shinichi OYAIZU, Kengo ISHIHARA, and Tohru FUSHIKI†

Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School
of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan

Received November 5, 2001; Accepted January 18, 2002
We re-examined the methods used in an adjustable-current swimming pool for evaluating the endurance capacity of mice to improve the sensitivity and reproducibility. We found that the BALB/c strain was most suitable to minimize the variation in time taken to swim to the point of fatigue. We found that precise adjustment of the apparatus and the use of three primary swimming trials to select mice before a study made the swimming time more uniform. This procedure was used to establish a more precise evaluation protocol.
Key words: endurance capacity; adjustable-current swimming pool; improved protocol

-33-
Note
Cloning and Sequencing of the Serine Dehydrogenase Gene
from Agrobacterium tumefaciens

Hisae FUJISAWA, Shinji NAGATA, Emran Kabir CHOWDHURY†,
Miki MATSUMOTO, and Haruo MISONO＊

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

Received November 7, 2001; Accepted December 19, 2001
The structural gene for NADP＋-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.
Key words: serine dehydrogenase; Agrobacterium tumefaciens; short-chain alcohol dehydrogenase; nucleotide sequence; amino acid sequence

-34-
Note
Cloning and Characterization of cDNAs for the Jasmonic Acid-responsive
Genes RRJ1 and RRJ2 in Suspension-cultured Rice Cells

Miho SUGIMORI,1 Kyoko KIRIBUCHI,1 Chiharu AKIMOTO,2 Takeshi YAMAGUCHI,2
Eiichi MINAMI,2 Naoto SHIBUYA,2 Hiroyuki SOBAJIMA,1 Eun-Min CHO,1
Nobuyuki KOBASHI,1 Hideaki NOJIRI,1 Toshio OMORI,1 Makoto NISHIYAMA,1
and Hisakazu YAMANE1,†

1Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan
2Department of Biochemistry, National Institute of Agrobiological Science, Tsukuba,
Ibaraki 305-8602, Japan

Received November 7, 2001; Accepted December 26, 2001
Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine γ-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.
Key words: jamonic acid-responsive gene; Oryza sativa L.; putative cystathionine γ-lyase; pyruvate decarboxylase

-35-
Note
Functional Expression of Chitinase and Chitosanase, and Their Effects
on Morphologies in the Yeast Schizosaccharomyces pombe

Kumiko SHIMONO, Hideyuki MATSUDA, and Makoto KAWAMUKAI†

Department of Life Science and Biotechnology, Faculty of Life ＆ Environmental Science,
Shimane University, Matsue 690-8504, Japan

Received November 12, 2001; Accepted December 7, 2001
The chitinase A (chiA) gene from Enterobacter sp. G-1 and the chitosanase A (choA) gene from Matsuebacter chitosanotabidus 3001 were expressed separately and simultaneously in the yeast Schizosaccharomyces pombe. The chiA gene was placed under the transcriptional control of the nmt1 promoter with the glutathione S transferase (GST), and the choA gene was expressed by the human cytomegalovirus (hCMV) promoter with the P factor secretion signal (P3). The expressed proteins of ChoA and GST-ChiA were enzymatical active and were detected as 34-kDa and 80-kDa, respectively, by Western blot analysis. The transformant that expressed the choA gene was able to secrete ChoA efficiently into the culture medium with the specific activity of 102.36 U/mg protein. When the chiA gene was expressed in S. pombe, yeast cells grew slowly and cells became elongated, but when the choA gene was expressed, cells became swollen. Expression of both the chiA and the choA genes resulted in elongated and fat cells.
Key words: chitosanase; chitinase; Schizosaccharomyces pombe; Matsuebacter chitosanotabidus; Enterobacter sp. G-1

-36-
Note
Green Fluorescent Protein-labeled Recombinant Fluobody
for Detecting the Picloram Herbicide

In Seon KIM,1,† Jae Han SHIM,2 Yong Tack SUH,2 Kerrm Y. F. YAU,3
J. Christopher HALL,3 Jack T. TREVORS,3 and Hung LEE3

1Department of Chemical and Materials Engineering, University of Alberta, Edmonton,
Alberta T6G 2G6, Canada
2Department of Biological and Environmental Chemistry, Institute of Agricultural Science and Technology,
Chonnam National University, Kwang-Ju 500-757, South Korea
3Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).
Key words: green fluorescent protein; herbicide; immunoassay; picloram

-37-
Note
An Enzymatic Fluorometric Assay for Pyridoxal
with High Specificity and Sensitivity

Yanee TRONGPANICH, Mitsunori MITO, and Toshiharu YAGI†

Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe-Otsu 200,
Nankoku, Kochi 783-8502, Japan

Received November 15, 2001; Accepted January 4, 2001
An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r＝0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5′-phosphate by column chromatography.
Key words: pyridoxal; enzymatic assay; pyridoxal dehydrogenase; Microbacterium luteolum

-38-
Note
Analysis of the Molecular Construction of Xylogalacturonan Isolated
from Soluble Soybean Polysaccharides

Akihiro NAKAMURA,† Hitoshi FURUTA, Hirokazu MAEDA, Toshifumi TAKAO,1
and Yasunori NAGAMATSU2

Tsukuba R＆D Center, Fuji Oil Co. Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun, Ibaraki 300-2497, Japan
1Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan

Received November 19, 2001; Accepted January 16, 2002
Soluble soybean polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides, and exhibited a pectin-like structure. After digesting galacturonan with polygalacturonase, two novel galacturonan (GN) fragments, which were directly linked to xylosyl oligosaccharides, were obtained. One consisted of (β-D-Xyl)7 branched at the C-3 site of 1,4-linked (α-D-GalA)4, and the other consisted of (β-D-Xyl)4 branched at the C-3 site of 1,4-linked (α-D-GalA)3.
Key words: soybean; polysaccharide; pectin; xylose

-39-
Note
Molecular Phylogeny of Symbiotic Basidiomycetes of Fungus-growing
Termites in Thailand and Their Relationship with the Host

Yaovapa TAPRAB,1,3 Moriya OHKUMA,1,2,† Toru JOHJIMA,1,2 Yoshimasa MAEDA,1
Shigeharu MORIYA,1,2,4 Tetsushi INOUE,1,2 Poonpilai SUWANARIT,3
Napavarn NOPARATNARAPORN,3 and Toshiaki KUDO1,2,4

1Microbiology Laboratory, RIKEN and
2Japan Science and Technology Corporation, Wako, Saitama 351-0198, Japan
3Department of Microbiology, Kasetsart University, Bangkok 10900, Thailand
4Graduate School of Integrated Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japan

Received November 27, 2001; Accepted January 9, 2002
Termitomyces-related symbiotic basidiomycetes in the nests of fungus-growing termites (Macrotermitinae) of several genera in Thailand were cultivated and analyzed phylogenetically based on the DNA sequence of nuclear ribosomal RNA genes. The relationships of the symbiotic fungi with host termites and their locality were apparently complex, supporting intricate mechanisms for the termites to acquire the symbionts.
Key words: symbiosis; termite; basidiomycete; Termitomyces; phylogeny

-40-
Note
Syntheses of Four Methyl-branched Secondary Acetates and a Methyl-branched
Ketone as Possible Candidates for the Female Pheromone
of the Screwworm Fly, Cochliomyia hominivorax†

Ayako FURUKAWA, Chi<Oh><0225><Wa>e SHIBATA and Kenji MORI††

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan

Received November 28, 2001; Accepted January 21, 2002
6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.
Key words: Cochliomyia hominivorax; methyl-branched ketone; methyl-branched secondary acetate; pheromone; screwworm fly

-41-
Note
Technical Improvement to 2D-PAGE of Rice Organelle Membrane Proteins

Satoshi MIKAMI, Tadashi KISHIMOTO, Hidetaka HORI, and Toshiaki MITSUI†

Laboratories of Molecular Life Science, Graduate School of Science and Technology, Niigata University,
Niigata 950-2181, Japan

Received December 3, 2001; Accepted January 15, 2002
Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.
Key words: Oryza sativa L.; membrane protein; 2D-PAGE; NEPHGE

-42-
Note
Method for Purification of Fluorescence-Labeled Oligosaccharides
by Pyridylamination

Shunji NATSUKA,† Jiro ADACHI, Masahumi KAWAGUCHI, Akira ICHIKAWA, and Koji IKURA

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology,
Goshokaigo-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Received December 10, 2001; Accepted January 19, 2002
We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.
Key words: pyridylamination; oligosaccharide; fluorescence; glycome

-43-
Note
The groESL Operon of the Halophilic Lactic Acid Bacterium
Tetragenococcus halophila

Daisuke FUKUDA, Maki WATANABE, Yuji ASO, Kenji SONOMOTO†, and Ayaaki ISHIZAKI

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

Received December 17, 2001; Accepted January 15, 2002
The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45°C), and 4-fold by high NaCl (3--4 M). The Bacillus subtilis σA-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.
Key words: heat shock protein; groESL operon; CIRCE; halophile; Tetragenococcus

-44-
Note
Purification and Characterization of Heparinase That Degrades
Both Heparin and Heparan Sulfate from Bacillus circulans

Eiichi YOSHIDA,1 Kazuya SAKAI,1 Shinji TOKUYAMA,1 Hirofumi MIYAZONO,2
Hiroshi MARUYAMA,2 Kiyoshi MORIKAWA,2 Keiichi YOSHIDA,2 and Yasutaka TAHARA1,†

1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Shizuoka 422-8529, Japan
2Central Research Laboratories, Seikagaku Corp., Higashiyamato, Tokyo 207-0021, Japan

Received December 17, 2001; Accepted January 21, 2002
A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45°C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both haparin and HS, similar to heparinase II from Flavobacterium heparinum.
Key words: heparinase; heparin; heparan sulfate; Bacillus circulans; glycosaminoglycan