Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.3 2001)

Hepatic Mitochondrial Proteins in Congenitally Hyperammonemic spf Mice:
Effect of Acetyl-L-carnitine
Rachid BENZERROUK and Ijaz A. QURESHI 　p.495

Purification, Characterization, and Molecular Cloning of a Chitinase
from the Seeds of Benincasa hispida
Chao-Yun T. SHIH,1,・・・ Anwar A. KHAN,2 Shifang JIA,1,＊ Junlin WU,2,・・・ and Ding S. SHIH2　p.501

DNA Synthesis and Fragmentation in Bacteroids during Astragalus sinicus
Root Nodule Development
Hajime KOBAYASHI, Michihiro SUNAKO, Makoto HAYASHI＊, and Yoshikatsu MUROOKA　p.510

Natto Mucilage Containing Poly-γ-glutamic Acid Increases Soluble Calcium
in the Rat Small Intestine
Hiroyuki TANIMOTO,1,・・・ Masato MORI,2 Masao MOTOKI,1 Kunio TORII,2
Motoni KADOWAKI,3,＊ and Tadashi NOGUCHI3　p.516

Improving the Freeze Tolerance of Bakers′ Yeast by Loading with Trehalose
Reiko HIRASAWA, Kumio YOKOIGAWA,＊ Yuka ISOBE, and Hiroyasu KAWAI　p.522

Purification and Characterization of an β-D-Xylosidase
from Candida utilis IFO 0639
Takaaki YANAI and Michikatsu SATO・・・　p.527

Involvement of Caspase-3 in Apoptosis Induced by Viscum album var.
coloratum Agglutinin in HL-60 Cells
Su Yun LYU, Won Bong PARK,・・・ Kyung Hee CHOI,＊ and Won Ho KIM＊　p.534

Inhibitory Effects of Ellagi- and Gallotannins on Rat Intestinal
α-Glucosidase Complexes
Miou TODA, Jun KAWABATA,・・・ and Takanori KASAI　p.542

Sequence of the Clostridium thermocellum Mannanase Gene man26B
and Characterization of the Translated Product
Junji KUROKAWA, Eiakalak HEMJINDA, Takamitsu ARAI, Shuichi KARITA,＊
Tetsuya KIMURA, Kazuo SAKKA,・・・ and Kunio OHMIYA　p.548

Mechanism of Growth Inhibition by Tungsten in Acidithiobacillus ferrooxidans
Tsuyoshi SUGIO,1,・・・ Hiroyuki KUWANO,1 Atsunori NEGISHI,2 Terunobu MAEDA,2
Fumiaki TAKEUCHI,3 and Kazuo KAMIMURA4　p.555

Cloning and Expression of cDNA Encoding the Complete Prepro-Form
of an Isoform of Der f 1, the Major Group 1 Allergen
from House Dust Mite Dermatophagoides farinae
Takaomi YASUHARA, Toshiro TAKAI,・・・ Toshifumi YUUKI, Hirokazu OKUDAIRA,＊
and Yasushi OKUMURA　p.563

Investigation of Various Genotype Characteristics for Inosine Accumulation
in Escherichia coli W3110
Hiroshi MATSUI,・・・ Hisashi KAWASAKI, Megumi SHIMAOKA, and Osamu KURAHASHI　p.570

Green Marker for Colonies of Bacillus subtilis
Mitsuhiro ITAYA, Syed M. SHAHEDUZZAMAN,・・・ Kuniko MATSUI, Akira OMORI,
and Takashi TSUJI　p.579

Purification and Characterization of Goose Type Lysozyme
from Cassowary (Casuarius casuarius) Egg White
Sompong THAMMASIRIRAK, Takao TORIKATA, Kazutoshi TAKAMI,1
Koichi MURATA,2 and Tomohiro ARAKI・・・　p.584

Oxidative Stress by Visible Light Irradiation Suppresses Immunoglobulin
Production in Mouse Spleen Lymphocytes
Yoshiyuki MIYAZAKI, Masao YAMASAKI, Hiroko MISHIMA, Keiko MANSHO,
Hirofumi TACHIBANA, and Koji YAMADA・・・　p.593

Effects of 2,3-Diketo-L-gulonic Acid on the Oxidation of Yolk Lipoprotein
Meihua LI,a Emiko SUZUKI,b and Tadao KURATAa,・・・　p.599

Expression Pattern of the CsPK3 Auxin-Responsive Protein Kinase Gene
Makiko CHONO,1,2 Yoshihito SUZUKI,1 Keisuke NEMOTO,3 Hisakazu YAMANE,4
Noboru MUROFUSHI,5 and Isomaro YAMAGUCHI1　p.605

Improving Effect of Feeding with a Phosphorylated Guar Gum Hydrolysate
on Calcium Absorption Impaired by Ovariectomy in Rats
Osamu WATANABE,1,・・・ Hiroshi HARA,2 Yoritaka AOYAMA,2 and Takanori KASAI2　p.613

Structures of Thermoactinomyces vulgaris R-47 α-Amylase II Complexed
with Substrate Analogues
Takehiro YOKOTA,1 Takashi TONOZUKA,1 Yoichiro SHIMURA,1 Kazuhiro ICHIKAWA,1
Shigehiro KAMITORI,2 and Yoshiyuki SAKANO1,・・・　p.619

Characterization and High-level Production of D-Amino Acid Oxidase
in Candida boidinii
Hiroya YURIMOTO, Tetsuya HASEGAWA, Yasuyoshi SAKAI,・・・ and Nobuo KATO　p.627

Resolution and Synthesis of Optically Active Alcohols with Immobilized
Ovalbumin and Pea Protein as New Bio-catalysts
Hiroyuki NAGAOKA,1 Hiroshi KAYAHARA,2 and Yasushi WAKABAYASHI2　p.634

Capsaicin Increases Modulation of Sympathetic Nerve Activity in Rats:
Measurement Using Power Spectral Analysis of Heart Rate Fluctuations
Koichiro OHNUKI, Toshio MORITANI, Kengo ISHIHARA, and Tohru FUSHIKI　p.638

ATP Production from Adenine by a Self-coupling Enzymatic Process:
High-level Accumulation under Ammonium-limited Conditions
Akihiko MARUYAMA and Tatsuro FUJIO＊　p.644

Note
Inhibition of Telomerase Activity by Fungus Metabolites, CRM646-A
and Thielavin B
Ken-ichi TOGASHI,1,・・・・・・ Hack-Ryong KO,1,2 Jong-Seog AHN,2 and Hiroyuki OSADA1,・・・　p.651

Note
Isolation and Characterizartion of Alginic Acid from Commercially
Cultured Nemacystus decipiens (Itomozuku)
Masakuni TAKO,・・・ Seiki KIYUNA, Shuntoku UECHI, and Fujiya HONGO　p.654

Note
Purification and Characterization of Mannose Isomerase
from Agrobacterium radiobacter M-1
Jun HIROSE,・・・ Kazuhiko MAEDA, Haruhiko YOKOI, and Yoshiyuki TAKASAKI　p.568

Note
Polymorphism in Rice Amylases at an Early Stage of Seed Germination
Shin-ichiro MITSUNAGA, Osamu KAWAKAMI，Tomoya NUMATA，Junji YAMAGUCHI，
Kiichi FUKUI，and Toshiaki MITSUI　p.662

Note
Characterization of the Gene Encoding the β-Lactamase of the Psychrophilic
Marine Bacterium Moritella marina strain MP-1
Mika TANAKA，Hidetoshi OKUYAMA，and Naoki MORITA　p.666

Note
Suppression of Lipopolysaccharide-induced Liver Injury by Various Types
of Tea and Coffee in D-Galactosamine-sensitized Rats
Puming HE, Yasuhiro NODA, and Kimio SUGIYAMA・・・　p.670

Note
Suppressive Effect of Caffeine on Hepatitis and Apoptosis Induced by Tumor
Necrosis Factor-α, but Not by the Anti-Fas Antibody, in Mice
Kimio SUGIYAMA,・・・ Yasuhiro NODA, and Puming HE　p.674

Note
Molecular Cloning of Three Genes Encoding G Protein Alpha Subunits
in the White Root Rot Fungus, Rosellinia necatrix
Tadanori AIMI, Sanae KANO, Qian WANG, and Tsutomu MORINAGA・・・　p.678

Note
Antiviral Activity of Fattiviracin FV-8 against Human Immunodeficiency
Virus Type 1 (HIV-1)
El-Sayed E. HABIB,1 Kazumi YOKOMIZO,1 Kazuhiko NAGAO,2
Shinji HARADA,2 and Masaru UYEDA1,・・・　p.683

Note
A Lectin from an Ascomycete Mushroom, Melastiza chateri:
No Synthesis of the Lectin in Mycelial Isolate
Shigeru OGAWA, Yumi OTTA, Akikazu ANDO, and Yoshiho NAGATA・・・　p.686

Note
Piezoresponse of the cyo-Operon Coding for Quinol Oxidase Subunits
in a Deep-sea Piezophilic Bacterium, Shewanella violacea
Kaoru NAKASONE,＊,・・・ Mitsunori YAMADA,＊,＊＊ Mohammad Hassan QURESHI,＊,＊＊＊
Chiaki KATO,＊ and Koki HORIKOSHI＊　p.690

Note
Reactivities of Mutants of a Major House Dust Mite Allergen Der f 2 to Mouse
Anti-Der f 2 Monoclonal Antibodies Analyzed by Immunoblotting
Toshiro TAKAI,・・・ Midori AKAGAWA-CHIHARA, Toyokazu YOKOTA, and Yasushi OKUMURA　p.694

Note
Thermally Induced Changes of Lipoate Acetyltransferase Inner Core Isolated
from the Bacillus stearothermophilus Pyruvate Dehydrogenase Complex
Yoichi ASO, Akihiro NAKAJIMA, Kohji MENO, and Masatsune ISHIGURO　p.698

Note
Autoproteolytic Processing of Aspartic Proteinase from Sunflower Seeds
Hyekyeong PARK, Isao KUSAKABE, Yoshikiyo SAKAKIBARA,＊ and Hideyuki KOBAYASHI＊,・・・　p.702

Note
Chemical and Nutritional Properties of Hypoallergenic Wheat Flour
Megumi MORIYAMA,＊ Chiyoko TOKUE,＊ Hideko OGIWARA,＊＊
Hiroko KIMURA,＊＊ and Soichi ARAI＊,・・・　p.706

Note
New Quinoline Alkaloids from the Leaves and Stems of Orixa japonica,
Orijanone, Isopteleflorine and 3'-O-Methylorixine
Toshiro NOSHITA, Masumi TANDO, Kadzuo SUZUKI, Kiyoshi MURATA,§ and Shinji FUNAYAMA・・・　p.710

Note
Unique Occurrence of the 1CF11 Carbohydrate Epitope in Primate Saliva
Shan-Ming YANG,1 Naoko YOKOI,1 Yoshihiro KANAMARU,1,・・・ Osamu TAKENAKA,2
Yasuro ATOJI,3 Yasuo BUNAI,4 Isao OHYA,4 Xong-Guang SONG,1 Satoshi NAGAOKA,1
Makoto SHIMIZU,5 and Goverdhan P. SACHDEV6　p.714

Note
Primary Structure and Phylogenetic Analysis of the Coat Protein
of a Toyama Isolate of Tobacco Necrosis Virus
Kazuhiko SAEKI,・・・ Yasuhiro TAKAHASHI, Hirozo OH-OKA, Tatsumi UMEOKA,
Yutaka ODA, and Keiichi FUKUYAMA　p.719

Note
The est1 Regulation Depends on the Oxygen Concentration
in Acetobacter pasteurianus
Yasuhiro KASHIMA,1,・・・ Yusuke NAKAJIMA,1 Akihiko KOSUGI,1 Kenji TAYAMA,2
Yukimichi KOIZUMI,1 Shigezo UDAKA,1 and Fujiharu YANAGIDA1　p.725

Note
MUP1, High Affinity Methionine Permease, is Involved in Cysteine Uptake
by Saccharomyces cerevisiae
Akihiko KOSUGI,＊ Yukimichi KOIZUMI, Fujiharu YANAGIDA, and Shigezo UDAKA　p.728

Note
Synthesis of Gibbilimbols A-D, Cytotoxic and Antibacterial Alkenylphenols
Isolated from Piper gibbilimbum
Yumi ABE,a Hirosato TAKIKAWA,b and Kenji MORIa,b・・・　p.732

Preliminary Communication
Designing Potent Derivatives of Ovokinin(2--7), an Anti-hypertensive
Peptide Derived from Ovalbumin
Nobuyuki MATOBA, Yuko YAMADA, Hachiro USUI, Ryusuke NAKAGIRI,・・・
and Masaaki YOSHIKAWA　p.736

Preliminary Communication
Structures of Azaspiracid Analogs, Azaspiracid-4 and Azaspiracid-5,
Causative Toxins of Azaspiracid Poisoning in Europe
Katsuya OFUJI,1 Masayuki SATAKE,1,・・・ Terry MCMAHON,2 Kevin J. JAMES,3
Hideo NAOKI,4 Yasukatsu OSHIMA,1 and Takeshi YASUMOTO1　p.740

-1-
Cloning, Expression, and Characterization of a Family 52 β-Xylosidase Gene
(xysB) of a Multiple-xylanase-producing Bacterium, Aeromonas caviae ME-1

Tohru SUZUKI,＊,1 Emiko KITAGAWA,2 Fukumitsu SAKAKIBARA,2 Keiji IBATA,2
Kengo USUI,2 and Keiichi KAWAI2

1Molecular Genetics Reseaech Center,
2Department of Biotechnology, Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

Received March 27, 2000; Accepted November 10, 2000
A λ phage genomic library of Aeromonas caviae ME-1, a multiple-xylanase-producing bacterium, was screened for xylan degradation activities. We isolated one clone, B65, which had weak xylanase activity, by the DNS method, but gave no visible bands on zymogram assay using SDS-xylan-PAGE. Based on TLC analyses of enzymatic products and some glycosidase assays using p-nitrophenyl substrates, we established that pB65 encodes a β-xylosidase gene. In the nucleotide sequence analysis, we found a 2190-bp open reading frame (ORF) named xysB. XysB protein is similar to some β-xylosidases, which are categorized in the glycosyl hydrolase family 52. Another ORF (xyg), that showed similarity to the family 67 α-glucuronidase, was also found downstream of the xysB gene. The xysB ORF and its promoter region were cloned into the pT7-Blue vector and the transformant cells had β-xylosidase activity. The relative molecular mass were estimated to be 75 kDa by SDS-PAGE and 159 kDa by gel filtration. These data showed that XysB has a dimeric structure of 80,697 Da subunits. This enzyme showed optimal activity at 50°C and pH 6.0. It was stable below 40°C and pH 5--8. The Km and Vmax were calculated to be 0.34 mM and 33 nmol・min−1・μg--1, respectively. This enzyme also showed transglycosylation activity against X3 and produced X4 and X5.
Key words: Aeromonas caviae ME-1; β-Xylosidase; xysB; glycosyl hydrolase family 52; α-glucuronidase

-2-
Hepatic Mitochondrial Proteins in Congenitally Hyperammonemic spf Mice:
Effect of Acetyl-L-carnitine

Rachid BENZERROUK and Ijaz A. QURESHI

Division of Medical Genetics, Sainte-Justine Hospital and Department of Pediatrics,
University of Montreal, Montreal, Quebec, H3T 1C5, Canada

Received April 18, 2000; Accepted October 30, 2000
The sparse-fur (spf) mutant mouse has an X-linked deficiency of hepatic ornithine transcarbamylase (OTC), and develops hyperammonemia immediately after weaning and maintains it throughout its life span. We have studied the effects of acetyl-L-carnitine (ALCAR) on the hepatic mitochondrial proteins of the chronically hyperammonemic spf mice. Two different age groups of mice were studied, the weanlings (3 weeks) and the adult mice (8 weeks). Our results indicate that in the mitochondrial matrix, the untreated chronic hyperammonemia induced a significant increase in the quantity of 54.4-kDa protein in spf adult mice. After ALCAR treatment, in spf adult mice, the quantities of the 54.4-kDa, 63.8-kDa, and 129-kDa matrix proteins were significantly increased. In the mitochondrial inner membrane fraction of the spf weanling mice, a 53.5-kDa protein was significantly increased by ALCAR treatment. Our results show that: (a) chronic hyperammonemia has altered the mitochondrial matrix protein profile in spf mice, that (b) ALCAR has a modulating effect on various matrix and inner membrane proteins, and that (c) there was no effect of hyperammonemia or ALCAR treatment on the outer membrane proteins.
Key words: sparse-fur mouse; congenital hyperammonemia; mitochondrial matrix proteins; mitochondrial membrane proteins; acetyl-L-carnitine

-3-
Purification, Characterization, and Molecular Cloning of a Chitinase
from the Seeds of Benincasa hispida

Chao-Yun T. SHIH,1,・・・ Anwar A. KHAN,2 Shifang JIA,1,＊ Junlin WU,2,・・・ and Ding S. SHIH2

1RCMI Program, Health Research Center, Department of Biology, Southern University,
Baton Rouge, LA 70813, U.S.A.
2Department of Biological Sciences, Louisiana State University and LSU Agricultural Center,
Baton Rouge, LA 70803 U.S.A.

Received July 3, 2000; Accepted November 15, 2000
A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.
Key words: chitinase; chitinase gene; pathogenesis-related protein; white gourd; Benincasa hispida

-4-
DNA Synthesis and Fragmentation in Bacteroids during Astragalus sinicus
Root Nodule Development

Hajime KOBAYASHI, Michihiro SUNAKO, Makoto HAYASHI＊, and Yoshikatsu MUROOKA

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka,
Suita, Osaka 565-0871, Japan

Received July 18, 2000; Accepted October 26, 2000
Histo- and cytochemical techniques were used to study the DNA replication and fragmentation patterns in bacteroids formed by Mesorhizobium huakuii subsp. rengei in nodules of Astragalus sinicus. DNA replication was detected by the incorporation of 5-bromo deoxy-uridine. Signals denoting DNA synthesis were observed in plant nuclei within the nodule meristem and in bacteroids near the meristem. The TUNEL (TdT-mediated dUTP nick-end labeling) assay was used to measure DNA fragmentation. In nutrient-depleted 1-mpi (month(s) post inoculation) nodule sections, some bacteroids were in vacuoles, and DNA fragmentation signals were observed only in such bacteroids. In contrast, 1-mpi nodule sections without nutrient depletion showed neither bacteroid localization in vacuoles nor DNA fragmentation signals. The bacteroid translocation into vacuoles upon nutrient starvation might results from autophagy of the plant. In 2-mpi nodule sections, bacteroids with DNA fragmentation signals appeared within the cytoplasm of some nodule cells in the senescence zone.
Key words: Astragalus sinicus (renge-soh); bacteroid; nodule; Mesorhizobium huakuii subsp. rengei; symbiosis

-5-
Natto Mucilage Containing Poly-γ-glutamic Acid Increases Soluble Calcium
in the Rat Small Intestine

Hiroyuki TANIMOTO,1,・・・ Masato MORI,2 Masao MOTOKI,1 Kunio TORII,2
Motoni KADOWAKI,3,＊ and Tadashi NOGUCHI3

1Food Research ＆ Development Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku,
Kawasaki 210-8681, Japan
2Basic Research Laboratories, Central Research Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho,
Kawasaki-ku, Kawasaki 210-8681, Japan
3Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received July 19, 2000; Accepted November 27, 2000
We prepared natto (fermented soybeans) mucilage containing poly-γ-glutamic acid (γ-PGA) from commercial natto. The effect of natto mucilage on calcium (Ca) solubility in vitro and in vivo was investigated. Ca solubility in vitro increased with an increase in the amount of natto mucilage, due to inhibition of the formation of an insoluble complex of Ca with phosphate by natto mucilage.
Rats were fed with 5 g of soybean protein isolate, natto, mucilage-free natto, or natto mucilage diet for 1.5 h. Small intestinal contents were collected 2.5 h after ingestion. In the lower half of the small intestine, both the amount and the percentage of soluble Ca of intestinal contents were significantly higher (P<0.001) in rats fed with natto mucilage diet than in those fed with the other diets. Natto mucilage also increased Ca solubility in vivo. These results suggested that γ-PGA is responsible for the increasing effect of natto mucilage on Ca solubility.
Key words: poly-γ-glutamic acid; natto mucilage; calcium solubility; calcium absorption; rat small intestine

-6-
Improving the Freeze Tolerance of Bakers′ Yeast by Loading with Trehalose

Reiko HIRASAWA, Kumio YOKOIGAWA,＊ Yuka ISOBE, and Hiroyasu KAWAI

Department of Food Science and Nutrition, Nara Women′s University, Nara 630-8263, Japan

Received July 31, 2000; Accepted November 6, 2000
We examined the freeze tolerance of bakers′ yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers′ yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers′ yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200--250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers′ yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers′ yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers′ yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers′ yeast was more efficiently improved than that of the freeze-tolerant type.
Key words: bakers′ yeast; trehalose; freeze-tolerance; leavening ability

-7-
Purification and Characterization of an β-D-Xylosidase
from Candida utilis IFO 0639

Takaaki YANAI and Michikatsu SATO・・・

Mercian Corporation, Wine ＆ Spirits Research Institute, 9-1, Johnan 4-chome, Fujisawa 251-0057, Japan

Received August 31, 2000; Accepted November 14, 2000
An intracellular β-D-xylosidase from Candida utilis IFO 0639 was purified to homogeneity through four chromatographic steps. The molecular mass of the enzyme was estimated to be 92 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.6, and was most active at pH 6.0 and at around 40°C. Ethanol at an optimal concentration (10％, v/v) stimulated the initial enzyme activity by 57％. D-Xylose, the product of the β-D-xylosidase, has no effect on the enzyme activity at 300 mM. The β-D-xylosidase was highly specific to the β-D-xylopyranoside configuration. The enzyme hydrolyzed β-1, 4-linked xylo-oligosaccharides with chain lengths from 2 to 5 by releasing xylose from the non-reducing end. It showed no activity against xylan. The enzyme efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. The fermentation of Muscat juice coupled with the enzyme addition produced a small increase in the concentration of monoterpenols.
Key words: β-D-xylosidase; Candida utilis; aroma improvement; monoterpenol

-8-
Involvement of Caspase-3 in Apoptosis Induced by Viscum album var.
coloratum Agglutinin in HL-60 Cells

Su Yun LYU, Won Bong PARK,・・・ Kyung Hee CHOI,＊ and Won Ho KIM＊

College of Natural Sciences, Seoul Women′s University, Seoul 139-774, Korea
＊College of Natural Sciences, Chung-Ang University, Seoul 156-756, Korea

Received September 1, 2000; Accepted November 7, 2000
A cytotoxic lectin (Viscum album L. coloratum agglutinin, VCA) from Korean mistletoe was isolated by affinity chromatography on Sepharose 4B immobilized with asialofetuin. In HL-60 cells, addition of VCA resulted in a dose- and time-dependent growth suppression, morphological changes of apoptotic nuclei, and DNA fragmentation characteristics of apoptosis. To investigate how caspase-3 activation during VCA-induced apoptosis induces cleavages of PARP, the expression of PARP and the pattern of caspase-3 activation in HL-60 cells were investigated. The native and processed PARP forms typically seen in apoptotic cells were observed, and a decrease in expression of the 32-kDa form of caspase-3 in a dose-dependent manner was observed. The VCA-induced apoptosis was significantly inhibited by a caspase-3 specific inhibitor, z-DEVD-FMK, and the PARP processing and caspase-3 activation were also inhibited by the inhibitor. A possible involvement of cell cycle arrest in VCA-induced apoptosis was investigated by flow cytometry and the results suggested that the apoptotic effect of VCA is not involved in the induction of cell cycle arrest.
Key words: Korean mistletoe; lectin; apoptosis; caspase-3; poly(ADP-ribose)polymerase

-9-
Inhibitory Effects of Ellagi- and Gallotannins on Rat Intestinal
α-Glucosidase Complexes

Miou TODA, Jun KAWABATA,・・・ and Takanori KASAI

Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kitaku, Sapporo 060-8589, Japan

Received September 4, 2000; Accepted October 26, 2000
The clove ellagitannins and their related polygalloylglucoses inhibited maltase activity of rat intestinal α-glucosidases. The structure-activity relationship study of those galloylglucoses, varying the extent of galloylation on the glucose core, with the ellagitannins, indicated that an increasing number of galloyl units in the molecule lead to an increase in the inhibitory activity. Penta-O-galloyl-β-D-glucose, with five galloyl groups showed the highest inhibitory activity. On the other hand, hexahydroxydiphenoyl units contained in the ellagitannins had little effect on the activity. After separation of maltase-glucoamylase and sucrase-isomaltase complexes from the crude mixture of the rat α-glucosidases, the inhibitory activities of the galloylglucose derivatives against each complex were examined. The inhibitory influence on the maltase-glucoamylase complex was more potent than on the sucrase-isomaltase complex.
Key words: α-glucosidase; inhibitor; galloyl; hexahydroxydiphenoyl; hydrolyzable tannin

-10-
Sequence of the Clostridium thermocellum Mannanase Gene man26B
and Characterization of the Translated Product

Junji KUROKAWA, Eiakalak HEMJINDA, Takamitsu ARAI, Shuichi KARITA,＊
Tetsuya KIMURA, Kazuo SAKKA,・・・ and Kunio OHMIYA

Faculty of Bioresources and ＊Center for Molecular Biology and Genetics, Mie University, Tsu 514-8507, Japan

Received September 4, 2000; Accepted November 1, 2000
The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.
Key words: Clostridium thermocellum; mannanase; cellulosome

-11-
Mechanism of Growth Inhibition by Tungsten in Acidithiobacillus ferrooxidans

Tsuyoshi SUGIO,1,・・・ Hiroyuki KUWANO,1 Atsunori NEGISHI,2 Terunobu MAEDA,2
Fumiaki TAKEUCHI,3 and Kazuo KAMIMURA4

1Science and Technology for Energy Converwion, Okayama University, Tsushima Naka,
Okayama 700-8530, Japan
2Technical Research Institute, Hazama Corporation, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan
3Administration Center for Environmental Science and Technology, Japan
4Faculty of Agriculture, Okayama University, Tsushima Naka, Okayama 700-8530, Japan

Received September 4, 2000; Accepted October 29, 2000
Cell growth of three hundred iron-oxidizing bacteria isolated from natural environments was inhibited strongly by 0.05 mM, and completely by 0.2 mM of sodium tungstate (Na2WO4), respectively. Since no great difference in the level of tungsten inhibition was observed among the 300 strains tested, the mechanism of inhibition by Na2WO4 was studied with Acidithiobacillus ferrooxidans strain AP19-3. When resting cells of AP19-3 were incubated in 0.1 M β-alanine-SO2−4 buffer (pH 3.0) with 0.1 mM Na2WO4 for 1 h, the amount of tungsten bound to the cells was 12 μg/mg protein. The optimum pH for tungsten binding to the resting cells was 2~3. Approximately 2 times more tungsten bound to the cells at pH 3.0 than at pH 6.0. The tungsten binding was specifically inhibited by sodium molybdenum. However, copper, nickel, cadmium, zinc, manganese, cobalt, and vanadate did not disturb tungsten binding to the resting cells. The iron-oxidizing activity of AP19-3 was inhibited 24, 62, and 77％ by 1, 5, and 10 mM of Na2WO4, respectively. Among the components of iron oxidation enzyme system, iron:cytochrome c oxidoreductase activity was not inhibited by 10 mM of Na2WO4. In contrast, the activity of cytochrome c oxidase purified highly from the strain was inhibited 50 and 72％, respectively, by 0.05 and 0.1 mM of Na2WO4. The amounts of tungsten bound to plasma membrane, cytosol fraction, and a purified cytochrome c oxidase were 8, 0.5, and 191 μg/mg protein, respectively. From the results, the growth inhibition by Na2WO4 observed in A. ferrooxidans is explained as follows: tungsten binds to cytochrome c oxidase in plasma membranes and inhibits cytochrome c oxidase activity, and as a results, the generation of energy needed for cell growth from the oxidation of Fe2+ is stopped.
Key words: tungsten; Acidithiobacillus ferrooxidans; inhibition site; cytochrome c oxidase

-12-
Cloning and Expression of cDNA Encoding the Complete Prepro-Form
of an Isoform of Der f 1, the Major Group 1 Allergen
from House Dust Mite Dermatophagoides farinae

Takaomi YASUHARA, Toshiro TAKAI,・・・ Toshifumi YUUKI, Hirokazu OKUDAIRA,＊
and Yasushi OKUMURA

Bioscience Research and Development Laboratory, Asahi Breweries, Ltd., 1-21, Midori 1-chome,
Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan
＊Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113, Japan

Received September 7, 2000; Accepted October 23, 2000
cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.
Key words: house dust mite; Der f 1; cDNA; isoform; recombinant allergen

-13-
Investigation of Various Genotype Characteristics for Inosine Accumulation
in Escherichia coli W3110

Hiroshi MATSUI,・・・ Hisashi KAWASAKI, Megumi SHIMAOKA, and Osamu KURAHASHI

Fermentation ＆ Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku,
Kawasaki-shi, Kanagawa 210-8681, Japan

Received September 7, 2000; Accepted October 29, 2000
For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential.
Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5?-monophosphate (AMP) and guanosine 5?-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains.
It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.
Key words: purF, purA, deoD, purR, add gene; phosphoribosylpyrophosphate amidotransferase; adenosine deaminase; Escherichia coli; inosine production

-14-
Green Marker for Colonies of Bacillus subtilis

Mitsuhiro ITAYA, Syed M. SHAHEDUZZAMAN,・・・ Kuniko MATSUI, Akira OMORI,
and Takashi TSUJI

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

Received September 13, 2000; Accepted November 21, 2000
A Bacillus subtilis plasmid encoding a green fluorescence protein gene (gfp) was constructed. The fluorescence of B. subtilis colonies having this plasmid on agar plates was so high that they could be readily discerned visually under UV light. The fluorescence could be effectively expressed in three ways (i) through use of a strong bsr promoter (blasticidin S resistance gene), (ii) by efficient translation with the bsr translation system, and (iii) through increase in the copy number per cell. The high stability of the GFP plasmid was demonstrated by using more complicated growth conditions without any antibiotic for selection.
Key words: Bacillus subtilis; green fluorescence protein; fused protein; multi-copy plasmid

-15-
Purification and Characterization of Goose Type Lysozyme
from Cassowary (Casuarius casuarius) Egg White

Sompong THAMMASIRIRAK, Takao TORIKATA, Kazutoshi TAKAMI,1
Koichi MURATA,2 and Tomohiro ARAKI・・・

Department of Biochemistry, School of Agriculture, Kyushu Tokai University, Aso,
Kumamoto 869-1404, Japan
1Osaka Tennoji Zoo, 1-108, Chausuyama, Tennoji-ku, Osaka 543-0063, Japan
2Kobe Oji Zoo, 3-1, Oji-cho, Nada-ku, Kobe 657-0838, Japan

Received September 13, 2000; Accepted October 31, 2000
A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30°C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30°C for lytic activity and the activity was completely abolished at 80°C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50°C and the enzyme was stable up to 40°C.
Key words: lysozyme; goose; cassowary; chitinase; goose type lysozyme

-16-
Oxidative Stress by Visible Light Irradiation Suppresses Immunoglobulin
Production in Mouse Spleen Lymphocytes

Yoshiyuki MIYAZAKI, Masao YAMASAKI, Hiroko MISHIMA, Keiko MANSHO,
Hirofumi TACHIBANA, and Koji YAMADA・・・

Laboratory of Food Chemistry, Institute of Applied Biological Chemistry, Department of Bioscience
and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School
of Kyushu University 46-09, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received September 14, 2000; Accepted October 26, 2000
In this study, we attempted to induce the oxidative stress in mouse spleen lymphocytes with visible light irradiation and examined the effects of lipid peroxidation on immunoglobulin (Ig) production. The spleen lymphocytes were isolated from 8-week-old male balb/c mice and irradiated with 300 W visible light. When the cells were cultured for 72 hr, Ig contents in culture supernatants were decreased gradually by irradiation for over 30 min. The cell viability was also lowered by the irradiation. Intracellular phosphatidylcholine hydroperoxide (PCOOH) levels and thiobarbituric acid-reactive substances (TBARS) values in culture supernatants were measured as indices of lipid peroxidation and we found that Ig production by mouse spleen lymphocytes was suppressed accompanied with the progress of peroxidation of intracellular phospholipids. Cell membrane fluidity was also significantly decreased, but the intracellular Ig level was not changed in the irradiated cells. These results suggest that the peroxidation of intracellular lipids is a cause of the suppression of Ig production by mouse spleen lymphocytes via lowering cell viability and suppressing Ig synthesis and secretion.
Key words: immunoglobulin production; mouse spleen lymphocytes; visible light irradiation; lipid peroxidation; membrane fluidity

-17-
Effects of 2,3-Diketo-L-gulonic Acid on the Oxidation of Yolk Lipoprotein

Meihua LI,a Emiko SUZUKI,b and Tadao KURATAa,・・・

aInstitute of Environmental Science for Human Life, Ochanomizu University, bDepartment of Human
Biological Studies, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan

Received September 20, 2000; Accepted November 20, 2000
2,3-Diketo-L-gulonic acid (DKG) is an important intermediate product of oxidative degradation of L-ascorbic acid (AsA) in both biological and food systems, but the physiological function of DKG is still unclear. In this study, it was found that DKG had a strong antioxidative effect on copper-dependent oxidative modification of yolk lipoprotein (YLP), on the basis of both the decreased electrophoretic mobility and longer lag time of conjugated diene formation in a concentration-dependent manner. DKG is known to be very unstable and easily converts into two δ-lactones of DKG, the 3,4-enediol form of DKG δ-lactone (3,4-DKGL) and 2,3-enediol form of DKG δ-lactone (2,3-DKGL) depending on both pH and temperature. 3,4-DKGL was thought to be the first degradation product of DKG and could play an antioxidative role in the oxidation of lipoproteins induced by copper ion or peroxyl radicals in neutral aqueous solution.
Key words: 2,3-diketo-L-gulonic acid; 3,4-enediol or 2,3-enediol form of DKG δ-lactones; copper; oxidized YLP

-18-
Expression Pattern of the CsPK3 Auxin-Responsive Protein Kinase Gene

Makiko CHONO,1,2 Yoshihito SUZUKI,1 Keisuke NEMOTO,3 Hisakazu YAMANE,4
Noboru MUROFUSHI,5 and Isomaro YAMAGUCHI1

1Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
2Bio-oriented Technology Research Advancement Institution (BRAIN), 3-18-19 Toranomon, Minato-ku,
Tokyo, 105-0001, Japan
3Asian Natural Environmental Science Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
4Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
5Faculty of Bioresource Sciences, Akita Prefectural University, 241-7 Nakano, Shimoshinjyo, Akita-shi,
Akita 010-0195, Japan

Received September 22, 2000; Accepted November 10, 2000
We have previously cloned a cDNA of a putative serine/threonine protein kinase gene named CsPK3 from cucumber, the mRNA level of which was up-regulated by auxin and down-regulated by light irradiation. To examine the CsPK3 gene expression in detail, we cloned a genomic DNA of CsPK3 gene and made transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants containing the fused CsPK3 promoter-β-glucuronidase gene. The β-glucuronidase expression was detected in the shoot apex, vascular tissues, and the outermost layer of cortex. The histological distribution of CsPK3 mRNA in cucumber seedlings was supported by in situ hybridization, where the positive signals were observed in similar tissues as those observed by β-glucuronidase staining. The responsiveness of the CsPK3 gene to auxin and light was also confirmed for β-glucuronidase activity. The pattern of β-glucuronidase staining changed during the development of the tobacco seedlings. The results of our experiment showed that CsPK3 was expressed in a wide variety of tissues and cells in which the developmental and growth controls by auxin are suggested.
Key words: auxin; β-glucuronidase (GUS); Cucumis sativus L.; protein kinase

-19-
Improving Effect of Feeding with a Phosphorylated Guar Gum Hydrolysate
on Calcium Absorption Impaired by Ovariectomy in Rats

Osamu WATANABE,1,・・・ Hiroshi HARA,2 Yoritaka AOYAMA,2 and Takanori KASAI2

1Hokkaido Food Processing Research Center, Midorimachi 589-4, Bunkyodai, Ebetsu 069-0836, Japan
2Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9,
Kita-ku, Sapporo 060-8589, Japan

Received September 25, 2000; Accepted November 6, 2000
We have previously reported that a phosphorylated guar gum hydrolysate (P-GGH) promoted calcium absorption and the accumulation of bone calcium in rats. We now investigate the effect of P-GGH (50 g/kg of diet) on the intestinal calcium absorption and bones of ovariectomized (OVX) rats in comparison with sham-operated rats over a six-week ingestion period.
The apparent calcium absorption was decreased by aging and ovariectomy in the rats fed on the control and GGH diets (50 g/kg of diet), but not in the rats fed on the P-GGH diet. The absorption was higher in the P-GGH group than in the GGH and control diet groups in the fourth and sixth weeks after feeding the test diets to OVX rats.
Femoral calcium and strength were decreased by OVX in the rats fed on the control and GGH diets, but not in the rats fed on the P-GGH diet. The values of these parameters were higher in the P-GGH group than in either the control or GGH group of OVX rats.
The amount of soluble calcium in the ileal contents was higher in the P-GGH group than in the control and GGH groups. These results indicate that P-GGH may be useful for preventing the reduction of intestinal calcium absorption and bone in the condition of estrogen deficiency.
Key words: calcium; phosphorylation; guar gum hydrolysate; rat; ovariectomized

-20-
Structures of Thermoactinomyces vulgaris R-47 α-Amylase II Complexed
with Substrate Analogues

Takehiro YOKOTA,1 Takashi TONOZUKA,1 Yoichiro SHIMURA,1 Kazuhiro ICHIKAWA,1
Shigehiro KAMITORI,2 and Yoshiyuki SAKANO1,・・・

1Department of Applied Biological Science, Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan
2Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology,
2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan

Received September 27, 2000; Accepted December 1, 2000
The structures of Thermoactinomyces vulgaris R-47 α-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl β-cyclodextrin (mβ-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2 ・ and 3.3 ・ resolution, respectively. In d325nTVA II-mβ-CD complex, the orientation and binding-position of β-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to α-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.
Key words: crystal structure; α-amylase; TVA II; pullulan

-21-
Characterization and High-level Production of D-Amino Acid Oxidase
in Candida boidinii

Hiroya YURIMOTO, Tetsuya HASEGAWA, Yasuyoshi SAKAI,・・・ and Nobuo KATO

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

Received October 5, 2000; Accepted November 30, 2000
D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host. The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor. The enzyme was active toward several D-amino acids such as D-Ala, D-Met, and D-Ser. An alcohol oxidase-depleted strain (aod1Δ) was found to be a more suitable host for DAO production than the wild-type strain. Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1Δ strain. Finally, an aod1Δ strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30％ of the total soluble proteins.
Key words: Candida boidinii; D-amino acid oxidase; peroxisome; high-level production; gene expression

-22-
Resolution and Synthesis of Optically Active Alcohols with Immobilized
Ovalbumin and Pea Protein as New Bio-catalysts

Hiroyuki NAGAOKA,1 Hiroshi KAYAHARA,2 and Yasushi WAKABAYASHI2

1Sanyo Shokuhin Co., Ltd., 555-4 Asakura, Maebashi Gunma 371-0811, Japan
2Department of Bioscience and Biotechnology, Shinshu University, 8304 Minamiminowa, Kamiina,
Nagano 399-4598, Japan

Received October 11, 2000; Accepted November 28, 2000
It was found that ovalbumin stereoselectively oxidized one of the enantiomers of p-substituted racemic alcohols, thereby providing optically active alcohols with high optical purities. It was found out that, when used appropriately in combination with immobilized pea protein, immobilized ovalbumin made it possible to resolve and synthesize racemic 1-(2-naphthyl)ethanol,
1-phenylethanol, and 1-phenyl-1-propanol. Immobilized ovalbumin could be continuously recycled at least three times without lowering the yield and purity of the products. These results suggested that cereals, beans, and ovalbumin might have additional fourth function among conventional foods. Namely, there might contain nutritional, sensory, biologically regulatory and bio-catalytic functions in conventional foods.
Key words: immobilized ovalbmin; 1-(4-substitutedphenyl) ethanol; 1-(2-naphthyl)ethanol; 1-phenylethanol; 1-phenyl-1-propanol

-23-
Capsaicin Increases Modulation of Sympathetic Nerve Activity in Rats:
Measurement Using Power Spectral Analysis of Heart Rate Fluctuations

Koichiro OHNUKI, Toshio MORITANI, Kengo ISHIHARA, and Tohru FUSHIKI

Laboratory of Nutritional Chemistry, Department of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan

Received October 13, 2000; Accepted November 17, 2000
We assessed the sympatho-vagal activities of the heart after administration of capsaicin by measuring the power spectral analysis in rats. There were major two frequency components of heart rate variability, which we defined as high (1.0 Hz<, HF) and low (LF, <1.0 Hz) frequency components. Vagal blockade by atropine abolished the high frequency component, and lowered the amplitude of the low frequency component. On the other hand, under conditions of sympathetic blockade by propranolol, the low frequency component was reduced. Combined vagal and sympathetic blockade abolished all heart rate fluctuations. We analyzed the low and high frequency components by integrating the spectrum for the respective band width. The rats administered capsaicin had a higher heart rate and sympathetic nervous system index (LF/HF) than the control group of rats. These results suggest that power spectral analysis is an effective and noninvasive method for detecting subtle changes in autonomic activity in response to the intake of foods or drugs.
Key words: capsaicin; autonomic nerve; heart rate; telemetry system

-24-
ATP Production from Adenine by a Self-coupling Enzymatic Process:
High-level Accumulation under Ammonium-limited Conditions

Akihiko MARUYAMA and Tatsuro FUJIO＊

Bio-Chemicals Development Department, Bio-chemicals Division, and ＊Research and Development Division,
Kyowa Hakko Kogyo Co., Ltd., 1-6-1 Ohtemachi, Chiyoda-ku, Tokyo 100-8185, Japan

Received October 17, 2000; Accepted November 20, 2000
To improve ATP production from adenine, we optimized cultivation and reaction conditions for the ATP producing strain, Corynebacterium ammoniagenes KY13510. In the conventional method, 28％ NH4OH has been used both to adjust pH during cultivation and reaction, and to provide nitrogen for cell growth. In the ATP-producing reaction, high concentrations of inorganic phosphate and magnesium ion are needed, which form magnesium ammonium phosphate (MgNH4PO4) precipitate. To keep inorganic phosphate and magnesium ions soluble in the reaction mixture, it was indispensable to add phytic acid as a chelating agent of divalent metal ions. Under such conditions, 37 mg/ml (61.2 mM) ATP was accumulated in 13 h (Appl. Microbiol. Biotechnol. 21, 143 1985). If ammonium ion was depleted from the reaction mixture to avoid MgNH4PO4 formation, we expected that there was no need to add phytic acid and ATP accumulation might be improved. Therefore, we obtained the cultured broth of C. ammoniagenes KY13510 strain with low ammonium ion content (less than 1 mg/ml as NH3) by the method that a part of alkali solution (28％ NH4OH) for pH control was replaced with 10 N KOH. Using this culture broth, ATP producing reaction was done in 2-liter jar fermentor, controlling the pH of the reaction mixture with 10 N KOH. Under these conditions, the rate of ATP accumulation improved greatly, and 70.6 mg/ml (117 mM) ATP was accumulated in 28 h. The molar conversion ratio from adenine to ATP was about 82％. Phytic acid was slightly inhibitory to ATP formation under these ammonium-limited conditions.
Key words: ATP; enzymatic reaction; ammonium ion; phytic acid; Corynebacterium ammoniagenes

-25-
Note
Inhibition of Telomerase Activity by Fungus Metabolites, CRM646-A
and Thielavin B

Ken-ichi TOGASHI,1,・・・・・・ Hack-Ryong KO,1,2 Jong-Seog AHN,2 and Hiroyuki OSADA1,・・・

1Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan
2Cellular Response Modifier Research Unit, Korea Research Institute of Bioscience and Biotechnology,
PO Box 115, Yusong, Taejon 305-600, Korea

Received May 23, 2000; Accepted October 25, 2000
We performed a screening program to identify telomerase inhibitors from our drug source obtained from fungus fermentations, and found that two compounds, CRM646-A and thielavin B, inhibited telomerase activity at doses of 3.2 and 32 μM, respectively. These compounds also inhibited the activity of viral reverse transcriptase at almost the same dose levels which inhibited telomerase activity.
Key words: screening; telomerase; CRM646-A; thielavin B; reverse transcriptase

-26-
Note
Isolation and Characterizartion of Alginic Acid from Commercially
Cultured Nemacystus decipiens (Itomozuku)

Masakuni TAKO,・・・ Seiki KIYUNA, Shuntoku UECHI, and Fujiya HONGO

Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus,
Nishihara, Okinawa 903-0123, Japan

Received July 19, 2000; Accepted October 30, 2000
An alginate was isolated from commercially cultured Nemacystus decipiens which had been harvested in Yonashiro Town (Okinawa, Japan). The yield of the alginate was 1.6％ (w/w of wet alga), and the uronic acid, ash and moisture contents of the alginate were 86.0％, 12.0％, and 2.3％ (w/w), respectively. The molecular mass of the alginate was estimated to be about 1.5×105. The infrared spectrum and optical rotation of the alginate were in agreement with those of the standard alginate. D-Mannuronic acid and L-guluronic acid were identified by 1H- and 13C-NMR spectroscopy, the molar ratio of both sugar residues being estimated to be 0.72:1.00.
Key words: alginic acid; Itomozuku; Nemacystus decipiens

-27-
Note
Purification and Characterization of Mannose Isomerase
from Agrobacterium radiobacter M-1

Jun HIROSE,・・・ Kazuhiko MAEDA, Haruhiko YOKOI, and Yoshiyuki TAKASAKI

Department of Applied Chemistry, Faculty of Engineering, Miyazaki University, Miyazaki 889-2192, Japan

Received July 21, 2000; Accepted November 13, 2000
A mannose isomerase from Agrobacterium radiobacter M-1 (formerly Pseudomonas sp. MI) was purified to electrophoretic homogeneity and characterized. A cell-free extract was separated by ammonium sulfate fractionation, Butyl-Toyopearl 650M, DEAE-Sepharose and hydroxylapatite column chromatography. Its molecular mass was estimated to be 44 kDa by SDS-PAGE and 90 kDa by gel filtration, in which the enzyme is most likely a dimer composed of two identical subunits. The purified enzyme had an optimum pH at 8.0, an optimum temperature at 60°C, a pI of 5.2 and a Km of 20 mM, and specifically converted D-mannose and D-lyxose to ketose. The N-terminal amino acid sequence was identified.
Key words: D-mannose; mannose isomerase; Agrobacterium radiobacter; purification

-28-
Note
Polymorphism in Rice Amylases at an Early Stage of Seed Germination

Shin-ichiro MITSUNAGA, Osamu KAWAKAMI,＊ Tomoyo NUMATA, Junji YAMAGUCHI,＊＊
Kiichi FUKUI,＊＊＊ and Toshiaki MITSUI＊＊＊＊

Department of Life and Health Science, Joetsu University of Education, Joetsu, Niigata 943-8512, Japan
＊Niigata Agricultural Research Institute, Nagaoka, Niigata 940-0826, Japan
＊＊BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan
＊＊＊Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita,
Osaka 565-0871, Japan
＊＊＊＊Laboratory of Molecular Life Sciences, Graduate School of Science and Technology,
Niigata University, Niigata 950-2181, Japan

Received July 25, 2000; Accepted October 31, 2000
A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In nonglutinous cultivars of rice, α-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, β-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of α-amylases are repressed.
Key words: rice; α-amylase; β-amylase; zymogram

-29-
Note
Characterization of the Gene Encoding the β-Lactamase of the Psychrophilic
Marine Bacterium Moritella marina Strain MP-1

Mika TANAKA,1 Hidetoshi OKUYAMA,1,2 and Naoki MORITA1,・

1Bioscience and Chemistry Division, Hokkaido National Industrial Research Institute, AIST/MITI,
Toyohira-ku, Sapporo 062-8517, Japan
2Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science,
Hokkaido University, Kita-ku, Sapporo 060-0810, Japan

Received August 22, 2000; Accepted October 31, 2000
The β-lactamase gene (mbla) of the psychrophilic marine bacterium Moritella marina strain MP-1 was identified in a previously isolated genomic DNA fragment and it was expressed in Escherichia coli cells. The mbla gene encoded a protein consisting of 287 amino acid residues. Its predicted amino acid sequence showed approximately 50％ identity with that of a number of class A β-lactamases, especially with that of CARB/PSE type of β-lactamases (carbenicillinases). E. coli transformed with the plasmid containing mbla grew on an ampicillin-containing plate at 37°C but not at 42°C, suggesting that the β-lactamase of this bacterium is heat-labile.
Key words: β-lactamase; Moritella marina; psychrophile; heat-labile enzyme

-30-
Note
Suppression of Lipopolysaccharide-induced Liver Injury by Various Types
of Tea and Coffee in D-Galactosamine-sensitized Rats

Puming HE, Yasuhiro NODA, and Kimio SUGIYAMA・・・

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

Received September 4, 2000; Accepted October 26, 2000
Extracts of various types of tea and coffee significantly suppressed lipopolysaccharide (LPS)-induced liver injury, as assessed by the plasma enzyme activities, in D-galactosamine-sensitized rats when administered orally once before injecting the drugs. There was a significant negative correlation between the caffeine levels of these extracts and liver injury. Authentic caffeine also had a hepatoprotective effect. These results suggest that caffeine-containing beverages generally suppress LPS-induced liver injury according to their caffeine content.
Key words: tea; coffee; caffeine; lipopolysaccharide; liver injury

-31-
Note
Suppressive Effect of Caffeine on Hepatitis and Apoptosis Induced by Tumor
Necrosis Factor-α, but Not by the Anti-Fas Antibody, in Mice

Kimio SUGIYAMA,・・・ Yasuhiro NODA, and Puming HE

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

Received September 4, 2000; Accepted October 26, 2000
Tumor necrosis factor (TNF)-α-induced hepatitis and apoptosis, as respectively assessed by serum enzyme activities and hepatic DNA fragmentation were effectively suppressed by a single force-feeding of caffeine (100 mg/kg) 1.5 h before injecting the drug. In contrast, caffeine had no significant effect on anti-Fas antibody-induced hepatitis and apoptosis. These results suggest that caffeine differentially affected TNF-α receptor- and Fas-mediated hepatitis and apoptosis.
Key words: caffeine; tumor necrosis factor-α; anti-Fas antibody; hepatitis; apoptosis

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Note
Molecular Cloning of Three Genes Encoding G Protein Alpha Subunits
in the White Root Rot Fungus, Rosellinia necatrix

Tadanori AIMI, Sanae KANO, Qian WANG, and Tsutomu MORINAGA・・・

Department of Bioresource Development, Hiroshima Prefectural University, Nanatsukahara, Shobara-shi,
Hiroshima 727-0023, Japan

Received September 4, 2000; Accepted October 20, 2000
Three genes encoding G protein alpha subunits were cloned from the white root rot fungus, Rosellinia necatrix, and characterized. Only one copy of each gene was present in the genome. The protein sequences of Rga1, Rga2, and Rga3 are very similar to those of MagA, MagB and MagC of Magnaporthe grisea, respectively. Moreover, Rga1 is similar to Mod-D which is closely related to vegetative incompatibility in Podospora anserina, which suggests that Rga1 is important in the vegetative incompatibility reaction in R. necatrix. Reverse transcription PCR (RT-PCR) analysis of Rga1, Rga2, and Rga3 mRNA expression showed that the three genes were all transcribed in R. necatrix cells.
Key words: Rosellinia; G protein alpha subunits; vegetative incompatibility; white root rot fungus

-33-
Note
Antiviral Activity of Fattiviracin FV-8 against Human Immunodeficiency
Virus Type 1 (HIV-1)

El-Sayed E. HABIB,1 Kazumi YOKOMIZO,1 Kazuhiko NAGAO,2
Shinji HARADA,2 and Masaru UYEDA1,・・・

1Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto, 862-0973, Japan
2School of Medicine, and Center for AIDS Research, Kumamoto University, 2-2-1 Honjou,
Kumamoto 860-0811, Japan

Received September 6, 2000; Accepted October 23, 2000
A novel antiviral agent, fattiviracin FV-8, purified from the culture broth of Streptomyces microflavus strain No. 2445, showed potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV), and influenza A and B viruses. The action mechanism of fattiviracin FV-8 against HIV-1 was examined. As a result, the agent was thought to act on HIV-1 particles directly without lysis of the particles, and it affords the inhibition of viral entry into the host cells.
Key words: Streptomyces microflavus; fattiviracin; antiviral agent; human immunodeficiency virus type 1

-34-
Note
A Lectin from an Ascomycete Mushroom, Melastiza chateri:
No Synthesis of the Lectin in Mycelial Isolate

Shigeru OGAWA, Yumi OTTA, Akikazu ANDO, and Yoshiho NAGATA・・・

Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo 271-8510, Japan

Received September 13, 2000; Accepted October 23, 2000
Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.
Key words: ascomycete mushroom; L-fucose-specific lectin; Melastiza chateri; mycelial isolate

-35-
Note
Piezoresponse of the cyo-Operon Coding for Quinol Oxidase Subunits
in a Deep-sea Piezophilic Bacterium, Shewanella violacea

Kaoru NAKASONE,＊,・・・ Mitsunori YAMADA,＊,＊＊ Mohammad Hassan QURESHI,＊,＊＊＊
Chiaki KATO,＊ and Koki HORIKOSHI＊

＊The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho,
Yokosuka 237-0061, Japan
＊＊Department of BioScience, Faculty of Science, Tokyo Metropolitan University, Hachioji,
Tokyo 192-0316, Japan
＊＊＊Department of Biological Science, University of Calgary, Calgary, Alberta, Canada T2N IN4

Received September 18, 2000; Accepted November 8, 2000
We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea. Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase. Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure. Upstream in the cyo-operon, a σ54-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure.
Key words: cyo-operon; piezophilic bacterium; pressure-regulation; quinol oxidase; Shewanella violacea

-36-
Note
Reactivities of Mutants of a Major House Dust Mite Allergen Der f 2 to Mouse
Anti-Der f 2 Monoclonal Antibodies Analyzed by Immunoblotting

Toshiro TAKAI,・・・ Midori AKAGAWA-CHIHARA, Toyokazu YOKOTA, and Yasushi OKUMURA

Bioscience Research and Development Laboratory, Asahi Breweries, Ltd., 1-21, Midori 1-chome,
Moriya-machi, Kitasoma-gun, Ibaraki, 302-0106, Japan

Received September 22, 2000; Accepted November 6, 2000
A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli. The cells were solubilized and run on SDS-PAGE under reducing conditions. Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.
Key words: major house dust mite allergen; Der f 2; site-directed mutagenesis; epitope; immunoblotting

-37-
Note
Thermally Induced Changes of Lipoate Acetyltransferase Inner Core Isolated
from the Bacillus stearothermophilus Pyruvate Dehydrogenase Complex

Yoichi ASO, Akihiro NAKAJIMA, Kohji MENO, and Masatsune ISHIGURO

Laboratory of Protein Chemistry and Engineering, Graduate School of Genetic Resources Technology,
Kyushu University, Fukuoka 812-8581, Japan

Received September 22, 2000; Accepted November 6, 2000
Incubation at 70°C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate. The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2--2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.
Key words: Bacillus stearothermophilus; lipoate acetyltransferase; thermostability; pyruvate dehydrogenase complex

-38-
Note
Autoproteolytic Processing of Aspartic Proteinase from Sunflower Seeds

Hyekyeong PARK, Isao KUSAKABE, Yoshikiyo SAKAKIBARA,＊ and Hideyuki KOBAYASHI＊,・・・

Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305-0006, Japan
＊National Food Research Institute, Ministry of agriculture, Forestry and Fisheries, Ibaraki 305-8642, Japan

Received September 22, 2000; Accepted November 2, 2000
The autoproteolytic processing of mature aspartic proteinase from sunflower seeds was investigated. The mature aspartic proteinase (48 kDa) was processed at N65s-D66s in the plant-specific region of the enzyme to form 34-kDa and 14-kDa subunits. The next step was the hydrolysis of the A25s-Q26s and N97s-E98s bonds to form a 39-kDa enzyme that consisted of 29-kDa and 9-kDa disulfide-bonded subunits. Finally, bonds including V1s-M2s, M2s-S3s, C100s-D101s, and D101s-R102s were cleaved to form non-covalently bound subunits (29 kDa and 9 kDa) by eliminating the disulfide bonds in the plant-specific region of the protein.
Key words: aspartic proteinase; plant-specific region; autoproteolytic processing; sunflower seed

-39-
Note
Chemical and Nutritional Properties of Hypoallergenic Wheat Flour

Megumi MORIYAMA,＊ Chiyoko TOKUE,＊ Hideko OGIWARA,＊＊
Hiroko KIMURA,＊＊ and Soichi ARAI＊,・・・

＊Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture,
Setagaya-ku, Tokyo 156-0054, Japan
＊＊Kagawa Nutrition University, Toshima-ku, Tokyo 170-8481, Japan

Received September 22, 2000; Accepted November 2, 2000
The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that the absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.
Key words: hypoallergenic wheat flour; wheat protein; peptide absorption

-40-
Note
New Quinoline Alkaloids from the Leaves and Stems of Orixa japonica,
Orijanone, Isopteleflorine and 3'-O-Methylorixine

Toshiro NOSHITA, Masumi TANDO, Kadzuo SUZUKI, Kiyoshi MURATA,§ and Shinji FUNAYAMA・・・

Department of Bioscience and Biotechnology, Aomori University, 2-3-1 Kohbata, Aomori 030-0943, Japan
§Aoba Kasei Co. Ltd., 4-19-1 Ake-Dori, Sendai 981-3206, Japan

Received September 25, 2000; Accepted October 30, 2000
Orixa japonica (Rutaceae) is a shrub widely distributed in Japan, and has been found to contain various quinoline alkaloids.
We investigated the alkaloidal constituents of O. japonica, and four quinoline alkaloids were isolated and characterized. Three of these alkaloids are new natural products.
Key words: Orixa japonica; orijanone; isopteleflorine; 3?-O-methylorixine; quinoline alkaloid

-41-
Note
Unique Occurrence of the 1CF11 Carbohydrate Epitope in Primate Saliva

Shan-Ming YANG,1 Naoko YOKOI,1 Yoshihiro KANAMARU,1,・・・ Osamu TAKENAKA,2
Yasuro ATOJI,3 Yasuo BUNAI,4 Isao OHYA,4 Xong-Guang SONG,1 Satoshi NAGAOKA,1
Makoto SHIMIZU,5 and Goverdhan P. SACHDEV6

1Department of Food Science, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan
2Molecular Biology Section, Primate Research Institute, Kyoto University, Aichi 484-8506, Japan
3Division of Veterinary Medicine, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan
4Department of Forensic Medicine, Gifu University School of Medicine, Gifu 500-8705, Japan
5Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan
6College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, U.S.A

Received September 25, 2000; Accepted November 15, 2000
We examined a large number of individual human and animal saliva samples for the reactivity with 1CF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of 1CF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with 1CF11.
Key words: 1CF11; monoclonal antibody; saliva; humans; simians

-42-
Note
Primary Structure and Phylogenetic Analysis of the Coat Protein
of a Toyama Isolate of Tobacco Necrosis Virus

Kazuhiko SAEKI,・・・ Yasuhiro TAKAHASHI, Hirozo OH-OKA, Tatsumi UMEOKA,
Yutaka ODA, and Keiichi FUKUYAMA

Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

Received September 26, 2000; Accepted November 11, 2000
The amino acid sequence of the coat protein (CP) of a tobacco necrosis virus (TNV) strain, Toyama isolate, was determined by a combination of peptide and cDNA sequencing. The deduced sequence of 276 residues was compared with CPs of other TNV isolates and other plant virus isolates of Tombusviridae. It showed the highest similarity to the TNV Nebraska isolate with 92％ identity and moderate similarity to the TNV strain A with 51％ identity, confirming the previous serological analysis. It also showed overall similarity with CPs of mostly genera Necrovirus and Sobemovirus, and partial similarity with CPs of genera Tombusvirus and Carmovirus. Among 13 CPs that showed overall similarity, there were 10 completely conserved residues. These included three residues that participate in Ca2+ ligation at the interfaces of virion subunits in TNV crystal structure, suggesting that similar metal binding occur in the viruses of genera Necrovirus and Sobemovirus.
Key words: tobacco necrosis virus; plant virus; coat protein; amino acid sequence

-43-
Note
The est1 Regulation Depends on the Oxygen Concentration
in Acetobacter pasteurianus

Yasuhiro KASHIMA,1,・・・ Yusuke NAKAJIMA,1 Akihiko KOSUGI,1 Kenji TAYAMA,2
Yukimichi KOIZUMI,1 Shigezo UDAKA,1 and Fujiharu YANAGIDA1

1Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture,
1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
2Central Research Institute, Nakano Vinegar Co., Ltd., Nakamura-cho, Handa, Aichi 475-0873, Japan

Received October 10, 2000; Accepted November 9, 2000
The regulation mechanism for expression of the ethanol inducible esterase gene, est1, was investigated in A. pasteurianus. Deletion analysis of the 5? non coding region of est1 showed that the FNR-binding consensus sequence is important in the induction of est1 by ethanol. Cells grown under oxygen starvation produced esterase-1 in not only the presence but also the absence of ethanol. These results suggest that the induction of est1-expression depends on the oxygen concentration, and the gene may be induced by a FNR-like factor activated by a decrease in the intracellular oxygen concentration.
Key words: Acetobacter pasteurianus; FNR; esterase; transcriptional regulation; ethanol

-44-
Note
MUP1, High Affinity Methionine Permease, is Involved in Cysteine Uptake
by Saccharomyces cerevisiae

Akihiko KOSUGI,＊ Yukimichi KOIZUMI, Fujiharu YANAGIDA, and Shigezo UDAKA

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

Received October 10, 2000; Accepted November 20, 2000
Using a mutant defective in cysteine uptake, which is resistant to a toxic analog of cysteine, allylglycine, we searched for a gene that complements the defect in cysteine uptake in a yeast genomic library and found a DNA fragment causing the recovery of cysteine uptake and sensitivity to allylglycine. The gene in the fragment was identical to MUP1, the high affinity methionine permease gene. We conclude that Mup1 is a major permease in cysteine uptake.
Key words: cysteine permease; methionine permease; Saccharomyces cerevisiae; allylglycine

-45-
Note
Synthesis of Gibbilimbols A-D, Cytotoxic and Antibacterial Alkenylphenols
Isolated from Piper gibbilimbum

Yumi ABE,a Hirosato TAKIKAWA,b and Kenji MORIa,b・・・

aDepartment of Mathematics and Science Education, Graduate School of Science, and
bDepartment of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan

Received October 11, 2000; accepted October 20, 2000
Gibbilimbols A [(E)-4-(4-decenyl)phenol, 1], B [(E)-4-(3-decenyl)phenol, 2], C [(E)-4-(4-octenyl)phenol, 3] and D [(E)-4-(3-octenyl)phenol, 4] were synthesized by coupling the phenolic parts with the alkyne parts and then reducing the triple bond of the resulting alkynylphenols. These alkenylphenols (1--4) are the cytotoxic and antibacterial constituents of the leaves of a medicinal plant (Piper gibbilimbum) that is used as a traditional medicine in Papua New Guinea.
Key words: alkenyl phenols; antibacterial activity; cytotoxic activity; Piper gibbilimbum

-46-
Preliminary Communication
Designing Potent Derivatives of Ovokinin(2--7), an Anti-hypertensive
Peptide Derived from Ovalbumin

Nobuyuki MATOBA, Yuko YAMADA, Hachiro USUI, Ryusuke NAKAGIRI,・・・
and Masaaki YOSHIKAWA

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

Received October 16, 2000; Accepted November 28, 2000
We obtained a potent anti-hypertensive peptide, RPFHPF, by replacing the amino acid residues of ovokinin(2--7) (RADHPF), an orally active anti-hypertensive peptide derived from ovalbumin. After intravenous administration in anesthetized Wistar rats, the designed peptide [Pro2, Phe3]-ovokinin(2--7) had a long-lasting hypotensive activity at a dose of 10 mg/kg, while that of ovokinin(2--7) was only transient even at a dose of 100 mg/kg. After oral administration in conscious spontaneously hypertensive rats (SHRs), [Pro2, Phe3]-ovokinin(2--7) significantly lowered the systolic blood pressure in a dose-dependent manner. It is noteworthy that the minimum effective dose of [Pro2, Phe3]-ovokinin(2--7) was 0.3 mg/kg, about one-thirtieth of that of ovokinin(2--7). On the other hand, orally administered [Pro2, Phe3]-ovokinin(2--7) did not show any significant hypotensive effect in normotensive Wistar-Kyoto rats (WKYs) even at a dose of 3 mg/kg. Taken together, [Pro2, Phe3]-ovokinin(2--7) proved to be an ideal, potent anti-hypertensive peptide with little effect on normal blood pressure when given orally.
Key words: ovalbumin; ovokinin; anti-hypertensive peptide; peptide design; spontaneously hypertensive rat (SHR)

-47-
Preliminary Communication
Structures of Azaspiracid Analogs, Azaspiracid-4 and Azaspiracid-5,
Causative Toxins of Azaspiracid Poisoning in Europe

Katsuya OFUJI,1 Masayuki SATAKE,1,・・・ Terry MCMAHON,2 Kevin J. JAMES,3
Hideo NAOKI,4 Yasukatsu OSHIMA,1 and Takeshi YASUMOTO1

1Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku,
Sendai 981-8555, Japan
2Marine Institute, Fisheries Research Centre, Dublin, Ireland
3Chemistry Department, Cork Institute of Technology, Cork, Ireland
4Suntory Institute for Bioorganic Research, Osaka, Japan

Received October 23, 2000; Accepted December 15, 2000
Two new analogs of azaspiracid, azaspiracid-4 and azaspiracid-5, isolated from the mussel Mytilus edulis, involved in a newly emerged shellfish poisoning in Europe were determined to be 3-hydroxy-22-
demethylazaspiracid and 23-hydroxy-22-demethylazaspiracid, respectively.
Key words: azaspiracid; shellfish poisoning; azaspiracid analogs