Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.4 2001)

The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation
of 6-Deoxyerythronolide B
Choonkeun KIM, Haeyoung KIM, and Oksoo HAN・ p.752

Reaction Kinetics and Modeling of the Enzyme-catalyzed Production
of Lactosucrose using β-Fructofuranosidase from Arthrobacter sp. K-1
Axel PILGRIM,1 Motoaki KAWASE,1,・ Masayasu OHASHI,1 Koki FUJITA,2
Kazufumi MURAKAMI,2 and Kenji HASHIMOTO1,・・ p.758

Molecular Cloning and Characterization of the Fructooligosaccharide-
Producing β-Fructofuranosidase Gene from Aspergillus niger ATCC 20611
Koji YANAI,・ Akitaka NAKANE, Akemi KAWATE, and Masao HIRAYAMA p.766

Effects of Brewer′s Yeast Cell Wall on Constipation and Defecation
in Experimentally Constipated Rats
Tomohiko NAKAMURA,1,・ Kazue AGATA,1 Mai MIZUTANI,1 and Hisakazu IINO2
p.774

Fibrinolytic and Antithrombotic Protease from Spirodela polyrhiza
Hye-Seon CHOI・ and You-Seon SA p.781

ESR Imaging on a Solid-tumor-bearing Mouse Using Spin-labeled Dextran
Kieko SAITO,1,・ Shunsuke KAZAMA,2 Hisayuki TANIZAWA,2 Tomohiro ITO,3
Mika TADA,3 Tateaki OGATA,3 and Hisashi YOSHIOKA1 p.787

S-Adenosyl-L-methionine-dependent O-Methylation
of 2-Hydroxy-3-alkylpyrazine in Wine Grapes:
A Putative Final step of Methoxypyrazine Biosynthesis
Katsumi HASHIZUME,・ Kazuyuki TOZAWA, Masashi ENDO, and Isao ARAMAKI p.795

Thermostable Chitosanase from Bacillus sp. Strain CK4: Its Purification,
Characterization, and Reaction Patterns
Ho-Geun YOON, Hee-Yun KIM,＊ Hye-Kyung KIM,＊＊ Bum-Shik HONG,
Dong-Hoon SHIN, and Hong-Yon CHO・ p.802

Identification of 2,3-Dihydro-γ-ionylideneethanol in Cercospora cruenta
Hirotaka YAMAMOTO,・ Masahiro INOMATA, Takeo UCHIYAMA, and Takayuki ORITANI＊
p.810

Biological Evaluation of 5-Substituted Pyrimidine Derivatives as Inhibitors
of Brassinosteroid Biosynthesis
Jing-Ming WANG,1・ Tadao ASAMI,2 Shigeo YOSHIDA,2 and Noboru MUROFUSHI1 p.817

Incorporation of the Whole Chromosomal DNA in Protoplast Lysates
into Competent Cells of Bacillus subtilis
Takashi AKAMATSU・ and Hisataka TAGUCHI p.823

Cloning, Sequence Analysis, and Expression in Escherichia coli of the
Gene Encoding Monovalent Cation-activated Levodione Reductase
from Corynebacterium aquaticum M-13
Ayumi YOSHISUMI,1 Masaru WADA,・1 Hiroshi TAKAGI,1 Sakayu SHIMIZU,2
and Shigeru NAKAMORI1 p.830

Purification of Soluble β-Glucan with Immune-enhancing Activity
from the Cell Wall of Yeast
Jung-Nam LEE, Do-Youn LEE, In-Hye JI, Gi-Eun KIM,1 Hye Nam KIM,
Jeongwon SOHN,2 Sangduk KIM, and Chan-Wha KIM・ p.837

Purification and Properties of a Galacturonic Acid-releasing
Exopolygalacturonase from a Strain of Bacillus
Tohru KOBAYASHI,・ Norihiko HIGAKI, Noriyuki YAJIMA, Atsushi SUZUMATSU,
Hiroshi HAGIHARA, Shuji KAWAI, and Susumu ITO p.842

Non-involvement of the K-ras Mutation in Colon Carcinogenesis Promoted
by Dietary Deoxycholate in Azoxymethane-treated Rats
Ryuhei KANAMOTO,1・ Naoyuki AZUMA,1 Yasunari TSUCHIHASHI,2 Hitoshi SUDA,1
Tohru SAEKI,1 and Kimikazu IWAMI1 p.848

Increases of Secondary Metabolite Production in Various Plant Cell Cultures
by Co-cultivation with Cork
Hirobumi YAMAMOTO,1 Atsushi YATO,2 Kazufumi YAZAKI,3 Hiroaki HAYASHI,2
Goro TAGUCHI,4 and Kenichiro INOUE2 p.853

Biosynthesis of Fattiviracin FV-8, an Antiviral Agent
EL-Sayed E. HABIB, Kazumi YOKOMIZO, Keitarou SUZUKI, and Masaru UYEDA＊
p.861

Increase in the Stability of Serine Acetyltransferase from Escherichia coli
against Cold Inactivation and Proteolysis by Forming a Bienzyme Complex
Koshiki MINO, Koreyoshi IMAMURA, Takaharu SAKIYAMA, Naoki EISAKI,＊
Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・ p.865

Transglycosylation to Ginseng Saponins by Cyclomaltodextrin
Glucanotransferases
Young-Hoi KIM, Young-Gu LEE, Kang-Ju CHOI, Kei UCHIDA,＊ and Yukio SUZUKI＊,・
p.875

Novel Method Using Phytase for Separating Soybean
β-Conglycinin and Glycinin
Tsutomu SAITO,＊ Mitsutaka KOHNO,＊ Kazunobu TSUMURA,＊ Wataru KUGIMIYA,＊
and Makoto KITO＊＊ p.884

A Novel Cryoprotective Protein (CRP) with High Activity
from the Ice-nucleating Bacterium, Pantoea agglomerans IFO12686
Noriko KODA,＊,＊＊ Toshiaki ASAEDA,＊ Kazuhiro YAMADE,＊ Hidehisa KAWAHARA,＊,＊＊
and Hitoshi OBATA＊,＊＊ p.888

Characterization of Soluble Protein Extracts from Keratinized Tissues:
Identification of Ubiquitin Universally Distributed in Hair,
Nail, and Stratum Corneum
Takafumi INOUE, Kenji KIZAWA, and Mayumi ITO p.895

Structures of the N-Linked Sugar Chains in PAS-7 Glycoprotein Sharing
the Same Protein Core with PAS-6 Glycoprotein
from the Bovine Milk Fat Globule Membrane
Jin-Seok SEOK,・ Michiko SHIMODA, Norihiro AZUMA, and Choemon KANNO・・
p.901

Controlled Trial of the Effects of Milk Basic Protein (MBP) Supplementation
on Bone Metabolism in Healthy Adult Women
Seicihiro AOE,1 Yasuhiro TOBA,2 Jun-ichi YAMAMURA,2 Hiroshi KAWAKAMI,2
Masatoshi YAHIRO,1 Masayoshi KUMEGAWA,3 Akira ITABASHI,4 and Yukihiro TAKADA2
p.913

Structural Elucidation of Twelve Novel Esters Composed of Five Fatty Acids
and Three New Branched Alcohols Together with Four Monoterpenoids
from Sancassania shanghaiensis (Acari: Acaridae)・
Tomoyo SAKATA,1 Kimiko OKABE,2 and Yasumasa KUWAHARA1,・・ p.919

Greater Effect of Dietary Potassium Tripolyphosphate than of Potassium
Dihydrogenphosphate on the Nephrocalcinosis and Proximal Tubular Function
in Female Rats from the Intake of a High-phosphorus Diet
Hiroshi MATSUZAKI,1 Ritsuko MASUYAMA,2 Mariko UEHARA,2 Kahoru NAKAMURA,1
and Kazuharu SUZUKI2＊ p.928

Note
Different Response to Choline Deficiency of the Serum Ornithine
Carbamoyltransferase Activity in Four Strains of Rats
Komang Ayu NOCIANITRI and Yoritaka AOYAMA・ p.935

Note
Detection of Antifungal Activity in Belamcanda chinensis by a Single-cell
Bioassay Method and Isolation of Its Active Compound, Tectorigenin
Ki-Bong OH,1＊ Heonjoong KANG,2 and Hideaki MATSUOKA3 p.939

Note
Microbial Hydroxylation of (±)- and (--)-(2Z,4E)-5-(1?,2?-Epoxy-2?,6?,6?-
trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid into
(±)- and (−)-Xanthoxin Acid by Cunninghamella echinulata
Ryou OKAZAKI, Takayuki ORITANI,・ Yoshihiro HARA and Hirotaka YAMAMOTO1 p.943

Note
Anti-tumor Promoting Activity of Bufadienolides from Kalanchoe pinnata
and K. daigremontiana×tubiflora
Unang SUPRATMAN,1 Tomoyuki FUJITA,1 Kohki AKIYAMA,1 Hideo HAYASHI,1・
Akira MURAKAMI,2 Hirofumi SAKAI,2 Koichi KOSHIMIZU,2 and Hajime OHIGASHI3
p.947

Note
Isolation and Identification of trans-2- and trans-3-Hydroxy-1,8-cineole
Glucosides from Alpinia galanga
Yuki SOMEYA, Akio KOBAYASHI and Kikue KUBOTA・ p.950

Note
Wine Has Activity against Entero-pathogenic Bacteria in Vitro but not in Vivo
Yoshiko SUGITA-KONISHI, Yukiko HARA-KUDO, Tamami IWAMOTO,＊ and Kazuo KONDO＊＊
p.954

Note
Rapid Dephosphorylation of eIF4E by Dietary Protein in the Skeletal Muscle
and Liver of Food-deprived Rats
Fumiaki YOSHIZAWA,・ Taketoshi KIDO,＊ and Takashi NAGASAWA＊ p.958

Note
Effect of Zinc Deficiency on Betacyanin Production in a Cell Suspension
Culture of Table Beet (Beta vulgaris L.)
Toru AKITA,・ Yasuhiko HINA, and Toyoyuki NISHI p.962

Note
Antibacterial Activity of S-Methyl Methanethiosulfinate and S-Methyl
2-Propene-1-thiosulfinate from Chinese Chive toward Escherichia coli O157:H7
Kwon Il SEO,1 Yea Hwang MOON,2 Sang Uk CHOI,3 and Ki Hun PARK3・ p.966

Note
Molecular Cloning and Functional Expression of cDNA Encoding
the Cysteine Proteinase Inhibitor Sca from Sunflower Seeds
Yoshiaki KOUZUMA, Keiko TSUKIGATA, Hideko INANAGA, Keiko DOI-KAWANO,
Nobuyuki YAMASAKI, and Makoto KIMURA p.969

Note
Facile Synthesis of (R)-4-Mercaptopyrrolidine-2-thione from L-Aspartic Acid
Masahiko SEKI・ and Toshiaki SHIMIZU p.973

Note
Cloning and Characterization of a Chitosanase Gene from the Koji Mold
Aspergillus oryzae Strain IAM 2660
Xiao-Yong ZHANG, An-Lan DAI, Kouji KUROIWA, Ritsuko KODAIRA, Masahiro NOGAWA,
Makoto SHIMOSAKA,・ and Mitsuo OKAZAKI＊ p.977

Note
Improvement of Shark Type I Collagen with Microbial
Transglutaminase in Urea
Yoshihiro NOMURA,・ Shinzi TOKI, Yasuhiro ISHII, and Kunio SHIRAI p.982

Note
Human Milk Bile-salt-stimulated Lipase Is Extremely Reactive
with the Monoclonal Antibody 1CF11 which Recognizes
a Human-specific Carbohydrate Antigen
Shan-Ming YANG,1 Yoshihiro KANAMARU,1・ Makoto SHIMOYAMADA,1 Fumiyuki ASANO,1
Satoshi NAGAOKA,1 Makoto SHIMIZU,2 and Goverdhan P. SACHDEV3 p.986

Note
7-Hydroxyphthalide: A New Natural Salicylaldehyde Analog from Oulenzia sp.
(Astigmata: Winterschmitiidae)・
Nobuhiro SHIMIZU and Yasumasa KUWAHARA＊ p.990

Note
Expression of marY1, a gypsy-type LTR-retroelement from the
Ectomycorrhizal Homobasidiomycete Tricholoma matsutake,
in the Budding Yeast Saccharomyces cerevisiae
Hitoshi MURATA and Yasumasa MIYAZAKI p.993

Note
Co-existing Proteins Interfere with the Action of Nordihydroguaiaretic Acid
on Retrograde Golgi-to-ER Protein Trafficking in NRK Cells
and α-Glucosidase Reaction in vitro
Kazuhito SATO,＊・ Makoto MUROI,＊ Machiko NAKAMURA,＊ Makoto FUJIMURA,・
and Akira TAKATSUKI＊・ p.996

Preliminary Communication
Soybean Resistant Proteins Interrupt an Enterohepatic Circulation of Bile Acids
and Suppress Liver Tumorigenesis Induced by Azoxymethane
and Dietary Deoxycholate in Rats
Ryuhei KANAMOTO,1,・ Naoyuki AZUMA,1 Tomokazu MIYAMOTO,1 Tohru SAEKI,1
Yasunari TSUCHIHASHI,2 and Kimikazu IWAMI1 p.999

Preliminary Communication
Small-Angle X-Ray Scattering Analysis of Stearic Acid Modified Lipase
Tatsuo MARUYAMA,1,2 Mitsutoshi NAKAJIMA,・,1 Sosaku ICHIKAWA,3 Yoh SANO,1
Hiroshi NABETANI,1 Shintaro FURUSAKI,2 and Minoru SEKI2 p.1003

-1-
Effects of Medium-Chain Fatty Acids on Intracellular Calcium Levels
and the Cytoskeleton in Human Intestinal (Caco-2) Cell Monolayers

Yukitaka KIMURA, Yasuo HOSODA, Masayo YAMAGUCHI, Hiroshi NAGANO,
Motohiro SHIMA, Shuji ADACHI, and Ryuichi MATSUNO

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

Received April 20, 2000; Accepted December 22, 2000
The effects of medium-chain fatty acids (MCFA) on intracellular calcium (Ca2+) levels and actin filaments in the Caco-2 monolayer were investigated. A site-dependent increase in intracellular Ca2+ levels caused by decanoic acid (C10) at 13 mM was observed by confocal laser scanning microscopy. The area in which the intracellular Ca2+ levels was increased was measured by image analysis, and increased to 11％ of the total area of the monolayer within 1 minute. This was maintained for 5 minutes, and decreased thereafter. The other MCFAs did not significantly increase the intracellular Ca2+ levels. Obvious morphological changes of actin filaments were induced by only C10 among C8--C14. The area in which actin filaments were depleted was also quantified, and the increase in area became significant after 40 minutes. The area of the actin-depleted spot corresponded to the area occupied by 5 to 10 cells as well as that in which the intracellular Ca2+ level was increased. The effectiveness of only C10 suggested that the mechanism of the absorption enhancement by C10 would be different from that by the other MCFAs, or that C10 has some additional physiological functions although the mechanism of the enhancement is the same as for the other MCFAs.
Key words: medium chain fatty acid; intracellular calcium levels; actin filaments; absorption enhancement; Caco-2

-2-
The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation
of 6-Deoxyerythronolide B

Choonkeun KIM, Haeyoung KIM, and Oksoo HAN・

Department of Genetic Engineering, Biotechnology Research Institute, Institute of Agricultural Science and
Technology, College of Agriculture, Chonnam National University, Kwangju, 500-757, Republic of Korea

Received July 10, 2000; Accepted November 22, 2000
A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.
Key words: cytochrome P450; 6-deoxyerythronolide B hydroxylase; site-directed mutagenesis; proton delivery system; uncoupling of cytochrome P450-catalyzed reaction

-3-
Reaction Kinetics and Modeling of the Enzyme-catalyzed Production
of Lactosucrose using β-Fructofuranosidase from Arthrobacter sp. K-1

Axel PILGRIM,1 Motoaki KAWASE,1,・ Masayasu OHASHI,1 Koki FUJITA,2
Kazufumi MURAKAMI,2 and Kenji HASHIMOTO1,・・

1Department of Chemical Engineering, Faculty of Engineering, Kyoto University, Yoshida-Honmachi,
Sakyo-ku, Kyoto 606-8501, Japan
2Bio Research Corporation of Yokohama, 13-46 Daikoku-cho, Tsurumi-ku, Yokohama 230-0053, Japan

Received July 17, 2000; Accepted November 27, 2000
Lactosucrose synthesis from sucrose and lactose was carried out by using β-fructofuranosidase from Arthrobacter sp. K-1. The transfructosylation mechanism was found to be of an ordered bi-bi type in which sucrose was bound first to the enzyme and lactosucrose was released last. Hydrolysis side-reaction experiments indicated that the reactions were uncompetitively inhibited by glucose and lactose, while no inhibition by fructose was apparent. The overall reaction rates were formulated. The reaction rate constants, equilibrium constant, and dissociation and Michaelis constants were determined at 35°C and 50°C by fitting the experimental concentration changes with the calculated values by a nonlinear least-square method. The average relative derivation for the concentrations was 9.67％. The kinetic parameters were also calculated for 43°C and 60°C by assuming the Arrhenius law, and the course of reaction was predicted. The obtained reaction rate equations well represented the concentration changes during the experiment at all temperatures.
Key words: lactosucrose; β-fructofuranosidase; transfructosylation; kinetic analysis; oligosaccharide

-4-
Molecular Cloning and Characterization of the Fructooligosaccharide-
Producing β-Fructofuranosidase Gene from Aspergillus niger ATCC 20611

Koji YANAI,・ Akitaka NAKANE, Akemi KAWATE, and Masao HIRAYAMA

Bio Science Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1, Chiyoda, Sakado-shi, Saitama 350-0289, Japan

Received July 28, 2000; Accepted November 30, 2000
The fopA gene encoding a fructooligosaccharide-producing β-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other β-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the β-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any β-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-β-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.
Key words: β-fructofuranosidase; fructooligosaccharide; Aspergillus niger

-5-
Effects of Brewer′s Yeast Cell Wall on Constipation and Defecation
in Experimentally Constipated Rats

Tomohiko NAKAMURA,1,・ Kazue AGATA,1 Mai MIZUTANI,1 and Hisakazu IINO2

1Applied Bioresearch Center, Corporate Research and Development Department, Kirin Brewery Co., Ltd.,
Miyahara Machi 3, Takasaki, Gunma 370-1295, Japan
2Showa Women′s University, 1-7 Taishido, Setagaya-ku, Tokyo 154-0004, Japan

Received August 2, 2000; Accepted December 4, 2000
Brewer′s yeast cell wall (BYC) was tested on constipated male Sprague-Dawley rats that had been induced by loperamide (2 mg/kg of body weight). The preventive effect of BYC on constipation was examined and compared with that of a non-fiber diet (NF) as the control. The dose-response of BYC and the effect on defecation by constipated experimental rats were also compared with the characteristics of cellulose diet (CE) group which served as a control. Defecation was observed to be greater by the rats fed with BYC than by those fed with NF or CE. The fecal water content and level of volatile fatty acids (VFA) in the cecal contents were likewise higher in the rats fed with BYC. These results indicate that the administration of BYC was effective for improving defecation and other parameters related to defecation. These favorable effects of BYC supplemented to the diet are attributed to the fermentation ability, water holding capacity and swelling force in the large intestine.
Key words: yeast cell wall; constipation; defecation; dietary fiber; rat

-6-
Fibrinolytic and Antithrombotic Protease from Spirodela polyrhiza

Hye-Seon CHOI・ and You-Seon SA

Department of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea

Received August 29, 2000; Accepted November 28, 2000
A fibrinolytic protease was purified from a Chinese herb (Spirodela polyrhiza). The protease has a molecular mass of 145 kDa and 70 kDa in gel filtration and SDS-polyacrlamide gel electrophoresis (PAGE), respectively, implying it is a dimer. Its optimum pH was 4.5--5.0. The enzyme was stable below 42°C and after lyophilization. The enzyme activity was inhibited significantly by leupeptin and aprotinin. The protease hydrolyzed not only fibrin but also fibrinogen, cleaving Aα and Bβ without affecting the γ chain of fibrinogen. It preferentially cleaved the peptide bond of Arg or Lys of synthetic substrates (P1 position). The enzyme had an anticoagulating activity measured with activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) tests. It delayed APTT, TT, and PT two times at the concentration of 36, 39, and 128 nM, respectively and this was drastically reduced after heat treatment.
Key words: anticoagulatory; antithrombotic; fibrinolytic protease; Spirodela polyrhiza; thrombin time

-7-
ESR Imaging on a Solid-tumor-bearing Mouse Using Spin-labeled Dextran

Kieko SAITO,1,・ Shunsuke KAZAMA,2 Hisayuki TANIZAWA,2 Tomohiro ITO,3
Mika TADA,3 Tateaki OGATA,3 and Hisashi YOSHIOKA1

1Institute for Environmental Sciences, 2School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
3Graduate School of Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa 992-8510, Japan

Received September 4, 2000; Accepted December 18, 2000
Imaging of a tumor with ESR was tried using two different types of spin probes, a low molecular weight spin probe, CPROXYL, and a polymer spin probe, TEMPO-DX. Spin probes were administered to a mouse bearing a solid tumor that was a transplanted Ehrlich′s ascites carcinoma in the back, using two methods, conventional intraperitoneal injection and continuous intravenous injection with a micro-feeder. First, the accumulation of the probe was examined by X-band ESR. CPROXYL, which was administered to a mouse intraperitoneally, was exclusively retained in urine, showing that it was rapidly excreted into the bladder, while TEMPO-DX was absorbed from the peritoneal cavity with difficulty to the vessel. Using continuous intravenous injection, CPROXYL was also rapidly excreted, but it was confirmed that TEMPO-DX concentrated in tumor tissue because it has a long half-life in vivo. In addition, measurement of ESR imaging was done to measure the distribution of spin probes with continuous intravenous injection. The strongest spot of CPROXYL was observed on ESR images, showing the accumulation at the bladder, while the spot of TEMPO-DX was observed in the solid tumor of the back of the mouse. These results suggest that TEMPO-DX could stay much longer than a low molecular weight spin probe in vivo and concentrate at the tumor. TEMPO-DX may be useful for developing specific ESR imaging agents for tumor.
Key words: ESR imaging; dextran; tumor; TEMPO; spin probe

-8-
S-Adenosyl-L-methionine-dependent O-Methylation
of 2-Hydroxy-3-alkylpyrazine in Wine Grapes:
A Putative Final step of Methoxypyrazine Biosynthesis

Katsumi HASHIZUME,・ Kazuyuki TOZAWA, Masashi ENDO, and Isao ARAMAKI

National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima 739-0046, Japan

Received September 18, 2000; Accepted December 4, 2000
The final step of 2-methoxy-3-alkylpyrazine (MP) biosynthesis has been presumed to involve O-methylation of 2-hydroxy-3-alkylpyarzine (HP), although this reaction has never been demonstrated in organisms. We detected 2-hydroxy-3-isobutylpyrazine (IBHP) and 2-hydroxy-3-isopropylpyrazine (IPHP) in unripe grapes, and S-adenosyl-L-methionine-dependent O-methyltransferase (OMT) activity toward HP in crude extracts from young shoots and unripe grapes that accumulate MP at different levels. The levels of HP in the berries and stems were estimated by using 2-hydroxy-3-sec-butylpyrazine as an internal standard. The OMT activity observed in the crude extracts from young shoot and berries was extremely low, but no MP-decomposing activity was detected in the solutions. The levels of HP and OMT activity were closely related with the level of MP in the grapes. These results indicate that the predicted final step of MP biosynthesis exists in wine grapes.
Key words: methoxypyrazine; methyltransferase; |||wine grape; hydroxypyrazine

-9-
Thermostable Chitosanase from Bacillus sp. Strain CK4: Its Purification,
Characterization, and Reaction Patterns

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

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
＊Department of Food Additives, Korea Food and Administration, Seoul 122-020, Korea
＊＊Department of Food and Biotechnology, Hanseo University, Chungnam 352-800, Korea

Received September 19, 2000; Accepted November 29, 2000
A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4％, has a molecular weight of about 29,000±2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other β-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co2+ and 1.4-fold by Mn2+. However, Cu2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60°C and 6.5, respectively. The enzyme was stable after heat treatment at 80°C for 30 min or 70°C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN)4 as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN)3--(GlcN)6 were increased about 30％ (w/w) in DAC 99 soluble chitosan containing 10％ ammonium sulfate, and (GlcN)1 was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10％ EtOH also showed no formation of (GlcN)1, however, the yield of (GlcN)3~(GlcN)6 was lower than DAC 99 soluble chitosan-10％ ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20％. At this concentration, the amount of hexamer was increased by over 12％ compared to the water-salt free system.
Key words: Thermostable chitosanase; Bacillus sp. strain CK4; Chitosan oligosaccharide

-10-
Identification of 2,3-Dihydro-γ-ionylideneethanol in Cercospora cruenta

Hirotaka YAMAMOTO,・ Masahiro INOMATA, Takeo UCHIYAMA, and Takayuki ORITANI＊
Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan

＊Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan

Received September 20, 2000; Accepted November 9, 2000
During our scrutiny of GC-EI-MS date for C15 alcohols as putative intermediates on the ABA biosynthetic pathway in Cercospora cruenta, a trace amount of
5 - [2?,2? - dimethyl - 6? - methylene - 1? - cyclohexyl] - 3-methyl-4-penten-1-ol (2,3-dihydro-γ-ionylideneethanol) was identified. Feeding experiments indicated that this compound was not an intermediate to ABA, but a catabolite that originated from γ-ionylideneacetaldehyde. The stereochemistry of 2,3-dihydro-γ-ionylideneethanol was deduced to be (3R,1?S) from a comparison with an authentic specimen prepared via baker′s yeast asymmetric reduction.
Key words: abscisic acid; carotenoid pathway; (2Z,4E)-γ-ionylideneethanol; Cercospora cruenta

-11-
Biological Evaluation of 5-Substituted Pyrimidine Derivatives as Inhibitors
of Brassinosteroid Biosynthesis

Jing-Ming WANG,1・ Tadao ASAMI,2 Shigeo YOSHIDA,2 and Noboru MUROFUSHI1

1Department of Biotechnology, Akita Prefectural University, Akita-shi, Akita 010-0195, Japan
2The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan

Received September 20, 2000; Accepted December 8, 2000
A series of 5-substituted pyrimidine derivatives was synthesized, and their ability to inhibit brassinosteroid biosynthesis was tested. The biological activity of these compounds was evaluated by the cress stem elongation method. Among the synthesized compounds, α-(4-chlorophenyl)-α-phenyl-5-pyrimidinemethanol (DPPM 4) exhibited potent inhibitory activity for retarding cress stem elongation in the light. This inhibition was reversed by the application of 10 nM brassinolide, but not by 1 μM GA_{3}. DPPM 4 also affected Arabidopsis growth in the dark. DPPM 4-treated Arabidopsis had phenotypes like those of brassinosteroid-deficient mutants, with short hypocotyls and open cotyledons, in the dark. These biological changes were restored by the co-application of 10 nM brassinolide, but not by 1 μM GA_{3}, suggesting that the primary site of action of DPPM 4 was the brassinosteroid biosynthetic pathway.
Key words: brassinosteroids biosynthesis; inhibitor; pyrimidine derivative

-12-
Incorporation of the Whole Chromosomal DNA in Protoplast Lysates
into Competent Cells of Bacillus subtilis

Takashi AKAMATSU・ and Hisataka TAGUCHI

Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan

Received September 22, 2000; Accepted December 7, 2000
Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade＾{+}, Trp＾{+}, or Ade＾{+} Trp＾{+} with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The cotransfer ratio of purB to trpC was constant at 7--9％ (Ade＾{+} Trp＾{+}/Trp＾{+}) or 3％ (Ade＾{+} Trp＾{+}/Ade＾{+}) at protoplast concentrations of 2.7×10＾{3}~2.7×10＾{6} per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7×10＾{3} protoplasts per ml is about 100 times lower than 3.2×10＾{5} competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.
Key words: Bacillus subtilis; transformation; competence; DNA; protoplast

-13-
Cloning, Sequence Analysis, and Expression in Escherichia coli of the
Gene Encoding Monovalent Cation-activated Levodione Reductase
from Corynebacterium aquaticum M-13

Ayumi YOSHISUMI,1 Masaru WADA,・1 Hiroshi TAKAGI,1 Sakayu SHIMIZU,2
and Shigeru NAKAMORI1

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

Received September 22, 2000; Accepted December 18, 2000
The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35％ identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH- binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two- column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K＾{+}, Na＾{+}, and NH_{4}＾{+}, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.
Key words: stereoselective reduction; short-chain alcohol dehydrogenase; cation activation

-14-
Purification of Soluble β-Glucan with Immune-enhancing Activity
from the Cell Wall of Yeast

Jung-Nam LEE, Do-Youn LEE, In-Hye JI, Gi-Eun KIM,1 Hye Nam KIM,
Jeongwon SOHN,2 Sangduk KIM, and Chan-Wha KIM・

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
1Department of Biotechnology, Seokyeong University, Seoul 136-704, Korea
2Department of Biochemistry Korea University College of Medicine, Seoul 136-701, Korea

Received September 22, 2000 ; Accepted November 7, 2000
β-Glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions, especially by activating macrophages. However, a major obstacle to the clinical application of β-(1→3)-glucan is its low solubility in aqueous media. In this study, soluble β-glucan, free of mannoprotein, was prepared, and its effects on TNF-α secretion and phagocytosis by macrophages were evaluated. β-Glucan was first rendered soluble from the yeast cell wall by alkaline extraction (glucan-p1). The extract contained 2.8％ of protein which was subsequently removed by successive DEAE-cellulose and ConA chromatography. β-Glucan thus prepared was completely free of mannoprotein and was soluble at neutral pH (glucan-p3). The effects of β-glucan on phagocytosis and TNF-α release activity were investigated. While glucan-p1 moderately induced TNF-α secretion at 200 μg/ml (550 pg of TNF-α/5×10＾{5} cells), glucan-p3 markedly stimulated macrophages at 200 μg/ml (2,860 pg of TNF-α/5×10＾{5} cells). Furthermore, glucan-p3 stimulated phagocytosis about 20％ more than glucan-p1 did. In conclusion, we purified water-soluble β-glucan which was completely devoid of mannoprotein and effectively stimulated the macrophage function, enabling it to be used as an intravenous injection for sepsis.
Key words: β-glucan; mannoprotein; macrophage function; TNF-α; phagocytosis

-15-
Purification and Properties of a Galacturonic Acid-releasing
Exopolygalacturonase from a Strain of Bacillus

Tohru KOBAYASHI,・ Norihiko HIGAKI, Noriyuki YAJIMA, Atsushi SUZUMATSU,
Hiroshi HAGIHARA, Shuji KAWAI, and Susumu ITO

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

Received September 25, 2000; Accepted December 7, 2000
An exopolygalacturonase [exo-PGase; poly (1,4-α-D-galacturonide) galacturonohydrolase, EC 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, through sequential column chromatographies. The enzyme had a molecular weight of approximately 45,000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp - Glu - Ile - Leu - Lys - Thr - Leu - Lys - Val - Pro - Val - Phe and had no sequence similarity to those of other pectinolytic enzymes reported to date. Maximum activity toward polygalacturonic acid (PGA) was observed at 60°C and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of oligogalacturonic acids increased, the apparent Km for them decreased, but the kcat values increased. This is the first bacterial exo-PGase that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.
Key words: exopolygalacturonase; pectic enzyme; Bacillus

-16-
Non-involvement of the K-ras Mutation in Colon Carcinogenesis Promoted
by Dietary Deoxycholate in Azoxymethane-treated Rats

Ryuhei KANAMOTO,1・ Naoyuki AZUMA,1 Yasunari TSUCHIHASHI,2 Hitoshi SUDA,1
Tohru SAEKI,1 and Kimikazu IWAMI1

1Department of Biological Resource Chemistry, Kyoto Prefectural University, Sakyo-ku,
Kyoto 606-8522, Japan
2Hospital Department of Pathology, Kyoto Prefectural University of Medicine, Kamigyo-ku,
Kyoto 602-8500, Japan

Received September 25, 2000; Accepted November 27, 2000
Fisher-344 rats, whose ileum or jejunum had been surgically removed to change the influx of bile acids into the colon, were intraperitoneally administered with azoxymethane and fed on a diet containing deoxycholate for 39 weeks to induce colon cancer. Fecal bile acids in the ileum-resected group were 1.5-times and serum bile acids were about half of those in the jejunum-resected group. As a result, the incidence and number of tumors were higher in the ileum-resected group. In the total of 59 colon tumors (40 were in the ileum-resected group and 19 in the jejunum-resected group), 56 were carcinomas, including two well-differentiated invasive and two mucinous carcinomas found in the ileum-resected rats. However, only three carcinomas, two invasive and one non-invasive, had the K-ras mutation. These results demonstrate that the K-ras mutation was not essentially involved in deoxycholate-promoted colon carcinogenesis.
Key words: aberrant crypt foci; adenoma-carcinoma sequence; bile acids; colon cancer; K-ras

-17-
Increases of Secondary Metabolite Production in Various Plant Cell Cultures
by Co-cultivation with Cork

Hirobumi YAMAMOTO,1 Atsushi YATO,2 Kazufumi YAZAKI,3 Hiroaki HAYASHI,2
Goro TAGUCHI,4 and Kenichiro INOUE2

1Medicinal Plant Garden, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan
2Laboratory of Pharmacognosy, Gifu Pharmaceutical University, 6-1, Mitahora-higashi 5-chome,
Gifu 502-8585, Japan
3Laboratory of Molecular and Cellular Biology Totipotency, Division of Integrated Life Science,
Graduate School of Biostudies, Kyoto Univ., Kitashirakawa, Sakyo, Kyoto 606-8502, Japan
4Gene Research Center, Shinshu University 3-15-1, Tokida, Ueda, Nagano 386-8567, Japan

Received September 25, 2000; Accepted December 8, 2000
Cork tissues increased secondary metabolite production of various plant cell cultures in a different manner from those of conventional elicitors. In Sophora flavescens and Glycyrrhiza glabra cultured cells, cork tissues increased the amounts of both lipophilic and hydrophilic flavonoids without affecting the cell growth, although elicitors such as copper ion and yeast extracts showed a clear inhibition of cell growth with the increasing amount of these lipophilic ones. The validity of this effect of cork tissues covered a wide range of aromatic compounds produced by suspension cell cultures derived from diverse plant species. Woody tissues of Japanese cypress had a very similar effect to that of cork. Partial purification of cork tissues suggested that the production-stimulating factor was present in the hemicellulose B fraction that was not included in the dedifferentiated cultured tissues.
Key words: cork tissues; cell walls; plant cell cultures; secondary metabolites; production-stimulating effect

-18-
Biosynthesis of Fattiviracin FV-8, an Antiviral Agent

EL-Sayed E. HABIB, Kazumi YOKOMIZO, Keitarou SUZUKI, and Masaru UYEDA＊

Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan

Received September 26, 2000; Accepted November 29, 2000
Streptomyces microflavus strain No. 2445 produces many derivatives of fattiviracin antibiotics. The major product of these derivatives is fattiviracin FV-8, which consists of four glucose and two trihydroxy fatty acid residues. We found that this strain has the ability to convert several sugars in the culture medium to glucose, and the glucose added to the medium is directly incorporated into the FV-8 molecule. Two trihydroxy fatty acid residues in the FV-8 molecule are derived from acetic acid, and production of FV-8 is inhibited by the addition of cerulenin, which is an inhibitor of fatty acid biosynthesis.
Key words: Streptomyces microflavus; fattiviracin; biosynthesis; sugar conversion; antiviral agent

-19-
Increase in the Stability of Serine Acetyltransferase from Escherichia coli
against Cold Inactivation and Proteolysis by Forming a Bienzyme Complex

Koshiki MINO, Koreyoshi IMAMURA, Takaharu SAKIYAMA, Naoki EISAKI,＊
Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University,
3-1-1 Tsushima-naka, Okayama 700-8530, Japan
＊Research and Development Division, Kikkoman Corporation, 399 Noda, Chiba 278-0037,
Japan/Research Institute of Innovative Technology for the Earth (RITE), 2-8-11 Nishi-shinbashi, Minato-ku,
Tokyo 105-0003, Japan

Received October 2, 2000; Accepted December 18, 2000
Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS). The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0°C. Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0°C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits. In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack by α-chymotrypsin and V8 protease by forming the complex with OASS. On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.
Key words: serine acetyltransferase; cysteine synthetase; enzyme complex; cold inactivation; proteolysis

-20-
Transglycosylation to Ginseng Saponins by Cyclomaltodextrin
Glucanotransferases

Young-Hoi KIM, Young-Gu LEE, Kang-Ju CHOI, Kei UCHIDA,＊ and Yukio SUZUKI＊,・

Korea Ginseng and Tobacco Research Institute, 302 Shinsong-dong, Yusong-ku, Taejon 305-345, Korea
＊Research Institute for Bioresources, Okayama University, 2-20-1, Chuo, Kurashiki 710-0046, Japan

Received October 2, 2000; Accepted December 18, 2000
Ten new α-glucosylginsenosides were found to be synthesized from dextrin and four ginsenosides, -Rb1, -Rc, -Re, and -Rg1, by the successive actions of B. stearothermophilus cyclomaltodextrin glucanotransferase and Rhizopus glucoamylase. Seven of them were isolated in the pure state by extraction with n-butanol saturated with water, silica gel column chromatography, and high pressure liquid chromatography, and identified as 3-O-
[α - D - glcp - (1→4) - β - D - glcp - (1→2) - β-D - glcp] - 20 - O - [β-D-glcp-(1→6)-β-D-glcp]-20(S)-protopanaxadiol, 3-O-[β-D-glcp - (1→2) - β - D - glcp] - 20 - O - [α - D - glcp - (1→4) - β - D - glcp-(1→6)-β-D-glcp]-20(S)-protopanaxadiol, 3-O-[α-D-glcp-(1→4) - β - D - glcp - (1→2) - β - D - glcp] - 20 - O - [α - L - araf - (1→6)-
β-D-glcp]-20(S)-protopanaxadiol, 3-O-[β-D-glcp-(1→2)-
β-D-glcp]-20-O-[(4G-α-D-glcp)-α-L-araf-(1→6)-β-D-glcp]-
20(S)-protopanaxadiol, 6-O-[α-L-rhap-(1→2)-β-D-glcp]-
20 - O - [α - D - glcp - (1→4) - β - D - glcp] - 20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, and 6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, by spectroscopy (FAB-MS, IR, 1H-NMR and 13C-NMR) and hydrolysis products in 50％ acetic acid. The bitterness of these α-glucosyl-ginsenosides was less than that of ginsenosides.
Key words: α-glucosylginsenoside Rb1; α-glucosylginsenoside Rc; α-glucosylginsenoside Re; α-glucosylginsenoside Rg1; Bacillus stearothermophilus cyclomaltodextrin glucanotransferase

-21-
Novel Method Using Phytase for Separating Soybean
β-Conglycinin and Glycinin

Tsutomu SAITO,＊ Mitsutaka KOHNO,＊ Kazunobu TSUMURA,＊ Wataru KUGIMIYA,＊
and Makoto KITO＊＊

＊New Ingredients Research Institute, Tsukuba R＆D Center, Fuji Oil Co. Ltd., 4-3 Kinunodai, Yawara,
Tsukuba-gun, Ibaraki 300-2497, Japan
＊＊Emeritus Professor of Kyoto University

Received October 4, 2000; Accepted November 30, 2000
A novel method for separating soybean β-conglycinin and glycinin from defatted soymilk by a phytase treatment was developed. Phytase was added to defatted soymilk (1000FYT/100 g of protein) at pH 6.0, and the mixture incubated for 1 h at 40°C. This procedure separated β-conglycinin and glycinin without needing a reducing agent or cooling into the soluble and insoluble fractions, respectively. Simultaneously, most of the phytate in both proteins was removed.
Key words: separation; soy protein; β-conglycinin; glycinin; phytase

-22-
A Novel Cryoprotective Protein (CRP) with High Activity
from the Ice-nucleating Bacterium, Pantoea agglomerans IFO12686

Noriko KODA,＊,＊＊ Toshiaki ASAEDA,＊ Kazuhiro YAMADE,＊ Hidehisa KAWAHARA,＊,＊＊
and Hitoshi OBATA＊,＊＊

＊Department of Biotechnology, Faculty of Engineering, and ＊＊High Technology Research Center,
Kansai University, 3-3-35, Yamatecho, Suita-shi, Osaka 564-8680, Japan

Received October 12, 2000; Accepted December 6, 2000
The ice-nucleating bacterium, Pantoea agglomerans IFO12686, induces the cryoprotective protein (CRP) by cold acclimation at 12°C. The CRP was purified to apparent homogeneity by various chromatographies. We found that the purified CRP was a monomer of approximately 29,000 according to gel filtration chromatography and SDS-PAGE, and was a heat-stable protein. The CRP could protect freeze-labile enzymes, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and isocitrate dehydrogenase (iCDH), against freezing-thawing denaturation. The activity of the CRP was about 3.5×104 times more effective than bovine serum albumin (BSA) and 2×106 times than COR26 from the ice-nucleating bacterium Pseudomonas fluorescens KUIN-1. We confirmed that the CRP was a novel protein, as judged by the a different molecule mass from the already-known cryoprotectants, and has an extremely high cryoprotective activity.
Key words: Pantoea; cryoprotective protein; lactate dehydrogenase

-23-
Characterization of Soluble Protein Extracts from Keratinized Tissues:
Identification of Ubiquitin Universally Distributed in Hair,
Nail, and Stratum Corneum

Takafumi INOUE, Kenji KIZAWA, and Mayumi ITO

Basic Research Laboratory, Kanebo Ltd., Kotobuki-cho 5-3-28, Odawara 250-0002, Japan

Received October 16, 2000; Accepted November 28, 2000
Partial protein extracts were prepared from hair, nail, and stratum corneum in the absence of urea and interfacial surfactant. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoreses of these extracts showed low-molecular weight protein-rich patterns apparently different from those of whole protein extracts, which mainly consist of keratin bands. Several protein bands characterized each keratinized tissue or its derived species. In addition, we identified a major band of approximately 7 kDa as ubiquitin, a ubiquitously distributed protein that mediates non-lysosomal protein degradation, through direct amino acid sequence analysis of the electro-blotted protein band. The partial extraction is useful for investigation of soluble proteins retained in the keratinized tissues.
Key words: ubiquitin; S100A3; hair; nail; stratum corneum

-24-
Structures of the N-Linked Sugar Chains in PAS-7 Glycoprotein Sharing
the Same Protein Core with PAS-6 Glycoprotein
from the Bovine Milk Fat Globule Membrane

Jin-Seok SEOK,・ Michiko SHIMODA, Norihiro AZUMA, and Choemon KANNO・・

Department of Applied Biochemistry, Utsunomiya University, Utsunomiya 321-8505, Japan

Received October 17, 2000; Accepted December 7, 2000
Glycoproteins PAS-6 (50 kDa) and -7 (47 kDa) from the bovine milk fat globule membrane share a common protein core but differ in their carbohydrate moiety. We here analyzed and proposed the structures of the N-linked sugar chains of PAS-7. The N-linked sugar chains were liberated from PAS-7 by hydrazinolysis and, after modifying the reducing ends with 2-aminopyridine (PA), were separated into one neutral (7N, 55％) and two acidic (7M, mono-, 43％; 7D, di-, 2％) sugar chain groups. The latter were converted into neutral groups (7MN and 7DN) by sialidase digestion. 7N was finally separated into 5 chains (7N1A, 7N1B-1, 7N1B-2, 7N2A, and 7N2B), and 7MN and 7DN were separated into 3 (7MN1, 7MN2, and 7MN3) and 2 (7DN1 and 7DN2) chains, respectively. The structure of each of these PA-neutral sugar chains was determined by sugar analysis, sequential exoglycosidase digestion, partial acetolysis, and 1H-NMR spectroscopy. The results show that the 10 sugar chains were of the biantennary complex type with and without fucose. The structure of 7N2A, one of the major sugar chains, was proposed as;
Galβ1--4GlcNAcβ1--2Manα1Galβ1--4GlcNAcβ1--2Manα1・・63 Manβ1--4GlcNAcβ1--4GlcNAc-PA
A structural comparison between PAS-6 and -7 indicated that, although they shared the same protein core, their sugar moiety was markedly different, involving the existence of a different pathway during the post-transcriptional modification.
Key words: PAS-7 glycoprotein; milk fat globule membrane; bovine; N-linked sugar chain

-25-
Controlled Trial of the Effects of Milk Basic Protein (MBP) Supplementation
on Bone Metabolism in Healthy Adult Women

Seicihiro AOE,1 Yasuhiro TOBA,2 Jun-ichi YAMAMURA,2 Hiroshi KAWAKAMI,2
Masatoshi YAHIRO,1 Masayoshi KUMEGAWA,3 Akira ITABASHI,4 and Yukihiro TAKADA2

1Product Planning Department, Snow Brand Milk Products Co. Ltd., 1-1-2 Minamidai, Kawagoe,
Saitama 350-1165, Japan
2Technology ＆ Research Institute, Snow Brand Milk Products Co. Ltd., 1-1-2 Minamidai, Kawagoe,
Saitama 350-1165, Japan
3Department of Oral Anatomy, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado,
Saitama 350-0283, Japan
4Central Clinical Laboratory, Saitama Medical School, 38 Morohongo, Moroyama, Saitama 350-0495, Japan

Received October 26, 2000; Accepted December 15, 2000
Milk has more beneficial effects on bone health compared to other food sources. Recent in vitro and in vivo studies showed that milk whey protein, especially its basic protein fraction, contains several components capable of both promoting bone formation and inhibiting bone resorption. However, the effects of milk basic protein (MBP) on bone metabolism of humans are not known. The object of this study was to examine the effects of MBP on bone metabolism of healthy adult women. Thirty-three normal healthy women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for six months. The bone mineral density (BMD) of the left calcaneus of each subject was measured at the beginning of the study and after six months of treatment, by dual-energy x-ray absorptiometry. Serum and urine indices of bone metabolism were measured at the base line, three-month intervals, and the end of the study. Daily intake of nutrients was monitored by a three-day food record made at three and six months. The mean (±SD) rate of left calcaneus BMD gain of women in the MBP group (3.42±2.05％) was significantly higher than that of women in the placebo group (2.01±1.75％, P=0.042). As compared with the placebo group, urinary cross-linked N-teleopeptides of type-I collagen/creatinine and deoxypyridinoline/creatinine were significantly decreased in the MBP group (p<0.05), while no significant differences between the two groups were observed in serum osteocalcin and bone-specific alkaline phosphatase concentrations. A daily MBP supplementation of 40 mg in healthy adult women can significantly increase their BMD independent of dietary intake of minerals and vitamins. This increase in BMD might be primarily mediated through inhibition of osteoclast-mediated bone resorption by the MBP supplementation.
milk basic protein; bone mineral density; bone resorption; healthy adult women

-26-
Structural Elucidation of Twelve Novel Esters Composed of Five Fatty Acids
and Three New Branched Alcohols Together with Four Monoterpenoids
from Sancassania shanghaiensis (Acari: Acaridae)・

Tomoyo SAKATA,1 Kimiko OKABE,2 and Yasumasa KUWAHARA1,・・

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan
2Forest Insect Management Laboratory, Forestry and Forest Products Research Institute, Tsukuba Norin
Kenkyu Danchi, P.O.Box 16, Ibaraki 305-8687, Japan

Received October 27, 2000; Accepted November 22, 2000
A total of 12 novel esters and four monoterpenoids (rosefuran, (2R,3R)-epoxyneral, and α- and β-acaridials) were detected by GC/MS analyses as the opisthonotal gland components of Sancassania shanghaiensis. The acidic fraction after hydrolysis was composed of five common fatty acids (palmitic, stearic, oleic, linoleic and arachidic acid), while the alcoholic fraction consisted of two major components (C6 and C8 alcohols with branched methyls), together with a trace amount of C9 alcohol. The two major alcohols were identified as new alcohols [(S)-2-methylpentanol and (2S,4S)-2,4-dimethylhexanol] by comparing the physico-chemical data of their 3,5-dinitrobenzoates with those of regio-selectively synthesized alcohols. The C9 alcohol was suggested as (2S,4S)-2,4-dimethylheptanol, based on a structural and biogenetic analogy to the C6 and C8 alcohols. Five of the compounds were each identified by GC to be (S)-2-methylpentyl esters from five fatty acids, and the other five components likewise as (2S,4S)-2,4-dimethylhexyl esters. The remaining two were suggested as (2S,4S)-2,4-dimethylheptyl stearate and linolate.
Key words: astigmatid mite; Sancassania shanghaiensis; (S)-2-methylpentanol; (2S,4S)-2,4-dimethylhexanol; fatty acid ester of (S)-2-methylpentanol and (2S,4S)-2,4-dimethylhexanol

-27-
Greater Effect of Dietary Potassium Tripolyphosphate than of Potassium
Dihydrogenphosphate on the Nephrocalcinosis and Proximal Tubular Function
in Female Rats from the Intake of a High-phosphorus Diet

Hiroshi MATSUZAKI,1 Ritsuko MASUYAMA,2 Mariko UEHARA,2 Kahoru NAKAMURA,1
and Kazuharu SUZUKI2＊

1Department of Nutrition, Junior College of Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku,
Tokyo 156-8502, Japan
2Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1
Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received Octorber 30, 2000; Accepted December 4, 2000
We examined whether a difference in potassium dihydrogenphosphate (KH2PO4) and potassium tripolyphosphate (K5P3O10) as dietary phosphorus sources could differentially effect the nephrocalcinosis and proximal tubular function in female rats. Rats were fed on a diet containing KH2PO4 or K5P3O10, at the normal phosphorus level (normal phosphorus diet) or at a high phosphorus level (high-phosphorus diet) for 21 d. Nephrocalcinosis, as confirmed by a histological examination, was apparent in all rats fed on the high-phosphorus diet, and this condition was more severe in those rats fed on K5P3O10 than in those fed on KH2PO4. As indicators of the proximal tubular function, the N-acetyl-β-D-glucosaminidase activity in urine and the urinary β2-microglobulin excretion were significantly increased in those rats fed on the high-phosphorus diet containing K5P3O10. These results indicate that the intake of a high-phosphorus diet, more strongly influenced the nephrocalcinosis and proximal tubular function when K5P3O10 rather than KH2PO4 was used as the dietary phosphorus source.
Key words: potassium dihydrogenphosphate; potassium tripolyphosphate; nephrocalcinosis; proximal tubular function; female rats

-28-
Note
Different Response to Choline Deficiency of the Serum Ornithine
Carbamoyltransferase Activity in Four Strains of Rats

Komang Ayu NOCIANITRI and Yoritaka AOYAMA・

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Nishi-9, Kita-9,
Kita-ku, Sapporo 060-8589, Japan

Received April 10, 2000; Accepted December 11, 2000
Rats of the Donryu, Wistar, Fischer, and Sprague-Dawley strains were examined for the effects of choline deficiency on liver lipids, serum lipids, and serum ornithine carbamoyltransferase. The liver total lipid, triacylglycerol, cholesterol and phospholipid contents in the choline-deficient rats were significantly higher than those in choline-sufficient rats. The contents of total lipids and phospholipids in the liver of the Wistar and Fischer rats fed on a choline-deficient diet were significantly higher than those of the Donryu and Sprague-Dawley rats. The levels of triacylglycerol, cholesterol and phospholipids in the serum were significantly decreased by feeding with the choline-deficient diet. The serum ornithine carbamoyltransferase activity was increased in the Wistar and Fischer strains by feeding with the choline-deficient diet. The Wistar and Fischer strains were consequently the most sensitive to both lipid accumulation and liver lesions induced by the choline deficiency.
Key words: choline-deficiency; fatty liver; strain; ornithine carbamoyltransferase

-29-
Note
Detection of Antifungal Activity in Belamcanda chinensis by a Single-cell
Bioassay Method and Isolation of Its Active Compound, Tectorigenin

Ki-Bong OH,1＊ Heonjoong KANG,2 and Hideaki MATSUOKA3

1Natural Products Research Institute, Seoul National University, 28 Yungun, Jongro, Seoul 110-460, Korea
2MBCL, Oceanographic Program, School of Earth and Environmental Sciences, Seoul National University,
San 56-1, Shinlim, Kwanak, Seoul 151-742, Korea
3Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology,
2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan

Received July 28, 2000; Accepted November 14, 2000
The antifungal activity of Belamcanda chinensis was evaluated by a single-cell bioassay method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The isolated compound was found to be identical to tectorigenin (5,7-dihydroxy-3-(4-hydroxy phenyl)-6-methoxy-4H-1-benzopyran-4-one) which has formerly appeared in the literature without any remarks on its antimicrobial activity. Antimicrobial activity was investigated against 17 strains of fungi and 6 strains of bacteria. This compound showed marked antifungal activity against dermatophytes of the genera Trichophyton, the minimum inhibitory concentration (MIC) being in the range of 3.12--6.25 mg/ml.
Key words: medicinal plant; Belamcanda chinensis; antimicrobial activity; single-cell bioassay; tectorigenin

-30-
Note
Microbial Hydroxylation of (±)- and (--)-(2Z,4E)-5-(1?,2?-Epoxy-2?,6?,6?-
trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid into
(±)- and (−)-Xanthoxin Acid by Cunninghamella echinulata

Ryou OKAZAKI, Takayuki ORITANI,・ Yoshihiro HARA and Hirotaka YAMAMOTO1

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, Sendai 981-8555, Japan
1Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarasih,
Niigata 950-2181, Japan

Received August 30, 2000: Accepted November 16, 2000
Microbial hydroxylation of (±)-(2Z,4E)-5-(1?,2?-epoxy - 2?,6?,6? - trimethylcyclohexyl) - 3 - methyl - 2,4 - pentadienoic acid (3a) with Cercospora cruenta, a fungus producing (+)-abscisic acid, gave a four- stereoisomeric mixture consisting of (+)- and (--)-xanthoxin acid (4a), and (+)- and (--)-epi-xanthoxin acid (5a) by an HPLC analysis with a chiral column. Screening of the microorganisms capable of oxidizing (±)-3a showed that Cunninghamella echinulata stereoselectively oxidized (±)-3a to xanthoxin acid (4a) with the some degree of enantioselectivity as (--)-3a to (--)-4a.
Key words: xanthoxin; xanthoxin acid; Cunninghamella echinulata; Cercospora cruenta; microbial hydroxylation

-31-
Note
Anti-tumor Promoting Activity of Bufadienolides from Kalanchoe pinnata
and K. daigremontiana×tubiflora

Unang SUPRATMAN,1 Tomoyuki FUJITA,1 Kohki AKIYAMA,1 Hideo HAYASHI,1・
Akira MURAKAMI,2 Hirofumi SAKAI,2 Koichi KOSHIMIZU,2 and Hajime OHIGASHI3

1Division of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, Osaka Prefecture
University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
2Department of Biotechnological Science, Faculty of Biology-oriented Science and Technology,
Kinki University, Iwade-Uchita, Wakayama 649-6493, Japan
3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received August 31, 2000; Accepted November 11, 2000
Five bufadienolides (1--5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana×tubiflora (Crassulaceae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC_{50}=0.4 μM) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.
Key words: bufadienolide; Epstein-Barr virus; anti-tumor promoter; Kalanchoe pinnata; Kalanchoe daigremontiana×tubiflora

-32-
Note
Isolation and Identification of trans-2- and trans-3-Hydroxy-1,8-cineole
Glucosides from Alpinia galanga

Yuki SOMEYA, Akio KOBAYASHI and Kikue KUBOTA・

Laboratory of Food Chemistry, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan

Received September 11, 2000; Accepted November 7, 2000
Three hydroxy-1,8-cineole glucopyranosides,
(1R, 2R, 4S)- and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole β-D-glucopyranosides, and (1R, 3S, 4S)-trans-3-hydroxy-1,8-cineole β-D-glucopyranoside, which are possible precursors of acetoxy-1,8-cineoles as unique aroma components, were isolated from the rhizomes of greater galangal (Alpinia galanga W.). Their structures were analyzed by FAB-MS and NMR spectrometry, and the absolute configulation of each aglycone was determined by using a GC-MS analysis with a capillary column coated with a chiral stationary phase. The composition of the diastereomers of (1R, 2R, 4S)- and (1S, 2S, 4R)- trans-2-hydroxy-1,8-cineole β-D-glucopyranosides in the rhizomes was determined as 3:7 by a GC-MS analysis after preparing the trifluoroacetate derivatives of the glucosides.
Key words: Alpinia galanga; trans-2-hydroxy-1,8-cineole glucopyranoside; trans-3-hydroxy-1,8-cineole glucopyranoside; absolute configuration

-33-
Note
Wine Has Activity against Entero-pathogenic Bacteria in Vitro but not in Vivo

Yoshiko SUGITA-KONISHI, Yukiko HARA-KUDO, Tamami IWAMOTO,＊ and Kazuo KONDO＊＊

Department of Biomedical Food Research, National Institute of Infectious Diseases, 1-23-1, Toyama,
Shinjuku-ku, Tokyo 162-8640, Japan
＊Department of Food Science, National Institute of Health and Nutrition, Japan 1-23-1, Toyama,
Shinjuku-ku, Tokyo 162-8640, Japan
＊＊Faculty of Human Life and Environmental Sciences, Ochanomizu University, 2-1-1, Otuka,
Bunkyou-ku, Tokyo 112-8610, Japan

Received September 22, 2000; Accepted December 15, 2000
We studied the activity of wine against entero-pathogenic bacteria both in vitro and in vivo. The food-borne bacteria were killed in both red and white wine within 30 min. However, the results of a Salmonella infection experiment using mice suggested that wine was not effective in preventing food-borne diseases in vivo.
Key words: red wine; white wine; Salmonella enteritidis; E. coli O 157:H7; alcohol

-34-
Note
Rapid Dephosphorylation of eIF4E by Dietary Protein in the Skeletal Muscle
and Liver of Food-deprived Rats

Fumiaki YOSHIZAWA,・ Taketoshi KIDO,＊ and Takashi NAGASAWA＊

Department of Animal Science, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan
＊Department of Agro-bioscience, Iwate University, Morioka, Iwate 020-8550, Japan

Received September 22, 2000; Accepted November 22, 2000
The effect of dietary protein on eIF4E phosphorylation was examined in rats starved for 18 h and then fed on either a 20％ casein diet (20C) or a protein-free diet (0C). Refeeding with the 20C diet, but not the 0C diet, resulted in partial dephosphorylation of eIF4E in both the skeletal muscle and liver. The results suggest that the dephosphorylation of eIF4E in response to food intake was regulated by the increase in plasma amino acid concentration that occurred after feeding with the 20C diet.
Key words: protein synthesis; eIF4E phosphorylation; food intake; dietary protein; rat

-35-
Note
Effect of Zinc Deficiency on Betacyanin Production in a Cell Suspension
Culture of Table Beet (Beta vulgaris L.)

Toru AKITA,・ Yasuhiko HINA, and Toyoyuki NISHI

The Nippon Shinyaku Institute for Botanical Research, 39 Sakanotsuji-cho, Ohyake, Yamashina-ku,Kyoto 607-8182, Japan

Received October 4, 2000; Accepted December 4, 2000
The effect of microelements in the Linsmaier-Skoog (LS) medium on betacyanin production was investigated in suspension cultures of table beet (Beta vulgaris L.).
Removing zinc from the medium resulted in a high
betacyanin content of the cells, the betacyanin content of
the cells decreasing with increasing zinc concentration in the medium. The betacyanin content of cells cultured in the medium without zinc was twice as high as that in the medium containing 0.03 mM of zinc. In the revised LS medium without zinc, the maximum betacyanin yield was obtained of 590 mg/l from a 21-day culture.
Key words: Beta vulgaris L.; betacyanin; cell suspension culture; zinc

-36-
Note
Antibacterial Activity of S-Methyl Methanethiosulfinate and S-Methyl
2-Propene-1-thiosulfinate from Chinese Chive toward Escherichia coli O157:H7

Kwon Il SEO,1 Yea Hwang MOON,2 Sang Uk CHOI,3 and Ki Hun PARK3・

1Department of Food and Nutrition, Sunchon National University, Sunchon 540-742, Korea
2Department of Animal Science, Chinju National University, Chinju 660-758, Korea
3Department of Agricultural Chemistry, Gyeongsang National University, Chinju 660-701, Korea

Received October 4, 2000; Accepted December 7, 2000
S-Methyl methanethiosufinate (1) and S-methyl 2-propene-1-thiosulfinate (2) were easily seperated from Chinese chive (Allium tuberosum L.) using simple column chromatography. Both compounds showed significant antibacterial activities against E. coli O-157:H7 including spoilage microorganism in food. Structural assignment was based on Mass and NMR-spectroscopic methods.
Key words: Escherichia coli O-157:H7; Chinese chive; S-methyl methanethiosulfinate; S-methyl 2-propene-1-thiosulfinate

-37-
Note
Molecular Cloning and Functional Expression of cDNA Encoding
the Cysteine Proteinase Inhibitor Sca from Sunflower Seeds

Yoshiaki KOUZUMA, Keiko TSUKIGATA, Hideko INANAGA, Keiko DOI-KAWANO,
Nobuyuki YAMASAKI, and Makoto KIMURA

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

Received October 17, 2000; Accepted November 28, 2000
Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.
Key words: cDNA cloning; cysteine proteinase; overexpression; phytocystatin; sunflower

-38-
Note
Facile Synthesis of (R)-4-Mercaptopyrrolidine-2-thione from L-Aspartic Acid

Masahiko SEKI・ and Toshiaki SHIMIZU

Product ＆ Technology Development Laboratory, Tanabe Seiyaku Co. Ltd. 3-16-89 Kashima, Yodogawa-ku,
Osaka 532-8505, Japan

Received October 19, 2000; Accepted December 6, 2000
An SN2-type of substitution of (S)-bromide 4, which had been prepared from L-aspartic acid, with potassium thiobenzoate provided (R)-benzoylthio derivative 5 with complete inversion of the configuration. Compound 5 was converted, via iodide 6c, to (R)-4-amino-3-benzoylthiobutyric acid 8b. (R)-4-Mercapto pyrrolidine-2-thione 1 was readily obtained from 8b through cyclization with acetic anhydride, thionation with Lawesson′s reagent and facile removal of the S-benzoyl group with sodium methoxide.
Key words: L-aspartic acid; α-amino acid; carbapenem; thioester

-39-
Note
Cloning and Characterization of a Chitosanase Gene from the Koji Mold
Aspergillus oryzae Strain IAM 2660

Xiao-Yong ZHANG, An-Lan DAI, Kouji KUROIWA, Ritsuko KODAIRA, Masahiro NOGAWA,
Makoto SHIMOSAKA,・ and Mitsuo OKAZAKI＊

Department of Applied Biology, Faculty of Textile Science and Technology, and ＊Gene Research Center,
Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan

Received November 6, 2000; Accepted December 13, 2000
A genomic copy of the gene coding for chitosanase (csnA) was isolated from Aspergillus oryzae IAM 2660. A. oryzae csnA contains an open reading frame that encodes a polypeptide of 245 amino acids with a calculated molecular mass of 26,500 Da. The deduced amino acid sequence of A. oryzae csnA indicates extensive similarities to those of other fungal chitosanases.
Key words: chitosanase; Aspergillus; gene cloning; koji mold

-40-
Note
Improvement of Shark Type I Collagen with Microbial
Transglutaminase in Urea

Yoshihiro NOMURA,・ Shinzi TOKI, 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 November 6, 2000; Accepted January 15, 2001
In the presence of urea, type I collagen could form a gel with crosslinks with microbial transglutaminase (MTGase). Collagen self-assembly was accelerated with the addition of MTGase. The proportion of reconstructed collagen fibrils was raised with the addition of MTGase. MTGase-treated collagen gel remained gelled at high temperatures at which collagen denatured. By treatment with MTGase, collagen could form the gel under impossible condition to collagen self-assembly, and that denaturation temperature was raised.
Key words: type I collagen; transglutaminase; self-assembly; shark; dynamic viscoelasticity

-41-
Note
Human Milk Bile-salt-stimulated Lipase Is Extremely Reactive
with the Monoclonal Antibody 1CF11 which Recognizes
a Human-specific Carbohydrate Antigen

Shan-Ming YANG,1 Yoshihiro KANAMARU,1・ Makoto SHIMOYAMADA,1 Fumiyuki ASANO,1
Satoshi NAGAOKA,1 Makoto SHIMIZU,2 and Goverdhan P. SACHDEV3

1Department of Food Science, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan
2Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan
3College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, U.S.A

Received November 6, 2000; Accepted December 14, 2000
1CF11 (Kanamaru, Y. et al.; Biochem. Biophys. Res. Commun., 249, 618--623, 1998) is a monoclonal antibody obtained after being raised in a mouse by injection of human milk MUC1 mucin as the antigen. Its reactivity was found to be unique in that it only reacts with a carbohydrate epitope shared by glycoproteins in human secretions, while its chemical nature is still unknown. Since a glycoprotein of Mr 135,000 (135K) in human milk was found to react extremely strongly with this antibody, we intended in this study to isolate the glycoprotein by a combination of various chromatographic techniques and identify it. It is a human milk bile-salt-stimulated lipase. By comparison of its immunoreactivity and glycan structures so far reported with those of lactoferrin from human milk, it is suggested that the epitope recognized by mAb 1CF11 could be a human-specific novel glycan.
Key words: 1CF11; human milk; glycoprotein; BSSL; lactoferrin

-42-
Note
7-Hydroxyphthalide: A New Natural Salicylaldehyde Analog from Oulenzia sp.
(Astigmata: Winterschmitiidae)・

Nobuhiro SHIMIZU and Yasumasa KUWAHARA＊

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

Received November 9, 2000; Accepted December 8, 2000
A new salicyl lactone was detected from the unidentified Oulenzia sp. and its chemical structure was elucidated as 7-hydroxyphthalide (7-hydroxy-3H-isobenzofuran-1-one), based on its GC/MS and GC/FT-IR data and SiO_{2} column behavior. The compound was synthesized by NaBH_{4} reduction of 3-hydroxyphthalic anhydride. The GC/MS and GC/FT-IR spectra of the natural compound were consistent with those of the synthetic product. Although the compound is known as a medical material, this is the first example of its presence in nature.
Key words: Oulenzia sp.; 7-hydroxy-3H-isobenzofuran-1-one; 7-hydroxyphthalide; Winterschmitiidae

-43-
Note
Expression of marY1, a gypsy-type LTR-retroelement from the
Ectomycorrhizal Homobasidiomycete Tricholoma matsutake,
in the Budding Yeast Saccharomyces cerevisiae

Hitoshi MURATA and Yasumasa MIYAZAKI

Division of Bio-Resource Development, Forestry ＆ Forest Products Research Institute, P.O.Box 16,
Tsukuba-Norin, 305-8687, Japan

Received November 13, 2000; Accepted December 19, 2000
marY1 is a gypsy-type LTR-retroelement present in the genome of the ectomycorrhizal homobasidiomycete Tricholoma matsutake. We document here that a marY1-lacZ gene fusion was expressed in the budding yeast Saccharomyces cerevisiae. The finding strongly suggests that marY1 is activated by trans-regulatory factors common to higher fungi, and may be useful for the development of new recombinant systems in ectomycorrhizal fungi and homobasidiomycetes.
Key words: ectomycorrhizal fungi; heterologous gene expression; homobasidiomycetes; LTR-retroelement; Tricholoma matsutake

-44-
Note
Co-existing Proteins Interfere with the Action of Nordihydroguaiaretic Acid
on Retrograde Golgi-to-ER Protein Trafficking in NRK Cells
and α-Glucosidase Reaction in vitro

Kazuhito SATO,＊・ Makoto MUROI,＊ Machiko NAKAMURA,＊ Makoto FUJIMURA,・
and Akira TAKATSUKI＊・

＊Animal and Cellular Systems Laboratory, RIKEN (The Institute of Physical and Chemical Research)
Hirosawa, Wako-shi, Saitama 351-0198, Japan
・Faculty of Life Science, Toyo University, Izumino, Itakura, Gunma 374-0193, Japan

Received November 21, 2000; Accepted December 25, 2000
Induction of retrograde trafficking of mannosidase II and TGN38 in NRK cells and inhibition of α-glucosidase in vitro by nordihydroguaiaretic acid (NDGA) were strongly interfered with by serum, serum albumin, or other unrelated proteins added to the medium or incubation mixture. These observations indicate that NDGA interacts with diverse kinds of proteins, and therefore, pharmacological effects of NDGA at cellular levels should be carefully interpreted.
Key words: nordihydroguaiaretic acid; retrograde trafficking; Golgi apparatus; α-glucosidase

-45-
Preliminary Communication
Soybean Resistant Proteins Interrupt an Enterohepatic Circulation of Bile Acids
and Suppress Liver Tumorigenesis Induced by Azoxymethane
and Dietary Deoxycholate in Rats

Ryuhei KANAMOTO,1,・ Naoyuki AZUMA,1 Tomokazu MIYAMOTO,1 Tohru SAEKI,1
Yasunari TSUCHIHASHI,2 and Kimikazu IWAMI1

1Department of Biological Resource Chemistry, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan and 2Hospital Department of Pathology, Kyoto Prefectural University of Medicine, Kamigyo-ku,
Kyoto 602-8500, Japan

Received September 11, 2000; Accepted January 9, 2000
We found that azoxymethane and dietary deoxycholate induced liver tumors in rats. The incidence and the development of the tumor were closely related to the enterohepatic circulation of bile acids. The feeding of a high-molecular-weight fraction of soy protein digest (HMF) suppressed the tumorigenesis, probably due to the inhibitory effect of soybean resistant protein on reabsorption of bile acids in the intestine.
Key words: soybean resistant proteins; bile acid; enterohepatic circulation; colon tumorigenesis; liver tumorigenesis

-46-
Preliminary Communication
Small-Angle X-Ray Scattering Analysis of Stearic Acid Modified Lipase

Tatsuo MARUYAMA,1,2 Mitsutoshi NAKAJIMA,・,1 Sosaku ICHIKAWA,3 Yoh SANO,1
Hiroshi NABETANI,1 Shintaro FURUSAKI,2 and Minoru SEKI2

1National Food Research Institute, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8642, Japan
2Department of Chemistry and Biotechnology, The University of Tokyo, Hongo 7-3-1, Bunkyoku,
Tokyo 113-8656, Japan
3Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan

Received December 8, 2000; Accepted January 19, 2001
Stearic acid modified lipase (from Rhizopus japonicus) exhibited remarkable interesterification activity in n-hexane, but crude native lipase did not. The structure of the fatty acid modified lipase had not been analyzed until now. We analyzed the modified lipase by small-angle X-ray scattering (SAXS) measurements in order to clarify the structure. SAXS measurements showed that the modified lipase consisted of a lipid lamellar structure and implied that the lipase was incorporated into the lamellar structure of stearic acid. The long spacings in the lamellar structures of the modified lipase and stearic acid were measured.
Key words: interesterification; modified lipase; small-angle X-ray scattering