Contents and Abstracts of Latest Issue of BBB

(Vol.63 No.3 1999)

Susceptibility to Hydrogen Peroxide and Catalase Activity of Root Nodule Bacteria
Takuji OHWADA, Yukika SHIRAKAWA, Masayuki KUSUMOTO,
Hiroshi MASUDA, and Tetsuya SATO

Purification and Some Chemical Properties of 30 kDa Ginkgo biloba
Glycoprotein, which Reacts with Antiserum Against
β1→2 Xylose-Containing N-Glycans＊
Yoshinobu KIMURA,1,・ Takuo HARADA,1 Sayuri MATSUO,1 and Masami YONEKURA2

Reduced Urination Rate while Drinking Beer with an Unpleasant Taste and Off-flavor
Yoko NAGAO,1 Hozue KODAMA,1 Tomomi YAMAGUCHI,1 Toshihiko YONEZAWA,2
Ayako TAGUCHI,3 Seiji FUJINO,3 Keiichi MORIMOTO,4 and Tohru FUSHIKI1,＊

Strain Dependency of the Immunopotentiating Activity
of Lactobacillus delbrueckii subsp. bulgaricus
Shinya NAGAFUCHI,＊ Takeshi TAKAHASHI,＊ Takaji YAJIMA,＊ Tamotsu KUWATA,＊
Kazuhiro HIRAYAMA,＊＊ and Kikuji ITOH＊＊

Effects of Ionic Compositions of the Medium on Monosodium Glutamate Binding
to Taste Epithelial Cells
Yukako HAYASHI, Takashi TSUNENARI,・ and Tomohiko MORI＊

Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction
of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase
Hiroyuki UKEDA,＊ Daisuke KAWANA, Susumu MAEDA, and Masayoshi SAWAMURA

9-Oxooctadeca-10,12-dienoic Acids as Acetyl-CoA Carboxylase Inhibitors
from Red Pepper (Capsicum annuum L.)
Jun WATANEBE, Jun KAWABATA,＊ and Takanori KASAI

Synthesis and Fungicidal Activities of 4,5-Dihydro-7H-pyrano[3,4-c]isoxazole Derivatives
Hyung Jin KIM,・ Ju Young JANG, Kun Hoe CHUNG,＊ and Jae Hyun LEE＊

Removal of Cadmium from Scallop Hepatopancreas by Microbial Processes
Toshiyuki OBARA,・ Takuji SAWAYA,＊ Katsutoshi HOKARI,＊＊ Yasuo UMEHARA,＊＊
Masakatsu MIZUKAMI, and Fusao TOMITA＊＊＊

Role of Ornithine in the N-Acetylglutamate Turnover in the Liver of Rats
Kazutoshi HAYASE1 and Akira YOSHIDA＊

Reducing-agents-mediated Solubilization and Activation
of Debranching Enzyme (Pullulanase) in Rice Flour
Wakako TAKEUCHI,1,・ Hironori MASUI,2 and Junji YAMAGUCHI3

Biosynthetic Relationship between Acutumine and Dechloroacutumine
in Menispermum dauricum Root Cultures
Hind A. A. BABIKER, Yukihiro SUGIMOTO,・ Tomoki SAISHO, Shinobu INANAGA,
Masayuki HASHIMOTO,＊ and Akira ISOGAI＊＊

A Rapid Method for Transformation of Carrot (Daucus carota L.)
by Using Direct Somatic Embryogenesis
Yoshihiko TOKUJI・ and Hiroo FUKUDA

New Bitter Diterpenes, Rabdosianone I and II, Isolated from Isodon japonicus Hara
Yasumasa YAMADA,＊,・ Noritaka SAKO,＊＊ Eriko ANDO,＊ Manami YAMADA,＊
Hiroe KIKUZAKI,＊＊＊ and Takashi YAMAMOTO＊＊

Involvement in Denitrification of the napKEFDABC Genes Encoding
the Periplasmic Nitrate Reductase System in the Denitrifying Phototrophic
Bacterium Rhodobacter sphaeroides f. sp. denitrificans
Hui-Ping LIU, Susumu TAKIO,・ Toshio SATOH, and Isamu YAMAMOTO・・

Antimutagenicity of Sweetpotato (Ipomoea batatas) Roots
Makoto YOSHIMOTO, Shigenori OKUNO, Masaru YOSHINAGA, Osamu YAMAKAWA,
Masaatu YAMAGUCHI,＊ and Jiro YAMADA＊＊

Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid
by Penicillium citrinum
Yan-Jun JIA,・ Yukiko KAKUTA, Makihiro SUGAWARA, Toshinori IGARASHI,
Nobuko OKI, Miho KISAKI, Toshihiko SHOJI, Yoshinori KANETUNA,
Tomomi HORITA, Hirokazu MATSUI, Mamoru HONMA

Non-proteolytic Activation of Human Prorenin by Anti-prorenin Prosegment
(pf＃1: 1P--15P) Antiserum
Fumiaki SUZUKI,＊,・ Yasuhiko HATANO,＊＊ Tsutomu NAKAGAWA,＊＊＊ Kazuhiro TERAZAWA,＊＊＊
Akiko GOTOH,＊＊＊ Uddin Mohammad NASIR,＊＊＊＊ Yuichi ISHIDA,＊＊ and Yukio NAKAMURA＊＊＊

Isolation of Peptides from an Enzymatic Hydrolysate of Food Proteins
and Characterization of Their Taste Properties
Kenji MAEHASHI, Masato MATSUZAKI, Yasushi YAMAMOTO, and Shigezo UDAKA・

Note
Breeding of Yeast Strains Able to Grow at 42°C
Daizo KAWAMURA

Note
Cloning of Oxetanocin A Biosynthetic and Resistance Genes that Reside
on a Plasmid of Bacillus megaterium Strain NK84-0128
Makoto MORITA,1 Katsuhisa TOMITA,1 Masaru ISHIZAWA,1 Kenkichi TAKAGI,1
Fujio KAWAMURA,2,＃ Hideo TAKAHASHI,2 and Tomio MORINO1,・

Note
Decreased IgE-binding with Wheat Gluten by Deamidation
Nobuyuki MARUYAMA, Futoshi SUGIURA,1 Toru KISHIMOTO, Keiko ICHISE,
Yoshihiro TAKEUCHI,2 Tadashi SAWADA,2 Atsushi TSUDA1 and Shigeru UTSUMI・

Note
Suppression of D-Galactosamine-Induced Rat Liver Injury by Glycosidic
Flavonoids-Rich Fraction from Green Tea
Shingo WADA, Puming HE, Naoharu WATANABE, Kanzo SAKATA, and Kimio SUGIYAMA・

Note
Purification and Properties of NAD＋-dependent Sorbitol Dehydrogenase
from Bacillus fructosus
Takayuki UWAJIMA＊

Note
Effect of Dietary Antioxidants on Serum Lipid Contents and Immunoglobulin
Productivity of Lymphocytes in Sprague-Dawley Rats
Shihoko KAKU, Shin-ichi YUNOKI, Mitsuo MORI, Ken-ichi OHKURA,
Michiko NONAKA, Michihiro SUGANO,＊ and Koji YAMADA・

Note
Improvement of Peroxyl Radical Scavenging Bioassay Based
on Bactericidal Action of tert-Butyl Peroxyl Radical
Toshiyuki KAWASUMI,・,＊ Masami SATO,＊＊ Yoshihiro TSURIYA,＊＊ and Shigenori UENO＊＊

Note
Antioxidant Activity of a Medicine Based on Aspergillus oryzae NK Koji
Measured by a Modified t-Butyl Peroxyl Radical Scavenging Assay
Toshiyuki KAWASUMI,＊,・ Masami SATO,＊＊ Yoshihiro TSURIYA,＊＊ and Shigenori UENO＊＊

Note
Apoptosis-inducing Activity of Polyphenol Compounds Derived
from Tea Catechins in Human Histiolytic Lymphoma U937 Cells
Kouichi SAEKI, Mitsuaki SANO,＊ Toshio MIYASE,＊ Yoshiyuki NAKAMURA,＊
Yukihiko HARA,＊＊ Yutaka AOYAGI,＊＊＊ and Mamoru ISEMURA・

Note
An Organosulfur Compound Isolated from Oil-Macerated Garlic Extract,
and Its Antimicrobial Effect
Hisae YOSHIDA, Hirotaka KATSUZAKI,＊ Rie OHTA, Keiko ISHIKAWA,
Hiroyuki FUKUDA, Tsuchiyoshi FUJINO,・ and Atsushi SUZUKI

Note
Antimicrobial Activity of the Thiosulfinates Isolated
from Oil-Macerated Garlic Extract
Hisae YOSHIDA, Hirotaka KATSUZAKI,＊ Rie OHTA, Keiko ISHIKAWA,
Hiroyuki FUKUDA, Tsuchiyoshi FUJINO,・ and Atsushi SUZUKI

Note
Effects of Castor Oil on Lipid Metabolism in Rats
Miyuki IHARA-WATANABE,・ Takashi SHIROYAMA, Gen KONDA, Hayato UMEKAWA,
Takao TAKAHASHI, Yoshihiro YAMADA,＊ and Yukio FURUICHI

Note
Asymmetric Reduction of Ethyl 2-methyl 3-oxobutanoate by Chlorella
Takayuki KURAMOTO,1 Kenzo IWAMOTO,1 Minoru IZUMI,2 Mitsunori KIRIHATA,3
and Fumiki YOSHIZAKO,1,・

Note
Effective Cloning of Unmarked DNA Fragments in the Bacillus subtilis 168 Genome
Mitsuhiro ITAYA・

Note
Fucoxanthin as the Major Antioxidant in Hijikia fusiformis,
a Common Edible Seaweed
Xiaojun YAN, Yoshihiro CHUDA, Masahiro SUZUKI, and Tadahiro NAGATA・

Note
Brachyury Regulatory Region Active in Embryonal Carcinoma P19 Cells
Hirotake YAMAGUCHI,1 Yasuo KITAGAWA,1,2 and Kiyoshi MIKI1,2,・

Note
Autodegradation of Protein Disulfide Isomerase
Reiko URADE,・ Ayako YASUNISHI, Hirokazu OKUDO, Tatsuya MORIYAMA,
and Makoto KITO


-1-
Susceptibility to Hydrogen Peroxide and Catalase Activity
of Root Nodule Bacteria

Takuji OHWADA, Yukika SHIRAKAWA, Masayuki KUSUMOTO,
Hiroshi MASUDA, and Tetsuya SATO

Applied Molecular Biology, Department of Bioresource Science, Obihiro University
of Agriculture and Veterinary Medicine, Inada-cho, Obihiro City, Hokkaido 080-8555 Japan

Received July 15, 1998; Accepted November 27, 1998
The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested.
root nodule bacteria; hydrogen peroxide (H2O2); catalase

-2-
Purification and Some Chemical Properties of 30 kDa Ginkgo biloba
Glycoprotein, which Reacts with Antiserum Against
β1→2 Xylose-Containing N-Glycans＊

Yoshinobu KIMURA,1,・ Takuo HARADA,1 Sayuri MATSUO,1 and Masami YONEKURA2

1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
2Department of Applied Biological Resource Science, School of Agriculture, Ibaraki University,
Ami-machi, Ibaraki 300-0300, Japan

Received July 29, 1998; Accepted November 4, 1998
From the seeds of Ginkgo biloba, a glycoprotein, which is a major component that reacts with an antiserum against β1→2 xylose-containing N-glycans, has been purified and characterized. The N-terminal amino acid sequence of the purified glycoprotein was H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-L-L-F-G-Q-. The molecular mass was estimated to be 17 kDa and 16 kDa by SDS-PAGE under reducing conditions, however, the molecular mass of this glycoprotein in the native state was 30,762 by MALDI-TOF MS, suggesting that this glycoprotein consists of two subunits; one is glycosylated and the other is not. The structure of N-glycan linked to this glycoprotein (designated 30 kDa GBGP) was identified as Man3Fuc1Xyl1GlcNAc2, which is the predominant N-glycan linked to the storage glycoproteins in the same seeds (Kimura, Y et al. (1998) Biosci. Biotechnol. Biochem. 62, 253--261). From the peptic digest of the carboxymethylated glycosylated subunit, one glycopeptide was purified by RP-HPLC and the amino acid sequence was identified as H-K-A-N-N(Man3Fuc1Xyl1GlcNAc2)-V-T-V-A-F, which corresponded to the N-terminal amino acid sequence.
antigenic N-glycan; antiserum against plant N-glycan; plant glycoprotein; immunoblotting; Ginkgo biloba

-3-
Reduced Urination Rate while Drinking Beer with an Unpleasant Taste
and Off-flavor

Yoko NAGAO,1 Hozue KODAMA,1 Tomomi YAMAGUCHI,1 Toshihiko YONEZAWA,2
Ayako TAGUCHI,3 Seiji FUJINO,3 Keiichi MORIMOTO,4 and Tohru FUSHIKI1,＊

1Laboratory of Nutrition Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan
2Kirin-Seagram Ltd., 970 Shibanuta, Gotemba 412-0003, Japan
3Research Laboratory for New Products, Kirin Brewery Co. Ltd., 6-26-1 Jingumae, Shibuya-ku,
Tokyo 150-8011, Japan
4Research and Development Division, Kirin Brewery Co. Ltd., 6-26-1 Jingumae, Shibuya-ku,
Tokyo 150-8011, Japan

Received August 10, 1998; Accepted October 29, 1998
A lowered subjective evaluation of the taste and flavor of beer due to staleness or to the addition of an unpleasant taste and flavor was found to be closely correlated with the urination rate. Beer in the same lot was compared immediately after shipment from the brewery and after leaving at room temperature for 1 month or 5 months. Each beer sample was given to volunteers at the rate of 3 ml/kg/15 min for 2 hours, and the urine volume was measured every 30 minutes. The urination rate was highest from the volunteers who drank fresh beer and lowest from those who drank 5-month-old beer. The subjective evaluation of both the taste and drinkability of 5-month-old beer was significantly lower than that of fresh beer. Beer samples with various unpleasant taste and flavor substances added lowered the urination rate. The results suggest that the perception of an unpleasant taste and off-flavor would lower the urination rate.
beer; taste; off-flavor; urination rate; drinkability

-4-
Strain Dependency of the Immunopotentiating Activity
of Lactobacillus delbrueckii subsp. bulgaricus

Shinya NAGAFUCHI,＊ Takeshi TAKAHASHI,＊ Takaji YAJIMA,＊ Tamotsu KUWATA,＊
Kazuhiro HIRAYAMA,＊＊ and Kikuji ITOH＊＊

＊Nutrition Science Institute, Meiji Milk Products Co., Ltd., 540, Naruda, Odawara, Kanagawa 250-0862, Japan
＊＊Department of Veterinary Public Health, Faculty of Agriculture, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan

Received August 11, 1998; Accepted November 4, 1998
To obtain strains of Lactobacillus delbrueckii subsp. bulgaricus with high immunopotentiating activity, we screened 90 strains of this bacterial species for the proliferative response of murine spleen and β-lactoglobulin-primed lymph node cells. In this screening, certain strains showed strong immunopotentiating activity. Among them, strain 1023 had the strongest mitogenic activity for murine Peyer′s patch (PP) cells. Furthermore, strain 1023 induced IgA antibody production by PP cells as strongly as Bifidobacterium longum 6001, which had adjuvant activity when orally administered. Also in the assays using immune cells from human-flora-associated mice a few strains including 1023 showed strong immunopotentiating activity comparable to B. longum 6001. These results suggest that L. delbrueckii subsp. bulgaricus strains such as 1023 may be useful for the production of fermented milk with a more beneficial effect on the systemic and mucosal immune system.
Lactobacillus delbrueckii subsp. bulgaricus; immunopotentiating activity; human-flora-associated mouse

-5-
Effects of Ionic Compositions of the Medium on Monosodium Glutamate Binding
to Taste Epithelial Cells

Yukako HAYASHI, Takashi TSUNENARI,・ and Tomohiko MORI＊

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan

Received August 12, 1998; Accepted November 20, 1998
Monosodium glutamate and nucleotides are umami taste substances in animals and have a synergistic effect on each other. We studied the ligand-binding properties of the glutamate receptors in taste epithelial cells isolated from bovine tongue. Specific glutamate binding was observed in an enriched suspension of taste receptor cells in Hanks′ balanced salt solution, while no specific glutamate binding was apparent in the absence of divalent ions or when the cells had been depolarized by a high content of potassium in Hanks′ balanced salt solution. There was no significant difference between the release of glutamate under depolarized or divalent ion-free conditions and under normal conditions. However, glutamate was easily released from the depolarized cells in the absence of divalent ions. These data suggest that the binding of glutamate to receptors depends on divalent ions, which also have an effect on maintaining binding between glutamate and receptors.
umami taste; glutamate; bovine; binding assay; epithelium

-6-
Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction
of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase

Hiroyuki UKEDA,＊ Daisuke KAWANA, Susumu MAEDA, and Masayoshi SAWAMURA

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

Received September 7, 1998; Accepted November 17, 1998
Two novel highly water-soluble tetrazolium salts, WST-1||(4 - [3 - (4 - iodophenyl) - 2 - (4 - nitrophenyl) - 2H - 5 - tetrazolio]-||||1,3-benzene disulfonate sodium salt) and WST-8 (4-[3-(2-||||methoxy - 4 - nitrophenyl) - 2 - (4 - nitrophenyl) - 2H - 5 - tetrazolio]-||1,3-benzene disulfonate sodium salt) were applied to the assay of superoxide dismutase (SOD). The superoxide anion generated by xanthine/xanthine oxidase (XO) reduced WST-1 and WST-8 to water-soluble formazans which exhibited absorbance maxima at 438 and 460 nm, respectively. The rates of reduction were linearly related to the XO activity, and reduction was inhibited by SOD. Complete inhibition by SOD of the reduction of both WST-1 and WST-8 was achieved, suggesting that these WSTs were not reduced with XO. WST-1 was found more useful than WST-8 because it had shown higher sensitivity which was apparently not dependent on the assay pH value in the range pH 8.0--10.2. These properties of WST-1 are ideal for the spectrophotometric assay of SOD in an aqueous system.
superoxide dismutase; assay; water-soluble tetrazolium; xanthine oxidase; WST

-7-
9-Oxooctadeca-10,12-dienoic Acids as Acetyl-CoA Carboxylase Inhibitors
from Red Pepper (Capsicum annuum L.)

Jun WATANEBE, Jun KAWABATA,＊ and Takanori KASAI

Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University,
Kita-ku, Sapporo 060-8589, Japan

Received September 8, 1998; Accepted November 4, 1998
A methanol extract of red pepper showed potent acetyl-CoA carboxylase inhibitory activity. The active principles were isolated and identified as (E, E)- and (E, Z)-9-oxooctadeca-10,12-dienoic acids by instrumental analyses. The IC50 values of the compounds were 1.4×10−6 and 1.5×10−6 M, respectively, their activity being nearly sixty-times higher than that of the common fatty acids themselves. A comparative study of the structure-activity relationship among their related compounds showed that the inhibitory activity was influenced neither by the position and species of the oxygen functional group in the middle of the alkyl chain nor by the configurations of the double bonds. However, it was found that the presence of double bonds between the terminal carboxyl and the mid-chain oxygen functional group lowered the inhibitory activity which could be recovered by hydrogenation of the double bonds.
acetyl-CoA carboxylase inhibitor; red pepper; oxooctadecadienoic acid

-8-
Synthesis and Fungicidal Activities of 4,5-Dihydro-7H-pyrano[3,4-c]isoxazole Derivatives

Hyung Jin KIM,・ Ju Young JANG, Kun Hoe CHUNG,＊ and Jae Hyun LEE＊

Department of Chemical Technology, Chonnam National University, Kwangju 500-757, Korea
＊Bio-Organic Division, Korea Research Institute of Chemical Technology, Daejeon 305-606, Korea

Received September 10, 1998; Accepted October 30, 1998
4,5-Dihydro-7H-pyrano[3,4-c]isoxazoles (II and III) with an o-chlorophenyl or p-chlorophenyl group at C-7 were synthesized and the effect of substitution at C-3 of II and III on fungicidal activity was investigated in vivo. When the substituent at C-3 of II and III was CH2Br, CH＝NOMe, CH＝NOEt or CH＝NO-allyl, the fungicidal effect was significant and selectively high on wheat leaf rust and barley powdery mildew at 250 ppm. Compound IId with the CH2Br substituent at C-7 showed high fungicidal activity against rice blast, providing more than 90％ control of the disease at 2 ppm.
pyrano[3,4-c]isoxazole; fungicidal activity; plant pathogen; rice blast; wheat leaf rust

-9-
Removal of Cadmium from Scallop Hepatopancreas by Microbial Processes

Toshiyuki OBARA,・ Takuji SAWAYA,＊ Katsutoshi HOKARI,＊＊ Yasuo UMEHARA,＊＊
Masakatsu MIZUKAMI, and Fusao TOMITA＊＊＊

Hakodate National College of Technology, Tokura-cho 14-1, Hakodate 042-0953, Hokkaido, Japan
＊Hokkaido Industrial Technology Center, Kikyo-cho 379, Hakodate 041-0801, Hokkaido, Japan
＊＊Nippon Chemical Feed Co. Ltd, Asano-cho 3-6, Hakodate 040-0076, Hokkaido, Japan
＊＊＊Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University,
Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Hokkaido, Japan

Received September 18, 1998; Accepted November 4, 1998
A microbial process for removing cadmium from a homogenate of hepatopancreas, a waste of scallop processing, was devised to use this waste for value-added protein resources. Microorganisms were screened on the basis of the ability to remove cadmium from a medium with the initial concentration of 10 mg/l of cadmium. One soil isolate, identified as Xanthomonas sp. UR No. 2 by its taxonomical characteristics, removed 98％ of the cadmium in the medium in 2 d. During cultivation of this strain in the homogenates of hepatopancreas digested by endopeptidases, 90％ of cadmium was removed, while this strain had little effect on the simple non-digested homogenates. The mass balance of cadmium during homogenizations of the hepatopancreas tissues and cultivations in the protease-treated homogenate were examined. The content of crude proteins of culture supernatant treated by Xanthomonas sp. UR No. 2 was equivalent to those of various feedstuffs on the market.
microbial removal of cadmium; scallop hepatopancreas; Xanthomonas sp.; waste tissue; protease

-10-
Role of Ornithine in the N-Acetylglutamate Turnover in the Liver of Rats

Kazutoshi HAYASE1 and Akira YOSHIDA＊

Department of Home Economics, Aichi University of Education, Kariya, Aichi 448-8542, Japan
＊Department of Food and Nutritional Sciences, Nagoya Bunri Junior College, Nagoya 451-0077, Japan

Received September 18, 1998; Accepted November 4, 1998
We determined whether the synthesis and degradation of N-acetylglutamate would regulate urea synthesis when the ornithine status was manipulated. Experiments were done on two groups of rats, each being treated with ornithine or saline (control). The plasma concentration of urea and the liver concentration of N-acetylglutamate in rats given ornithine were each significantly higher than in the control rats. Compared with the control rats, the liver N-acetylglutamate degradation was significantly lower in those rats treated with ornithine. Treatment of the rats with ornithine did not affect N-acetylglutamate synthesis in the liver. An inverse correlation between the liver N-acetylglutamate degradation and liver concentration of N-acetylglutamate was found. These results suggest that the lower degradation of N-acetylglutamate in the ornithine treatment group would be likely to increase the hepatic concentration of this compound and stimulate urea synthesis.
ornithine; urea synthesis; N-acetylglutamate synthesis; N-acetylglutamate degradation

-11-
Reducing-agents-mediated Solubilization and Activation
of Debranching Enzyme (Pullulanase) in Rice Flour

Wakako TAKEUCHI,1,・ Hironori MASUI,2 and Junji YAMAGUCHI3

1Faculty of Domestic Science, Nagoya Women′s University, Mizuho-ku, Nagoya 467-8610, Japan
2Mukogawa Women′s University, Junior College Division, Department of Dietary Life Science,
Nishinomiya 663-8558, Japan
3Bioscience Center and Graduate School of Bioagricultural Sciences, Chikusa, Nagoya 464-8601, Japan

Received September 21, 1998; Accepted November 27, 1998
The effects of reducing agents on solubilization and activation of the debranching enzyme (pullulanase) were examined using rice flour. The activity of the debranching enzyme was observed in a buffer solution (pH 7.5) in which||rice flour was incubated together with thiol-reducing rea||||gents, (dithiothreitol, 2-mercaptoethanol etc.), but there||was only low activity in the absence of reducing agents. Immunochemical measurement and the specific activity of the enzyme showed that the activation caused by the reductant was due to solubilization of the enzyme protein besides the enzyme activation.
pullulanase; starch degradation; dithiothreitol; endosperm

-12-
Biosynthetic Relationship between Acutumine and Dechloroacutumine
in Menispermum dauricum Root Cultures

Hind A. A. BABIKER, Yukihiro SUGIMOTO,・ Tomoki SAISHO, Shinobu INANAGA,
Masayuki HASHIMOTO,＊ and Akira ISOGAI＊＊

Arid Land Research Center, Tottori University, 1390 Hamasaka, Tottori 680-0001, Japan
＊Nitto Technical Information Center Co., Ltd., 1-1-2 Shimohozumi, Ibaraki, Osaka 567-0041, Japan
＊＊Graduate School of Biological Science, Nara Institute of Science and Technology,
8916-5 Takayama-cho, Ikoma 630-0101, Japan

Received September 24, 1998; Accepted November 7, 1998
The biosynthetic relationship between acutumine 1 and dechloroacutumine 2 was studied using 13C-labeled tyrosine and 3H-labeled 2 as tracers. 13C-NMR spectra of 13C-labeled 1 and 2 showed that the alkaloids, each composed of two molecules of tyrosine, are derived from the same biosynthetic pathway. Feeding Menispermum dauricum (Menispermaceae) roots, cultured in a chloride-enriched medium, with 3H-labeled 2 demonstrated that 1 is the only alkaloid metabolite of 2. Conversion (5％) of the exogenously applied 2, taken up by the roots, into 1 showed that 2 is the precursor of 1. Incomplete conversion of 2 into 1 suggests accumulation of the exogenously applied 2 in cell organelles and/or compartmentation of the enzymes involved in the biosynthesis of 1.
Menispermum dauricum; root cultures; biosynthesis; acutumine; dechloroacutumine

-13-
A Rapid Method for Transformation of Carrot (Daucus carota L.)
by Using Direct Somatic Embryogenesis

Yoshihiko TOKUJI・ and Hiroo FUKUDA

Department of Biological Sciences, Graduate School of Science, University of Tokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

Received September 28, 1998; Accepted November 18, 1998
Several genes that may be involved in embryogenesis have been isolated from somatic embryos of carrot by many workers. However, the function of these genes has not been discovered yet. As the first step toward finding the function of these genes, we established a rapid and efficient method for transformation of carrot by using direct embryogenesis from hypocotyl segments treated with 2,4-dichlorophenoxyacetic acid (2,4-D) for a short period.
transgenic plant; somatic embryo; carrot; Agrobacterium tumefaciens

-14-
New Bitter Diterpenes, Rabdosianone I and II, Isolated
from Isodon japonicus Hara

Yasumasa YAMADA,＊,・ Noritaka SAKO,＊＊ Eriko ANDO,＊ Manami YAMADA,＊
Hiroe KIKUZAKI,＊＊＊ and Takashi YAMAMOTO＊＊

＊Department of Food Science and Nutrition, Doshisha Women′s College,
Teramachi-Imadegawa, Kamigyo-ku, Kyoto 602-0893, Japan
＊＊Department of Behavioral Physiology, Faculty of Human Sciences, Osaka University,
Suita, Osaka 565-0871, Japan
＊＊＊Department of Food and Nutrition, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan

Received September 28, 1998; Accepted November 20, 1998
The new bitter diterpenes, rabdosianone I (C20H24O5) and II (C22H28O6), were isolated from Isodon japonicus (Japanese name, enmeiso), and their structures were elucidated by spectroscopic methods. Electrophysiological experiments were performed to compare rabdosianone I with quinine. The taste responses of chorda tympani nerves to rabdosianone I were smaller than those to quinine in Wistar rats.
bitterness; rabdosianone I; rabdosianone II; Isodon japonicus; electrophysiology

-15-
Involvement in Denitrification of the napKEFDABC Genes Encoding
the Periplasmic Nitrate Reductase System in the Denitrifying Phototrophic
Bacterium Rhodobacter sphaeroides f. sp. denitrificans

Hui-Ping LIU, Susumu TAKIO,・ Toshio SATOH, and Isamu YAMAMOTO・・

Department of Biological Science, Faculty of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan

Received October 1, 1998; Accepted November 12, 1998
Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by Ω-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.
periplasmic nitrate reductase; napKEFDABC; denitrification; phototrophic bacterium; Rhodobacter sphaeroides f. sp. denitrificans

-16-
Antimutagenicity of Sweetpotato (Ipomoea batatas) Roots

Makoto YOSHIMOTO, Shigenori OKUNO, Masaru YOSHINAGA, Osamu YAMAKAWA,
Masaatu YAMAGUCHI,＊ and Jiro YAMADA＊＊

Department of Upland Farming, Kyushu National Agricultural Experiment Station, Miyakonojo,
Miyazaki 885-0091, Japan
＊College of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0001, Japan
＊＊Laboratory of Food Science, Faculty of Education, Yamaguchi University, 1677-1 Yoshida,
Yamaguchi 753-0841, Japan

Received October 1, 1998; Accepted November 4, 1998
Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweetpotato, 3-(6,6′-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.
antimutagenicity; sweetpotato storage root; anthocyanin; phenolics

-17-
Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid
by Penicillium citrinum

Yan-Jun JIA,・ Yukiko KAKUTA, Makihiro SUGAWARA, Toshinori IGARASHI,
Nobuko OKI, Miho KISAKI, Toshihiko SHOJI, Yoshinori KANETUNA,
Tomomi HORITA, Hirokazu MATSUI, Mamoru HONMA

Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received October 2, 1998; Accepted November 16, 1998
1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05％ L-methionine and ACC deaminase was done from cells incubated in a medium containing 1％ 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of Mr 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s−1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of Mr 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s−1. The presence of 7 mM Cu2＋ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.
1-aminocyclopropane-1-carboxylic acid (ACC); ACC synthase; ACC deaminase; Penicillium citrinum

-18-
Non-proteolytic Activation of Human Prorenin by Anti-prorenin Prosegment
(pf＃1: 1P--15P) Antiserum

Fumiaki SUZUKI,＊,・ Yasuhiko HATANO,＊＊ Tsutomu NAKAGAWA,＊＊＊ Kazuhiro TERAZAWA,＊＊＊
Akiko GOTOH,＊＊＊ Uddin Mohammad NASIR,＊＊＊＊ Yuichi ISHIDA,＊＊ and Yukio NAKAMURA＊＊＊

＊Molecular Genetics Research Center, ＊＊＊Department of Biotechnology, and ＊＊＊＊United Graduate School
of Agricultural Science, Gifu University, Gifu 501-1193, Japan
＊＊Tokiwa Chemical Co., 4-16-22 Kami-ikebukuro Toshima-ku, Tokyo 170-0012, Japan

Received October 14, 1998; Accepted November 10, 1998
Recombinant human prorenin was activated by incubation with anti-prorenin prosegment (L1PPTDTTTFKRIFLKR15P) antiserum at 4°C. This activation was dependent on the concentration of the antiserum and incubation time. After the activation no molecular weight alteration of prorenin was observed by immunoblotting analysis. A peptide of L1PPTDTTTF8P as well as L1PPTDTTTFKRIFLKR15P potently interfered with the activation. Most of the activated prorenin bound to Protein A Sepharose CL 4B. The Km and Vmax values of the activated prorenin were 0.2 μM and 23.7 μg Ang I/ml/h, respectively, which were similar in level to those of mature renin obtained by trypsinization.
human prorenin; immuno-activation; non-proteolytic activation; proenzyme activation

-19-
Isolation of Peptides from an Enzymatic Hydrolysate of Food Proteins
and Characterization of Their Taste Properties

Kenji MAEHASHI, Masato MATSUZAKI, Yasushi YAMAMOTO, and Shigezo UDAKA・

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

Received October 29, 1998; Accepted November 24, 1998
Soybean protein, casein, bonito protein and chicken protein, each as foodstuff protein, were hydrolyzed with four proteinases; namely, pepsin, trypsin, α-chymotrypsin and bromelain. Since the chicken protein hydrolysate with bromelain possessed the most favorable umami taste, eleven peptides were isolated from the chicken protein hydrolysate by successive chromatography on ODS, Amberlite IR-120B, Amberlite IRA-410 and AG-50W; their structures were Asp-Ala, Asp-Val, Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Asp-Glu-Glu, Asp-Glu-Ser, Glu-Glu-Asn, Ser-Pro-Glu, and Glu-Pro-Ala-Asp. Many of them did not show any umami taste by themselves, but Glu-Glu, Glu-Val, Ala-Asp-Glu, Ala-Glu-Asp, Asp-Glu-Glu, and Ser-Pro-Glu were recognized to enhance the umami taste of 0.02％ 5′-inosine monophosphate (IMP). A combination of these peptides, especially 0.5％ each of Glu-Glu, Glu-Val, Asp-Glu-Glu and Glu-Glu-Asn, with 0.02％ IMP produced a delicious ``full′′ umami taste.
||taste peptide; protein hydrolysate; bromelain; ||umami

-20-
Note
Breeding of Yeast Strains Able to Grow at 42°C

Daizo KAWAMURA

Hiroshima Prefectural Food Technological Research Center, 12-70 Hijiyama-honmachi,
Minami-ku, Hiroshima 732-0816, Japan

Received June 10, 1998; Accepted November 4, 1998
Saccharomyces cerevisiae strain �A-39/10A is able to ferment alcohol at 42°C. The ability of various yeast strains, including �A-39/10A, to grow at high temperatures was compared. The strain �A-39/10A was able to grow at 42°C and the high temperature growth was found to be governed by more than one gene. The yeast strains that can grow at 42°C were bred by crossing the haploid strains, which are inherently unable to grow at high temperatures.
Saccharomyces cerevisiae; high temperature; breeding

-21-
Note
Cloning of Oxetanocin A Biosynthetic and Resistance Genes that Reside
on a Plasmid of Bacillus megaterium Strain NK84-0128

Makoto MORITA,1 Katsuhisa TOMITA,1 Masaru ISHIZAWA,1 Kenkichi TAKAGI,1
Fujio KAWAMURA,2,＃ Hideo TAKAHASHI,2 and Tomio MORINO1,・

1Research and Development Division, Pharmaceuticals Group, Nippon Kayaku Co. Ltd.,
3-31-12, Shimo, Kita-ku, Tokyo 115, Japan
2Institute of Molecular and Cellular Biosciences, The University of Tokyo,
1-1-1, Yayoi, Bunkyo-ku, Tokyo 113, Japan

Received July 7, 1998; Accepted November 20, 1998
Bacillus megaterium strain NK84-0218 produces a potent antiviral antibiotic, oxetanocin A, which has an oxetanosyl-N-glycoside linkage to an adenine moiety. However, the oxetanocin A productivity of the original strain was unstable and low. In this study, oxetanocin A productivity and resistance was shown to be lost simultaneously when a 51.5-kb plasmid, pOXT1, was cured during cultivation. The deficiency of oxetanocin A productivity and resistance was restored by re-introduction of the pOXT1 plasmid into the cured strain. By a cloning experiment it was shown that a 6.8-kb BglII-D fragment of the pOXT1 plasmid was responsible for oxetanocin A productivity and resistance.
antibiotic production; antibiotic resistance; Bacillus megaterium; oxetanocin A; plasmid transformation

-22-
Note
Decreased IgE-binding with Wheat Gluten by Deamidation

Nobuyuki MARUYAMA, Futoshi SUGIURA,1 Toru KISHIMOTO, Keiko ICHISE,
Yoshihiro TAKEUCHI,2 Tadashi SAWADA,2 Atsushi TSUDA1 and Shigeru UTSUMI・

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
1Glico Food Co. Ltd, Takatsuki, Osaka 569-0053, Japan
2Department of Pediatrics, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan

Received July 13, 1998; Accepted October 28, 1998
The major wheat allergens contain a number of glutamine residues, suggesting that deamidation would be a promising method to produce hypoallergenic wheat proteins. Gluten was deamidated by acid under various conditions. The 30％-, 50％- and 90％-deamidated glutens were reacted with sera of patients allergic to wheat proteins. The results indicate that the reactivity was dramatically decreased according to the degree of deamidation.
deamidation; gluten; hypoallergenic wheat proteins

-23-
Note
Suppression of D-Galactosamine-Induced Rat Liver Injury by Glycosidic
Flavonoids-Rich Fraction from Green Tea

Shingo WADA, Puming HE, Naoharu WATANABE, Kanzo SAKATA, and Kimio SUGIYAMA・

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Shizuoka 422-8529, Japan

Received August 12, 1998; Accepted November 14, 1998
Tea constituents that had a preventive effect on D-galactosamine-induced liver injury in rats were partially purified by column chromatography from a n-butanol-soluble fraction of green tea. The fraction containing glycosidic flavonoids was found to suppress the D-galactosamine-induced increase of plasma alanine aminotransferase and aspartate aminotransferase activities. These results indicate that glycosidic flavonoids contribute, at least in part, to the liver injury-preventive effect of green tea.
green tea; flavonoids; glycosides; liver injury; D-galactosamine

-24-
Note
Purification and Properties of NAD＋-dependent Sorbitol Dehydrogenase
from Bacillus fructosus

Takayuki UWAJIMA＊

Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-0023, Japan

Received September 3, 1998; Accepted November 2, 1998
Sorbitol dehydrogenase (EC 1.1.1.14), which catalyzes the NAD＋-linked interconversion of D-sorbitol and D-fructose, was purified and crystallized from cell-free extracts of Bacillus fructosus grown on D-sorbitol as a sole carbon source. The crystalline enzyme was homogeneous on disc electrophoresis and ultracentrifugation. The molecular weight was 102,000 by the sedimentation equilibrium method. The enzyme acted specifically on D-sorbitol, and showed an optimum pH at 9.0. The Km values for D-sorbitol and NAD＋ were 1.1×10−2 M and 2.2×10−4 M, respectively. The enzyme activity was inhibited by p-chloromercuribenzoate, Ag＋, Hg2＋, and Cu2＋.
sorbitol dehydrogenase; NAD＋; Bacillus fructosus; D-sorbitol; D-fructose

-25-
Note
Effect of Dietary Antioxidants on Serum Lipid Contents and Immunoglobulin
Productivity of Lymphocytes in Sprague-Dawley Rats

Shihoko KAKU, Shin-ichi YUNOKI, Mitsuo MORI, Ken-ichi OHKURA,
Michiko NONAKA, Michihiro SUGANO,＊ and Koji YAMADA・

Laboratory of Food Science, Department of Food Science and Technology, Faculty of Agriculture,
Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
＊Department of Food Sciences and Nutrition, Prefectural University of Kumamoto,
3-1-100 Tsukide, Kumamoto 862-0092, Japan

Received September 9, 1998; Accepted November 6, 1998
Sprague-Dawley rats were fed α-tocopherol, tocotrienol, or quercetin to examine their dietary effects on serum lipid contents and immunoglobulin productivity. In tocotrienol or quercetin groups, serum triglyceride was lower than in the none group. Moreover, in the α-tocopherol group, serum IgA level and IgA productivity of MLN lymphocytes were high, while in the tocotrienol group, IgM productivity of spleen lymphocytes and IgA, IgG, and IgM productivity of MLN lymphocytes were high. Thus, we suggested each antioxidant had different effects in rats.
antioxidants; quercetin; tocotrienol; triglyceride; immunoglobulin

-26-
Note
Improvement of Peroxyl Radical Scavenging Bioassay Based
on Bactericidal Action of tert-Butyl Peroxyl Radical

Toshiyuki KAWASUMI,・,＊ Masami SATO,＊＊ Yoshihiro TSURIYA,＊＊ and Shigenori UENO＊＊

＊Department of Food and Nutrition, Faculty of Home Economics, Japan Women′s University,
2-8-1 Mejirodai, Bunkyo, Tokyo 112-8681, Japan
＊＊OTC Drug R＆D Department, Wakamoto Pharmaceutical Co., Ltd., 1-6-1 Ohtemachi, Chiyoda,
Tokyo 100-0004, Japan

Received September 10, 1998; Accepted November 6, 1998
An improved bioassay for measurement of peroxyl radical scavenging activity1,2) is described. The modifications included the use of (1) Enterococcus faecium strain S, (2) 25％ dimethyl sulfoxide, (3) L-ascorbic acid for the termination of the bactericidal reaction, and (4) the concentration of antioxidants that gives 75％ of the log (colony forming unit/ml) of the control reaction (without methemoglobin or tert-butyl hydroperoxide), for the expression of antioxidant activity.
bioassay; antioxidant; t-butyl peroxyl radical scavenging; synergism

-27-
Note
Antioxidant Activity of a Medicine Based on Aspergillus oryzae NK Koji
Measured by a Modified t-Butyl Peroxyl Radical Scavenging Assay

Toshiyuki KAWASUMI,＊,・ Masami SATO,＊＊ Yoshihiro TSURIYA,＊＊ and Shigenori UENO＊＊

＊Department of Food and Nutrition, Faculty of Home Economics, Japan Women′s University,
2-8-1 Mejirodai, Bunkyo, Tokyo 112-8681, Japan
＊＊OTC Drug R＆D Department, Wakamoto Pharmaceutical Co., Ltd., 1-6-1 Ohtemachi, Chiyoda,
Tokyo 100-0004, Japan

Received September 10, 1998; Accepted November 20, 1998
A koji-based medicine composed of powder of Aspergillus oryzae NK koji, dried yeast, and lactobacilli koji had high antioxidant activity measured by a modified t-butyl peroxyl radical scavenging assay. This activity was mainly derived from A. oryzae NK koji. Digestion of koji-making grain germ medium with several commercial enzymes also increased antioxidant activity. By two weeks of oral administration of A. oryzae NK koji, the serum lipid peroxide levels elevated in STZ-induced diabetic rats could be decreased significantly.
antioxidant; t-butyl peroxyl radical scavengers; medicine; Aspergillus oryzae; koji

-28-
Note
Apoptosis-inducing Activity of Polyphenol Compounds Derived
from Tea Catechins in Human Histiolytic Lymphoma U937 Cells

Kouichi SAEKI, Mitsuaki SANO,＊ Toshio MIYASE,＊ Yoshiyuki NAKAMURA,＊
Yukihiko HARA,＊＊ Yutaka AOYAGI,＊＊＊ and Mamoru ISEMURA・

School of Food and Nutritional Sciences, ＊School of Pharmaceutical Sciences, University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan
＊＊Food Research Laboratories, Mitsui Norin Co., Ltd., 2-1-17 Fujieda, Shizuoka 426-2001, Japan
＊＊＊Department of Third Internal Medicine, Niigata University School of Medicine, Niigata 951-8122, Japan

Received September 24, 1998; Accepted November 10, 1998
Polyphenolic compounds derived from tea catechins were examined for apoptosis-inducing activity in human histiolytic lymphoma U937 cells. (−)-Epigallocatechin gallate, theasinensin D, compound OH-5, theaflavin, and theaflavin digallate induced apoptosis as evidenced by DNA ladder formation, its inhibition by a caspase inhibitor, and chromatin condensation. Theasinensin D was the most potent inducer and the data suggest the importance of the number and three dimensional localization of their phenolic groups in this activity. These apoptosis-inducible compounds may be useful as a cancer chemopreventive and chemotherapeutic agent.
apoptosis; catechin; epigallocatechin gallate; theaflavin; theasinensin D

-29-
Note
An Organosulfur Compound Isolated from Oil-Macerated Garlic Extract,
and Its Antimicrobial Effect

Hisae YOSHIDA, Hirotaka KATSUZAKI,＊ Rie OHTA, Keiko ISHIKAWA,
Hiroyuki FUKUDA, Tsuchiyoshi FUJINO,・ and Atsushi SUZUKI

Biodevelopment Division, Central Institute, Nagoya Seiraku Co. Ltd., 310 Nakasuna-cho,
Tempaku-ku, Nagoya, Aichi 468-0065, Japan
＊Faculty of Bioresources, Mie University, 1515 Kamihama-cho, Tsu, Mie 514-8507, Japan

Received September 25, 1998; Accepted November 17, 1998
An organosulfur compound was isolated from oil-macerated garlic extract by silica gel column chromatography and preparative TLC. From the results of NMR, IR, and MS analyses, its structure was determined as E-4,5,9-trithiadeca-1,7-diene-9-oxide (iso-E-10-devinylajoene, iso-E-10-DA). This compound was different from E-4,5,9-trithiadeca-1,6-diene-9-oxide (E-10-devinylajoene, E-10-DA) only in the position of a double bond. Iso-E-10-DA had antimicrobial activity against Gram-positive bacteria, such as Bacillus cereus, B. subtilis, and Staphylococcus aureus, and yeasts at the concentration lower than 100 μg/ml, but Gram-negative bacteria were not inhibited at the same concentration. The antimicrobial activity of iso-E-10-DA was inferior to those of similar oil-macerated garlic extract compounds such as E-ajoene, Z-ajoene, and Z-10-DA. From these results, it was suggested that trans structure and/or the position of double bond of iso-E-10-DA reduce the antimicrobial activity.
organosulfur compound; garlic; antimicrobial effect; oil-macerated garlic extract; Allium sativum

-30-
Note
Antimicrobial Activity of the Thiosulfinates Isolated
from Oil-Macerated Garlic Extract

Hisae YOSHIDA, Hirotaka KATSUZAKI,＊ Rie OHTA, Keiko ISHIKAWA,
Hiroyuki FUKUDA, Tsuchiyoshi FUJINO,・ and Atsushi SUZUKI

Biodevelopment Division, Central Institute, Nagoya Seiraku Co., Ltd., 310 Nakasuna-cho,
Tempaku-ku, Nagoya, Aichi 468-0065, Japan
＊Faculty of Bioresources, Mie University, 1515 Kamihama-cho, Tsu, Mie 514-8507, Japan

Received September 25, 1998; Accepted November 17, 1998
Three thiosulfinates were isolated from oil-macerated garlic extract, and their structures were identified as 2-propene-1-sulfinothioic acid S-(Z,E)-1-propenyl ester [AllS(O)SPn-(Z,E)], 2-propenesulfinothioic acid S-methyl ester [AllS(O)SMe], and methanesulfinothioic acid S-(Z,E)-1-propenyl ester [MeS(O)SPn-(Z,E)]. This is the first report of isolating these thiosulfinates from oil-macerated garlic extract. Antimicrobial activities of AllS(O)SPn-(Z,E) and AllS(O)SMe against Gram-positive and negative bacteria and yeasts were compared with 2-propene-1-sulfinothioic acid S-2-propenyl ester [AllS(O)SAll, allicin] which is well-known as the major thiosulfinate in garlic. Antimicrobial activity of AllS(O)SMe and AllS(O)SPn-(Z,E) were comparable and inferior to that of allicin, respectively. This result suggested that the antimicrobial activity of 2-propene sulfinothioic acid S-alk(en)yl esters were affected by alk(en)yl groups. The order for antimicrobial activity was: allyl・methyl>propenyl.
antimicrobial effect; garlic; thiosulfinates; oil-macerated garlic extract; Allium sativum

-31-
Note
Effects of Castor Oil on Lipid Metabolism in Rats

Miyuki IHARA-WATANABE,・ Takashi SHIROYAMA, Gen KONDA, Hayato UMEKAWA,
Takao TAKAHASHI, Yoshihiro YAMADA,＊ and Yukio FURUICHI

Laboratory of Nutritional Chemistry, Department of Agricultural Chemistry, Faculty of Bioresources,
Mie University, Tsu, Mie 514-8507, Japan
＊Ito Oil Chemicals Co., Ltd., 13-26 Suehiro-cho, Yokkaichi 510-0052, Japan

Received October 2, 1998; Accepted November 12, 1998
Weanling rats were given diets contained castor oil (CAO-diet), coconut oil (CO-diet), or high-oleic safflower oil (HO-diet) each 10％ (wt). No growth retardations were observed on the CAO-diets. The CAO-diet group showed significantly lower serum cholesterol and hepatic triacylglycerols than the HO-diet group. Ricinoleic acid was found at an extremely low level in perirenal adipose tissue.
castor oil; ricinoleic acid; high-oleic safflower oil; cholesterol; fatty acid composition

-32-
Note
Asymmetric Reduction of Ethyl 2-methyl 3-oxobutanoate by Chlorella

Takayuki KURAMOTO,1 Kenzo IWAMOTO,1 Minoru IZUMI,2 Mitsunori KIRIHATA,3
and Fumiki YOSHIZAKO,1,・

1Research Institute for Advanced Science and Technology, Osaka Prefecture University,
1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan
2Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama-cho,
1-1, Toyonaka, Osaka, 560-0043, Japan
3Department of Applied Biochemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received October 5, 1998; Accepted November 2, 1998
Chlorella pyrenoidosa Chick reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the diastereomer (anti/syn) ratio of 53/47. The enantiomer excesses of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters were 89 and >99ee％ respectively. C. vulgaris and C. regularis afforded predominantly the syn-isomer, contrary to C. pyrenoidosa. The differences in the activity of reducing ethyl 2-methyl 3-oxobutanoate were observed among three strains of Chlorella. Addition of 2％ metal salts slightly increased the chemical yield of the hydroxy ester.
Chlorella; asymmetric reduction; ethyl 2-methyl 3-oxobutanoate; (2S, 3S)-ethyl 2-methyl 3-hydroxybutanoate; (2S, 3R)-ethyl 2-methyl 3-hydroxybutanoate

-33-
Note
Effective Cloning of Unmarked DNA Fragments in the Bacillus subtilis
168 Genome

Mitsuhiro ITAYA・

Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan

Received October 6, 1998; Accepted November 9, 1998
The Bacillus subtilis 168 genome is being used to clone DNA segments, particularly unmarked DNA segments between the two available clones. To facilitate this cloning, a counter selection method was developed in which loss of a cI repressor gene rendered the host strain resistant to neomycin. This method is promoted to use the B. subtilis genome as a general cloning vehicle.
positive selection; repressor binding; inducible promoter

-34-
Note
Fucoxanthin as the Major Antioxidant in Hijikia fusiformis,
a Common Edible Seaweed

Xiaojun YAN, Yoshihiro CHUDA, Masahiro SUZUKI, and Tadahiro NAGATA・

National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries,
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received Octorber 12, 1998; Accepted November 9, 1998
The radical scavenging activity of Japanese edible seaweeds was screened by the DPPH (1-diphenyl-2-picrylhydrazyl) assay to evaluate the DPPH radical scavenging activity in organic extracts. The fresh brown alga Hijikia fusiformis showed the strongest DPPH radical scavenging activity, followed by Undaria pinnatifida and Sargassum fulvellum. The major active compound from Hijikia fusiformis in its acetone extract was identified as fucoxanthin by 13C-NMR spectroscopy.
edible seaweed; antioxidant; free radical; fucoxanthin; Hijikia fusiformis

-35-
Note
Brachyury Regulatory Region Active in Embryonal Carcinoma P19 Cells

Hirotake YAMAGUCHI,1 Yasuo KITAGAWA,1,2 and Kiyoshi MIKI1,2,・

1Graduate Program for Biochemical Regulation, Graduate School of Bioagricultural Sciences, Nagoya University and 2Nagoya University Bioscience Center, Chikusa, Nagoya 464-8601, Japan

Recieved October 27, 1998; Accepted November 25, 1998
Brachyury (T) is involved in mesoderm induction during early mouse development. We analyzed the region regulating expression of T in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro. Transfection of plasmids encoding reporter genes under the control of the 5′-flanking region showed positive regulatory elements between −298 and −129 bp are responsible for driving T expression in mesodermal cells.
Brachyury (T); embryonal carcinoma; gene expression; mesoderm

-36-
Note
Autodegradation of Protein Disulfide Isomerase

Reiko URADE,・ Ayako YASUNISHI, Hirokazu OKUDO, Tatsuya MORIYAMA,
and Makoto KITO

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan

Received November 9, 1998; Accepted November 13, 1998
Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.
protein disulfide isomerase; endoplasmic reticulum; proteolytic activity
　

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