Contents and Abstracts of Latest Issue of BBB

(Vol.62 No.3 1998)

Immunological Detection and Quantification of Oxidized Proteins by Labelling with Digoxigenin
Juan BAUTISTA and Mar´ia D. MATEOS-NEVADO 419

Cysteine Uptake for Accumulation of Glutathione by the Cyanobacterium Synechocystis strain PCC 6803
Katsuaki SUGINAKA,・ Keiko YAMAMOTO, Hiroyuki ASHIIDA,＊ Yasuhisa KONO,Yoshihiro SAW, and Hitoshi SHIBATA・・ 424

HPLC Profile Analysis of Oleanene-Glucuronides in Several Edible Beans＊
Junei KINJO, Misa HATAKEYAMA, Manabu UDAYAMA, Yukiko TSUTANAGA,
Masami YAMASHITA, Toshihiro NOHARA,＊＊ Yumiko YOSHIKI,＊＊＊
and Kazuyoshi OKUBO＊＊＊ 429

Polysaccharides from Agaricus blazei Stimulate Lymphocyte T-Cell Subsets in Mice
Masashi MIZUNO, Mikio MORIMOTO, Ken-ichiro MINATO, and Hironobu TSUCHIDA 434

Novel Bioactive Oxazolomycin Isomers Produced by Streptomyces albus JA3453
Hiroshi KANZAKI,・ Ken-ichi WADA, Teruhiko NITODA, and Kazuyoshi KAWAZU 438

Physico-chemical Properties of Fatty Acids for Assessing the Threshold Concentration to Enhance the Absorption of a Hydrophilic Substance
Yukitaka KIMURA, Yasuo HOSODA, Motohiro SHIMA, Shuji ADACHI,
and Ryuichi MATSUNO 443

Catalase Catalyzes of Peroxynitrite-mediated Phenolic Nitration
Yasuhisa KONO, Tomoaki YAMASAKI, Akane UEDA, and Hitoshi SHIBATA 448

Facile Synthesis of Cyanogen Glycosides (R)-Prunasin, Linamarin, Linamarin and (S)-Heterodendrin
Noriyuki NAKAJIMA and Makoto UBUKATA 453

A Very-high-density Lipoprotein with Clotting Ability from Hemolymph of Sand Crayfish, Ibacus ciliatus
Masaharu KOMATSU＊ and Seiichi ANDO＊＊ 459

The Increased Effect of Kneading on the Formation of Inclusion Complexes between d-Limonene and β-Cyclodextrin at Low Water Content
Hidefumi YOSHII,・ Takeshi FURUTA, Eiji OKITA, Akira TOYOMI,＊
Yu-Yen LINKO,＊＊ and Pekka LINKO＊＊ 464

Characterization of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida＃
Osao ADACHI,＊ Tatsuro KUBOTA, Ayse HACISALIHOGLU,＊＊ Hirohide TOYAMA,
Emiko SHINAGAWA,＊＊＊ Johannis A. DUINE,＊＊ and Kazunobu MATSUSHITA 469

Effect of Dibromochloropropane (DBCP) on the Hormone Receptors of the Male Rat Reproductive System
Seiichi YOSHIDA, Hiroyuki YAMADA,＊ Isamu SUGAWARA,＊,・ and Ken TAKEDA 479

Cell Age Distribution of Erythrocytes at the Incidence of Cerebral Stroke in Stroke-Prone Spontaneously Hypertensive Rats, and Their Glutathione Peroxidase Activity
Tetsuo MURAKAMI,＊ Kumiko TAKEMORI,＊ Norifumi SHIRASAkA,＊ Hajime YOSHIZUMI,＊
and Hiroyuki ITO＊＊ 484

Effects of Synthetic Hydroxy Isothiocyanates on Microbial Systems
Hirokuni TAJIMA, Hisashi KIMOTO,＊ Yoriko TAKETO,＊＊ and Akira TAKETO＊ 491

New Limonoids from Melia toosendan
Jian-Bo ZHOU, Kenjiro TADERA, Yuji MINAMI, Fumio YAGI,
Junichi KURAWAKI,＊ Ken TAKEZAKI,＊＊ and Munehiro NAKATANI＊ 496

Purification and Characterization of a Novel Cysteine Synthase Isozyme from Spinach Hydrated Seeds
Takayuki YAMAGUCHI,1,2 Xia ZHU,1 and Masahiro MASADA1,＊ 501

Association between Hepatic Triacylglycerol Accumulation Inducedby Administering Orotic Acid and Enhanced Phosphatidate
Phosphohydrolase Activity in Rats
Jae-Young CHA, Yuji MAMEDA, Kyosuke YAMAMOTO,＊ Kazuhiro OOGAMI,＊＊
and Teruyoshi YANAGITA 508

Cloning, Sequence Analysis, and Expression in Escherichia coli of a Gene Coding for an Enzyme from Bacillus circulans K-1 that Degrades Guar Gum
Seiji YOSHIDA, Yoshihiko SAKO,＊ and Aritsune UCHIDA＊ 514

Stereoselective Synthesis of (2S,3S)-2-Benzyl-2-hydroxy-3-(3,4-methylenedioxybenzyl)-γ-butyrolactone from L-(＋)-Arabinose via a Carissanol-type of Lignan
Satoshi YAMAUCHI and Yoshiro KINOSHITA 521

Identification of Brassinosteroids with Epimerized Substituents and/or the 23-Oxo Group in Pollen and Anthers of Japanese Cedar
Takao YOKOTA,1,・ Kyoko HIGUCHI,2 Nobutaka TAKAHASHI,2 Yasuo KAMURO,3
Tsuyoshi WATANABE,4 and Suguru TAKATSUTO5 526

Isolation and Identification of Acetyl-CoA Carboxylase Inhibitors from Green Tea (Camellia sinensis)
Jun WATANABE, Jun KAWABATA,・ and Ryoya NIKI 532

Role of N-Acetylglutamate Turnover in Urea Synthesis by Rats Treated with the Thyroid Hormone
Kazutoshi HAYASE1, Yaeko NAGANUMA, Miho KOIE and Akira YOSHIDA＊ 535

Suppression of the Lethal Effect of Acidic-Phospholipid Deficiency in
Escherichia coli by Bacillus subtilis Chromosomal Locus ypoP
Koichi INOUE, Atsuhiro KISHIMOTO,・ Motoo SUZUKI, Hiroshi MATSUZAKI,
Kouji MATSUMOTO, and Isao SHIBUYA・・ 540

α2-Adrenoceptor-Mediated Antisecretory Effect of Hypoxia in Conscious Rats
Ryoichi YAMAJI, Yasuyo OHNISHI, Miki SAKAMOTO, Makoto TAKENOSHITA,
Mitsuaki OHTA,＊ Shingo TSUYAMA,＊＊ Fumio WATANABE,＊＊＊ Hiroshi INUI,
Kazutaka MIYATAKE, and Yoshihisa NAKANO・ 546

Characterization of cDNAs Encoding Small and Large Subunits of ADP-Glucose Pyrophosphorylases from Watermelon (Citrullus vulgaris S.)・
In-Jung KIM, Hyung-Yeel KAHNG, and Won-I1 CHUNG＊ 550

Note
Tomatine Content in Host and Transgenic Tomatoes by Absorptiometric
Measurement
Hiroyasu FURUI, Takahiro INAKUMA, Yukio ISHIGURO, and Makoto KISO＊ 556

Note
Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen,
via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene
in Transgenic Tobacco Plants
Kyu Whan CHOI, Oksoo HAN,＊,・ Hee Jae LEE,＊＊ Young Chae YUN,
Young Ho MOON, Myojeoung KIM,＊ Yong In KUK,＊＊＊ Sung Uk HAN,＊＊＊
and Ja Ock GUH＊＊＊ 558

Note
Occurrence of Anserine as an Antioxidative Dipeptide in a Red Alga,
Porphyra yezoensis
Yoshiyuki TAMURA, Shigeo TAKENAKA, Sumi SUGIYAMA, and Reiko NAKAYAMA 561

Note
cDNA Cloning, Expression, and Mutagenesis of Scytalone Dehydratase Needed for Pathogenicity of the Rice Blast Fungus, Pyricularia oryzae
Takayuki MOTOYAMA, Kinya IMANISHI,＊ and Isamu YAMAGUCHI・ 564

Note
A Non-radioassay of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase
by Anion-Exchange Chromatography
Koichi UEMURA,＊,・ Ayako SHIMOIDE,＊ and Akiho YOKOTA＊,＊＊,・・ 567

Note
Biological Activities of (1→3)-β-D-Glucans with Reducing Glucose Side Chains
Tadashi KIHO,＊ Mitsuhiro MATSUSHITA, Shigeyuki USUI, and Shigeo UKAI 570

Note
Sequence of a cDNA Encoding Mouse Ribosomal Protein S14
Mijoung LEE, Intaek HWANG, Yunjaie CHOI,＊ and Myunggi BAIK1 573

Note
Inhibition of HIV-1 Protease by Water-Soluble Lignin-Like Substance
from an Edible Mushroom, Fuscoporia obliqua
Toshiaki ICHIMURA, Osamu WATANABE,＊ and Susumu MARUYAMA 575

Note
Increasing Effect of Dietary Taurine on the Serum HDL-cholesterol
Concentration in Rats
Hideki MOCHIZUKI, Hiroaki ODA,＊ and Hidehiko YOKOGOSHI 578

Note
Increased Conversion Ratio of Tryptophan to Niacin in Severe Food Restriction
Katsumi SHIBATA, Takako KONDO, and Atsuyo MIKI 580

Note
Isolation and Characterization of Chitinase Isoforms from the Bulbs of
Four Species of the Genus Tulipa
Takeshi YAMAGAMI,・ Toki TAIRA, Yoichi ASO, and Masatsune ISHIGURO 584

Note
Enzymatic Synthesis of 6-O-α-D-Galactopyranosyl-1-deoxynojirimycin
Using α-Galactosidase from Green Coffee Beans
Nam Soo PAEK,・ Dae Jung KANG, Hong Sub LEE, Jung Joon LEE,＊
Yong Jin CHOI,＊＊ Tae Han KIM, and Kee Won KIM 588

Note
A Convenient and Efficient Synthesis of Sialyl Lewis X・
Kunihisa BABA, Noriyuki IWATA, Hitoshi HAMAJIMA, Takao IKAMI,＊
Hideharu ISHIDA,＊＊ Akira HASEGAWA,＊＊ and Makoto KISO＊＊590

Note
Involvement of Ovotransferrin in the Thermally Induced Gelation
of Egg White at around 65°C
Honami YAMASHITA, Junko ISHIBASHI, Youn-Ho HONG,＊ and Masaaki HIROSE＊＊ 593

Note
Structural Diversity of the Membrane Core Lipids of Extreme Halophiles
Mikio MORITA, Noriaki YAMAUCHI, Tadashi EGUCHI, and Katsumi KAKINUMA・ 596

Note
Improved Synthesis of the C8--C23 Segment of Aplysiatoxins via Asymmetric
Dihydroxylation of the Chiral Homoallylic Alcohol
Hiroaki TOSHIMA and Akitami ICHIHARA 599

Note
Effects of Hydrodynamic Volume of Anionic Lipopolysaccharide,
Emulsan, on Emulsifying Activity
Pil KIM, Seon Won KIM, Sang Yong KIM,＊ and Jung Hoe KIM・ 603

Short Communication
Sweetness of Lysozymes
Kenji MAEHASHI and Shigezo UDAKA・ 605

Short Communication
Field and Electroantennogram Responses of the Pine Sawfly,
Diprion nipponica, to Chiral Synthetic Pheromone Candidates
Akira TAI,・ Yasutomo HIGASHIURA,＊ Masashi KAKIZAKI,＊＊
Tikahiko NAITO,＊＊＊ Kazuki TANAKA, Morifumi FUJITA,
Takashi SUGIMURA, Hideho HARA,＊ and Naotaka HAYASHI＊ 607

Short Communication
Green Tea Suppresses D-Galactosamine-Induced Liver Injury in Rats
Kimio SUGIYAMA,＃ Puming HE, Shingo WADA, Fumi TAMAKI,
and Shigeru SAEKI＊ 609

-1-
Immunological Detection and Quantification of Oxidized Proteins by Labelling with Digoxigenin

Juan BAUTISTA and Mar´ia D. MATEOS-NEVADO

Department of Biochemistry, Bromatology and Toxicology, Faculty of Pharmacy, University of Sevilla,
41012-Sevilla, Spain

Received June 3, 1997
An immunological assay, based on the digoxigenin/anti-digoxigenin system, was developed to detect and quantify carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin (BSA) was oxidized by a hydroxyl radical-generating system consisting of ascorbate/Fe(III)/O2. The resulting albumin-derived carbonyls were labelled with digoxigenin-hydrazide and detected by dot blotting with an anti-digoxigenin antibody conjugated to alkaline phosphatase. Quantification was carried out by a densitometric analysi. This system allows the detection of a pmole-amount of carbonyl groups on blots. The assay covers a range of sensitivity from 1.26 to 126 pmoles. Another feature of this method is its application to a complex protein mixture (homogenate) to analyze the oxidative status of individual proteins, as are shown for intestinal brush border membrane homogenate of a rat.
oxidative modification; protein oxidation; immunological detection; digoxigenin; aging

-2-
Cysteine Uptake for Accumulation of Glutathione by the Cyanobacterium
Synechocystis strain PCC 6803

Katsuaki SUGINAKA,・ Keiko YAMAMOTO, Hiroyuki ASHIIDA,＊ Yasuhisa KONO.Yoshihiro SAW, and Hitoshi SHIBATA・・

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science,
Shimane University, Matsue, Shimane 690, Japan
＊Institute for Molecular Genetics, Shimane University, Matsue, Shimane 690, Japan

Received June 4, 1997
By incubation with precursor amino acids, L-glutamate, L-cysteine, and glycine, or with L-cysteine, the unicellular cyanobacterium Synechocystis strain PCC 6803 increased intracellular glutathione levels, under illumination with white light. L-Cystine could not serve as the precursor. Two transport systems for L-cysteine were evident in the strain: a high (Ks; L-cysteine concentration giving one-half of the maximum uptake, 21~35 μM) and a low (Ks, 1.0~1.8 mM) affinity transport system. The latter, responsible for glutathione accumulation, was an energy-requiring process. L-Cysteine uptake by the two transport systems were Na＋-independent, and inhibited by the presence of neutral amino acids and less inhibited by basic or acidic amino acids. These results suggested that the neutral amino acid symport(s) is involved in L-cysteine uptake for the GSH accumulation in this strain.
amino acid transport; cyanobacteria; cysteine uptake; glutathione; Synechocystis PCC 6803

-3-
HPLC Profile Analysis of Oleanene-Glucuronides in
Several Edible Beans＊

Junei KINJO, Misa HATAKEYAMA, Manabu UDAYAMA, Yukiko TSUTANAGA,
Masami YAMASHITA, Toshihiro NOHARA,＊＊ Yumiko YOSHIKI,＊＊＊
and Kazuyoshi OKUBO＊＊＊

Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862, Japan
＊＊＊Department of Environmental Bioremediation, Graduate School of Agriculture, Tohoku University,
Tsutsumidori, 1-1 Amamiya-machi, Sendai 981 Japan

Received July 9, 1997
HPLC analysis and yield of oleanene-glucuronide (OG) was done on some commercially available edible beans: seeds of Glycine max, Glycine max cv. Kuromame, Phaseolus vulgaris cv. Torosuku, Phaseolus vulgaris cv. Toramame, Phaseolus vulgaris cv. Taishokintoki, Phaseolus coccineus cv. Ooshirobana, Phaseolus coccineus cv. Murasakihanamame, Vigna unguiculata cv. Chuguro, Vigna angularis cv. Dainagon, Arachis hypogaea, Pisum sativum, and Vicia faba. All the beans listed above have OG, though in varying the amounts. Furthermore the HPLC profiles of beans belonging to the same genus were very similar except for that of the Vigna genus. There was no great difference of the HPLC profiles with respect to the cultivated varieties. The structures of the major OGs in each type of beans were identified as soyasaponins I (1) and V (2), and phaseoside I (3) (Phaseolus vulgaris and P. coccineus); 1 and soyasaponin II (4) (Glycine max); 1 and 2 (Vigna unguiculata); 1 (Pisum sativum, Arachis hypogaea and Vicia faba). In contrast, those in Dainagon (Vigna angularis cv. Dainagon) were identified as azukisaponins II (5) and VI (6).
HPLC analysis; oleanene glucuronide; triterpene saponin; bean; Leguminosae

-4-
Polysaccharides from Agaricus blazei Stimulate Lymphocyte T-Cell Subsets
in Mice

Masashi MIZUNO, Mikio MORIMOTO, Ken-ichiro MINATO, and Hironobu TSUCHIDA

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

Received July 14, 1997
Subset analysis of splenic lymphocytes using flow cytometry showed that the percentages of Thy1.2-(pan T-cells), L3T4-(CD4, helper T-cells), and Lyt2-(CD8, cytotoxic T-cells) positive cell populations were significantly increased in mice orally administered a hot water-soluble fraction from Agaricus blazei as compared with mice treated only with saline. 13C-NMR data indicates that the main component in the active polysaccharide is the complex of α-1,6- and α-1,4-glucan, which had already been shown to have anti-tumor activity against Sarcoma 180. It seems that the polysaccharide from Agaricus blazei may be an effective prophylactic, protecting humans against cancer by stimulating lymphocytes such as cytotoxic T-cells.
Agaricus blazei; T-cell subsets; flow cytometry; immunomodulating activity; mushrooms

-5-
Novel Bioactive Oxazolomycin Isomers Produced by Streptomyces albus JA3453

Hiroshi KANZAKI,・ Ken-ichi WADA, Teruhiko NITODA, and Kazuyoshi KAWAZU

Laboratory of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700, Japan

Received July 17, 1997
Two novel oxazolomycin isomers, oxazolomycins B (2) and C (3), were isolated from the fermentation broth of an oxazolomycin-producing strain, Streptomyces albus JA3453. Both compounds are geometrical isomers of oxazolomycin (1), the configurations of their triene moieties being (4′E, 6′E, 8′E) (2) and (4′Z, 6′E, 8′E) (3) while that of oxazolomycin (1) is (4′Z, 6′Z, 8′E). Compounds 2 and 3 exhibited potent inhibitory activity against crown gall formation with the same MIC (0.8 μg/disk) as oxazolomycin. Compounds 2 and 3 showed no antibacterial activity against Agrobacterium tumefaciens, in contrast to oxazolomycin which has specific anti-A. tumefaciens activity.crown gall formation; Agrobacterium tumefaciens; potato tuber disk assay; plant transformation

-6-
Physico-chemical Properties of Fatty Acids for Assessing the Threshold
Concentration to Enhance the Absorption of a Hydrophilic Substance

Yukitaka KIMURA, Yasuo HOSODA, Motohiro SHIMA, Shuji ADACHI,
and Ryuichi MATSUNO

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

Received July 28, 1997
The effects of natural and non-natural fatty acids on enhancing the absorption of a hydrophilic marker through a human epithelial cell (Caco-2) monolayer were measured to elucidate the properties of the fatty acids. Fatty acids from C9 to C14 enhanced the absorption depending on the concentration and the carbon chain length. Those fatty acids with longer chains gave a higher permeability coefficient at low concentrations and a lower toxicity than those with shorter chains. The surface energy lowering coefficient (SELC), an intrinsic physico-chemical property, and the critical micellar concentration (CMC) were good criteria for identifying the threshold concentrations of a fatty acid to significantly enhance absorption.
absorption enhancement; physico-chemical property; Caco-2; fatty acid

-7-
Catalase Catalyzes of Peroxynitrite-mediated Phenolic Nitration

Yasuhisa KONO, Tomoaki YAMASAKI, Akane UEDA, and Hitoshi SHIBATA

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

Received July 29, 1997
Catalase catalyzed the peroxynitrite-mediated nitration of 4-hydroxyphenylacetic acid. The curve for the pH dependence of nitration was similar to that for the reaction between peroxynitrite and phenol. Cyanide, azide, and 3-amino-1,2,4-triazole inhibited the nitration in a dose-dependent way. When catalase was mixed with peroxynitrite, Compound I was detected as an intermediate. Because azide was an electron donor for the peroxidatic action of catalase, and because 3-amino-1,2,4-triazole inhibited catalase activity by binding with Compound I, peroxynitrite-mediated phenolic nitration was probably accompanied by Compound I formation. Both catalase and superoxide dismutase protected Escherichia coli from peroxynitrite toxicity.
bacterial killing; catalase; peroxynitrite; phenolic nitration; superoxide dismutase

-8-
Facile Synthesis of Cyanogen Glycosides (R)-Prunasin, Linamarin
and (S)-Heterodendrin

Noriyuki NAKAJIMA and Makoto UBUKATA

Biotechnology Research Center, Toyama Prefectural University 5180 Kosugi, Imizu, Toyama 939-03, Japan

Received August 8, 1997
A facile synthetic route is described to cyanogenic glycosides (R)-prunasin, linamarin and (S)-heterodendrin from||O　　-　　(2,3,4,6　　-　　tetra　　--　　O　　--　　acetyl　　-　　α　　-　　D　　- glucopyranosyl)trichloroace-||timidate and the corresponding α-hydroxyamides by a 3-step reaction of glycosylation, cyanohydrin formation by dehydration of carboxamides,and deprotection.
facile synthesis; cyanogenic glycoside, cyanohydrin formation; dehydration of carboxamide; glycosylation

-9-
A Very-high-density Lipoprotein with Clotting Ability from Hemolymph of Sand Crayfish, Ibacus ciliatus

Masaharu KOMATSU＊ and Seiichi ANDO＊＊

＊Laboratory of Food Chemistry and ＊＊Lipoprotein Research Laboratory, Faculty of Fisheries,
Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890, Japan

Received August 11, 1997
A very-high-density lipoprotein (VHDL) with a density of 1.27--1.29 g/ml was the most abundant lipoprotein in the hemolymph of the sand crayfish Ibacus ciliatus. The VHDL isolated by a density gradient ultracentrifugation consisted of 94％ protein and 6％ lipid reflecting its high density, and phospholipid was a predominant lipid component. The VHDL had an apolipoprotein of molecular mass 195 kDa and its N-terminal amino acid sequence was identified as follows: LQPGLEYQYRYNGRVAA. This sequence was similar to those of clotting proteins from the spiny lobster Panulirus interruptus and the freshwater crayfish Pacifastacus leniusculus. Transglutaminase and Ca2＋ also induced the VHDL to clot. Considering large amounts of VHDL in the hemolymph of sand crayfish, the VHDL not only functions as lipid carrier but plays an important role in the defense process of crustacea.
clotting protein; crustacea; fibrinogen; sand crayfish; very-high-density lipoprotein

-10-
The Increased Effect of Kneading on the Formation of Inclusion Complexes
between d-Limonene and β-Cyclodextrin at Low Water Content

Hidefumi YOSHII,・ Takeshi FURUTA, Eiji OKITA, Akira TOYOMI,＊
Yu-Yen LINKO,＊＊ and Pekka LINKO＊＊

Department of Biotechnology, Tottori University Tottori 680, Japan
＊Kurimoto Co., Ltd., Osaka 559, Japan
＊＊Department of Chemical Technology, Helsinki University of Technology, P.O. Box 6100,
FIN-02015 HUT (Espoo), Finland

Received August 11, 1997
The molecular inclusion powder of d-limonene in β-cyclodextrin was prepared by using a twin-screw kneader at a low water content. The influence of water and alcohol content on the formation of the inclusion complex was studied in comparison with the inclusion complex obtained by the micro-aqueous method. The inclusion fraction of d-limonene in the complex powder in β-cyclodextrin was much higher than that made by the micro-aqueous method. The marked differences in the inclusion fraction were observed particularly at the molar water ratio to β-cyclodextrin of less than 10. The inclusion fraction increased sharply with the increase in the kneading torque. This means that the energy of kneading increased the inclusion of d-limonene. A mathematical model for estimating the inclusion fraction was derived by combining the effects of water and the kneading energy on the formation of the inclusion complex.
cyclodextrin; inclusion complex powder; kneading; d-limonene

-11-
Characterization of Quinohemoprotein Amine Dehydrogenase
from Pseudomonas putida＃

Osao ADACHI,＊ Tatsuro KUBOTA, Ayse HACISALIHOGLU,＊＊ Hirohide TOYAMA,
Emiko SHINAGAWA,＊＊＊ Johannis A. DUINE,＊＊ and Kazunobu MATSUSHITA

Laboratory of Applied Microbiology, Department of Biological Chemistry, Faculty of Agriculture,
Yamaguchi University, Yamaguchi 753-0841, Japan
＊＊Kluyver Laboratory of Biotechnology, Department of Microbiology and Enzymology,
Delft University of Technology, 2628 BC Delft, The Netherlands
＊＊＊Department of Biotechnology, Ube Technical College, Ube, Yamaguchi 755-0031, Japan

Received August 11, 1997
Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystallized from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultracentrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing maxima at 554, 522, 420, and 320 nm in the reduced form and one peak at 410 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxidized form. The oxidized enzyme was specifically reduced by the addition of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two moles of heme c were detected per mole of AMDH and the 60-kDa subunit was found to be the heme c-carrying subunit. By redox-cycling quinone staining, a positive reaction band corresponding to the 20-kDa subunit was detected after developed by SDS-PAGE, but the 20 kDa band was scarcely stained by conventional protein staining. Only a silver staining method was possible to detect the subunit after the protein was developed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 455 nm, indicating Schiff base formation between the carbonyl cofactor in AMDH and the carbonyl reagent. Thus, AMDH is different from nonheme quinoprotein methylamine dehydrogenase and aromatic amine dehydrogenase in many respects. The presence of an azurin-like blue protein was identified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during AMDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstituted successfully only by AMDH, the blue protein, and the cytoplasmic membranes of the organism. The function of the 40-kDa subunit is unknown at the moment. The properties of AMDH were compared with other bacterial amine dehydrogenases so far reported.
amine dehydrogenase; blue protein in amine oxidation; Pseudomonas putida; quinohemoprotein

-12-
Effect of Dibromochloropropane (DBCP) on the Hormone Receptors
of the Male Rat Reproductive System

Seiichi YOSHIDA, Hiroyuki YAMADA,＊ Isamu SUGAWARA,＊,・ and Ken TAKEDA

Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Ichigaya-funagawara-machi,
Shinjuku, Tokyo 162, Japan
＊Department of Molecular Pathology, The Research Institute of Tuberculosis, 3-1-24 Matsuyama,
Kiyose, Tokyo 204, Japan

Received August 15, 1997
The effects of dibromochloropropane (DBCP), a pesticide, on the male rat reproductive system were examined at morphological and hormonal expression levels. Changes of Leydig cells and seminiferous tubules were examined in rats treated with one subcutaneous (s.c.) injection of DBCP at doses of 10, 50, 75 and 100 mg/kg of body weight. The Leydig cells degenerated and decreased in number in those rats with an s.c. injection of DBCP at a dose of 50 mg/kg of body weight. However, these morphological changes were not significant in those rats that were treated with four separate s.c. injections of DBCP at 10 mg/kg of body weight. The expression of luteinizing hormone receptor mRNA, which is specifically expressed in Leydig cells, was decreased significantly in the DBCP-treated rats as compared with the normal-control rats. The expression of follicle-stimulating hormone receptor mRNA was decreased to a mild degree in DBCP-treated rats. At a dose of 75 mg/kg of body weight, seminiferous tubules as well as Leydig cells were severely damaged morphologically and at hormonal receptor expression levels. Multinucleated giant cells appeared at a dose of 75 mg/kg of body weight. All the rats died at a dose of 100 mg/kg of body weight. Our data indicate that DBCP had a cytotoxic effect on the male rat reproductive system in a dose-dependent manner.
dibromochloropropane; rat testis; Leydig cells; hormone receptor expression

-13-
Cell Age Distribution of Erythrocytes at the Incidence of Cerebral Stroke
in Stroke-Prone Spontaneously Hypertensive Rats, and Their Glutathione
Peroxidase Activity

Tetsuo MURAKAMI,＊ Kumiko TAKEMORI,＊ Norifumi SHIRASAkA,＊ Hajime YOSHIZUMI,＊
and Hiroyuki ITO＊＊

＊Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, Nara, Japan
＊＊Division of Pathology, Research Institute of Hypertension, Kinki University, Osaka, Japan

Received August 22, 1997
To study the mechanism of the fall of glutathione peroxidase (GSH-Px) activity in erythrocyte after cerebral strokes in stroke-prone spontaneously hypertensive rats (SHRSP), erythrocytes were fractionated into low density erythrocytes (LD-E) and high density erythrocytes (HD-E) by a density gradient centrifugal method using Percoll solution, and fluctuation of the distribution ratio and changes of GSH-Px activity in fractionated erythrocytes were investigated. The distribution ratio of LD-E and HD-E in erythrocytes of SHRSP was about 4:1 at 5 weeks of age (n＝6), and the distribution to HD-E increased along with aging. While the distribution ratio was changed, however, there was no change in the GSH-Px activity in both LD-E and HD-E of erythrocytes. In senile, 30-week-old SHRSP (n＝4) with advanced hypertension, the GSH-Px activity in the HD-E was lower, in proportion to the increase of the distribution rate against HD-E. On the other hand, in SHRSP (n＝5) having cerebral stroke, the distribution ratio of LD-E and HD-E was about 1:4. The GSH-Px activity was 31.4±2.9 units/1010 erythrocytes in LD-E, which was hardly different from the value of SHRSP without stroke (35.7±3.3 units/1010 erythrocytes). In HD-E, however, the activity was 18.2±2.2 units/1010 erythrocytes, being lower than the activity of SHRSP without stroke. At the moment when the GSH-Px activity had dropped to 17 units/mg hemoglobin, and the control diet was changed to one based on fish or a hydralazine treatment given, the activity recovered, and an increase in body weight and the distribution rate of the LD-E over HD-E was increased. It is clear from these experiments that the fall of erythrocyte GSH-Px activity observed after cerebral stroke is due to a decrease of LD-E and increase of HD-E, which has lowered activity. However, nothing definite is known on the relationship between the fall of GSH-Px activity in erythrocytes and disorder in cerebral tissue. It appears that the fall of the GSH-Px activity causes at least functional and structural changes in erythrocytes, which interfere with the delivery of oxygen to peripheral tissues, triggering oxidation stress in cerebral tissues.
SHRSP; glutathione peroxidase; cerebral stroke; erythrocytes; cell age distribution

-14-
Effects of Synthetic Hydroxy Isothiocyanates on Microbial Systems

Hirokuni TAJIMA, Hisashi KIMOTO,＊ Yoriko TAKETO,＊＊ and Akira TAKETO＊

Fukui Research Laboratory, Rengo Co., Ltd., 10-8-1, Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui 919-0604, Japan
＊Department of Biochemistry I, Fukui Medical School, Matsuoka, Fukui 910-1103, Japan
＊＊Department of Pharmacology, Kanazawa University School of Medicine, Kanazawa 920-0934, Japan

Received August 29, 1997
Hydroxy isothiocyanates (ITCs), including some new derivatives of naturally occurring compounds, were synthesized and their minimum inhibitory, minimum fungicidal, and minimum bactericidal concentrations for Aspergillus niger, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli were estimated. These compounds were strongly antimicrobial; for example, 2-(4-hydroxyphenyl)ethyl ITC inhibited growth of all strains examined at concentrations of 7.8 to 15.6 μg/ml. The ATP concentration in E. coli was markedly reduced when cells were treated with 2-(4-hydroxyphenyl)ethyl ITC.
Inhibition of the growth of E. coli by 2-(4-hydroxyphenyl)ethyl ITC was decreased in the presence of cysteine. Streptolysin S production in washed cells of Streptococcus equisimilis was extremely sensitive to this ITC derivative and this inhibition also was counteracted by cysteine. The results showed that the ITC compounds had antimicrobial effects by blocking sulfhydryl groups.
hydroxy isothiocyanate; 2-(4-hydroxyphenyl)ethyl isothiocyanate; antimicrobial activity; sulfhydryl-blocking reagent

-15-
New Limonoids from Melia toosendan

Jian-Bo ZHOU, Kenjiro TADERA, Yuji MINAMI, Fumio YAGI,
Junichi KURAWAKI,＊ Ken TAKEZAKI,＊＊ and Munehiro NAKATANI＊

Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
Korimoto 1-21-24, Kagoshima 890, Japan
＊Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University,
Korimoto 1-21-35, Kagoshima 890, Japan
＊＊Kagoshima Prefectural Agricultural Experiment Station, Kamifukumoto 5500, Kagoshima 890, Japan

Received September 1, 1997
The root bark of Melia toosendan afforded four new limonoids with a C-19/C-29 bridged acetal structure, together with two known limonoids, 12α-hydroxyamoorastatone and its 12-acetate. The new compounds were elucidated as 1--O--acetyltrichilin H and 29--O--substituted amoorastatone derivatives, named neoazedarachins A, B and D, by spectroscopic and chemical means. Their antifeedant activity was also studied.
limonoid; antifeedant; Melia toosendan; 1--O--acetyltrichilin H; neoazedarachins

-16-
Purification and Characterization of a Novel Cysteine Synthase Isozyme
from Spinach Hydrated Seeds

Takayuki YAMAGUCHI,1,2 Xia ZHU,1 and Masahiro MASADA1,＊

1Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University,
Matsudo 648, Chiba 271, Japan
2Engineering Department, Kazami Co., Ltd., Sukedo 1-26, Tochigi 326, Japan

Received September 2, 1997
A novel type of cysteine synthase (CSase, EC 4.2.99.8) isozyme, designated as CSase 1′, was purified to homogeneity from hydrated spinach seeds. The enzyme had a molecular weight of 68,000 and consisted of two identical subunits of Mr 34,000. The apparent Km for O-acetyl-L-serine was 8.33 mM and that for sulfide was 0.66 mM. The activity of CSase 1′ was maintained when it was treated at 60°C for 1 min. This novel enzyme was similar to CSases 1, 2, and 3 already purified from spinach leaves, in results of double immunodiffusion, molecular weight, subunit composition, Km values for O-acetyl-L-serine and sulfide, and heat stability. On the other hand, N-terminal amino acid sequence, effects of immunotitration, pH optimum, and effects of hydroxylamine on purified CSase 1′ were different from those of the other CSases. Furthermore, it was found that CSases 2S and 3S isolated from hydrated spinach seeds were identical with the CSases 2 and 3 reported previously. It was also disclosed that CSases 1, 2, and 3 were localized in chloroplasts, cytosol, and mitochondria, respectively.
cysteine biosynthesis; cysteine synthase; O-acetylserine(thiol)lyase; O-acetyl-L-serine; sulfate assimilation

-17-
Association between Hepatic Triacylglycerol Accumulation Induced
by Administering Orotic Acid and Enhanced Phosphatidate
Phosphohydrolase Activity in Rats

Jae-Young CHA, Yuji MAMEDA, Kyosuke YAMAMOTO,＊ Kazuhiro OOGAMI,＊＊
and Teruyoshi YANAGITA

Department of Applied Biological Sciences, Saga University, Saga 840, Japan
＊Department of Internal Medicine, Saga Medical School, Saga 849, Japan

Received September 3, 1997
　Orotic acid is known to cause fatty liver, but it is unclear whether this is caused partly by stimulation of the enzymes for triacylglycerol (TG) synthesis. To understand the change of hepatic TG metabolism in fatty liver induced by orotic acid, we determined the liver tissue TG level and phosphatidate phosphohydrolase (PAP) activity over time in rats fed on a diet containing orotic acid (OA). A dietary lipid content of 10％ was achieved by using n-6 fatty acid-rich corn oil in experiment 1, and n-6 fatty acid-rich safflower oil (SO) and n-3 fatty acid-rich fish oil (FO) with the same polyunsaturated fatty acid/monounsaturated fatty acid/saturated fatty acid (P/M/S) ratio in experiment 2. In experiment 1, an increase in the hepatic TG level due to OA intake was observed from day 5 onwards, the level rising approximately 6-fold by day 10. The activity of hepatic microsomal PAP, the rate-limiting enzyme in TG synthesis, increased markedly from day 5 onwards, concurrent with the liver diacylglycerol concentration. A strong correlation (r＝0.974) was observed between the hepatic TG level and microsome-bound PAP activity. In experiment 2, we investigated the effects of dietary fatty acid on OA-induced fatty liver. Compared with the n-6 fatty acid-rich vegetable oil diet, the relative increase in hepatic TG was smaller with the n-3 fatty acid-rich FO diet, and hepatic PAP activity fell markedly to the level for an OA-free diet. In addition, the hepatic TG accumulation and serum TG concentration were lower in the FO group than in the SO group. Nevertheless, because the hepatic TG level was low, it seems that the inhibition of liver PAP activity by FO possibly had a strong influence on the accumulation of TG in the liver.
　In conclusion, enhanced TG synthesis mediated by changes in liver PAP activity was involved in the hepatic TG accumulation induced by OA administration, this change being markedly suppressed by dietary n-3 fatty acids.
fatty liver; triacylglycerol; phosphatidate phosphohydrolase; orotic acid; n-3 and n-6 fatty acids

-18-
Cloning, Sequence Analysis, and Expression in Escherichia coli of a Gene Coding for an Enzyme from Bacillus circulans K-1 that Degrades Guar Gum

Seiji YOSHIDA, Yoshihiko SAKO,＊ and Aritsune UCHIDA＊

Research Institute of Technology, Konoike Construction Co., Ltd., 1-20-1 Sakura, Tukuba, Ibaraki, 305-0003, Japan
＊Laboratory of Marine Microbiology, Department of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

Received September 4, 1997
A 2,048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking. This G-gene consisted of 1,551 nucleotides coding for a protein with Mr 55,242. The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19. The molecular mass of recombinant G-enzyme estimated by SDS-PAGE was 62 KDa, close to that from strain K-1. Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, and Gal to account for 1.7％, 14.4％, 6.1％, 3.2％, 54.2％, and 10.4％, respectively, of the total monosaccharides. Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band. The results suggested that the sugars accounted for the differences in the molecular masses. The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe-||Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala-||Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr. Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme. Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E. coli after translation. The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.
guar gum; mannanase; gene; expression; glycoprotein

-19-
Stereoselective Synthesis of (2S,3S)-2-Benzyl-2-hydroxy-3-
(3,4-methylenedioxybenzyl)-γ-butyrolactone from
L-(＋)-Arabinose via a Carissanol-type of Lignan

Satoshi YAMAUCHI and Yoshiro KINOSHITA

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

Received September 12, 1997
As a model experiment for the synthesis of optically active α,β-dibenzyl-α-hydroxy-γ-butyrolactone (1), (2S,3S)-||2　　-　　benzyl　　-　　2　　- hydroxy　　-　　3　　-　　(3,4　　-　　methylenedioxybenzyl)　　-　　γ　　-　　buty||||rolactone (3) was stereoselectively synthesized from||||L-(＋)-arabinose via the carissanol-type of lignan, (2R/||||S,3S,4S)-3　　-　　benzyl　　-　　2,3　　-　　dihydroxy　　-　　4　　-　　(3,4　　- methylenedio||xybenzyl)tetrahydrofuran (4).
lignan; α,β-dibenzyl-γ-butyrolactone; carissanol-type lignan

-20-
Identification of Brassinosteroids with Epimerized Substituents and/or the 23-Oxo Group in Pollen and Anthers of Japanese Cedar

Takao YOKOTA,1,・ Kyoko HIGUCHI,2 Nobutaka TAKAHASHI,2 Yasuo KAMURO,3
Tsuyoshi WATANABE,4 and Suguru TAKATSUTO5

1Department of Biosciences, Teikyo University, Utsunomiya 320, Japan
2Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
3BAL Planning Co. Ltd., 2-15-16 Hanaike, Ichinomiya-shi, Aichi 491, Japan
4Tama Biochemical Co. Ltd., 2-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 163, Japan
5Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943, Japan

Received September 12, 1997
Brassinosteroids in Japanese cedar (Cryptomeria japonica) were analyzed by GC-MS after being purified by reversed-phase HPLC. The occurrence of four stereoisomers of 23-dehydrobrassinolide, three stereoisomers of 28-homobrassinolide, and an isomer of homodolicholide was revealed. The stereochemical structures of these brassinosteroids remain undetermined. Among these, the 23-dehydrobrassinolide stereoisomers are the first brassinosteroids identified with an oxo group located at the C23 position. In addition, known brassinosteroids 3-dehydroteasterone, typhasterol and dolicholide were identified.
Cryptomeria japonica; 23-dehydrobrassinolide; 28-homobrassinolide; dolicholide; 28-homodolicholide

-21-
Isolation and Identification of Acetyl-CoA Carboxylase Inhibitors
from Green Tea (Camellia sinensis)

Jun WATANABE, Jun KAWABATA,・ and Ryoya NIKI

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

Received September 16, 1997
An aqueous methanol extract from green tea showed potent acetyl-CoA carboxylase inhibitory activity. An active compound was isolated from the extract and identified as (−)-epigallocatechin gallate by instrumental analyses. The IC50 value of (−)-epigallocatechin gallate was 3.1×10-4 M. Among tea catechins and related compounds, nearly equal activity was found in (−)-epigallocatechin gallate and (−)-epicatechin gallate, whereas (＋)-catechin, (−)-epicatechin, (−)-epigallocatechin, gallic acid and methyl gallate each had no inhibitory activity. These results indicate that the 3-O-gallate group of the catechin structure was necessary for this activity. (−)-Epigallocatechin gallate inhibited triglyceride accumulation in 3T3-L1 cells at a concentration of 1.0×10-7 M or higher.
acetyl-CoA carboxylase inhibitor; green tea; catechins

-22-
Role of N-Acetylglutamate Turnover in Urea Synthesis by Rats Treated
with the Thyroid Hormone

Kazutoshi HAYASE1, Yaeko NAGANUMA, Miho KOIE 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 16, 1997
We determined whether the synthesis and degradation of N-acetylglutamate would regulate urea synthesis when the thyroid status was manipulated. Experiments were done on three groups of rats, each being given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without a triiodothyronine (T3) treatment, treated with PTU＋T3, or receiving neither PTU nor T3 (control). The plasma concentration and urinary excretion of urea, the liver concentration of N-acetylglutamate, and the liver N-acetylglutamate synthesis in rats given PTU alone 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 given PTU without the T3 treatment. Treatment of the PTU-treated rats with T3 reversed the effects of PTU to the values of the control rats. N-Acetylglutamate synthesis in the liver was closely correlated with the excretion of urea, and inverse correlation between the liver N-acetylglutamate degradation and urea excretion was found. These results suggest that the greater synthesis and lower degradation of N-acetylglutamate in the hypothyroid (PTU alone) rats would be likely to increase the hepatic concentration of this compound and stimulate urea synthesis.
triiodothyronine; urea synthesis; N-acetylglutamate synthesis; N-acetylglutamate degradation

-23-
Suppression of the Lethal Effect of Acidic-Phospholipid Deficiency in
Escherichia coli by Bacillus subtilis Chromosomal Locus ypoP

Koichi INOUE, Atsuhiro KISHIMOTO,・ Motoo SUZUKI, Hiroshi MATSUZAKI,
Kouji MATSUMOTO, and Isao SHIBUYA・・

Department of Biochemistry and Molecular Biology, Saitama University, Shimo-Ohkubo, Urawa 338, Japan

Received September 18, 1997
An acidic-phospholipid deficiency caused by the pgsA3 allele that encodes a defective phosphatidylglycerophosphate synthase in Escherichia coli is lethal. The only known mutations that suppress this lethality fully have been related to the major outer-membrane lipoprotein. We isolated a Bacillus subtilis chromosomal locus that suppresses the lethality when harbored in a low copy-number plasmid, without restoring the synthase activity or phospholipid composition to normal. The locus was first recognized to suppress the conditional lethality of E. coli YA5512 (pgsA3) that harbored an unidentified mutation(s), allowing its growth in LB medium but not in media of lower osmolarities. The locus was then found to suppress the lethality of pgsA3 in wild-type E. coli W3110. This locus, named ypoP in the database, had 37％ nucleotide identity with the E. coli mprA gene, but the amplification of mprA had no suppressive effect. Plasmid pPOP1 containing ypoP completely prevented the decrease in the amount of a porin protein, OmpF, in the outer membrane and also cell mucoidy caused by pgsA3. The mechanisms underlying these unusual effects are discussed in relation to a putative stress signal(s) generated by the acidic-phospholipid deficiency.
acidic phospholipid; Bacillus subtilis; Escherichia coli; OmpF; phenotypic suppression

-24-
α2-Adrenoceptor-Mediated Antisecretory Effect of Hypoxia in Conscious Rats

Ryoichi YAMAJI, Yasuyo OHNISHI, Miki SAKAMOTO, Makoto TAKENOSHITA,
Mitsuaki OHTA,＊ Shingo TSUYAMA,＊＊ Fumio WATANABE,＊＊＊ Hiroshi INUI,
Kazutaka MIYATAKE, and Yoshihisa NAKANO・

Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
＊Department of Veterinary Physiology, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
＊＊Department of Veterinary Molecular Biology, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
＊＊＊Department of Foods and Nutrition, Kochi Women′s University, Kochi 780-8515, Japan

Received October 1, 1997
Gastric acid secretion is suppressed, resulting in a significant rise in gastric pH, when conscious rats are exposed to hypoxia (Yamaji et al., 1996). When adrenal medullectomized rats were exposed to moderate (10.5％ O2) hypoxia for 3 h, gastric acid secretion was restored to nearly the level in normoxia by the adrenal medullectomy. In severe (7.6％ O2) hypoxia, the operation also caused an increase in the level of gastric acid output, although the extent was lower than that under 10.5％ O2 hypoxic conditions. Gastric pH was normalized by the operation even with 7.6％ O2 hypoxia. Similar results were obtained when reserpine, which causes an adrenergic discharge, was administered. When an α2-adrenoceptor blocking agent, yohimbine, was administered, the inhibitory effect of 10.5％ and 7.6％ O2 hypoxia on gastric acid secretion was almost completely removed. However, neither prazosin (an α1-adrenoceptor blocker) nor propranolol (a β-adrenoceptor blocker) showed any significant effects on gastric acid output in hypoxia. These results indicate that acute hypoxia stimulated the adrenergic response from the adrenal medulla, and that gastric acid secretion was consequently suppressed through α2-adrenoceptor.
hypoxia; gastric acid secretion; α2-adrenoceptor; adrenal medullectomy; rats

-25-
Characterization of cDNAs Encoding Small and Large Subunits of ADP-Glucose Pyrophosphorylases from Watermelon (Citrullus vulgaris S.)・

In-Jung KIM, Hyung-Yeel KAHNG, and Won-I1 CHUNG＊

Department of Biological Sciences, Korea Advanced Institute of Science and Technology,
373-1, Kusong-dong, Yusong-gu, Taejon 305-701, Korea

Received November 11, 1997
Three cDNA clones encoding ADP-glucose pyrophosphorylases were isolated from a full red fruit cDNA library of watermelon (Citrullus vulgaris S.). Sequence analyses indicated that one clone, wms1, corresponds to the small subunit, and two clones, wml1 and wml2 (a partial gene), are the large subunits of AGPase. The presumed AGPase proteins encoded by wms1, wml1, and wml2 have 526, 526, and 481 amino acids, respectively. The protein sequences have the conserved amino acids important for the substrate or regulator binding site, with some variation.
　Developmental changes in the amounts of wms1, wml1, and wml2 transcripts in fruits were measured by northern blot analysis. Their expression levels decreased from the small green to medium green stages, then increased in accordance with fruit ripening, which was different from those of tomato and oriental melon.
ADP-glucose pyrophosphorylase; isoforms; developmental expression; watermelon (Citrullus vulgaris S.)

-26-
Note
Tomatine Content in Host and Transgenic Tomatoes by Absorptiometric
Measurement

Hiroyasu FURUI, Takahiro INAKUMA, Yukio ISHIGURO, and Makoto KISO＊

Research Institute, Kagome Co. Ltd., 17 Nishi-Tomiyama, Nishi-Nasuno-machi, Nasu-gun, Tochigi 329-27, Japan
＊Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-11, Japan

Received June 17, 1997
Tomatine is a steroidal glycoalkaloid in tomato plants (Lycopersicon esculentum) and other Lycopersicon and Solanum Species. Tomatine is used as an indicator to evaluate the safety of transgenic tomatoes by FDA in U.S.A. We have developed a facile and rapid method for absorptiometric measurement of the tomatine content. This method was used to measure the tomatine content of fruits of a transgenic tomato cultivar (Lycopersicon esculentum) which contained an antisense polygalacturonase gene (anti-PG). We found that the tomatine content in the fruits of transgenic and non-transgenic tomatoes was very similar. The data were also compared with those of other tomato cultivars, ``KAGOME 77′′ (L. esculentum) and ``KAGOME 88′′ (L. esculentum).
tomatine; transgenic tomato; absorptiometric measurement

-27-
Note
Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen,
via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene
in Transgenic Tobacco Plants

Kyu Whan CHOI, Oksoo HAN,＊,・ Hee Jae LEE,＊＊ Young Chae YUN,
Young Ho MOON, Myojeoung KIM,＊ Yong In KUK,＊＊＊ Sung Uk HAN,＊＊＊ and Ja Ock GUH＊＊＊

Plant Biotechnology Laboratory, Jinro Central Research Institute, Yongin, 449-910, Korea
＊Institute of Agricultural Science and Technology, Chonnam National University, Kwangju, 500-757, Korea
＊＊Institute of Biotechnology, Chonnam National University, Kwangju, 500-757, Korea
＊＊＊Department of Agronomy, Chonnam National University, Kwangju, 500-757, Korea

Received June 23, 1997
In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.
diphenyl ether herbicide; protoporphyrinogen oxidase; Bacillus subtilis; oxyfluorfen; transgenic tobacco plant

-28-
Note
Occurrence of Anserine as an Antioxidative Dipeptide in a Red Alga,
Porphyra yezoensis

Yoshiyuki TAMURA, Shigeo TAKENAKA, Sumi SUGIYAMA, and Reiko NAKAYAMA

Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, Sakai, Osaka 592, Japan

Received July 7, 1997
To examine the antioxidative compounds of nonprotein amino acids in a red alga, Porphyra yezoensis, an ethanol extract of the cultured thalli was fractionated with Dowex columns. The basic fraction V showed strong antioxidative capacities with ferric thiocyanate and TBARS measurements. In this basic fraction, histidine, 3-methylhistidine, carnosine, and anserine were detected beside ornithine, lysine, and arginine by amino acid analysis. The occurrence of carnosine and anserine suggests that the histidine-related compounds also contribute to the antioxidative reactions in P. yezoensis.
red alga; Porphyra yezoensis; antioxidant; carnosine; anserine

-29-
Note
cDNA Cloning, Expression, and Mutagenesis of Scytalone Dehydratase Needed for Pathogenicity of the Rice Blast Fungus, Pyricularia oryzae

Takayuki MOTOYAMA, Kinya IMANISHI,＊ and Isamu YAMAGUCHI・

The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
＊Yuki Research Centre, Nihon Bayer Agrochem Co., Ltd., Yuki, Ibaraki 307-0001, Japan

Received August 8, 1997
We purified scytalone dehydratase from the rice blast fungus, Pyricularia oryzae, and cloned its cDNA on the basis of its amino acid sequence. The deduced amino acid sequence was 62％ identical to the scytalone dehydratase from Colletotrichum lagenarium. The expression of this gene was induced transcriptionally in the stationary phase when melanin synthesis occurs. We constructed a heterologous expression system for the enzyme in Escherichia coli, did deletion analysis with this system, and found that a C-terminal region is essential for the enzyme function.
rice blast fungus; Pyricularia oryzae; melanin; scytalone dehydratase; pathogenicity

-30-
Note
A Non-radioassay of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase
by Anion-Exchange Chromatography

Koichi UEMURA,＊,・ Ayako SHIMOIDE,＊ and Akiho YOKOTA＊,＊＊,・・

＊Plant Molecular Physiology Laboratory, Research Institute of Innovative Technology for the Earth (RITE),
Kizu, Kyoto 619-02, Japan
＊＊Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST),
Ikoma, Nara 630-01, Japan

Received August 8, 1997
A non-radioisotopic assay method for ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is described. 3-Phosphoglycerate produced by the RuBisCO reaction was measured using an anion-exchange chromatography. The reaction course of spinach RuBisCO measured by this method was completely the same as that measured with 14CO2.
anion-exchange chromatography; assay; phosphoglycerate; ribulose 1,5-bisphosphate; ribulose 1,5-bisphosphate carboxylase/oxygenase

-31-
Note
Biological Activities of (1→3)-β-D-Glucans with Reducing Glucose Side Chains

Tadashi KIHO,＊ Mitsuhiro MATSUSHITA, Shigeyuki USUI, and Shigeo UKAI

Department of Hygienic Chemistry, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi,
Gifu 502, Japan

Received August 11, 1997
Newly synthesized (1→3)-β-D-glucans with reducing glucose side chains (6-O-glucopyranosylated curdlan and 3-O-glucopyranosylated curdlan, with glucose linked directly (except for anomeric carbon) had antitumor activity against mice sarcoma 180 in mice. The two glucans potentiated the reticuloendotheliai system and activated macrophages (increased their glucose consumption). The activity inducing tumor regressing factor of the glucan derivatives was stronger than a linear (1→3)-β-D-glucan (curdlan).
curdlan; 3-O-glucopyranosylated glucan; antitumor activity; immunomodulator

-32-
Note
Sequence of a cDNA Encoding Mouse Ribosomal Protein S14

Mijoung LEE, Intaek HWANG, Yunjaie CHOI,＊ and Myunggi BAIK1

Department of Genetic Engineering, Institute of Biotechnology, and Hormone Research Center,
College of Agriculture, Chonnam National University, Kwangju 500-757, Korea
＊Department of Animal Science and Technology, College of Agriculture and Life Sciences,
Seoul National University, Suwon 441-744, Korea

Received August 12, 1997
An involution-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as full-length cDNA encoding the 40S subunit of ribosomal protein S14 (rps14). Comparison of the deduced amino acid sequence to sequences of rps14 from humans, hamsters, and rats showed a conservation.
ribosomal protein S14 cDNA; mammary gland; mouse

-33-
Note
Inhibition of HIV-1 Protease by Water-Soluble Lignin-Like Substance
from an Edible Mushroom, Fuscoporia obliqua

Toshiaki ICHIMURA, Osamu WATANABE,＊ and Susumu MARUYAMA

National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology,
1-1 Higashi, Tsukuba, Ibaraki 305, Japan
＊Hokkaido Food Processing Research Center, Midorimachi, Bunkyodai, Ebetsu, Hokkaido 069, Japan

Received August 22, 1997
Activity that inhibited protease of human immunodeficiency virus type 1 was found in boiling water extracts of an edible mushroom, Fuscoporia obliqua. The active component was identified as a water-soluble lignin derivative of high molecular weight. Other polyphenols of low molecular weight and monomeric components of lignin did not inhibit the protease.
HIV-1 protease; lignin; mushroom

-34-
Note
Increasing Effect of Dietary Taurine on the Serum HDL-cholesterol
Concentration in Rats

Hideki MOCHIZUKI, Hiroaki ODA,＊ and Hidehiko YOKOGOSHI

School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
＊Department of Applied Biological Sciences, Nagoya University, Nogoya 464-8601, Japan

Received August 29, 1997
Taurine, 2-amino ethanesulfonic acid, is the major free intracellular amino acid present in many tissues and plays an important role in lipid metabolism such as that of bile acid conjugation for fat absorption. The effect of taurine on the serum cholesterol level in normal rats was investigated. Taurine enhanced the serum HDL-cholesterol concentration in a dose-dependent manner without any change in total cholesterol.
taurine; serum cholesterol; HDL-cholesterol; normal rat

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Note
Increased Conversion Ratio of Tryptophan to Niacin in Severe Food Restriction

Katsumi SHIBATA, Takako KONDO, and Atsuyo MIKI

Department of Human Health Science, Faculty of Human Sciences, Osaka International University for Women, Moriguchi, Osaka 570, Japan

Received September 3, 1997
The effect of food restriction on the conversion ratio of tryptophan to niacin was investigated, because it is known that the conversion ratio is influenced by nutritional factors. A 20％ casein diet was fed to rats ad libitum (control), 1/2 the food of the control. 1/4 the food of the control, or starved for 9 days, and urine samples were collected to measure the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, and nicotinamide. The conversion ratio in the 1/2, 1/4, or starving group increased at day 1 of the experiment, but returned to the original value from day 2. Only in the starving group did the conversion ratio extremely increase from day 6 to day 9, being about 5-times higher than that of the original value on day 9. The possible mechanism by which the conversion ratio increased during food restriction is discussed.
tryptophan metabolism; nicotinamide; energy restriction; niacin; rat

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Note
Isolation and Characterization of Chitinase Isoforms from the Bulbs of
Four Species of the Genus Tulipa

Takeshi YAMAGAMI,・ Toki TAIRA, Yoichi ASO, and Masatsune ISHIGURO

Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

Received September 22, 1997
Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15--17 Cys residues/mol), II (5--8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.
chitinase; Tulipa bakeri; Tulipa tarda; Tulipa turkestanica; Tulipa praestans

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Note
Enzymatic Synthesis of 6-O-α-D-Galactopyranosyl-1-deoxynojirimycin
Using α-Galactosidase from Green Coffee Beans

Nam Soo PAEK,・ Dae Jung KANG, Hong Sub LEE, Jung Joon LEE,＊
Yong Jin CHOI,＊＊ Tae Han KIM, and Kee Won KIM

IlDong Pharmaceutical Co., Research Laboratories, Shinkeonjiri, Ansung-eup, Ansung-kun,
Kyongki-do 60-1, Korea
＊Korea Research Institute of Bioscience and Biotechnology, KIST Taejion 305-600, Korea
＊＊Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea

Received September 26, 1997
A transgalactosylation reaction from p-nitrophenyl-α-D-galactopyranoside to 1-deoxynojirimycin was done using α-galactosidase [EC 3.2.1.22] from green coffee beans. The enzyme formed 6-O-α-D-galactopyranosyl-1-deoxynojirimycin as the major product.
1-deoxynojirimycin;　　α　　-galactosidase;　　6-O-　　α-D-galactopyranosyl-1-deoxynojirimycin

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Note
A Convenient and Efficient Synthesis of Sialyl Lewis X・

Kunihisa BABA, Noriyuki IWATA, Hitoshi HAMAJIMA, Takao IKAMI,＊
Hideharu ISHIDA,＊＊ Akira HASEGAWA,＊＊ and Makoto KISO＊＊

Drug Discovery Research Laboratory, Sanwa Kagaku Kenkyusho Co. Ltd., 363 Shiosaki, Hokusei-cho,
Inabe-gun, Mie 511-04, Japan
＊＊Department of Applied Bioorganic Chemistry, Gifu University, Gifu 501-11, Japan

Received September 29, 1997
A convenient synthesis of the sialyl Lewis X (sLex) tetrasaccharide, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc (8), as a carbohydrate ligand for selectins is described. The key step is the reaction between NeuAcα2-3GalSMe (5) as a glycosyl donor and the suitably protected Fucα1-3GlcNAc derivative (4) as the glycosyl acceptor by using dimethyl(methylthio)sulfonium triflate (DMTST) as the promoter.
selectin; glycosylation; sialyl Lewis X

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Note
Involvement of Ovotransferrin in the Thermally Induced Gelation
of Egg White at around 65°C

Honami YAMASHITA, Junko ISHIBASHI, Youn-Ho HONG,＊ and Masaaki HIROSE＊＊

The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

Received September 30, 1997
Egg white forms a soft opaque gel at around 65°C. An analysis of the turbidity appearance in the presence or absence of iron and polyacrylamide gel electrophoresis of the precipitated proteins after thermal treatment revealed ovotransferrin to be the major component involved in thermal gelation at around 65°C. The storage modulus of the thermally induced gel of purified ovotransferrin reinforced this conclusion.
ovotransferrin; egg white gelation; thermal gelation; ovotransferrin gelation; egg white protein

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Note
Structural Diversity of the Membrane Core Lipids of Extreme Halophiles

Mikio MORITA, Noriaki YAMAUCHI, Tadashi EGUCHI, and Katsumi KAKINUMA・

Department of Chemistry, Tokyo Institute of Technology O-okayama, Meguro-ku, Tokyo 152-8551, Japan

Received Octorber 3, 1997
The structural diversity of the core lipids of extreme halophiles Haloarcula japonica and Halobacterium halobium was investigated. The most significant difference is that Ha. japonica contains sn-2,3-di-O-phytanylglycerol exclusively as the core lipid, whereas Hb. halobium contains both sn-2,3-di-O-phytanylglycerol and sn-2-O-sesterterpanyl (3,7,11,15,19-pentamethyleicosanyl)-3-O-phytanylglycerol.
archaea; halophile; membrane lipid

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Note
Improved Synthesis of the C8--C23 Segment of Aplysiatoxins via Asymmetric
Dihydroxylation of the Chiral Homoallylic Alcohol

Hiroaki TOSHIMA and Akitami ICHIHARA
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

Received October 3, 1997
Important intermediate 2 of the segment (3) possessing four sequential asymmetric centers (C9--C12) of aplysiatoxins was synthesized via dihydroxylation of 5 in only 7 steps from methyl (S)-3-hydroxy-2-methylpropionate. Another promising compound (10a) was also synthesized in only 6 steps via asymmetric dihydroxylation of chiral homoallylic alcohol 4 as the key step.
aplysiatoxin; tumor promoter; protein kinase C; crotylboration; asymmetric dihydroxylation

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Note
Effects of Hydrodynamic Volume of Anionic Lipopolysaccharide,
Emulsan, on Emulsifying Activity

Pil KIM, Seon Won KIM, Sang Yong KIM,＊ and Jung Hoe KIM・

Department of Biological Sciences, Korea Advanced Institute of Science and Technology,
Taejon 305-701, Korea
＊R＆D Center, Dong Yang Confectionery Corp., Seoul 140-715, Korea

Received October 9, 1997
To understand the structure-function relationship of an anionic lipopolysaccharide emulsan, the effect of hydrodynamic volume on the emulsifying activity was investigated. As a result, it was found that the hydrodynamic volume of emulsan was an important factor in its emulsifying activity. The hydrodynamic volume was decreased by the addition of a positively charged polypeptide, and the emulsifying activity was decreased, but negatively charged or uncharged polypeptide had little effect. These results suggest that the conformation of the backbone of emulsan helps to govern its emulsifying activity.
emulsan; biosurfactant; lipopolysaccharide; conformation; emulsifying activity

-43-
Short Communication
Sweetness of Lysozymes

Kenji MAEHASHI and Shigezo UDAKA・

Department of Brewing and Fermentation, Tokyo University of Agriculture, 1-1 Sakuragaoka,
Setagaya-ku, Tokyo 156, Japan

Received September 29, 1997
While examining the taste of various proteins, we found that hen egg-white lysozyme, a bacteriolytic enzyme, had sweetness. Lysozymes from other sources such as turkey and soft-shelled turtle also showed sweetness with different tastes, heavy or light. In contrast, human lysozyme was tasteless. The amino acid sequences of the various lysozymes were similar to that of hen lysozyme, but hen lysozyme did not show significant homology to sweet proteins.
lysozyme; sweet protein; sweet taste

-44-
Short Communication
Field and Electroantennogram Responses of the Pine Sawfly,
Diprion nipponica, to Chiral Synthetic Pheromone Candidates

Akira TAI,・ Yasutomo HIGASHIURA,＊ Masashi KAKIZAKI,＊＊
Tikahiko NAITO,＊＊＊ Kazuki TANAKA, Morifumi FUJITA,
Takashi SUGIMURA, Hideho HARA,＊ and Naotaka HAYASHI＊

Faculty of Science, Himeji Institute of Technology, 1475-2 Kanaji, Kamigori, Ako-gun,
Hyogo 678-1297, Japan
＊Hokkaido Forestry Research Institute, Koshunai, Bibai, Hokkaido 079-0198, Japan
＊＊Hokkaido Central Agricultural Experiment Station, Naganuma, Hokkaido 069-1300, Japan
＊＊＊Faculty of Agriculture, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-0013, Japan

Received September 29, 1997
(1S, 2R, 6RS)-1,2,6-Trimethyldecyl propionate, a lower homolog of the sex pheromone of known sawflies, strongly attracted Diprion nipponica, a popular species in Japan.
pine sawfly; Diprion nipponica; EAD; field assay; pheromone

-45-
Short Communication
Green Tea Suppresses D-Galactosamine-Induced Liver Injury in Rats

Kimio SUGIYAMA,＃ Puming HE, Shingo WADA, Fumi TAMAKI,
and Shigeru SAEKI＊

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422, Japan
＊Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Osaka 558, Japan

Received October 14, 1997
Dietary supplementation with powder of a green tea extract suppressed the enhancement of plasma alanine aminotransferase and aspartate aminotransferase activities induced by D-galactosamine, but not by carbon tetrachloride, in a dose-dependent manner in rats. The minimum dose to cause a significant effect was 1 to 2％. Drinking green tea also suppressed plasma enzyme activities. These results indicate that green tea had a liver injury-preventive effect.
green tea; liver injury; D-galactosamine; carbon tetrachloride; rat

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